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For genes that encode one component of a multimeric protein complex, measuring only one phenotype often… Kalas, Pamela 2008

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FOR GENES THAT ENCODE ONE COMPONENT OF A MULTIMERIC PROTEIN COMPLEX, MEASURING ONLY ONE PHENOTYPE OFTEN GIVES A BIASED VIEW OF FUNCTION: SU(VAR)3-9 AND CHROMATIN ARCHITECTURE AS AN EXAMPLE. by Pamela Kalas  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2008  Pamela Kalas, 2008  ABSTRACT Eukaryotic genomes are organized into chromatin, a highly dynamic complex of DNA and proteins, which plays a critical role in the regulation of genes expression. This thesis focuses on the study of a non-histone chromatin protein, the SET domain-containing H3K9 methyltransferase (HMTase) SU(VAR)3-9, and its role in the packaging and regulation of a euchromatic locus, the histone genes cluster (HIS-C). SU(VAR)3-9 was discovered in Drosophila melanogaster, but it is highly conserved from yeast to mammals. It has two conserved domains, the chromo- and the SET domains, and both are required for its function in gene silencing. The SET domain is responsible for the catalytic activity of SU(VAR)3-9, while the exact function of the chromo domain is still unclear. To gain an insight on the role(s) of SU(VAR)3-9 in the regulation of gene silencing, we first characterized a collection of Su(var)3-9 EMS-induced mutants that had been isolated in a genetic screen for strong, dominant suppressors of position-effect variegation (PEV). These mutants were characterized at the molecular, enzymatic, and cellular level, and their effect on gene silencing was also examined. We found that all mutants have single amino acid substitutions in the conserved preSET/SET/postSET domain, and that they all display a dramatic or complete loss of HMTase activity, strongly suggesting that suppression of PEV is linked to SU(VAR)3-9’s ability to methylate H3K9. The HIS-C is a natural, euchromatic target of SU(VAR)3-9, and mutations in Su(var)3-9 can alter its chromatin structure. To investigate the exact role(s) of SU(VAR)3-9 in the regulation of this locus, we analyzed the effects of a series of  ii  Su(var)3-9 missense mutants on the chromatin architecture of the HIS-C and on the expression of the histone genes. We detected a drastic reduction in the levels of H3K9me2 and HP1 associated with the his genes in all Su(var)3-9 missense mutants, although the mutant SU(VAR)3-9 still associate with the HIS-C. In addition, these mutants have elevated amounts of histone H2A and histone H3 RNA, suggesting that the enzyme function of SU(VAR)3-9 is critical for the regulation of the histone genes.  iii  TABLE OF CONTENTS Abstract…………………………………………………………………………………. ii Table of contents………………………………………………………………………. iv List of tables……………………………………………………………………………. vi List of figures……………………………………………………………………….….. vii List of abbreviations…………………………………………………………………… ix Co-Authorship statement……………………………………………………………… x  CHAPTER 1 INTRODUCTION 1.1. Chromatin and gene regulation: an overview………………………….. 1 1.2. The components of chromatin…………………………………………… 2 1.3. SU(VAR)3-9 and heterochromatin assembly…………………………. 28 1.4. The histone gene cluster (HIS-C)…………………………………….…33 1.5. Subject(s) of this study………………………………………………….. 38 1.5. References……………………………………………………………….. 42  CHAPTER 2 ALTERED HISTONE H3 METHYLTRANSFERASE ACTIVITY OF SU(VAR)3-9 IMPAIRS GENE SILENCING 2.1. Introduction……………………………………………………………… 57 2.2. Results…………………………………………………………………… 59 2.3. Discussion……………………………………………………………….. 89 2.4. Materials and methods………………………………………………….102 2.5. References……………………………………………………………….111  iv  CHAPTER 3 THE ROLE OF SU(VAR)3-9 IN THE REGULATION OF DROSOPHILA’S HISTONE GENE CLUSTER (HIS-C) 3.1. Introduction……………………………………………………………… 117 3.2. Results……………………………………………………………………124 3.3. Discussion………………………………………………………………..152 3.4. Materials and methods………………………………………………….166 3.5. References……………………………………………………………….171  CHAPTER 4 GENERAL SUMMARY AND DISCUSSION 4.1. Summary of results…………………………………………………….. 176 4.2. The function(s) of SU(VAR)3-9 in PEV and in the regulation of the HIS-C…………………………………………………………………………..178 4.3. Drosophila SU(VAR)3-9 has a unique N-terminal region………….. 190 4.4. References……………………………………………………………… 193  APPENDICES Appendix 1…………………………………………………………………….195 Appendix 2…………………………………………………………………….196 Appendix 3…………………………………………………………………….197 Appendix 4…………………………………………………………………….199 Appendix 5…………………………………………………………………… 201 Appendices 6 and 7…………………………………………………………. 202  v  LIST OF TABLES Table 1.1. Post-translational modifications of canonical histones that have formally been detected in Drosophila………………………………………………… 6 Table 1.2. A list of characterized Drosophila SET domain-containing proteins……………………………………………………………………..19 Table 1.3. Drosophila proteins identified through screens for strong, dominant suppressors of PEV……………………………………………………………………24 Table 1.4. Additional Su(var)s……………………………………………………….. 26 Table 2.1. Putative Su(var)3-9 alleles that were mapped and sequenced in this study……………………………………………………………………………………. 62 Table 2.2. Summary of the molecular, biochemical, and Su(var) phenotypes of five Su(var)3-9 mutant alleles examined in this study…………………………… 101 Supplementary table 2.1……………………………………………………………. 109 Supplementary table 2.2……………………………………………………………. 110 Supplementary table 3.1……………………………………………………………. 170 Table 4.1. Summary of the characteristics of the Su(var)3-9 mutants analyzed…………………………………………………………………..189  vi  LIST OF FIGURES Figure 1.1. A schematic of the nucleosome core particle…………………………. 7 Figure 1.2. Histone H3 and H4 post-translational modifications………………….12 Figure 1.3. Structure of a SET domain, in this case that of SET7/9…………….. 17 Figure 1.4. A simplified diagram showing the assembly of heterochromatin……31 Figure 1.5. A schematic of Drosophila’s histone unit…………………………….. 35 Figure 2.1. Su(var)3-9 mutants are clustered in the preSET, SET and postSET domains………………………………………………………………………………... 66 Figure 2.2. Detection of Su(var)3-9 gene product in the EMS-induced Su(var)3-9 missense mutants, and in the null allele Su(var)3-906…………………………….. 69 Figure 2.3. Detection of H3K9me2 in Su(var)3-9 mutants……………………….. 74 Figure 2.4. The HMTase activity of variant SU(VAR)3-9 proteins………………. 78 Figure 2.5. HMTase activity of Su(var)3-9 alleles using unmodified and dimethylated H3 tail peptides…………………………………………………………82 Figure 2.6. Effect of several Su(var)3-9 alleles on PEV (here, wm4 variegation) in Su(var)3-9 hyperploid strains……………………………………………………...….87 Figure 2.7. Probable tertiary structure of the catalytic region of SU(VAR)3-9 and relative positions of the mutated residues………………………………………….. 95 Figure 3.1. A schematic of the histone unit……………………………………….. 122 Figure 3.2. Relative levels of SU(VAR)3-9 associated with 3 regions of the histone unit in wild type and Su(var)3-9 mutant embryos………………………..127 Figure 3.3. Relative levels of H3K9me2 associated with 3 regions of the histone unit in wild type and Su(var)3-9 mutant embryos………………………………  vii  131  Figure 3.4. Relative levels of HP1 associated with 4 regions of the histone unit in wild-type and Su(var)3-9 mutant embryos…………………………………………135 Figure 3.5. Relative quantifications of total histone H3 and histone H2A transcript in wild-type and Su(var)3-9 mutant embryos………………………………………140 Figure 3.6. Comparison of H2B transcript present in wild-type vs. Su(var)3-9330 mutant embryos……………………………………………………………………… 144 Figure 3.7. Relative levels of H3K9me2 associated with two intergenic regions of the histone unit in wild type and Su(var)3-9330 mutant embryos………………………………………………………………………………. 146 Figure 3.8. Relative quantifications of polyadenylated (polyA) histone H3 and histone H2A transcript in wild-type and Su(var)3-9 mutant embryos………………………………………………………………………………. 150 Supplementary figure 3.1………………………………………………………..…..168 Figure 4.1. A semi-quantitative summary of the phenotypes of four Su(var)3-9 mutants analyzed……………………………………………………………………. 185  viii  LIST OF ABBREVIATIONS ChIP  Chromatin immunoprecipitation  EGFP  Enhanced-green fluorescent protein  GST  Glutathione-S-transferase  H3K4  Lysine 4 of histone H3  H3K9  Lysine 9 of histone H3  H3K27  Lysine 27 of histone H3  H4K20  Lysine 20 of histone H4  HDAC1  Histone deacetylase 1  HIS-C  Histone Gene cluster  HP1  Heterochromatin Protein 1  MNase  Micrococcal nuclease  MOF  Males absent On the First  PEV  Position-effect variegation  PC  Polycomb  RTase  Reverse transcriptase  RT-PCR  Reverse transcription-PCR  S.E.M.  Standard error of the mean  Su(var)  Suppressor of position-effect variegation  ix  CO-AUTHORSHIP STATEMENT A version of chapters 2 and 3 of this thesis will be submitted for publication. I (P. Kalas) performed all the experiments, and the data analysis, presented in this thesis. Dr T.A. Grigliatti supervised the project and provided assistance with experimental design and editing of this manuscript.  x  1. INTRODUCTION 1.1.  Chromatin and gene regulation: an overview.  In metazoans, each somatic cell contains the same genetic information, but the fate and specificity of the cells comprising various tissue types is determined by and dependent upon the expression of different subsets of genes. While some genes go through "on" (expressed) and "off" (not expressed) states throughout the whole life of the organism, others remain permanently "off" in certain cell types, or after a certain stage of development. So, for example, nerve-specific genes are not expressed in muscle cells, and mitosis-specific genes are not expressed in cells that are not dividing anymore. Failure to express, or conversely, failure to silence a particular set of genes at the appropriate time in a specific cell type can have dramatic consequences such as congenital malformations, cancer, or death (reviewed for example by Ausio et al., 2003; Oligny, 2003; Jaffe, 2003; Moss and Wallrath, 2007; Nelson et al., 2007). Indeed, tight, accurate regulation of gene expression is absolutely crucial for the survival of any organism. Gene expression is regulated at many levels. At the transcriptional level, protein complexes assembled on regulatory sequences such as enhancers interact with the transcriptional machinery assembled on a gene’s promoter(s), stimulating gene transcription. However, in order for this to happen, the gene in question must first be "transcriptionally competent", or in other words, in a form that makes it accessible to transcription factors. In prokaryotes, the entire genome is transcriptionally competent, that is, the DNA is readily accessible to regulatory proteins. However, this is not the case in eukaryotes. 1  The eukaryotic genome is subdivided into chromosomes and organized and compacted into chromatin, a complex of DNA and proteins (see below, section 1.2.). Chromatin exists in different forms, depending on its level of compaction, the presence of specific proteins, and particular post-translational modifications of some of its components (recently reviewed by Ebert et al., 2006; Razin et al., 2007; Kouzarides, 2007). Some forms, often referred to as "open chromatin" or euchromatin, are more accessible to the transcriptional machinery, while "silent chromatin", or heterochromatin, in contrast, is typically refractory to transcription. Chromatin is extremely dynamic, having the ability to convert, under the appropriate circumstances, from an open to a silent conformation, and vice-versa). By modulating the chromatin architecture of certain regions, cells are able to make genes, and entire chromosome domains, transcriptionally competent or silenced (for a review see for example Struhl, 1999; Talbert and henikoff, 2006). Hence, in order to fully understand the biological process of gene regulation, it is necessary to uncover the mechanisms underlying chromatin biology and chromatin architecture. This thesis focuses on the study of SU(VAR)3-9, a key non-histone chromatin protein with a histone methyltransferase activity, and on its roles in modulating the packaging and thus the regulation of a euchromatic locus, the histone gene cluster.  1.2.  The components of chromatin.  Chromatin is a dynamic, highly organized complex of DNA and proteins. Histones are the most abundant chromatin proteins, but there are also numerous non-  2  histone chromatin proteins (NHCPs) that have crucial roles in regulating and maintaining chromatin architecture.  1.2.1. Histones The basic unit of chromatin is the nucleosome, 146 bp of DNA wrapped around an octamer consisting of two copies of each of the core histones, H2A, H2B, H3 and H4 (Finch et al., 1977; Klug et al., 1980; Luger et al., 1997), and chromatin at its simplest can be described as an array of nucleosomes (Kornberg, 1977). Although devoid of enzymatic activity, histones are far from being inert structural components of chromatin. Post-translational modifications of their N-terminal tails play an active role in recruiting and/or stabilizing the binding of non-histone chromatin proteins (NHCPs) to the chromatin fibre. To date, about a dozen histone modifications have been described, including methylation, acetylation, ubiquitylation and SUMOylation of lysines, phosphorylation of serines and threonines, methylation of arginines, and ADP-ribosylation (for a systematic review of the current nomenclature, see Turner, 2005). Table 1.1. summarizes the histone modifications thus far described in Drosophila, and the enzymes catalyzing them. The availability of antibodies specific for most histone modifications, in combination with high throughput analyses in yeast, Drosophila and mammals have been providing high resolution, genome-wide maps of histone posttranslational modifications (reviewed by Schones and Zhao, 2008). This has allowed the systematic study of the correlations between the chromatin structure and the transcriptional state of a locus (Crawford et al., 2006), as well as the  3  presence of particular histone modifications in that region. In all organisms analyzed, including yeast, flies and mammals, acetylated histone H3 (H3ac) and methylated lysines 4 and 36 of histone H3 (H3K4me, H3K36me) are enriched in regions corresponding to active promoters and transcribed genes (Roh et al., 2005; Schubeler et al., 2004; Liu et al., 2005; Bernsterin et al., 2005; Kim et al., 2005; Pokholok et al., 2005; Barski et al., 2007). In animals, the presence of phosphorylated histone H3 serine 10 (H3S10ph) is also characteristic of transcriptionally competent regions of the genome (Wang et al., 2001; Ebert et al., 2004; 2006). On the other hand, di- and tri-methylated histone H3 lysines 9 and 27, and methylated histone H4 lysine 20 (H3K9me2,3, H3K27me2,3, H4K20me3) are typically associated with large heterochromatic regions (Schotta et al., 2002; 2004; Peters et al., 2002; Ebert et al., 2004; 2006). Two main mechanisms have been proposed to be responsible for the "translation" of a given set of histone modifications into a particular transcriptional state of the region in question. One of them postulates that the addition of a charged group (such as an acetyl or a phosphate group) to the histone tail can cause a localized decondensation of the chromatin fibre, making it accessible to the transcriptional machinery (Turner, 2000). The other, more widely applicable, proposes that specific histone modifications, or combinations thereof, serve as a binding platforms for specific chromatin proteins (for example Lachner and Jenuwein, 2002; de la Cruz et al., 2005). Evidence exists in support of both models, suggesting that both mechanisms probably play a role. For instance, it has been demonstrated that in vitro reconstituted chromatin arrays can form compact  4  fibres, but this compaction is prevented when H4K16 is acetylated, suggesting that the acetyl group could directly affect chromatin architecture (Shogren-Knaak et al., 2006; Chodaparambil et al., 2007). However, histone modifications have also been widely shown to affect chromatin structure and gene regulation via the action of NHCPs. The chromodomain of HP1 has been shown to recognize and bind to H3K9me2,3 (Lachner et al., 2001; Jacobs et al., 2001; Fischle et al., 2003), while the chromodomain of Polycomb specifically recognizes H3K27me2,3 (Cao et al., 2002) and those of CHD1 and CHD3 proteins bind to H3K4me and H3K36me, respectively (reviewed by Mellor, 2006). In contrast, bromodomains typically recognize acetylated histone residues (reviewed by Yang, 2004; Mujtaba et al., 2007). The next section will focus on the roles of lysine methylation, since histone lysines are the target of SU(VAR)3-9’s methyltransferase activity, and have important functions in the regulation of gene silencing as well as transcriptional competence.  5  Table 1.1. Post-translational modifications of canonical histones that have been formally detected in Drosophila. MODIFICATION H3K4me3  PREDOMINANT LOCATIONS Euchromatin/interbands  ENZYME(S) RESPONSIBLE TRX TRR ASH1, ASH2  H3K9me  Euchromatic sites  dG9a, SU(VAR)3-9  H3K9me3  Heterochromatin  SU(VAR)3-9  H3K9ac  Euchromatin, bands  H3S10ph  Euchromatin, interbands  dADA2b-containing complex JIL-1  H3K14ac  Euchromatin/bands  dGCN5/dADA2b  H3K27me  Heterochromatin  H3K36me H3K79me  Euchromatin/interbands Euchromatin (puffs,  E(Z)/ESC/SU(Z)12 complex dSet2 GRAPPA  SELECTED REFERENCES Smith et al., 2002; Sedkov et al., 2003; Beisel et al., 2002; Byrd and Shearn, 2003; Beltran et al, 2007. Seum et al., 2007; Tzeng et al., 2007. Schotta et al., 2002; 2003; Eskeland et al., 2004; Mis et al., 2006; Stabell et al., 2006. Ner et al., 2002; Schotta et al., 2002; Stabell et al., 2006. Schotta et al., 2002; 2003; Eskeland et al., 2004. Pankotai et al., 2005; Ebert et al., 2006. Jin et al., 1999; Wang et al., 2001; Ebert et al., 2004; Zhang et al., 2006. Cheung et al., 2000; Pankotai et al., 2005. Muller et al., 2002; Ebert et al., 2006. Stabell et al., 2007. Shanower et al., 2005.  H3K9me  IV chromosome  DmSetDB1  H3K9me1,2  Heterochromatin  SU(VAR)3-9,  Undetermined  Ludlam et al., 2002.  Undetermined  Ludlam et al., 2002.  Undetermined  H4K16ac  Heterochromatin, particular distribution in embryonic nuclei. Euchromatin  H4K16ac  Male X chromosome  MOF  H4K20me  Euchromatin, bands and interbands Heterochromatin  PR-Set7, ASH1 SUV4-20  Turner et al., 1992; Ludlam et al., 2002; Swaminathan et al., 2005. Grienenberger et al., 2002, Miotto et al., 2006. Akhtar and Becker, 2000; Smith et al., 2001. Karachentsev et al., 2005; Beisel et al., 2002. Schotta et al., 2004.  dG9a  H4K5ac H4K8ac H4K12ac  H4K20me3  interbands, some bands) Synticial blastoderm nuclei. Ubiquitos in embryonic nuclei.  CHAMEAU  6  Histone H3 Histone H4 Histone H2B Histone H2A  Figure 1.1. A schematic of the nucleosome core particle.  7  Figure 1.1. A schematic of the nucleosome core particle. The histone proteins are represented as solid circles. Their unstructured N-terminal tails, which comprise many of the residues targeted by histone-modifying enzymes, are shown as «squiggles» protruding from the core particle. Histones H2A and H2B are represented in light and dark green, respectively, H3 is represented in yellow and H4 in red (due to the perspective, only one H4 molecule is visible). The DNA double helix is represented as a plain black line.  8  1.2.1.1. DISTRIBUTION AND FUNCTIONS OF METHYLATED HISTONE LYSINES  Over a dozen lysine residues have been shown to be susceptible to methylation (reviewed by Kouzarides, 2007). These lysines reside mainly within the H3 and H4 histone proteins, but also on H2A and H2B. Lysines can be mono-, di-, or trimethylated (me1, me2, me3, respectively), giving rise to a large number of possible methylation states for each nucleosome. Distinct methylation states are observed not only in different regions of the genome (i.e. transcriptionally active vs. inactive), but also within different portions of a given gene. In addition, methylation of certain lysines seems to be incompatible with that of others, suggesting that the co-ordinated action of histone methyltransferases (HMTases) and demethylases (DMTases) is a critical factor in the regulation of gene expression. Methylated H3K4 (H3K4me) is generally associated with transcriptionally active genes. It appears that H3K4me3 tends to be enriched within promoters and the 5’-most region of transcriptionally competent genes, while H3K4me2 is more abundant within the middle portion of active genes, and H3K4me1 is localized towards the 3’-most region (reviewed by Schones and Zhang, 2008). H3K36me3 is also associated with transcriptionally active chromatin (Barski et al., 2007). However, unlike the H3K4me "marks", it seems to be less localized, and spread over the entire length of the transcribed regions and peaking at their 3’ end (Bannister et al., 2005; Barski et al., 2007). Also, while H3K4me3 is detected at the start of genes that are transcriptionally competent, but not necessarily transcribed, the presence of H3K36me3 seems to be restricted, at least in mammals, to genes that are actively transcribed (Mikkelsen et al., 2007).  9  Trimethylated H3K9 and H4K20, on the other hand, are generally considered hallmarks of heterochromatin. In mammals, H3K9me3 and, to a lesser degree, H3K9me2, are associated with heterochromatin and also with silent regions of the genome, while H3K9me1 and H4K20me1 appear to be associated with active genes (Barski et al., 2007). In Drosophila, H3K9me1,2, H3K27me1,2,3 and H4K20me3 are all highly enriched in pericentric heterochromatin, but they are also present in other regions of the genome (Schotta et al., 2004; Ebert et al., 2004; 2006). For instance, H3K9me1,2,3 are associated with a number of euchromatic sites and with telomeres and, in polytene chromosomes, H3K27me1,2 are associated with virtually all bands, which represent regions of the genome that are somewhat condensed (Ebert et al., 2006). H3K9me3, on the other hand, does not appear to be very abundant, and is only detected within the core of the chromocentre and at a few other sites (Ebert et al., 2004; 2006). It is becoming increasingly apparent that these "methyl marks" may be involved in combinatorial, as well as in step-by-step mechanisms that regulate chromatin condensation/decondensation. In Drosophila, mutations in the H3K4 demethylase SU(VAR)3-3/dLSD1 are epistatic to the presence of additional copies of the H3K9 methyltransferase SU(VAR)3-9, indicating that demethylation of H3K4 must precede methylation of H3K9 (Rudolph et al., 2007). However, the fact that no "methyl mark" is completely restricted to transcriptionally active or inactive chromatin, suggests that none of these modifications is sufficient, by itself, to determine the transcriptional state of a chromatin region.  10  The addition of methyl groups to lysine residues is catalyzed by histone methyltransferase enzymes (HMTases). The vast majority of HMTases characterized so far are non-histone chromatin proteins (NHCPs) containing the signature SET domain (see below). Differences within key residues of their catalytic region confer SET domain-containing HMTases their substrate specificity. A wealth of information is available about SET domain-containing proteins, and it will be discussed in section 1.2.2.1. Much less is known about histone demethylases, as their discovery is much more recent (Wang et al., 2004; Cuthberg et al., 2004; Shi et al., 2004; Tsukada et al., 2006). So far, four histone lysine demethylases have been identified in Drosophila: SU(VAR)3-3/dLsd1, which demethylates H3K4me1,2, Lid, which appears to be specific for H3K4me3, and the JMJD2 homologs, dJMJD2(1) and (2) that demethylate H3K9me3 and H3K36me3, respectively (Rudolph et al., 2007; Di Stefano et al., 2007; Eissenberg et al., 2007; Lee et al., 2007; Secombe et al., 2007; Lloret-Llinares et al., 2008).  11  12  *  *  *  *  * * * * 1 tgrgkggkglgkggakrhrkvlrdniqgitkpairrlarrggvkrisgliyeetrgvlkvflenvirdavtytehak  78 rktvtamdvvyalkrqgrtlygfgg  78 rktvtamdvvyalkrqgrtlygfgg  Dm  Hs  Dm  *  * * * * 1 sgrgkggkglgkggakrhrkvlrdniqgitkpairrlarrggvkrisgliyeetrgvlkvflenvirdavtytehak  Hs  *  78 fktdlrfqssavmalqeaseaylvglfedtnlcaihakritimpkdiqlarrirgera  Dm  Histone H4  * 78 fktdlrfqssavmalqeaseaylvglfedtnlcaihakrvtimpkdiqlarrirgera  *  * 1 artkqtarkstggkaprkqlatkaarksapatggvkkphryrpgtvalreirryqkstellirklpfqrlvreiaqd  *  * 1 artkqtarkstggkaprkqlatkaarksapatggvkkphryrpgtvalreirryqkstellirklpfqrlvreiaqd  Hs  Dm  Hs  Histone H3  Figure 1.2. Histone H3 and H4 post-translational modifications. Amino acid sequence of human (H.s.) and Drosophila (D.m.) histones H3 and H4. The sequences corresponding to H3 and H4’s N-terminal tails are underlined in light blue, while yellow underlining denotes the sequences comprising the histone folds (according to Luger et al., 1997). Formally identified post-translational modifications are represented as blue squares (methylation), green circles (phosphorylation) and red asterisks (acetylation) above the residues concerned. More details about the modifications identified in Drosophila, including all relevant references, are listed in Table 1.4. The diagrams relative to human H3 and H4 modifications are based on the Abcam/Millipore histone modification map (http://www.histone.com).  13  1.2.2. Non-histone chromatin proteins Non-histone chromatin proteins (NHCPs) are generally defined as either "structural" components of chromatin or chromatin-modifying enzymes. The former include all the proteins that are physically associated with chromatin, but that do not appear to have catalytic functions, such as HP1, Drosophila’s SU(VAR)3-7 and S. pombe’s Rik1. Chromatin-modifying enzymes, in contrast, include histone acetyl-transferases and deacetylases (HATs and HDACs), HMTases and DMTases, histone kinases and phosphatases, and so forth. In many cases, these modifying enzymes also physically associate with chromatin and play a structural role in chromatin architecture. The next section will focus on HMTases and, in particular, SET domain-containing HMTases, since SU(VAR)3-9 belongs to this family of enzymes.  1.2.2.1.  SET DOMAIN-CONTAINING METHYLTRANSFERASES  The SET domain was originally identified as a ~140 amino acid region present and highly conserved in the gene products of Su(var)3-9, Enhancer of zeste and trithorax (Jones and Gelbart, 1993; Tschiersch et al., 1994). Dozens of SETcontaining proteins have since been described in eukaryotes, and many of them appear to have HMTase or, more generally, protein lysine MTase activities (reviewed by Qian and Zhou, 2006). Table 1.2. summarizes the known Drosophila SET domain-containing HMTases and their substrate specificities. SET domain-containing proteins can be classified into families based on phylogenetic analyses, on the presence of other protein domains (such as a  14  chromodomain, ankyrin repeats, zinc fingers, and so forth), and/or based on their substrate specificity. Most SET domain-containing HMTases can only catalyze the addition of methyl groups to one or two histone residues and, while some HMTases are strictly mono-methylases, others are able to catalyze the addition of multiple methyl groups (Eskeland et al., 2004; Xiao et al., 2005; Chin et al., 2006; Qian et al., 2006; Guo et al., 2007). The crystal structures of a dozen SET domains have been resolved (reviewed by Qian and Zhou, 2006), providing an explanation for their respective substrate specificities, and allowing investigators to rationalize the specificity of yet un-crystallized SET-containing HMTases. In most cases, the SET domain is surrounded by a preSET and a postSET regions, which contain a number of conserved cysteines. These residues are not involved in the catalytic process, but they coordinate a set of zinc ions and are necessary to stabilize the structure of the SET domain (Min et al., 2002; reviewed in Qian and Zhou, 2006). The substrate (histone tail) and the methyl group/methyl donor complex (S-adenosyl-methyl-methionine) bind to two distinct clefts located at opposite sides of the SET domain. These two clefts are connected by a hydrophobic channel, and the substrate specificity of SET domains is likely determined by the side chains of the residues forming this channel (Qian and Zhou, 2006). The combination of side chains is thought to recognize particular residues that flank the target lysine in the substrate, which would explain why several HMTases also display lysine MTase activity on non-histone proteins (Kouskouti et al., 2004; Couture et al., 2006; Chin et al., 2007; Sampath et al., 2007). In addition, the nature and size of the side chains in the hydrophobic  15  channel can determine the degree of methylation that can be achieved by a given SET domain. For example, the presence of a large, bulky side chain will not permit an already methylated histone tail access to the channel, thus precluding the transfer of multiple methyl groups on the target lysine. SET-containing HMTases also have an additional layer of specificity, as it is becoming apparent that different HMTases act within different chromatin regions. In Drosophila, for example, SU(VAR)3-9 is responsible for H3K9me2,3 within centric and pericentric heterochromatin, as well as some euchromatic sites. In contrast, DmSetDB1 mono- and dimethylates H3K9 almost exclusively on the mainly heterochromatic fourth chromosome (Schotta et al., 2002; Eskeland et al., 2004; Mis et al., 2006; Stabell et al., 2006; Seum et al., 2007; Tzeng et al., 2007; Ebert et al., 2006, and references therein). When this thesis was begun, the catalytic activity of one SET domain, that of SUV39H and CLR4 (the human and yeast homologs of SU(VAR)3-9, respectively) had just been discovered (Rea et al., 2000). Prior to that, SET domains were thought to have protein-protein interaction roles (for example Aagard et al., 1999; Cui et al., 1998), and no structure/function analyses had ever been performed.  16  Channel  Figure 1.3.  17  Figure 1.3. Structure of a SET domain, in this case SET7/9. The substrate (histone H3) binds to the shallow groove adjacent to the channel, on the side of the protein that is facing the viewer in this figure. The methyl donor (AdoMet) binds to a pocket on the opposite side of the protein (hidden in this figure). The narrow channel indicated with an arrow allows the H3 tail to come in contact with the AdoMet moiety. The blue shading indicates different levels of residue conservation relative to other SET domain proteins. This figure was modified from Kwon et al.: Mechanism of histone lysine methyl transfer revealed by the structure of SET7/9AdoMet. EMBO J 22, 292-303 (2003).  18  Table 1.2. A list of characterized Drosophila SET domain-containing proteins. Note that predicted/hypothetical proteins are not included. Protein name SU(VAR)3-9  Activity and specificity reported H3K9me (preferentially mono/dimethylation of H3K9me1)  E(Z)  H3K27me (as part of the E(Z)/ESC/SU(Z)12 complex)  TRITHORAX TRR ASH1 ASH2 SUV4-20 PR-Set7 dG9a  H3K4me3 H3K4me3 H3K4me3, H3K9me3 H4K20me3 H3K4me3 H4K20me3 H4K20me  DmSetDB1  H3K9me1,2 H3K27me, H4K20me (in vitro) H3K9me1,2  dSet2  H3K36me2  19  Selected references Ebert et al., 1994; Czermin et al., 2001; Schotta et al., 2002; Eskeland et al., 2004. Muller et al., 2002; Ebert et al., 2006. Smith et al., 2004 Sedkov et al., 2003. Beisel et al., 2002; Byrd and Shearn, 2003. Beltran et al, 2007. Schotta et al., 2004. Nishioka et al., 2002; Karachentsev et al., 2005. Mis et al., 2006; Stabell et al., 2006. Seum et al., 2007; Tzeng et al., 2007. Stabell et al., 2007.  1.2.2.2.  SU(VAR)3-9  In Drosophila, SU(VAR)3-9, is one of the most prominent HMTases. It appears to be very conserved, with homologs in virtually every eukaryote from the fission yeast to mammals and plants (Krauss et al., 2006). SU(VAR)3-9, as well as most of its homologs, has two conserved domains: a SET domain (see above) and a chromodomain, which is also found in other chromatin proteins such as HP1 and Polycomb (Paro and Hogness, 1991). Chromodomains recognize and bind methylated histone residues, and each chromodomain seems to be very specific for one particular methyl-residue. For example, the chromodomain of HP1 binds to H3K9me (Bannister et al., 2001; Lachner et al. 2001; Eskeland et al. 2007), that of PC binds to H3K27me, and it was recently shown that the chromodomain of CLR4, the yeast homolog of SU(VAR)3-9, binds to H3K9me (Zhang et al., 2008). Drosophila’s SU(VAR)3-9 is 635 amino acids long, which is larger than its known homologs, and it is unique in that it contains an N-terminal domain responsible for dimerization (Eskeland et al., 2004). The N-terminal moiety of SU(VAR)3-9 also comprises motifs that are required for interaction with two other chromatin proteins, HP1 and SU(VAR)3-7 (Schotta et al., 2002). In yeast, clr4 mutants are viable, but show impaired silencing at centromeres and at the mating type locus (Ivanova et al., 1998). In mouse, homozygosity for knockout mutations in either Suv39h1 or Suv39h2 doesn’t lead to any noticeable phenotypes (Peters et al., 2001). However, a double knockout for both Su(var)3-9 paralogs, Suv39h1 and Suv39h2, is semi-lethal, and those few homozygous double mutant mice that survive display growth retardation , a high incidence of B cell lymphomas and chromosomal instability (Peters et al., 2001). 20  In Drosophila, homozygous Su(var)3-9 mutant adults are viable and fertile, but the chance of Su(var)3-9 null embryos reaching adulthood is only about 50% of that of their wild-type counterparts (Mis et al., 2006). These observations suggest that SU(VAR)3-9 is not absolutely essential for development, possibly because other HMTases are able to mimic some of SU(VAR)3-9’s functions at least to some degree. On polytene chromosomes, SU(VAR)3-9 is detected at the chromocentre, as well as at some euchromatic sites on the chromosomal arms (Schotta et al., 2002), including the HIS-C (Ner et al., 2002). In addition to controlling methylation of H3K9 in heterochromatin, it interacts physically and functionally with a number of other chromatin proteins, including HP1 and the histone deacetylase HDAC1, which is responsible for deacetylating H3K9 (Schotta et al., 2002; Ebert et al., 2004; 2006; Czermin et al., 2001). These and additional pieces of evidence obtained in yeast, have given rise to the current models for SU(VAR)3-9’s role in chromatin-based gene silencing (see below, 1.3.1).  21  1.2.2.3.  OTHER NON-HISTONE CHROMATIN PROTEINS IN DROSOPHILA  Lysine HMTases, and SU(VAR)3-9 in particular, are thought to work in conjunction with other NHCPs to form and maintain chromatin architecture. In Drosophila, the genes encoding many of these proteins were originally identified in genetic screens for dominant suppressors of position-effect variegation (PEV, reviewed by Spofford, 1976; Weiler and Wakimoto, 1995). Table 1.3 summarizes the genes originally identified through screens for dominant suppressors of PEV and the characteristics of their products, most of which are NHCPs. These include SU(VAR)3-7 and HDAC1, both of which are known to interact with SU(VAR)3-9 (Reuter et al., 1990; Cléard et al., 1997; Mottus et al., 2000; Czermin et al., 2001; Schotta et al., 2002). SU(VAR)3-7 is a large, Drosophila-specific, zinc-finger protein that binds to repetitive DNA and physically interacts with both SU(VAR)3-9 and HP1 (Cléard and Spierer, 2001; Schotta et al., 2002; Jaquet et al., 2006). It is preferentially associated with heterochromatin and appears to be sufficient to induce SU(VAR)39-dependent heterochromatisation (Reuter et al., 1990; Cléard et al., 1995; Delattre et al., 2004). In contrast, localization of the histone deacetylase HDAC1, the homolog of S. cerevisiae Rpd3, does not appear to be heterochromatinspecific, as this protein is detected at hundreds of sites on polytene chromosomes (Chang et al, 2001; Tie et al., 2003). HDAC1 is an H3K9 deacetylase that has been shown to co-immunoprecipitate with SU(VAR)3-9, suggesting that the two proteins may be part of the same complex (Czermin et al., 2001; Rudolph et al., 2007). In addition, the deacetylation activity of HDAC1 is necessary for H3K9 methylation (Nakayama et al., 2001; Czermin et al., 2001; Vaute et al., 2002). 22  One NHCP, HP1, was originally identified as the specific target of a monoclonal antibody that associated preferentially with heterochromatin in polytene chromosomes (James and Elgin, 1986; James et al., 1989) and Su(var)25, the gene encoding HP1, was identified as a strong dominant suppressor of PEV (Eissenberg et al., 1990; 1992). HP1 has homologs in almost all eukaryotes from the fission yeast (Swi6) to mammals and plants; it is comprised of two conserved domains, the chromodomain and the chromo-shadow domain, linked by what is known as the "hinge" or "linker" region (reviewed by Lomberk et al., 2006). The chromodomain is responsible for specifically recognizing and binding H3K9me, while the chromo-shadow domain is required for protein-protein interactions and for dimerisation (Bannister et al., 2001; Lachner et al., 2001; Jacobs et al., 2001; Brasher et al., 2000; Jones et al., 2000; Nielsen et al., 2001; Cowieson et al., 2000). Both the chromo-shadow and the "hinge" domains are necessary for interaction with SU(VAR)3-9 and SU(VAR)3-7 (Schotta et al., 2002; Jaquet et al., 2002). With the advent of genome-wide sequencing efforts, genomic databases and bioinformatics tools, a number of genes encoding NHCPs were cloned and characterized based on sequence homologies and in silico searches. These include two HMTase-encoding genes, dG9a and Su(var)4-20, as well as several DMTases (Schotta et al., 2004; Mis et al., 2006; Stabell et al., 2006; Schotta et al., 2004; Secombe et al., 2007; Lloret-Llinares et al., 2008). Several NHCPs identified through an in silico approach have also been shown to suppress PEV, indicating that they are functionally involved in epigenetic silencing (Table 1.4).  23  24  Su(var)3-3  (Note that Su(var)3-1 is a hypermorph allele of JIL-1)  Su(var)3-1  77A3  68A5-A6  (Synonym: dLsd1)  SU(VAR)3-3  JIL-1  * multiple isomorphs  (Synonyms: Zimp, PIAS, SU(VAR)2-10)  dPIAS*  45A8-A9  Su(var)2-10  Gene product HP1 (Synonym: HP1a, C1A9 nuclear antigen)  Location 28F2-F3  (Sometimes referred to as Su(var)205)  Gene name Su(var)2-5  DNA binding, chromosome organization, structure and function, transcriptional regulation, regulation of the JAK-STAT cascade, haematopoiesis. Histone kinase (specific for H3S10), regulation of H3K9methylation, chromatin architecture, chromosome segregation (meiosis), transcriptional regulation. Histone demethylase (specific for H3K4me1,2), gametogenesis, heterochromatin formation, transcriptional regulation.  Attributed functions Chromatin silencing and assembly, telomere maintenance, transcriptional regulation, chromosome segregation, binding to H3K9me, RNA-binding.  LSD1 (mammals), SPR-5 (C. elegans), LSD1 (S. pombe).  Undetermined  **several paralogs present in mammals.  PIAS** (mammals)  Paralogs: HP1b, HP1c (Drosophila), HP1b, HP1g (mammals).  *M31 (mouse)  Homologs Orthologs: HP1a (mammals*), SWI6 (S. pombe), LHP1 (Arabidopsis),  Di Stefano et al., 2007; Rudolph et al., 2007.  Jin et al., 1999; Wang et al., 2001; Ebert et al., 2004; Bao et al., 2005; Zhang et al., 2006.  Mohr and Boswell, 1999; Evans et al., 2003; Hari et al., 2001; Muller at al., 2005;  Selected references James and Elgin, 1986; Eissenberg et al., 1992; Kellum and Alberts, 1995; Nielsen et al., 2001, Greil et al., 2003.  location to which the gene in question has been mapped. Note that, under «homologs», hypothetical/predicted proteins are not included.  Table 1.3. Drosophila proteins identified through screens for strong, dominant suppressors of PEV. «Location» refers to the cytological  25  88E6-E8  Su(var)3-9  Su(var)326/ Rpd3  64B12  87E3  Su(var)3-7  (Note: pitkin, a very strong E(var), is a hypermorph allele of Su(var)3-9)  Location 87B9-B10  Gene name Su(var)3-6  HDAC1/RPD3  SU(VAR)3-9  SU(VAR)3-7  (Synonyms: PP-1a, PP1-c, PP-1a, SU(VAR)3-6, CK19).  Gene product PP1-87B  Histone deacetylase (preference for H3K9ac), interaction with SU(VAR)3-9, gene regulation, chromatin architecture, silencing, cell cycle regulation.  Attributed functions Ser/Thr phosphatase, cell cycle regulation/mitosis, glycogen metabolism, gametogenesis, wing, eye, nervous system and muscle development. DNA-binding (preference for satellite sequences), interaction with HP1 and SU(VAR)3-9, chromatin compaction, architecture and silencing. HMTase (specific for H3K9), gene regulation, chromatin architecture and silencing, interaction with HP1, SU(VAR)37, SU(VAR)3-3, HDAC1.  SUVH3,4,5 (Arabidopsis). HDAC2/1 (mammals), Rpd3 (S. cerevisiae).  Paralogs: SUV39H2 (human), Suv39h2 (mouse).  Orthologs: SUV39H1 (human), Suv39h1 (mouse), CLR4/Clr4p (S. pombe); DIM-5 (N. crassa).  To date, no homologs identified outside the genus Drosophila.  Homologs PP1 (vertebrates), TOPP2 (Arabidopsis), Ppz1p (S. cerevisiae).  Maixner et al., 1998; Mottus et al., 2000; Huang and Kadonaga, 2001; Czermin et al., 2001.  Tschiersch et al., 1994; Ner et al., 2002; Schotta et al., 2002; Eskeland et al., 2004; Swaminathan et al., 2005.  Reuter et al., 1990; Cléard and Spierer, 2001; Jaquet et al., 2002; Delattre et al., 2004; Demakova et al., 2007.  Selected references Dombradi et al., 1990; Baksa et al., 1993; Bennett et al., 2003; Babu et al., 2005.  26  27F3-4  47F13-14  68A4  E(Pc)  Su(UR)  1B13-14  Suv4-20  chm  Location 1A1  Gene name dG9a  rearrangement, Sb .  V  SUUR  E(PC)  (Synonym: HAT1)  CHAMEAU  SUV4-20  Gene product dG9a  Chromatin architecture, regulation of genes arranged in clusters.  Major attributed functions Histone methyltransferase (preference for H3K9 and K27 in euchromatin), regulation of ecdysone pathways. Histone methyltransferase (preference for H4K20), gene regulation, chromatin architecture and silencing. HAT (preference for H4K16), chromatin architecture, silencing, regulation of the JNK cascade, transcriptional regulation. Subunit of the Tip60 chromatinremodeling complex.  Paralogs: EPC2 (human), Epc2 (mouse). Undetermined  Orthologs: EPC1 (human), Epc1 (mouse), Epl1 (S. cerevisiae).  Paralogs: Suv4-20h2 (mammals). HBO1 (human), Sas2 (S. cerevisiae).  Orthologs: Suv4-20h1 (mammals).  Homologs G9a (mammals).  Belyaeva et al., 1998; 2003; Belyakin et al., 2005.  Stankunas et al., 1998; Sinclair et al., 1998; Kusch et al., 2004.  Grienenberger et al., 2002; Miotto et al., 2006;  Schotta et al., 2004.  Selected references Tachibana et al., 2002; Mis et al., 2006; Stabell et al., 2006.  that, since dG9a and Suv4-20 are located on the X chromosome, their Su(var) phenotypes have so far only been tested on one variegating  different variegating rearrangements. Note that the Su(var) phenotype associated puc and dSas4 was determined in our lab (Toub, unpublished), and  Table 1.4. Additional Su(var)s. Mutations in the following Drosophila genes were also found to act as dominant suppressors of PEV on at least two  27  84E12-13  100E3  mod  Location 84C6-7  puc  (Previously known as S2214)  Gene name dSas-4  MODULO  PUCKERED  Gene product dSAS-4  Ser/Thr and Tyr phosphatase, regulation of JNK cascade, dorsal closure and metamorphosis. Transcriptional regulation, gametogenesis, cell growth and proliferation, chromatin architecture.  Major attributed functions Centriole replication, biogenesis of centrosomes, flagella and cilia.  Nucleolin (vertebrates), p67/NSR1 (S. cerevisiae).  Pyst1 (human); CEL-F081 (C. elegans).  (*Spindle assembly 4)  Homologs SAS-4* (C. elegans).  Krejci et al., 1989; Garzino et al., 1992; Perrin et al., 2003.  Glise and Noselli, 1997; Martin-Blanco et al., 1998; Agnès et al., 1999.  Selected references Basto et al., 2006.  1.3. SU(VAR)3-9 and heterochromatin assembly Heterochromatin is a transcriptionally inert form of chromatin that remains highly compacted throughout the cell cycle. Heterochromatin is characterized by the presence of repetitive DNA, transposable elements, highly ordered nucleosomal arrays (Wallrath and Elgin, 1995), specific subsets of histone modifications (e.g. H3K9me2,3, H4K20me3 ) and enrichment for particular NHCPs, such as SU(VAR)3-9, HP1, SU(VAR)3-7 (Eissenberg and Elgin, 2000; Schotta et al., 2002; Reuter et al., 1990; for a comprehensive review see Ebert et al., 2006). Heterochromatic regions of the genome tend to be replicated late during S phase and appear underreplicated in polytene chromosomes. Chromosomes’ centromeres and telomeres, the inactive X chromosome in female mammals and most of Drosophila’s fourth chromosome are heterochromatic. However, chromatin is highly dynamic, and its packaging status, as well as the histone modifications and NHCPs present at a given locus, are not fixed; instead they change throughout the cell cycle, and during development, as the cell responds to cellular and extracellular signals (de Wit et al., 2005; Ebert et al., 2004; Dormann et al., 2006). Results obtained from genetic and biochemical analyses in yeast, and from the study of polytene chromosomes in flies suggest that heterochromatin formation is a multi-step process. The model presented in section 1.3.1 focuses on the roles of SU(VAR)3-9, based on what is known from Drosophila and yeast.  28  1.3.1. Postulated mechanism for heterochromatin formation Demethylation of H3K4me by SU(VAR)3-3/dLSD1 is thought to be one of the first steps in heterochromatisation of a region, since a loss of function mutation in Su(var)3-3 is epistatic to the presence of extra copies of Su(var)3-9, and SU(VAR)3-9’s activity is absolutely necessary for the formation of heterochromatin (Rudolph et al., 2007; Schotta et al., 2002; 2003). Deacetylation of H3K9ac by HDAC1 also needs to occur prior to SU(VAR)3-9-dependent methylation, as H3K9 can’t be simultaneously acetylated and methylated. In addition, loss of function mutations in the gene encoding HDAC1 are also epistatic to the presence of extra copies of Su(var)3-9 (Czermin et al., 2001). Once H3K4 is demethylated and H3K9 is deacetylated, SU(VAR)3-9 can diand trimethylate H3K9. It should however be noted that, since SU(VAR)3-9 seems to preferentially di- and trimethylate H3K9me1, its action is probably preceded by that of an H3K9 monomethylase, possibly dG9a (Mis et al., 2006). How SU(VAR)39 is recruited to its target sites is not completely understood, but we know that its recruitment is at least partially dependent on SU(VAR)3-7, and that the presence of HP1 is required to prevent the binding of SU(VAR)3-9 to ectopic sites (Delattre et al., 2004; Schotta et al., 2002; 2003). Since SU(VAR)3-9 is able to physically interact with HDAC1, SU(VAR)3-7 and HP1, it is possible that these chromatin proteins form a complex (Delattre et al., 2000; Czermin et al., 2001, Schotta et al., 2002; 2003). The H3K9me2,3 probably acts as a binding platform for the chromodomain of HP1 (Bannister et al. 2001; Jacobs et al., 2001). There is also some evidence  29  showing that, at least in yeast, H3K9me2,3 helps recruit and stabilize the binding of Clr4, the yeast homolog of SU(VAR)3-9, to chromatin (Zhang et al., 2008). If the same is true in Drosophila, the presence of H3K9me2,3 may stabilize the association of SU(VAR)3-9 with chromatin, in addition to facilitating, with the help of auxiliary factors, the binding of HP1. This, in turn, is thought to recruit SU(VAR)4-20, an HMTase that trimethylates H4K20 (Ebert et al., 2006; Eskeland et al., 2007; Schotta et al., 2004). SU(VAR)3-9 has also been associated with euchromatic gene regulation (Vandel et al., 2001; Nielsen et al., 2001; Ner et al., 2002; Greil et al., 2003). Because of its role in heterochromatin formation, the presence of SU(VAR)3-9 at euchromatic loci is hypothesized to be associated with silencing of the genes within these loci. Whether this is always the case, or not, has not been well documented. In addition, it is presently unclear whether SU(VAR)3-9 acts through similar or completely distinct mechanisms at heterochromatic and euchromatic sites. Investigating its role in the regulation of the histone genes, which are located in euchromatin should provide useful information in this regard, and is the subject of this thesis.  30  31  Figure 1.4.  H3K4meK9ac  *  SILENCING COMPLEX  SU(VAR)3-3  H3K9ac  *  HP1  H3K9me2,3 H4K20me3  BONUS  SU(VAR)3-7  SU(VAR)3-9  BONUS  ?  HDAC1  ?  HP1  SU(VAR)4-20  SU(VAR)3-7  H3  dG9a  H3K9me2,3  factors  Auxiliary  SU(VAR)3-9  H3K9me1  Figure 1.4. A simplified diagram showing the assembly of heterochromatin. The nucleosome core is represented as a cylinder, with the dotted line representing the DNA wrapped around it. For simplicity, only 4 N-terminal histone tails are shown instead of 8; the histone H3 tail in yellow, H4 in red, H2A in light green and H2B in dark green. The blue squares indicate methyl groups, the red asterisks represent acetyl groups. The enzymes known to catalyze the addition and removal of these groups are shown. Proteins known to be associated with the HIS-C are highlighted in yellow.  32  1.4. The histone gene cluster (HIS-C) One of the targets of SU(VAR)3-9 in polytene chromosomes is the HIS-C, and some evidence exists that the regulation of the histone genes is, at least partially, dependent on SU(VAR)3-9 (Ner et al., 2002; see below), thus making the HIS-C a very good model system to start dissecting the SU(VAR)3-9’s mechanism of action at a euchromatic locus.  1.4.1. Structure of the HIS-C and characteristics of the histone genes Drosophila’s histone genes are organized into a “histone unit” consisting of one copy of each of the five histone genes (h2a, h2b, h3, h4 and h1). About 110 tandemly repeated copies of this histone unit form the Histone Gene cluster (HISC). The order of the histone genes and their direction of transcription are shown in Figure 1.2. Located on the left arm of the second chromosome, Drosophila’s HISC spans over 500 kb (Saigo et al., 1981) and has some peculiar characteristics. In spite of being a euchromatic locus, it displays some features that are usually associated with heterochromatin. It replicates slightly later in S-phase than other euchromatic loci, it is somewhat underreplicated in polytene chromososomes, nuclease sensitivity assays suggest that it is packaged as a higher order chromatin structure, and, at DNA level, it is a reiterated locus (Zhimulev and Belyaeva, 2003; Samal et al., 1981). In metazoans, histone genes differ from most genes in that they are intronless and, more importantly, their mRNAs are, for the most part, not polyadenylated in spite of being transcribed by RNA polymerase II. Instead, the  33  processing of histone pre-mRNAs depends on two elements located at their 3’ end: a highly conserved, 16 nucleotide stem-loop sequence, and a purine-rich sequence, known as HDE (histone downstream element). The processing also requires several trans-acting factors: the stem-loop binding protein (SLBP), which binds to the stem-loop structure, a U7 snRNP, which binds the HDE, plus some additional (poorly characterized) factors (for a review, see for example Dominski and Marzluff, 1999; 2007). Relatively little is known about the precise functions of SLBP, and much of the data available comes from studies in vertebrates. We know that SLBP remains associated with mature histone mRNAs as they are transported into the cytoplasm, where it is thought to play a role in the stability and translation of the transcripts (reviewed by Dominski and Marzluff, 1999; 2007). More importantly, SLBP is only present in large amounts during S-phase, possibly explaining why non-polyadenylated histone mRNAs only accumulate at this stage of the cell cycle. One of its main roles may be to stabilize and protect the histone mRNA from degradation (reviewed by Dominski and Marzluff, 1999; 2007).  34  35 1840  210 450 530  820  EcoRI  histone H1  PstI BamHI HindIII PstI  Figure 1.5.  0  BglII  BglII  histone H3  histone H4  histone H2A  histone H2B BglII  Figure 1.5. A schematic of Drosophila’s histone unit. The BglII fragment defining the histone unit is comprised of one copy of each histone gene. The histone gene cluster (HIS-C) consists of a tandem array of about 110 copies of the histone unit.The coding regions of the histone genes are represented in blue (H2A and H2B), red (H3 and H4), and yellow (H1). The arrowheads indicate the direction of transcription. Selected restriction sites, and their relative distances and positions along the histone unit, are indicated.  36  1.4.2. H3K9me and SU(VAR)3-9 distribution across the HIS-C Several pieces of evidence suggest that SU(VAR)3-9 must have a role in the regulation of the histone gene cluster. Based on MNase and DNaseI sensitivity assays, it appears that the HIS-C has an altered chromatin structure in Su(var)3-9 mutants, and at least three Su(var)3-9 mutants have increased levels of histone h1 and histone h4 transcripts (Ner et al., 2002). ChIP experiments have shown that SU(VAR)3-9 is associated with transcribed and non transcribed/ intergenic regions of the histone unit in staged embryos (Ner et al., 2002; Ner et al., in preparation), indicating that it probably plays a direct role in the regulation of the locus. The association of SU(VAR)3-9 with the HIS-C was confirmed in adults using a SU(VAR)3-9::DAM fusion protein (Ner et al., in preparation). In addition, localization of SU(VAR)3-9 at the HIS-C was also reported in Drosophila nurse cells (Koryakov et al., 2006). H3K9me2 follows the same distribution pattern as SU(VAR)3-9 along the histone unit, suggesting that SU(VAR)3-9 is not only present at the locus, but it is also enzymatically functional (Ner et al., 2002). Other NHCPs known to interact with SU(VAR)3-9, such as HP1 and HDAC1, have also been detected at the HIS-C (Greil et al., 2003; Koryakov et al., 2006; Ner et al., in preparation).  1.4.3. Possible function of SU(VAR)3-9 in the regulation of the histone genes What could be the function of SU(VAR)3-9 in the regulation of histone gene expression? First of all, a role at the transcriptional level seems most compatible  37  with its nature as a chromatin-associated H3K9 HMTase. Assuming that this is the case, SU(VAR)3-9 could be involved in modulating the chromatin structure, and therefore transcriptional competency, of the HIS-C. It could do so in at least three ways. The first possibility is that it acts on individual histone units, determining which/how many of the ~110 of them are available for transcription. This possibility implies that the five histone genes comprising a given unit are all either accessible, or inaccessible to the transcriptional machinery. Alternatively, SU(VAR)3-9 could work as a transcriptional regulator that modulates the expression level of each histone gene independently. In this case, genes belonging to the same histone unit may be transcribed at different rates. Finally, it is also possible that SU(VAR)3-9’s function is somehow involved in the coupling of histone genes expression with the cell cycle. Chapter 3 of this thesis will address some of these issues.  1.5. Subject(s) of this study Chromatin structure and dynamics play crucial roles in gene regulation. Thus, the importance of identifying chromatin components and understanding their functions and interaction is obvious. However, chromatin’s size and complexity make it necessary to study its components one, or a few, at a time. The focus of this study is a non-histone chromatin protein, the H3K9-specific HMTase SU(VAR)3-9, and its role in the packaging and regulation of a euchromatic locus, the histone genes cluster .  38  Highly conserved from yeast to mammals (Krauss et al., 2006), SU(VAR)3-9 is very well characterized. However, when this study was begun, the effects of single amino acid substitutions on the protein’s HMTase activity were not known. Subsequently, the enzymatic activity of a dozen missense mutants has been reported, but only with respect to a single synthetic substrate (a histone tail peptide), which does not allow for a detailed characterization of the catalytic characteristics of each mutant (Ebert et al., 2004). Also, the mutants’ phenotypes at the enzymatic and cellular level, and their effect on chromatin-based gene silencing, have never been systematically related to the position of the mutated residue within the three dimensional structure of SU(VAR)3-9. Part of the reason for this lack of detailed structure-function analyses of SU(VAR)3-9 may be that its crystal structure has not yet been resolved, so one needs to "extrapolate" from structural information relative to similar HMTases. Finally, this thesis represents the first attempt to determine whether the effect(s) of specific Su(var)3-9 mutations on one phenotype (for example, catalytic activity on a given substrate) are good predictors of how these mutations will affect other phenotypes (for example, overexpression of the histone genes). The first section of my thesis (chapter 2) is concerned with the characterization of a series of Su(var)3-9 missense alleles isolated in our laboratory as strong, dominant suppressors of position-effect variegation (PEV, see below, section 1.2.3.1.) at a number of different phenotypic levels, including: the molecular, enzymatic, and cellular levels, as well as at their morphological phenotype, suppression of PEV, the phenotype against which the mutants were  39  identified and isolated. Taking advantage of the crystal structure of CLR4 (Min et al., 2002), the yeast homolog of SU(VAR)3-9, and of the high level of identity between the primary sequences of the two proteins, the results are then discussed in terms of structure-function. This analysis allows us to propose roles for the various mutated amino acids in the HMTase function of SU(VAR)3-9. In addition, it highlights the functional biases of the original genetic screen that was used to identify Su(var)3-9 as well as the interpretive biases that result from characterizing mutants against a single molecular or cellular phenotype. Based on this molecular, biochemical and functional analyses we can postulate a possible mechanism by which SU(VAR)3-9 acts as a regulator of chromatin-based gene silencing (see "Discussion"). The second part of this thesis (chapter 3) deals with the role of SU(VAR)3-9 as a regulator of euchromatic gene expression. Although it was first identified as a chromatin protein, and found to be associated mainly with heterochromatic regions of the genome (Schotta et al., 2002; 2003; Ebert et al., 2004), there is reason to believe that SU(VAR)3-9 also plays an important role in the regulation of many euchromatic genes (Nielsen et al., 2001; Greil et al., 2003). Among the pieces of evidence supporting this idea is the relationship between Su(var)3-9 function and regulation of the HIS-C transcription. The HIS-C appears to be one of the major euchromatic targets of SU(VAR)3-9, and at least two Su(var)3-9 mutants show an altered chromatin structure at the HIS-C locus and elevated levels of histone transcripts (Ner et al., 2002).  40  Assuming that SU(VAR)3-9 does indeed play a direct role in the regulation of histone genes expression, its mechanism of action is not known. In order to gain some insights on the function(s) of SU(VAR)3-9 at the HIS-C, its distribution along this locus is studied in wild-type and particular Su(var)3-9 mutants. The distributions of dimethyl-H3K9 (H3K9me2), the histone modification elicited by SU(VAR)3-9, and that of HP1, another chromatin protein thought to bind to H3K9me2, are also analyzed. This study gives us a general picture of the HIS-C "landscape" in terms of SU(VAR)3-9, HP1 and H3K9me2, and allows us to determine the interdependence (or lack thereof) among these three factors. Finally, the relative amounts of histone transcripts are accurately quantified in the different Su(var)3-9 mutants and, by relating this information to the distribution of SU(VAR)3-9, HP1 and H3K9me2 at the HIS-C in each mutant, we can propose a mechanism for SU(VAR)3-9’s function at the HIS-C (chapter 3).  41  1.6. References Aagaard, L., Laible, G., Selenko, P., Schmid, M., Dorn, R., Schotta, G., Kuhfittig, S., Wolf, A., Lebersorger, A., Singh, P.B., Reuter, G., Jenuwein, T.: Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31. EMBO J 18, 1923-3 (1999) Agnès, F., Suzanne, M., Noselli, S.: The Drosophila JNK pathway controls the morphogenesis of imaginal discs during metamorphosis. Development 126, 545362 (1999) Akhtar, A., Becker, P.B.: Activation of transcription through histone H4 acetylation by MOF, an acetyltransferase essential for dosage compensation in Drosophila. Mol Cell 5, 367-75 (2000) Ausio, J., Levin, D.B., De Amorim, G.V., Bakker, S., Macleod. P.M.: Syndromes of disordered chromatin remodeling. Clin. Genet. 64, 83-95 (2003) Babu, K., Bahri, S., Alphey, L., Chia, W.: Bifocal and PP1 interaction regulates targeting of the R-cell growth cone in Drosophila. Dev Biol. 288, 372-86 (2005) Baksa, K., Morawietz, H., Dombradi, V., Axton, M., Taubert, H., Szabo, G., Torok, I., Udvardy, A., Gyurkovics, H., Szoor, B., et al.: Mutations in the protein phosphatase 1 gene at 87B can differentially affect suppression of position-effect variegation and mitosis in Drosophila melanogaster. Genetics 135, 117-25 (1993) Bannister, A.J., Zegerman, P., Partridge, J.F., Miska, E.A., Thomas, J.O., Allshire, R.C., Kouzarides, T.: Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120-4 (2001) Bannister, A.J., Schneider, R., Myers, F.A., Thorne, A.W., Crane-Robinson, C., Kouzarides, T.: Spatial distribution of di- and tri-methyl lysine 36 of histone H3 at active genes. J Biol Chem. 280, 17732-6 (2005) Bao, X., Zhang, W., Krencik, R., Deng, H., Wang, Y., Girton, J., Johansen, J., Johansen, K.M.: The JIL-1 kinase interacts with lamin Dm0 and regulates nuclear lamina morphology of Drosophila nurse cells. J Cell Sci. 118, 5079-87 (2005) Barski, A., Cuddapah, S., Cui, K., Roh, T.Y., Schones, D.E., Wang, Z., Wei, G., Chepelev, I., Zhao, K.: High-resolution profiling of histone methylations in the human genome. Cell 129, 823-37 (2007) Basto, R., Lau, J., Vinogradova, T., Gardiol, A., Woods, C.G., Khodjakov, A., Raff, J.W.: Flies without centrioles. Cell 125, 1375-86 (2006)  42  Beisel, C., Imhof, A., Greene, J., Kremmer, E., Sauer, F.: Histone methylation by the Drosophila epigenetic transcriptional regulator Ash1. Nature 419, 857-62 (2002) Beltran, S., Angulo, M., Pignatelli, M., Serras, F., Corominas, M.: Functional dissection of the ash2 and ash1 transcriptomes provides insights into the transcriptional basis of wing phenotypes and reveals conserved protein interactions. Genome Biol. 8, R67 (2007) Belyaeva, E.S., Zhimulev, I.F., Volkova, E.I., Alekseyenko, A.A., Moshkin, Y.M., Koryakov, D.E.: Su(UR)ES: a gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes. Proc Natl Acad Sci U S A 95, 7532-7 (1998) Belyaeva, E.S., Boldyreva, L.V., Volkova, E.I., Nanayev, R.A., Alekseyenko, A.A., Zhimulev, I.F.: Effect of the Suppressor of Underreplication (SuUR) gene on position-effect variegation silencing in Drosophila melanogaster. Genetics 165, 1209-20 (2003) Belyakin, S.N., Christophides, G.K., Alekseyenko, A.A., Kriventseva, E.V., Belyaeva, E.S., Nanayev, R.A., Makunin, I.V., Kafatos, F.C., Zhimulev, I.F.: Genomic analysis of Drosophila chromosome underreplication reveals a link between replication control and transcriptional territories. Proc Natl Acad Sci U S A 102, 8269-74 (2005) Bennett, D., Ször, B., Gross, S., Vereshchagina, N., Alphey, L.: Ectopic expression of inhibitors of Protein Phosphatase Type 1 (PP1) can be used to analyze roles of PP1 in Drosophila development. Genetics 164, 235-45 (2003) Bernstein, B.E., Humphrey, E.L., Erlich, R.L., Schneider, R., Bouman, P., Liu, J.S., Kouzarides, T., Schreiber, S.L.: Methylation of histone H3 Lys 4 in coding regions of active genes. Proc Natl Acad Sci U S A 99, 8695-700 (2002) Brasher, S.V., Smith, B.O., Fogh, R.H., Nietlispach, D., Thiru, A., Nielsen, P.R., Broadhurst, R.W., Ball, L.J., Murzina, N.V., Laue, E.D.: The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer. EMBO J 19, 1587-97 (2000) Byrd, K.N., Shearn, A.: ASH1, a Drosophila trithorax group protein, is required for methylation of lysine 4 residues on histone H3. Proc Natl Acad Sci U S A 100, 11535-40 (2003) Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H., Tempst, P., Jones, R.S., Zhang, Y.: Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039-43 (2002)  43  Chang, Y.L., Peng, Y.H., Pan, I.C., Sun, D.S., King, B., Huang, D.H.: Essential role of Drosophila Hdac1 in homeotic gene silencing. Proc Natl Acad Sci U S A 98, 9730-5 (2001) Cheung, P., Tanner, K.G., Cheung, W.L., Sassone-Corsi, P., Denu, J.M., Allis, C.D.: Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation. Mol Cell 5, 905–915 (2000) Chin, H.G., Estève, P.O., Pradhan, M., Benner, J., Patnaik, D., Carey, M.F., Pradhan, S.: Automethylation of G9a and its implication in wider substrate specificity and HP1 binding. Nucleic Acids Res 35, 7313-23 (2007) Chodaparambil, J.V., Barbera, A.J., Lu, X., Kaye, K.M., Hansen, J.C., Luger, K.: A charged and contoured surface on the nucleosome regulates chromatin compaction. Nat Struct Mol Biol 14, 1105-7 (2007) Cléard, F., Matsarskaia, M., Spierer, P.: The modifier of position-effect variegation Suvar(3)7 of Drosophila: there are two alternative transcripts and seven scattered zinc fingers, each preceded by a tryptophan box. Nucleic Acids Res 23, 796-802 (1995). Erratum in: Nucleic Acids Res 23, 3804 (1995) Cléard, F., Delattre, M., Spierer, P.: SU(VAR)3-7, a Drosophila heterochromatinassociated protein and companion of HP1 in the genomic silencing of positioneffect variegation. EMBO J. 16, 5280-8 (1997) Cléard, F., Spierer, P.: Position-effect variegation in Drosophila: the modifier Su(var)3-7 is a modular DNA-binding protein. EMBO Rep. 2, 1095-100 (2001) Couture, J.F., Hauk, G., Thompson, M.J., Blackburn, G.M., Trievel, R.C.: Catalytic roles for carbon-oxygen hydrogen bonding in SET domain lysine methyltransferases. J Biol Chem 281, 19280-7 (2006) Cowieson, N.P., Partridge, J.F., Allshire, R.C., McLaughlin, P.J.: Dimerisation of a chromo shadow domain and distinctions from the chromodomain as revealed by structural analysis. Curr Biol 10, 517-25 (2000) Crawford, G.E., Davis, S., Scacheri, P.C., Renaud, G., Halawi, M.J., Erdos, M.R., Green, R., Meltzer, P.S., Wolfsberg, T.G., Collins, F.S.: DNase-chip: a highresolution method to identify DNase I hypersensitive sites using tiled microarrays. Nat Methods 3, 503-9 (2006) Cui, X., De Vivo, I., Slany, R., Miyamoto, A., Firestein, R., Cleary, M.L.: Association of SET domain and myotubularin-related proteins modulates growth control. Nat Genet 18, 331-7 (1998) Cuthbert, G.L., Daujat, S., Snowden, A.W., Erdjument-Bromage, H., Hagiwara, T.,  44  Yamada, M., Schneider, R., Gregory, P.D., Tempst, P., Bannister, A.J., Kouzarides, T.: Histone deimination antagonizes arginine methylation. Cell 118, 545-53 (2004) Czermin, B., Schotta, G., Hulsmann, B.B., Brehm, A., Becker, P.B., Reuter, G., Imhof, A.: Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila. EMBO Rep. 2, 915-9 (2001) de la Cruz, X., Lois, S., Sánchez-Molina, S., Martínez-Balbás, M.A.: Do protein motifs read the histone code? Bioessays 27, 164-7 (2005) Delattre, M., Spierer, A., Tonka, C-H., Spierer, P.: The genomic silencing of position-effect variegation in Drosophila melanogaster: interaction between the heterochromatin-associated proteins Su(var)3-7 and HP1. J Cell Sci 113, 4253-61 (2000). Delattre, M., Spierer, A,. Jaquet, Y., Spierer, P.: Increased expression of Drosophila Su(var)3-7 triggers Su(var)3-9-dependent heterochromatin formation. J Cell Sci. 117, 6239-47 (2004) Demakova, O.V., Pokholkova, G.V., Kolesnikova, T.D., Demakov, S.A., Andreyeva, E.N., Belyaeva, E.S., Zhimulev, I.F.: The SU(VAR)3-9/HP1 complex differentially regulates the compaction state and degree of underreplication of X chromosome pericentric heterochromatin in Drosophila melanogaster. Genetics 175, 609-20 (2007) de Wit, E., Greil, F., van Steensel, B.: Genome-wide HP1 binding in Drosophila: developmental plasticity and genomic targeting signals. Genome Res. 15, 1265-73 (2005) Di Stefano, L., Ji, J.Y., Moon, N.S., Herr, A., Dyson, N.: Mutation of Drosophila Lsd1 disrupts H3-K4 methylation, resulting in tissue-specific defects during development. Curr Biol 17, 808-12 (2007) Dominski, Z., Marzluff, W.F.: Formation of the 3' end of histone mRNA. Gene 239, 1-14 (1999) Dominski, Z., Marzluff, W.F.: Formation of the 3' end of histone mRNA: getting closer to the end. Gene 396, 373-390 (2007) Dombradi, V., Axton, J.M., Brewis, N.D., da Cruz e Silva, E.F., Alphey, L., Cohen, P.T.: Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1. Eur J Biochem. 194, 739-45 (1990) Dormann, H.L., Tseng, B.S., Allis, C.D., Funabiki, H., Fischle, W.: Dynamic regulation of effector protein binding to histone modifications: the biology of HP1  45  switching. Cell Cycle 5, 2842-51 (2006) Ebert, A., Schotta, G., Lein, S., Kubicek, S., Krauss, V., Jenuwein, T., Reuter, G.: Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev 18, 2973-83 (2004) Ebert, A., Lein, S., Schotta, G., Reuter, G.: Histone modification and the control of heterochromatic gene silencing in Drosophila. Chromosome Res. 14, 377-92 (2006) Eskeland, R., Czermin, B., Boeke, J., Bonaldi, T., Regula, J.T., Imhof, A.: The Nterminus of Drosophila SU(VAR)3-9 mediates dimerization and regulates its methyltransferase activity. Biochemistry 43, 3740-9 (2004) Eskeland, R., Eberharter, A., Imhof, A.: HP1 binding to chromatin methylated at H3K9 is enhanced by auxiliary factors. Mol Cell Biol. 27, 453-65 (2007) Eissenberg, J.C., James, T.C., Foster-Hartnett, D.M., Hartnett, T., Ngan, V., Elgin, S.C.: Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster. Proc Natl Acad Sci U S A 87, 9923-7 (1990) Eissenberg JC, Morris GD, Reuter G, Hartnett T.: The heterochromatin-associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation. Genetics 131, 345-52 (1992) Eissenberg, J.C., Elgin, S.C.: The HP1 protein family: getting a grip on chromatin. Curr Opin Genet Dev 10, 204-10 (2000) Eissenberg, J.C., Lee, M.G., Schneider, J., Ilvarsonn, A., Shiekhattar, R., Shilatifard, A.: The trithorax-group gene in Drosophila little imaginal discs encodes a trimethylated histone H3 Lys4 demethylase. Nat Struct Mol Biol. 14, 344-6 (2007) Evans, C.J., Hartenstein, V., Banerjee, U.: Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis. Dev Cell. 5, 673-90 (2003) Finch, J.T., Rhodes, D., Brown, R.S., Rushton, B., Levitt, M., Klug, A.: Structure of nucleosome core particles of chromatin. Nature 269, 29-36 (1977) Fischle, W., Wang, Y., Jacobs, S.A., Kim, Y., Allis, C.D., Khorasanizadeh, S.: Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev. 17, 1870-81 (2003)  46  Garzino, V., Pereira, A., Laurenti, P., Graba, Y., Levis, R.W., Le Parco, Y., Pradel, J.: Cell lineage-specific expression of modulo, a dose-dependent modifier of variegation in Drosophila. EMBO J. 11, 4471-9 (1992) Glise, B., Noselli, S.: Coupling of Jun amino-terminal kinase and Decapentaplegic signaling pathways in Drosophila morphogenesis. Genes Dev. 11, 1738-47 (1997) Greil, F., van der Kraan, I., Delrow, J., Smothers, J.F., de Wit, E., Bussemaker, H.J., van Driel, R., Henikoff, S., van Steensel, B.: Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev. 17, 2825-38 (2003) Grienenberger, A., Miotto, B., Sagnier, T., Cavalli, G., Schramke, V., Geli, V., Mariol, M.C., Berenger, H., Graba, Y., Pradel, J.: The MYST domain acetyltransferase Chameau functions in epigenetic mechanisms of transcriptional repression. Curr Biol. 12, 762-6 (2002) Guo, H.B., Guo, H.: Mechanism of histone methylation catalyzed by protein lysine methyltransferase SET7/9 and origin of product specificity. Proc Natl Acad Sci U S A 104, 8797-80 (2007) Hari, K.L., Cook, K.R., Karpen, G.H.: The Drosophila Su(var)2-10 locus regulates chromosome structure and function and encodes a member of the PIAS protein family. Genes Dev. 15, 1334-48 (2001) Huang, X., Kadonaga, J.T.: Biochemical analysis of transcriptional repression by Drosophila histone deacetylase 1. J Biol Chem. 276, 12497-500 (2001) Jacobs, S.A., Taverna, S.D., Zhang, Y., Briggs, S.D., Li, J., Eissenberg, J.C., Allis, C.D., Khorasanizadeh, S.: Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3. EMBO J. 20, 5232-41 (2001) Jaffe, L.F.: Epigenetic theories of cancer initiation. Adv Cancer Res. 90, 209-30 (2003) James, T.C., Elgin, S.C.: Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene. Mol Cell Biol. 6, 3862-72 (1986) James, T.C., Eissenberg, J.C., Craig, C., Dietrich, V., Hobson, A., Elgin, S.C.: Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. Eur J Cell Biol 50, 170-80 (1989) Jaquet, Y., Delattre, M., Spierer, A., Spierer, P.: Functional dissection of the Drosophila modifier of variegation Su(var)3-7. Development 129, 3975-82 (2002)  47  Jaquet, Y., Delattre, M., Montoya-Burgos, J., Spierer, A., Spierer, P.: Conserved domains control heterochromatin localization and silencing properties of SU(VAR)3-7. Chromosoma 115, 139-5 (2006) Jin, Y., Wang, Y., Walker, D.L., Dong, H., Conley, C., Johansen, J., Johansen, K.M.: JIL-1: a novel chromosomal tandem kinase implicated in transcriptional regulation in Drosophila. Molec. Cell 4, 29-35 (1999) Jones, D.O., Cowell, I.G., Singh, P.B.: Mammalian chromodomain proteins: their role in genome organisation and expression. Bioessays 22, 124-37 (2000) Jones, R.S., Gelbart, W.M.: The Drosophila Polycomb-group gene Enhancer of zeste contains a region with sequence similarity to trithorax. Mol Cell Biol 13, 635766 (1993) Karachentsev, D., Sarma, K., Reinberg, D., Steward, R.: PR-Set7-dependent methylation of histone H4 Lys 20 functions in repression of gene expression and is essential for mitosis. Genes Dev. 19, 431-5 (2005) Kellum, R., Alberts, B.M.: Heterochromatin protein 1 is required for correct chromosome segregation in Drosophila embryos. J Cell Sci. 108, 1419-31 (1995) Kim, T.H., Barrera, L.O., Zheng, M., Qu, C., Singer, M.A., Richmond, T.A., Wu, Y., Green, R.D., Ren, B.: A high-resolution map of active promoters in the human genome. Nature 436, 876-80 (2005) Klug, A., Rhodes, D., Smith, J., Finch, J.T., Thomas, J.O.: A low resolution structure for the histone core of the nucleosome. Nature 287, 509-516 (1980) Kornberg, R.D.: Structure of chromatin. Annu Rev Biochem. 46, 931-54 (1977) Koryakov, D.E., Reuter, G., Dimitri, P., Zhimulev, I.F.: The SuUR gene influences the distribution of heterochromatic proteins HP1 and SU(VAR)3-9 on nurse cell polytene chromosomes of Drosophila melanogaster. Chromosoma 115, 296-310 (2006) Kouskouti A, Scheer E, Staub A, Tora L, Talianidis I.: Gene-specific modulation of TAF10 function by SET9-mediated methylation. Mol Cell 14, 175-82 (2004) Kouzarides, T.: Chromatin modifications and their function. Cell 128, 693-705 (2007) Krauss, V., Fassl, A., Fiebig, P., Patties, I., Sass, H.: The evolution of the histone methyltransferase gene Su(var)3-9 in metazoans includes a fusion with and a refission from a functionally unrelated gene. BMC Evol Biol 2, 6:18 (2006)  48  Krejci, E., Garzino, V., Mary, C., Bennani, N., Pradel, J.: Modulo, a new maternally expressed Drosophila gene encodes a DNA-binding protein with distinct acidic and basic regions. Nucleic Acids Res. 17, 8101-15 (1989) Kusch, T., Florens, L., Macdonald, W.H., Swanson, S.K., Glaser, R.L., Yates, J.R. 3rd, Abmayr, S.M., Washburn, M.P., Workman, J.L.: Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions. Science 306, 20847 (2004) Kwon, T., Chang, J.H., Kwak, E., Lee ,C.W., Joachimiak, A., Kim, Y.C., Lee, J., Cho, Y.: Mechanism of histone lysine methyl transfer revealed by the structure of SET7/9-AdoMet. EMBO J 22, 292-303 (2003) Lachner, M., O'Carroll, D., Rea, S., Mechtler, K., Jenuwein, T.: Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116-20 (2001) Lachner, M., Jenuwein, T.: The many faces of histone lysine methylation. Curr Opin Cell Biol 14, 286-98 (2002) Lee, N., Zhang, J., Klose, R.J., Erdjument-Bromage, H., Tempst, P., Jones, R.S., Zhang, Y.: The trithorax-group protein Lid is a histone H3 trimethyl-Lys4 demethylase. Nat Struct Mol Biol. 14, 341-3 (2007) Liu, C.L., Kaplan, T., Kim, M., Buratowski, S., Schreiber, S.L., Friedman, N., Rando, O.J.: Single-nucleosome mapping of histone modifications in S. cerevisiae. PLoS Biol. 3, e328 (2005) Lloret-Llinares, M., Carré, C., Vaquero, A., de Olano, N., Azorín, F.: Characterization of Drosophila melanogaster JmjC+N histone demethylases. Nucleic Acids Res 36, 2852-6 (2008) Lomberk G, Bensi D, Fernandez-Zapico ME, Urrutia R.: Evidence for the existence of an HP1-mediated subcode within the histone code. Nat Cell Biol 8, 407-15 (2006) Ludlam, W.H., Taylor, M.H., Tanner, K.G., Denu, J.M., Goodman, R.H., Smolik, S.M.: The acetyltransferase activity of CBP is required for wingless activation and H4 acetylation in Drosophila melanogaster. Mol Cell Biol. 22, 3832-41 (2002) Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., Richmond, T.J.: Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251-60 (1997)  49  Maixner, A., Hecker, T.P., Phan, Q.N., Wassarman, D.A.: A screen for mutations that prevent lethality caused by expression of activated Sevenless and Ras1 in the Drosophila embryo. Dev. Genet. 23, 347-61 (1998) Martin-Blanco, E., Gampel, A., Ring, J., Virdee, K., Kirov, N., Tolkovsky, A.M., Martinez-Arias, A.: puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila. Genes Dev. 12, 557-70 (1998) Mellor, J.: It takes a PHD to read the histone code. Cell 126, 22-4 (2006) Mikkelsen, T.S., Ku, M., Jaffe, D.B., Issac, B., Lieberman, E., Giannoukos, G., Alvarez, P., Brockman, W., Kim, T.K., Koche, R.P., Lee, W., Mendenhall, E., O'Donovan, A., Presser, A., Russ, C., Xie, X., Meissner, A., Wernig, M., Jaenisch, R., Nusbaum, C., Lander, E.S., Bernstein, B.E.: Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553-60 (2007) Min, J., Zhang, X., Cheng, X., Grewal, S.I., Xu, R.M.: Structure of the SET domain histone lysine methyltransferase Clr4. Nat Struct Biol. 9, 828-32 (2002) Min, J., Zhang, Y., Xu, R.M.: Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27. Genes Dev. 17, 1823-8 (2003) Miotto, B., Sagnier, T., Berenger, H., Bohmann, D., Pradel, J., Graba, Y.: Chameau HAT and DRpd3 HDAC function as antagonistic cofactors of JNK/AP-1dependent transcription during Drosophila metamorphosis. Genes Dev. 20, 101-12 (2006) Mis, J., Ner, S.S., Grigliatti, T.A.: Identification of three histone methyltransferases in Drosophila: dG9a is a suppressor of PEV and is required for gene silencing. Mol Genet Genomics 275, 513-26 (2006) Mohr, S.E., Boswell, R.E.: Zimp encodes a homologue of mouse Miz1 and PIAS3 and is an essential gene in Drosophila melanogaster. Gene 229, 109-16 (1999) Moss, T.J., Wallrath, L.L.: Connections between epigenetic gene silencing and human disease. Mutat Res. 618, 163-74 (2007) Mottus, R., Sobel, R.E., Grigliatti, T.A.: Mutational analysis of a histone deacetylase in Drosophila melanogaster: missense mutations suppress gene silencing associated with position effect variegation. Genetics 154, 657-68 (2000) Mujtaba, S., Zeng, L., Zhou, M.M..: Structure and acetyl-lysine recognition of the bromodomain. Oncogene 26, 5521-7 (2007)  50  Muller, P., Kuttenkeuler, D., Gesellchen, V., Zeidler, M.P., Boutros, M: Identification of JAK/STAT signalling components by genome-wide RNA interference. Nature 436, 871-75 (2005) Nakayama, J., Rice, J.C., Strahl, B.D., Allis, C.D., Grewal, S.I.: Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110-3 (2001) Nelson, W.G., Yegnasubramanian, S., Agoston, A.T., Bastian, P.J., Lee, B.H., Nakayama, M., De Marzo, A.M.: Abnormal DNA methylation, epigenetics, and prostate cancer. Front Biosci.12, 4254-66 (2007) Ner, S.S., Harrington, M.J., Grigliatti, T.A.: A role for the Drosophila SU(VAR)3-9 protein in chromatin organization at the histone gene cluster and in suppression of position-effect variegation. Genetics 162, 1763-74 (2002) Nielsen, S.J., Schneider, R., Bauer, U.M., Bannister, A.J., Morrison, A., O'Carroll, D., Firestein, R., Cleary, M., Jenuwein, T., Herrera, R.E., Kouzarides, T.: Rb targets histone H3 methylation and HP1 to promoters. Nature 412, 561-5 (2001) Nishioka, K., Rice, J.C., Sarma, K., Erdjument-Bromage, H., Werner, J., Wang, Y., Chuikov, S., Valenzuela, P., Tempst, P., Steward, R., Lis, J.T., Allis, C.D., Reinberg, D.: PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin. Mol Cell 9, 1201-13 (2002) Oligny, L.L.: Cancer and epigenesis: a developmental perspective. Adv Pediatr. 50, 59-80 (2003) Pankotai, T., Komonyi, O., Bodai, L., Ujfaludi, Z., Muratoglu, S., Ciurciu, A., Tora, L., Szabad, J., Boros, I.: The homologous Drosophila transcriptional adaptors ADA2a and ADA2b are both required for normal development but have different functions. Mol Cell Biol. 25, 8215-27 (2005) Paro, R., Hogness, D.S.: The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila. Proc Natl Acad Sci U S A 88, 263-7 (1991) Perrin, L., Benassayag, C., Morello, D., Pradel, J., Montagne, J.: Modulo is a target of Myc selectively required for growth of proliferative cells in Drosophila. Mech Dev. 120, 645-55 (2003) Peters, A.H., O'Carroll, D., Scherthan, H., Mechtler, K., Sauer, S., Schofer, C., Weipoltshammer, K., Pagani, M., Lachner, M., Kohlmaier, A., Opravil, S., Doyle, M., Sibilia, M., Jenuwein, T.: Loss of the Suv39h histone methyltransferases  51  impairs mammalian heterochromatin and genome stability. Cell 107, 323-37 (2001) Peters, A.H., Mermoud, J.E., O'Carroll, D., Pagani, M., Schweizer, D., Brockdorff, N., Jenuwein, T.: Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin. Nat Genet. 30, 77-80 (2002) Pokholok, D.K., Harbison, C.T., Levine, S., Cole, M., Hannett, N.M., Lee, T.I., Bell, G.W., Walker, K., Rolfe, P.A., Herbolsheimer, E., Zeitlinger, J., Lewitter, F., Gifford, D.K., Young, R.A.: Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122, 517-27 (2005) Qian, C., Wang, X., Manzur, K., Sachchidanand, Farooq, A., Zeng, L., Wang, R., Zhou, M.M.: Structural insights of the specificity and catalysis of a viral histone H3 lysine 27 methyltransferase. J Mol Biol 359, 86-96 (2006) Qian, C., Zhou, M.M.: SET domain protein lysine methyltransferases: Structure, specificity and catalysis. Cell Mol Life Sci 63, 2755-63 (2006) Razin, S.V., Iarovaia, O.V., Sjakste, N., Sjakste, T., Bagdoniene, L., Rynditch, A.V., Eivazova, E.R., Lipinski, M., Vassetzky, Y.S.: Chromatin domains and regulation of transcription. J Mol Biol. 369, 597-607 (2007) Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B.D., Sun, Z.W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C.P., Allis, C.D., Jenuwein, T.: Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593-9 (2000) Reuter G, Giarre M, Farah J, Gausz J, Spierer A, Spierer P.: Dependence of position-effect variegation in Drosophila on dose of a gene encoding an unusual zinc-finger protein. Nature 344, 219-23 (1990) Reuter, G., Spierer, P.: Position effect variegation and chromatin proteins. Bioessays 14, 605-12 (1992) Reuter, G., Wolff, I.: Isolation of dominant suppressor mutations for position-effect variegation in Drosophila melanogaster. Mol Gen Genet. 182, 516-9 (1981) Roh, T.Y., Cuddapah, S., Zhao, K.: Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping. Genes Dev 19, 542-52 (2005) Rudolph, T., Yonezawa, M., Lein, S., Heidrich, K., Kubicek, S., Schafer, C., Phalke, S., Walther, M., Schmidt, A., Jenuwein, T., Reuter, G.: Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3. Mol Cell. 26, 103-15 (2007) Saigo, K., Millstein, L., Thomas, C.A. Jr. The organization of Drosophila  52  melanogaster histone genes. Cold Spring Harb Symp Quant Biol. 45, 815-27 (1981) Samal, B., Worcel, A., Louis, C., Schedl, P.: Chromatin structure of the histone genes of D. melanogaster. Cell 23, 401-9 (1981) Sampath, S.C., Marazzi, I., Yap, K.L., Sampath, S.C., Krutchinsky, A.N., Mecklenbräuker, I., Viale, A., Rudensky, E., Zhou, M.M., Chait, B.T., Tarakhovsky, A.: Methylation of a histone mimic within the histone methyltransferase G9a regulates protein complex assembly. Mol Cell 27, 596-608 (2007) Schones, D.E., Zhao, K.: Genome-wide approaches to studying chromatin modifications. Nat Rev Genet. 9, 179-91 (2008) Schotta, G., Ebert, A., Krauss, V., Fischer, A., Hoffmann, J., Rea, S., Jenuwein, T., Dorn, R., Reuter, G.: Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. EMBO J. 21, 1121-31 (2002) Schotta, G., Ebert, A., Reuter, G.: SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing. Genetica 117, 149-58 (2003) Schotta, G., Ebert, A., Dorn, R., Reuter, G.: Position-effect variegation and the genetic dissection of chromatin regulation in Drosophila. Semin Cell Dev Biol. 14, 67-75 (2003) Schotta, G., Lachner, M., Sarma, K., Ebert, A., Sengupta, R., Reuter, G., Reinberg, D., Jenuwein, T.: A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. Genes Dev. 18, 1251-62 (2004) Schübeler, D., MacAlpine, D.M., Scalzo, D., Wirbelauer, C., Kooperberg, C., van Leeuwen, F., Gottschling, D.E., O'Neill, L.P., Turner, B.M., Delrow, J., Bell, S.P., Groudine M.: The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev.18, 1263-71 (2004) Secombe, J., Li, L., Carlos, L., Eisenman, R.N.: The Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth. Genes Dev. 21, 537-51 (2007) Sedkov, Y., Cho, E., Petruk, S., Cherbas, L., Smith, S.T., Jones, R.S., Cherbas, P., Canaani, E., Jaynes, J.B., Mazo, A.: Methylation at lysine 4 of histone H3 in ecdysone-dependent development of Drosophila. Nature 426, 78-83 (2003) Seum, C., Reo, E., Peng, H., Rauscher, F.J. 3rd, Spierer, P., Bontron, S.: Drosophila SETDB1 is required for chromosome 4 silencing. PLoS Genet. 3, e76 (2007)  53  Seum, C., Bontron, S., Reo, E., Delattre, M., Spierer, P.: Drosophila G9a is a nonessential gene. Genetics 177, 1955-7 (2007) Shanower, G.A., Muller, M., Blanton, J.L., Honti, V., Gyurkovics, H., Schedl, P.: Characterization of the grappa gene, the Drosophila histone H3 lysine 79 methyltransferase. Genetics 169, 173-84 (2005) Shi, Y., Lan, F., Matson, C., Mulligan, P., Whetstine, J.R., Cole, P.A., Casero, R.A., Shi, Y.: Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119, 941-53 (2004) Shogren-Knaak, M., Ishii, H., Sun, J.M., Pazin, M.J., Davie, J.R., Peterson, C.L.: Histone H4-K16 acetylation controls chromatin structure and protein interactions. Science 311, 844-7 (2006) Sinclair, D.A., Clegg, N.J., Antonchuk, J., Milne, T.A., Stankunas, K., Ruse, C., Grigliatti, T.A., Kassis, J.A., Brock, H.W.: Enhancer of Polycomb is a suppressor of position-effect variegation in Drosophila melanogaster. Genetics 148, 211-20 (1998) Smith, S.T., Petruk, S., Sedkov, Y., Cho, E., Tillib, S., Canaani, E., Mazo, A.: Modulation of heat shock gene expression by the TAC1 chromatin-modifying complex. Nat Cell Biol. 6, 162-7 (2004) Spofford, J.B.: Position effect variegation in Drosophila. In: The Genetics and Biology of Drosophila Vol. 1c, 955–1018. (1976) Edited by M. Ashburner & E. Novitski. Academic Press, New York. Stabell, M., Eskeland, R., Bjorkmo, M., Larsson, J., Aalen, R.B., Imhof, A., Lambertsson, A.: The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development. Nucleic Acids Res. 34, 460921 (2006) Stankunas, K., Berger, J., Ruse, C., Sinclair, D.A., Randazzo, F., Brock, H.W.: The enhancer of polycomb gene of Drosophila encodes a chromatin protein conserved in yeast and mammals. Development 125, 4055-66 (1998) Struhl, K.: Fundamentally different logic of gene regulation in eukaryotes and prokaryotes. Cell 98, 1-4 (1999) Swaminathan, J., Baxter, E.M., Corces, V.G.: The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin. Genes Dev. 19, 65-76 (2005) Tachibana, M., Sugimoto, K., Nozaki, M., Ueda, J., Ohta, T., Ohki, M., Fukuda, M.,  54  Takeda, N., Niida, H., Kato, H., Shinkai, Y.: G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 16, 1779-91 (2002) Talbert, P.B., Henikoff, S.: Spreading of silent chromatin: inaction at a distance. Nat Rev Genet. 10, 793-803 (2006) Tie F, Prasad-Sinha J, Birve A, Rasmuson-Lestander A, Harte PJ.: A 1megadalton ESC/E(Z) complex from Drosophila that contains polycomblike and RPD3. Mol Cell Biol 23, 3352-62 (2003) Tschiersch, B., Hofmann, A., Krauss, V., Dorn, R., Korge, G., Reuter, G.: The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J. 13, 3822-31 (1994) Tsukada, Y., Fang, J., Erdjument-Bromage, H., Warren, M.E., Borchers, C.H., Tempst, P., Zhang, Y.: Histone demethylation by a family of JmjC domaincontaining proteins. Nature 439, 811-6 (2006) Turner, B.M., Birley, A.J., Lavender, J.: Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell 69, 375-84 (1992) Turner, B.M.: Histone acetylation and an epigenetic code. Bioessays 22, 836-45 (2000) Turner, B.M.: Reading signals on the nucleosome with a new nomenclature for modified histones. Nat Struct Mol Biol. 12, 110-2 (2005) Tzeng, T.Y., Lee, C.H., Chan, L.W., Shen, C.K.: Epigenetic regulation of the Drosophila chromosome 4 by the histone H3K9 methyltransferase dSETDB1. Proc Natl Acad Sci U S A. 104, 12691-6 (2007) Vandel, L., Nicolas, E., Vaute, O., Ferreira, R., Ait-Si-Ali, S., Trouche, D.: Transcriptional repression by the retinoblastoma protein through the recruitment of a histone methyltransferase. Mol Cell Biol. 21, 6484-94 (2001) Vaute, O., Nicolas, E., Vandel, L., Trouche, D.: Functional and physical interaction between the histone methyl transferase Suv39H1 and histone deacetylases. Nucleic Acids Res 30, 475-81 (2002) Wallrath, L.L., Elgin, S.C.: Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 9, 1263-77 (1995) Wang, Y., Zhang, W., Jin, Y., Johansen, J., Johansen, K.M.: The JIL-1 tandem  55  kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila. Cell 105, 433-43 (2001) Wang, Y., Wysocka, J., Sayegh, J., Lee, Y.H., Perlin, J.R., Leonelli, L., Sonbuchner, L.S., McDonald, C.H., Cook, R.G., Dou, Y., Roeder, R.G., Clarke, S., Stallcup, M.R., Allis, C.D., Coonrod, S.A.: Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 306, 279–83 (2004) Weiler, K.S., Wakimoto, B.T.: Heterochromatin and gene expression in Drosophila. Annu Rev Genet 29, 577-605 (1995) Xiao, B., Jing, C., Kelly, G., Walker, P.A., Muskett, F.W., Frenkiel, T.A., Martin, S.R., Sarma, K., Reinberg, D., Gamblin, S.J., Wilson, J.R.: Specificity and mechanism of the histone methyltransferase Pr-Set7. Genes Dev 19, 1444-54 (2005) Yang, X.J.: Lysine acetylation and the bromodomain: a new partnership for signaling. Bioessays 26, 1076-87 (2004) Zhang, K., Mosch, K., Fischle, W., Grewal, S.I.: Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin. Nat Struct Mol Biol 15, 381-8 (2008) Zhang, W., Deng, H., Bao, X., Lerach, S., Girton, J., Johansen, J., Johansen, K.M.: The JIL-1 histone H3S10 kinase regulates dimethyl H3K9 modifications and heterochromatic spreading in Drosophila. Development 133, 229-35 (2006) Zhimulev, I.F., Belyaeva, E.S.: Intercalary heterochromatin and genetic silencing. Bioessays 25, 1040-51 (2003)  56  2. ALTERED HISTONE H3 METHYLTRANSFERASE ACTIVITY OF SU(VAR)3-9 IMPAIRS GENE SILENCING1.  2.1.  Introduction The chromatin of eukaryotic organisms is a dynamic complex of DNA and  proteins. The nucleosome, its basic structural unit, is comprised of 146 bp of DNA wrapped around an octamer of the core histones H3, H4, H2A and H2B (Finch et al. 1977; Klug et al., 1980, Luger et al., 1997). Higher order assemblies of nucleosomes package the genome into structured regions that typically have different functional properties. For example, regions of the genome surrounding the centromere have nucleosomes organized into highly condensed and tightly packaged chromatin, are transcriptionally inert, late replicating and are collectively termed heterochromatin (Gatti and Pimpinelli, 1992; Lohe and Hilliker, 1995). Regions that are less densely packaged, accessible to the transcriptional machinery, and which replicate earlier in S-phase, are collectively known as euchromatin (often also referred to as “open” chromatin). However, even euchromatin is a mosaic of silenced (repressed) and transcriptionally competent (open) domains. Numerous non-histone proteins and a range of covalent histone modifications influence nucleosome-nucleosome interactions and higher order packaging and thus control the transitions between transcriptionally competent (open) and transcriptionally repressed states of the euchromatic portion of the genome. The histone modifications, particularly at the N-terminal tails, include acetylation,  1  A version of this chapter will be submitted for publication. Kalas, P. and Grigliatti, T.A. Altered histone H3 methyltransferase activity of SU(VAR)3-9 impairs gene silencing.  57  Specific patterns of histone modifications correlate with gene activity and chromatin structure - the “histone code” hypothesis (Jenuwein and Allis, 2001). For example, acetylation of lysine 9 of histone H3 (H3K9ac) is typically associated with gene expression, while methylation of H3K9 (H3K9me) and H4K20me are usually associated with gene repression or silenced regions of the genome (Litt et al., 2001; Schotta et al., 2004; reviewed in Rice and Allis, 2001; Berger, 2002; Ebert et al., 2006). In Drosophila, methylation of H3K9 is catalyzed by SU(VAR)3-9, the archetypal SET domain-containing histone methyltransferase (HMTase) that is highly conserved in eukaryotes (Aagaard et al., 1999; Rea et al., 2000; Ivanova et al., 1998). Mutations in Su(var)3-9 were recovered in genetic screens (Reuter and Wolff, 1981; Sinclair et al., 1983; Donaldson et al., 2002) as strong dominant suppressors of the heterochromatinassociated gene silencing phenomenon, position-effect variegation (PEV) (Muller, 1930; Grigliatti, 1991; Lewis, 1950; Weiler and Wakimoto, 1995). All Su(var)3-9 mutants known to date are strong, dominant suppressors of wm4, SbV and bwV variegation (Sinclair et al., 1983; Reuter and Wolff, 1981). SU(VAR)3-9 has now been extensively characterized. It associates with heterochromatic regions (Schotta et al., 2002; 2003) and numerous euchromatic loci (Greil et al., 2003), including the tandemly reiterated histone gene cluster (HIS-C) (Ner et al., 2002), where it epigenetically modulates the expression of the histone genes by altering the chromatin structure of the locus via methylation of H3K9 and recruitment of HP1 (Ner et al., 2002; Ner et al., in preparation). The original EMS mutagenesis screen isolated over 2 dozen mutants that mapped near the Su(var)3-9 locus (Sinclair et al. 1983). This chapter focuses on a subset of these mutants. We demonstrate that these mutations are indeed Su(var)3-9  58  alleles, and examine their phenotypes at four different levels: molecular, cellular, biochemical and morphological (eye colour/PEV). At the molecular level, we found that all alleles have missense mutations resulting in single amino acid substitutions in the catalytic region of the protein, which consists of the evolutionarily conserved preSET, SET and postSET domains. In contrast, no mutations were recovered in the other highly conserved region of SU(VAR)3-9, the chromodomain, or in the Drosophila-specific Nterminus, suggesting alterations in these regions don’t influence the epigenetic silencing resulting from PEV. At the cellular level, the Su(var)3-9 mutants had reduced levels of H3K9me2 and HP1 at both the chromocentre and a euchromatic locus, the HIS-C. Biochemically, we showed that all amino acid substitutions either abolish or dramatically reduce the HMTase activity of SU(VAR)3-9 in vitro. Substitutions of conserved cysteine residues in the preSET domain directly correlated with a complete loss-of-HMTase function in vitro, while changes in the SET or postSET regions resulted in partial loss-offunction. Finally, at the level of morphological phenotypes, eye colour/PEV, all Su(var)39 mutants were able to suppress wm4 variegation in a strain that is hyperploid for Su(var)3-9, but we observed differences in their strength of suppression.  2.2. Results 2.2.1. Recombination mapping and DNA Sequence analysis In the early 1980s our lab recovered about 50 third chromosome EMS-induced mutants that were strong, dominant suppressors of PEV (Sinclair et al., 1983). Over twenty of these mutants are homozygous viable, clustered on the right arm of the third chromosome and circumstantial evidence strongly indicated that a large subset of these  59  were Su(var)3-9 alleles (Harrington, 2001). The mutations are homozygous viable, which obviated the use of complementation analyses to define allelism. Thus, to determine which of the mutations were Su(var)3-9 alleles, we performed recombination analysis using a recessive lethal P-element insert, Su(var)3-9P25 (Harrington, 2001). For each of the 20 putative Su(var)3-9 alleles, we generated heterozygous Su(var)39P25/Su(var)3-9putative females and crossed them to wild-type males, and examined the progeny. For 13 mutants we observed no wild-type recombinants (after screening > 2000 flies for each cross), indicating that the mutations they carry are very closely linked to Su(var)3-9P25 (<0.1 cM; Table 1). These 13 mutants were then characterized by DNA sequence analysis to determine the precise nature and position of the mutation. Nine out of the 12 were missense mutants in Su(var)3-9 (Table 1). Accordingly, we renamed these mutants as Su(var)3-9allele number (Table 1). Three mutants, Su(var)3-9317, Su(var)3-9327, and Su(var)3-9329, did not contain mutations in the coding region of Su(var)3-9. Since the estimated distance between the mutations in each of these three mutants and Su(var)3-9P25 is <0.1 cM, which represents about 50kb or less in the region surrounding the Su(var)3-9 locus (http://flybase.bio.indiana.edu), these mutants may still be alleles of Su(var)3-9 and represent alterations in the regulatory region of Su(var)3-9 or cause defects in mRNA processing. Alternatively, their Su(var) phenotype may be the result of mutations in one or more genes that are closely linked to Su(var)3-9, such as Set or Oscp. Indeed, Su(var) mutations are rather commonly found in clusters, closely mapping, but not contiguous (Sinclair et al., 1983). The sequence analysis also revealed single base-pair changes leading to ALA304PHE  and/or ILE375LEU in Su(var)305, Su(var)3-9324, Su(var)3-9329 and Su(var)331  60  (Table 2.1). Since Su(var)305, which carries both substitutions, and Su(var)331, which carries the I375L substitution, are not allelic to Su(var)3-9 (as determined by recombination mapping with Su(var)3-9P25, see Materials and Methods), we concluded that these substitutions are not the cause of the Su(var) phenotype in Su(var)3-9324 or Su(var)3-9329. In Su(var)3-9324, the C428Y is most likely the cause of the Su(var) effect, while for Su(var)3-9329 the mutation responsible for this phenotype may be located in one of the cis-regulatory elements of the gene, which are not known and were therefore not sequenced.  61  Table 2.1. Putative Su(var)3-9 alleles that were mapped and sequenced in this study. Allelism to Su(var)3-9 was determined by recombination mapping with the Pelement induced Su(var)3-9P25 mutant (Ner et al., 2002). Mutants that failed to yield wild-type recombinants were further analyzed by sequencing and, if allelism to Su(var)3-9 was confirmed, they were renamed accordingly.  MUTANT Su(var)301 Su(var)305  Su(var)3-9 allele (genetically)? NO NO  Allelism confirmed by sequencing? N/A (not tested) No  Su(var)306 Su(var)309 Su(var)311 Su(var)312 Su(var)314 Su(var)315 Su(var)317 Su(var)318 Su(var)319 Su(var)320 Su(var)324  NO a YES YES YES NO a NO YES YES YES NO YES  N/A Yes Yes Yes N/A N/A No Yes Yes N/A Yes  Su(var)325 Su(var)327 Su(var)329 Su(var)C76 Su(var)330 Su(var)331  YES YES YES YES YES NO  Yes No No Yes Yes N/A  a  Mutation N/A A304Fb, I375Lb N/A C462Y G521D C462Y N/A N/A N/A S616L S616L N/A C428Y, I375Lb P582Q N/A A304Fb C421S D536N I375Lb  New name  Su(var)3-9309 Su(var)3-9311 Su(var)3-9312  Su(var)3-9318 Su(var)3-9319 Su(var)3-9324 Su(var)3-9325 Su(var)3-9376 Su(var)3-9330  Harrington, 2001. These amino acid substitutions likely represent naturally occurring polymorphisms, and are not responsible for the Su(var) phenotype. b  62  2.2.2. Amino acid substitutions in Su(var)3-9 target the preSET/SET/postSET domain. SU(VAR)3-9 has two distinct, and highly conserved domains: a chromodomain, and a SET domain, which includes the flanking pre- and a postSET regions (Figure 2.1). The chromodomain of SU(VAR)3-9 is about 40 amino acids long and its function is not entirely clear, but it is speculated to have a role in protein-protein interactions that may control H3K9 methylation (Schotta et al., 2003). The preSET/SET/postSET region is ~250 amino acids long, represents less than 40% of the entire length of the protein, and constitutes the catalytic region of the SU(VAR)3-9 HMTase activity (Tschiersch et al., 1994; Rea et al., 2000; Schotta et al., 2002; Ner et al., 2002). Both domains are also found in other chromatin proteins, where they play critical roles in silencing (Ivanova et al., 1998; Nakayama et al., 2001; Schotta et al., 2002; Platero et al., 1995: Messmer et al., 1992; Ma et al., 2001; Akhtar et al., 2000; Jacobs et al., 2001; Bannister et al., 2001; Lachner et al., 2001; Bouazoune et al., 2002; Fischle et al., 2003; Min et al., 2003; PrayGrant et al., 2005). The Drosophila SU(VAR)3-9 has a longer N-terminus than its mammalian and yeast counterparts. This novel region of the protein appears to be required for interactions with chromatin proteins SU(VAR)3-7 and HP1, and for SU(VAR)39 dimerization (Schotta et al., 2002; 2003; Eskeland et al., 2004). Theoretically, mutations in the Drosophila specific N-terminus, the chromodomain, or the preSET/SET/postSET regions could lead to a loss of the protein’s silencing function, and to a Su(var) phenotype. Mutations in the N-terminus may do so by preventing SU(VAR)3-9 dimerization and/or its interaction with other chromatin proteins, such as HP1 and SU(VAR)3-7. Mutations in the chromodomain could affect the targeting of SU(VAR)3-9 and/or its interaction with other proteins. Finally, alterations in the  63  preSET/SET/postSET may impair its catalytic activity, with or without affecting the protein’s ability to participate in protein-protein interactions. In addition, mutations in any region could potentially affect the protein’s folding, thus rendering it inactive. All Su(var)3-9 mutations were recovered in an EMS screen. EMS causes mainly base-pair substitutions, principally G:C to A:T transitions and thus, effectively causes random mutations. Accordingly, we reasoned that 1) if the Su(var)3-9 mutations isolated in the original screen preferentially target certain regions of the protein, then these regions must be crucial for PEV-type gene silencing, and 2) if some of the EMS-induced lesions represent missense mutations, the affected residues are likely crucial for the silencing function of SU(VAR)3-9. Strikingly, we found that all 9 mutants are located solely in the preSET/SET/postSET domain. Not surprisingly, all are single base pair substitutions resulting in missense mutations. Since all the mutations cluster within the catalytic (preSET/SET/postSET) domain of SU(VAR)3-9, our results suggest the catalytic function of SU(VAR)3-9 is essential for the silencing associated with PEV, and perhaps, amino acid substitutions in the remainder of the protein do not influence the repression caused by PEV. The nature of the amino acid substitutions in the various Su(var)3-9 alleles was examined further. Su(var)3-9309 and Su(var)3-9312 arose independently, but have the same A-to-G transition, giving rise to a CYS to TYR substitution at position 462. CYS462 is part of the preSET domain, and is conserved in all homologues of SU(VAR)3-9, including SUV39H1, Suv39h1 and Clr4p. It corresponds to one of nine cysteine residues that coordinate three zinc ions (Min et al., 2002). Su(var)3-9324 (CYS428TYR) and Su(var)3-9376 (CYS421SER) also cause substitutions in this highly conserved group  64  of cysteines (Fig. 2.1). Three alleles, Su(var)3-9325, Su(var)3-9330 and Su(var)3-9311, cause amino acid substitutions in the SET domain. They correspond to PRO582GLU in Su(var)3-9325, GLY521ASP in Su(var)3-9311, and ASP536ASN in Su(var)3-9330. Both PRO582  and GLY521 are conserved in the mammalian homologues of SU(VAR)3-9, but  not in the yeast homologue, Clr4p. ASP536 is conserved in all H3K9-specific HMTases and plays a key role in the interaction with the N-terminal tail of H3 (Zhang et al., 2003). Su(var)3-9318 and Su(var)3-9319 represent a pair of independently induced mutations and each causes a SER616LEU substitution. This residue is part of the post-SET, but it is not conserved in SUV39H1, Suv39h1 or Clr4p. A similar screen performed by Reuter and colleagues (Reuter and Wolff, 1981; Ebert et al., 2004) also recovered multiple alleles of Su(var)3-9, but none of them correspond to the nine mutations we recovered (see Discussion). In summary, the screen yielded nine different Su(var)3-9 mutants of which 4 carry mutations in the preSET domain, 3 in the SET domain, and 2 in the postSET domain. In two cases (Su(var)3-9309 and Su(var)3-9312, and Su(var)3-9318 and Su(var)39319) the same residue was mutated independently, so the 9 different mutants represent 7 different, new alleles/mutations.  65  66  b.  a. N-terminal (fly specific)  eIF2γ chromo preSET  6 4 37 32 3  , 12  30  9 1 0 31 33  SET  3  25  31  post SET  3  , 18  9  Y SU(VAR)3-9324  SU(VAR)3-9311  D  SU(VAR)3-9330  N  . Figure 2.1.  SU(VAR)3-9325  Q  SU(VAR)3-9318/319  L  SU(VAR)3-9 550 ANYGNISHFINHSCDPNLAVFPCWIEHLNVALPHLVFFTLRPIKAGEELSFDY-IRADNEDVPYENLST-------------AVRVECRCGADNCRKVLFSUV39H1 313 AYYGNISHFVNHSCDPNLQVYNVFIDNLDERLPRIAFFATRTIRAGEELTFDYNMQVDPVDMESTRMDSNFGLAGLPGSPKKRVRIECKCGTESCRKYLFCLR4 399 QNYGDVSRFFNHSCSPNIAIYSAVRNHGFRTIYDLAFFAIKDIQPLEELTFDYAGAKDFSPVQSQKSQQN---------RISKLRRQCKCGSANCRGWLFG  SU(VAR)3-9309/312  Y  SU(VAR)3-9 452 AIYECNSRCSCDSSCSNRLVQHGRQVPLVLFKTANGSGWGVRAATALRKGEFVCEYIGEIITSDEANERGKAYDDNGRTYLFDLDYNTAQDSEYTIDA SUV39H1 215 PIYECNSRCRCGYDCPNRVVQKGIRYDLCIFRTDDGRGWGVRTLEKIRKNSFVMEYVGEIITSEEAERRGQIYDRQGATYLFDLDY---VEDVYTVDA CLR4 303 VIYECNSFCSCSMECPNRVVQRGRTLPLEIFKTKE-KGWGVRSLRFAPAGTFITCYLGEVITSAEAAKRDKNYDDDGITYLFDLDMFD-DASEYTVDA  SU(VAR)3-9376  S  SU(VAR)3-9 361 FEKRMNHVEKPSPPIRVENNIDLDTIDS-NFMYIHDNIIGKDVPKPEAGI-VGCKCTEDTEEC-TAST-KC-CARFAG--ELFAYERSTRRLRLRPGS SUV39H1 145 --------------ITVENEVDLDGFFR-AFVYINEYRVGEGITLNQVA--VGCEC----QDCLWAPTGGC-CPGASL--HKFAYNDQG-QVRLRAGL CLR4 220 --------------VTLVNEVDDEPCPSLDFQFISQYRLTQGVIPPDPNFQSGCNCSSLGG-C DLNNPSRCECLDDLDEPTHFAYDAQG-RVRADTGA  SU(VAR)3-9  interactions with HP1, SU(VAR)3-7, SU(VAR)3-9  catalytic region (HMTase)  635 412 490  549 312 398  451 214 302  Figure 2.1. Su(var)3-9 mutants are clustered in the preSET, SET and postSET domains. a) A schematic of the domain structure of SU(VAR)3-9 (635 amino acids). The relative positions of the chromodomain (red box), the pre- and postSET domains (green boxes), the SET domain (blue box), and the region that SU(VAR)3-9 shares with eIF2γ (eIF2γ) are shown. The region N-terminal to the chromodomain is longer in the Drosophila protein than the mammalian or yeast homologues and is involved in proteinprotein interactions with HP1, SU(VAR)3-7 and SU(VAR)3-9 (Schotta et al., 2003, Eskeland et al., 2004). The vertical bars mark the positions of amino acids substitutions in 9 Su(var)3-9 mutants identified by sequence analysis. b) Amino acid alignment of the catalytic domain of SU(VAR)3-9, SUV39H1 (human) and CLR4P (fission yeast) showing major conserved residues (red), conserved preSET cysteines that coordinate 3 zinc ions in CLR4P (yellow) (Min et al. 2002) and the conserved aspartate that directly interacts with the substrate (green) (Zhang et al., 2003). The amino acid changes observed in each EMS-induced mutant are shown below the alignment. Allele pairs Su(var)3-9309 and Su(var)3-9312, and Su(var)3-9318 and Su(var)3-9319, are independently generated mutants resulting in the same amino acid substitutions.  67  2.2.3. The missense alleles express (mutant) Su(var)3-9 gene products. At the morphological level (suppression of PEV in the wm4 strain), the phenotype of the EMS-induced Su(var)3-9 missense mutants is virtually indistinguishable from that of the "protein null", Su(var)3-906 (Reuter and Wolff, 1981; Sinclair et al., 1983; Tschiersch et al., 1994; Schotta et al., 2002). Su(var)3-906 is an X ray-induced, null allele that results in no RNA or protein products (Figures 2.2a and 2.2b, and Tschiersch et al., 1994; Schotta et al., 2002). Western blots were performed on each homozygous mutant strain to ensure that the EMS-induced Su(var)3-9 alleles are not effectively "protein nulls", as a consequence of, for example, an instability of their mutant gene products. All strains tested (Su(var)3-9309, Su(var)3-9330, Su(var)3-9318, Su(var)3-9319 and Su(var)3-9311) showed approximately the same level of SU(VAR)3-9 as the wild-type, confirming that the protein is still present in the mutant strains, in spite of single basepair substitutions. A representative blot is shown in Figure 2.2b.  68  Figure 2.2  a. Su(var)3-9 cDNA amplified RP49 strain wild-type Su(var)3-906 wild-type Su(var)3-906 + + + + RTase 1  3  4  5  6  7  8  -9 3 )3  ar  1  2  3  4  5  α−SU(VAR)3-9  69  30  09  -9 3 Su  (v  Su  (v  ar  )3  -9 0 )3 ar  (v  pe  Su  ild  ty  G w  c. re  b.  ST -S  U  6  (V AR  )3  -9  2  Figure 2.2. Detection of Su(var)3-9 gene products in the EMS-induced Su(var)3-9 missense mutants, and in the null allele Su(var)3-906. a) RT-PCR reactions using total RNA extracted from wild-type (lanes 1, 2, 5 and 6) or Su(var)3-906 female flies as starting material. RNA samples were reverse-transcribed with Su(var)3-9- and RP49-specific primers simultaneously, and the cDNA mixture obtained was PCR amplified separately using primers specific for RP49 (lanes 1-4) or Su(var)3-9 (lanes 5-8). The odd-numbered lanes represent PCR amplification of mock RT reactions (no reverse transcriptase). Identical results were obtained using total RNA from males. Note that primer 3906rt hybridizes to the first 17 nucleotides of Su(var)3-9’s third exon. b) Western blot analysis of wild-type and mutant embryo extracts. Lane 1: GST-SU(VAR)3-9[residues 310-635] recombinant protein (1µg), lane 2: wild-type embryo extract (150µg), lanes 3-5: extracts from homozygous Su(var)3-9 mutant embryos (150µg).  70  2.2.4. Su(var)3-9 mutants display a reduction in the levels of H3K9me and HP1 associated with the chromocentre and with the HIS-C. Since di- and trimethylated H3K9 (H3K9me2/H3K9me3) are the predominant products of SU(VAR)3-9 catalytic activity (Eskeland et al., 2004), we asked whether our EMS-induced Su(var)3-9 mutations had an effect on the level of H3K9me2 in vivo. First, a set of western blots was performed with an anti-H3K9me2 antibody, to compare the levels of H3K9me2 present in embryo extracts from the Su(var)3-9 homozygous mutant strains to the levels in wild-type and Su(var)3-906 ("protein null") extracts. The western blots were also probed with anti-tubulin antibody to establish equal loading of protein extracts, and the total histone H3 present in each extract was detected on parallel blots to ensure that any difference we observed in H3K9me2 levels between the extracts were meaningful (Figure 3a). The Su(var)3-9 mutant extracts had significantly reduced levels of H3K9me2 relative to the wild-type extract (Figure 3a, compare lane 2 with lanes 3-6). However, all Su(var)3-9 missense mutant extracts showed a residual level of H3K9me2. We also detected residual H3K9me2 in the protein null strain, Su(var)3-906. This was not unexpected since there are several other HMTases, including dG9a and DmSETDB1, that can methylate H3K9 (Ayyanathan et al., 2003; Mis et al., 2006; Seum et al., 2007). H3K9me2 is generally considered a mark of heterochromatin, but is also detected at many euchromatic regions (Nielsen et al., 2001; Schotta et al., 2002; Ner et al., 2002; Greil et al., 2003; Ebert et al., 2006). Thus, we next asked whether the reduction in the level of methylated H3K9 observed in the Su(var)3-9 mutants affects heterochromatin exclusively, or whether it can also be observed in some euchromatic  71  regions. For this purpose, we examined the distribution of H3K9me2 at a the chromocentre (heterochromatin) and at the histone genes cluster (HIS-C), a euchromatic target of SU(VAR)3-9 (Ner et al., 2002), in wild-type and Su(var)3-9 mutants. As expected, the chromocenter of wild-type nuclei was richly stained for H3K9me2 (Figure 2.3c, panels A-C). In contrast, the amount of H3K9me2 detected at the chromocentre was substantially reduced in the Su(var)3-9 mutants (Figure 2.3c, compare panel A with panels D, G, J and M). This reduction was particularly dramatic in Su(var)3-906, confirming previous observations (Schotta et al., 2002; Ebert et al., 2006). Note however that H3K9me2, despite its low level, was still detected at the chromocenter. The presence of H3K9me2 at the HIS-C was examined by chromatin immunoprecipitation (ChIP) of cross-linked extracts with α-H3K9me2. Extracts from 1216 hours old embryos of wild-type, Su(var)3-906 and the various Su(var)3-9 missense mutant strains were tested, and HIS-C DNA sequences were detected in all extracts (Figure 2.3b, lane 3). However, the proportion of HIS-C DNA pulled down by the antibody was lower in the Su(var)3-9 mutants than it was in the wild-type strain (Figure 2.3b, compare lanes 1 and 3 in the wild-type extracts (WT) with lanes 1 and 3 in the Su(var)3-9 mutants). We conclude that the missense Su(var)3-9 mutants, as well as the null mutant Su(var)3-906, are associated with reduced levels of H3K9me2 at both the highly compacted pericentric heterochromatin and at the largely silenced HIS-C, two natural targets of SU(VAR)3-9. Several pieces of evidence suggest that H3K9me2 serves as a substrate for HP1 in the formation of centric heterochromatin (Bannister et al. 2001; Lachner et al. 2001; Nakayama et al. 2001; Ebert et al., 2006), and HP1 has been shown to colocalize with  72  H3K9me2 in heterochromatin (Cryderman et al., 2005; Ebert et al., 2006). Colocalization of H3K9me2 and HP1 within euchromatic regions is much less frequent, but has been observed for a few loci, such as cdc2, the HIS-C and a few others (Cryderman et al., 2005; Greil et al., 2003; Ner et al., in preparation). Accordingly, we asked whether the drastic reductions in the levels of H3K9me2 at the chromocentre and at the HIS-C observed in the Su(var)3-9 mutants correlated with a disruption of HP1 targeting to these two regions. Consistent with previous results, we found that HP1 is still present at the chromocentre of Su(var)3-9 mutants, but in much lower amounts (Figure 2.3b, panels E, H, K, N). Similarly, ChIP analyses demonstrated that although HP1 is present at the HIS-C locus in both wild-type and Su(var)3-9 mutant embryos, it is considerably less abundant in the Su(var)3-9 mutant extracts (Figure 3b, compare lanes 4 and 6 in the wild-type to lanes 4 and 6 in the mutants). We conclude that, like in the case of H3K9me, Su(var)3-9 mutants display reduced levels of HP1 associated with pericentric heterochromatin and with the HIS-C.  73  Su(var)3-9 wild-type  1 2 3 4 5 P1 )  H3  tubulin 6  CHIP figure  06  Su(var)3-9309  Su(var)3-9330  Su(var)3-9318  6  Figure 2.3.  74  ild  st on  ty p  hi  8  es  e Su (v ar )3 -9 0 Su 6 (v ar )3 -9 3 Su 09 (v ar )3 -9 3 Su 30 (v ar )3 -9 3 1  w  co re  wild type  H3K9me2 c.  Su(var)3-9318 Su(var)3-9330 Su(var)3-9309 Su(var)3-906  (H  A b  )  5  IP  O  00  :1  )  e2  4  N  (1  9m  3  IN  b  3K  (H  A  2  IP  O  0)  b. :1  (1  1  N  IN  a. HP1 H3 K9me2 A B C  D E F  G H I  J K L  M N O  merge  Figure 2.3. Detection of H3K9me2 and HP1 in Su(var)3-9 mutants. a) Western blot analysis of wild-type and mutant embryo extracts. Lane 1: bulk core histones (control), lanes 2: wild-type embryo extracts, lanes 3-6: extracts from homozygous Su(var)3-9 mutant embryos. The allele numbers are indicated above the panel. The antibodies used in each case are indicated to the left of the panels. Each lane was loaded with the following: core histones (2 µg), extracts (15 µg for the H3 blot and 150 µg for the H3K9me2/tubulin blots). The H3K9me2 and tubulin panels represent the same western blot probed first with anti-H3K9me2 and subsequently with anti-tubulin. b) Chromatin immunoprecipitation of wild-type and Su(var)3-9 mutant embryo extracts. In each case, the recovered DNA was PCR amplified using primers specific for the coding region of the histone H3 gene (H3F and H3R, see Suppl.table 2). Lane 1: input DNA at a 1:10 dilution (IN (1:10)), lanes 2 and 5: mock IP, no antibody (NO Ab), lane 3: IP with antiH3K9me2 (IP (K9me2)), lane 4: input DNA at a 1: 100 dilution (IN 1:100), lane 6: IP with anti-HP1 (IP (HP1)). c) Immunostaining of polytene nuclei from salivary glands of wildtype and Su(var)3-9 mutant larvae with anti-H3K9me2 and anti-HP1 (HP1). The genotypes of the larvae are indicated to the left of each set of panels. The arrowheads point to the chromocenter region.  75  2.2.5. The HMTase activity of the SU(VAR)3-9 variants is impaired. Since all Su(var)3-9 missense alleles tested displayed a reduction in the levels of H3K9me2, and carried mutations in the catalytic region of SU(VAR)3-9, we next asked how much, if any, HMTase activity is retained by each of the mutant proteins, and whether the levels of residual activity correlate with the positions of the different amino acid substitutions. To address this, we tested the in vitro enzyme activity of five of our seven SU(VAR)3-9 mutant proteins. We chose two mutants with substitutions in the preSET (Su(var)3-9376 and Su(var)3-9309), two in the SET domain (Su(var)3-9311 and Su(var)3-9330), and one in the postSET domain (Su(var)3-9318). Su(var)3-9330 and Su(var)3-9311 were selected because they represent substitutions of a highly conserved (ASP536), and relatively un-conserved (GLY521) residues, respectively. We produced various GST-SU(VAR)3-9 recombinant proteins (referred to as SU(VAR)3-9allele number for simplicity) and tested their HMTase activity. As an additional control, we generated a GST-SU(VAR)3-9 fusion protein that carries the 2 polymorphisms (ALA304PHE, ILE375LEU) detected in some of our strains (Table 2.1). We named this recombinant protein SU(VAR)3-9pol, and expected it to retain wild-type levels of enzyme activity since these polymorphisms don’t result in suppression of PEV. HMTase assays were first performed on bulk histones. Although the natural substrate for SU(VAR)3-9 is histone H3, we used all the histones to rule out the possibility that the mutants had acquired activity towards the other histones. As expected, SU(VAR)3-9WT (Fig. 2.4b, lane 2) and SU(VAR)3-9pol (data not shown) efficiently catalyzed the transfer of the methyl moiety onto H3. All mutant SU(VAR)3-9 proteins showed either no activity or a dramatically reduced HMTase function compared to SU(VAR)3-9WT. SU(VAR)3-9309 (Fig.  76  2.4b, lane 4) and SU(VAR)3-9376 (data not shown) had no detectable HMTase activity. SU(VAR)3-9330, SU(VAR)3-9318 and SU(VAR)3-9311 showed partial loss of function and they varied in the level of residual enzyme activity (Fig 2.4b, lanes 5-7). Finally, none of the mutants showed any obvious activity on histones other than H3. Since bulk histones are prepared from nuclei, they contain extensively modified histones, which could have skewed our HMTase measurements. Therefore, we retested the mutants using unmodified recombinant histone H3. The results of this analysis were similar to those obtained for the bulk histones (Fig 2.4c and 2.4e). SU(VAR)3-9309 and SU(VAR)3-9376 showed no detectable HMTase function (Fig. 2.4d, lanes 11 and 15), while SU(VAR)3-9330, SU(VAR)3-9318 and SU(VAR)3-9311 had reduced levels of activity (Fig. 2.4d, compare lanes 12-14 with lane 9). The relative activities of the different mutants are shown in Figure 2.4e and Table 2.2. Finally, as expected, SU(VAR)3-9pol methylated H3 as efficiently as SU(VAR)3-9WT (99.0% +/-1.55%, data not shown). In summary, the in vitro HMTase data indicate that all 5 variant SU(VAR)3-9 proteins have dramatically reduced enzymatic activity. While preSET mutants displayed a complete loss-of-function, SET and postSET domain mutants were hypomorphs that retained a small fraction of the wild-type enzyme activity.  77  31 8, 31  9,  6  37  1  31  8  0  31  9  33  30  ST  G  fluorogram  r3-9  H3  14C  H3  w  t  1 31  8 31  0 33  9 30  G  t 14C  r3-9  GST-3-9 fusion (r3-9)  RECOMBINANT H3  d.  fluorogram  w  ST  BULK HISTONES  b.  3 31 12 331 0  GST  30  37  a.  6  SU(VAR)3-9 aa 182  9  Figure 2.4  H1  2  3  4  c.  6  7  80  24.3  20 0  0  wild type GST  14.3  12.8  318  311  <<1  309  330  8  9 10 11 12 13 14 15  100  60 40  H3  e.  Specific activity on core histones  100  Relative activity (%)  5  core histones  Relative activity (%)  1  r3-9  Coomassie  Coomassie  r3-9  78  Specific activity on recombinant H3  80  60 40  31.3  20 0  wild type  0  <<1  GST  309  330  24.7  22.7  318  311  Figure 2.4. The HMTase activity of variant SU(VAR)3-9 proteins. a) Each SU(VAR)3-9 variant protein was expressed and purified as a GST fusion polypeptide (r3-9). The position of the chromodomain (red box), the pre- and postSET (green box) and the SET domain (blue box) are indicated in the stick diagram. The vertical black bars indicate the positions of the amino acid substitutions in 5 Su(var)3-9 alleles that were selected for HMTase analysis. The numbers above each bar indicate the corresponding allele. b) and d) HMTase assay on bulk histones and recombinant H3 respectively. The enzyme activity of the variant proteins was tested using labeled AdoMet as the methyl donor. The fluorogram (top panel) and the corresponding Coomassie stained gel (bottom panel) are shown. Lanes 1 and 8: molecular weight marker, lanes 2 and 9: wild-type GST-SU(VAR)3-9, lanes 3 and 10: recombinant GST (negative control), lanes 4-7 and 11-15: GST-SU(VAR)3-9 mutant proteins. c) and e) Relative HMTase activity of the various mutants on bulk histones and recombinant H3, respectively. Presented as a bar graph. Following each assay, the methyl H3 radioactive signals and the Coomassie-stained bands corresponding to the appropriate GST-SU(VAR)3-9 proteins were quantified using the ImageQuant and NIH Image softwares respectively (see materials and methods). The bars represent the average of 3 independent assays and the error bars span two S.E.M. Both Su(var)3-9309 and Su(var)3-9376 displayed no detectable activity, but for simplicity only Su(var)3-9309 is shown on the graph.  79  2.2.6. A mutation in the postSET prevents the addition of a third methyl group to H3K9me2. There are a wide variety of SET domain-containing HMTases that differ in the lysine residues that they target and/or the number of methyl groups they transfer (Eskeland et al., 2004; Mis et al., 2006). Some methyltransferases can exclusively mono-methylate H3K9 while others, like SU(VAR)3-9, can (mono-), di- or trimethylate (Eskeland et al., 2004). X-ray crystal structural analyses have revealed the key amino acid residues in the preSET, SET and postSET domains that contribute to these activities. For example, the presence of VAL569 in the active site and PHE602 in the enzyme channel allows addition of multiple methyl groups to H3K9 by creating sufficient space to accommodate tri-methylated H3K9 (Xiao et al., 2003; Collins et al., 2005; Zhang et al., 2003). Since some of the mutations in our Su(var)3-9 alleles affect amino acids located in close proximity to residues in the active site, we asked if the variant SU(VAR)3-9 proteins, which show a partial loss of function, have an altered ability to mono/di- or trimethylate. The bulk histones and recombinant H3 assays did not allow us to distinguish between these three levels of K9 methylation. So, we addressed this issue using H3 tail peptides. We took advantage of an unmodified peptide corresponding to residues 1-20 of H3, and a peptide comprising amino acids 1-21 of H3 in which K9 is dimethylated. Both H3 peptides are very efficient substrates for SU(VAR)3-9WT and are readily methylated (Fig 2.5a and 2.5c, lanes 1 and 7), clearly showing that SU(VAR)3-9 is able to catalyze the addition of a third methyl group to H3K9me2.  80  The mutant protein SU(VAR)3-9309 showed no enzyme function on either the unmodified or the dimethylated peptides (Fig. 2.5a, lane 3, and 2.5c, lane 9). SU(VAR)39311 was a hypomorph, and it retained a fraction of the wild-type activity (Fig. 5a, lane 6, and 2.5c, lane 12, respectively) on both peptides. These results are similar to those obtained when full length (unmodified) H3 was used as a substrate. Thus the activity of the SU(VAR)3-9309 and SU(VAR)3-9311 mutant forms of the enzyme was consistent on all substrates tested. In contrast, SU(VAR)3-9318 behaved differently with the different substrates. While it had reduced activity towards the unmodified peptide and the full-length histone H3, it was completely inactive toward the H3K9me2 peptide (Fig. 2.5a and 2.5c, lanes 5 and 11). Thus, not only did SU(VAR)3-9318 have a reduced overall catalytic activity, but it also appeared to be unable to tri-methylate K9. SU(VAR)3-9318 has a SER616LEU substitution in the postSET domain. SU(VAR)3-9330 also showed a partial loss-of-enzyme function when either bulk histones or recombinant H3 were used as the substrate. However, rather surprisingly, this variant was unable to methylate the H3 peptides. It retained less than 1% and 4% activity towards the unmodified and H3K9me2 peptides, respectively (Fig. 2.5a and 2.5c, lanes 4 and 10). In summary, the SU(VAR)3-9 mutants displayed an array of severely reduced enzyme activities, ranging from complete abolition to ~25% of the wild-type activity. In addition, some of the mutants showed distinct HMTase characteristics (summarized in Table 2.2) depending on the substrate used.  81  Figure 2.5  3  4  5  6  HMTase activity on H3 tail peptide  80 60 40  18.3  20 wild type  0  <<1  1.3  GST  309  330  318  14.3  1 31  8 31  0 33  9  30  ST G  w  fluoroogram 14C  100 80  311  82  8  9  10  11 12  HMTase activity on H3K9me2 peptide  60  40 20 0  r3-9  H3 tail  7  d. Relative activity (%)  Relative activity (%)  t  1 31  8 31  33  30  9  ST G  t 14C  2  100  0  r3-9  H3 tail  1  b.  H3 K9me2 TAIL (aa 1-21)  c.  fluoroogram  w  0  H3 TAIL (aa 1-20)  a.  0 wild-type GST  <<1 309  4 330  <<1 318  11.5 311  Figure 2.5. HMTase activity of Su(var)3-9 alleles using unmodified and dimethylated H3 tail peptides. a) and c) Fluorograms of an HMTase assay on H3 peptide (H3 tail, aa 1-20) and dimethylK9H3 peptide (H3K9me2 tail, aa 1-21) respectively. Lanes 1 and 7: wild-type GST-SU(VAR)3-9, lanes 2 and 8: GST (negative control), lanes 3-6 and lanes 9-12: GST-SU(VAR)3-9 mutants. The allele numbers are indicated above the corresponding lanes. b) and d) Relative in vitro activity of the indicated Su(var)3-9 alleles on unmethylated (b) and dimethylK9 (d), H3K9me2 H3 tail peptides. The numbers used in the bar graphs were obtained as described in the materials and methods.  83  2.2.7. Effect of Su(var)3-9 missense and null alleles on PEV in the presence of one mutant and 2 wild type copies of Su(var)3-9. All Su(var)3-9 mutants were originally identified on the basis of a morphological phenotype, dominant suppression of PEV. Here, we examine the morphological phenotype of the Su(var)3-9 mutants in more detail. We specifically asked whether the mutant alleles differ in their ability to suppress heterochromatic gene silencing that results from PEV and, if so, whether these are correlated to the differences observed at the biochemical level (in vitro enzyme function). Direct examination of the Su(var)3-9 alleles with the “classical” variegating strain, wm4, is inadequate for this study since all the Su(var)3-9 mutants almost completely suppress w+ gene silencing. So, we used a variation on the wm4 assay system and examined the suppression effect in the presence of an extra copy of wild-type Su(var)3-9, that is, in flies with wm4; Su(var)39mutation/Su(var)3-9+, Su(var)3-9+ genotypes. We took advantage of a Su(var)3-9::eGFP transgene inserted in the third chromosome (pP{GS[ry+,(10kb Su(var)3-9)EGFP]}, (Schotta and Reuter, 2000), here referred to as P[3-9egfp] for simplicity). Crosses were set up, in triplicate, between males homozygous for this transgene and for the wild-type, endogenous Su(var)3-9 allele (wm4/Y; Su(var)3-9+,P[3-9egfp]/Su(var)3-9+, P[3-9egfp]) and females homozygous for each of the Su(var)3-9 alleles (see material and methods). The progeny of these crosses carry one maternally inherited mutant copy (or, in the case of the control cross, one wild-type copy) and two paternally derived wild-type copies of Su(var)3-9 (the endogenous Su(var)3-9+ and the ectopically inserted P[39egfp]). For each cross, the eyes of the male progeny were visually scored for the amount of eye pigment and thus the level of suppression of PEV.  84  Over 98% of the flies derived from the control cross, which have 3 copies of the Su(var)3-9+ allele (wm4/Y; Su(var)3-9+/Su(var)3-9+,P[3-9egfp]), displayed a strong E(var) phenotype, confirming that the Su(var)3-9::eGFP transgene is functional and overproduction of SU(VAR)3-9 enhances the gene silencing that results from PEV (Fig. 2.6). On the other hand, in the progeny bearing a mutant allele, a wild-type (endogenous) allele and an extra (transgenic) copy of Su(var)3-9+, we observed a broad range of variegating eye phenotypes. None of the individuals displayed the almost completely white eye phenotype of their control counterparts. However, we did note a significant proportion of flies with bilaterally unequal eye pigmentation, that is, one eye strongly suppressed and the other unsuppressed. For this reason, instead of performing standard pigment assays, we scored each eye visually and assigned it to one of three categories: strongly suppressed (~75-100% pigment), mildly suppressed (~20-75%), or unsuppressed (~5-20%) (Fig. 2.6b, panels A, B & C respectively). The proportion of strongly suppressed, mildly suppressed and unsuppressed eyes varied with the genotype (Fig. 2.6b). For the sake of simplicity, and since all these individuals have the identical wild type and transgenic alleles, and only differ in their mutant allele of Su(var)3-9, the description of their genotypes is limited to the identity of the mutant allele they carry. Over half of the individuals carrying the Su(var)3-906 allele had strongly suppressed eyes. Since Su(var)3-906 is a null allele for which no mRNA or protein products are detected (Fig. 2.2 and Tschiersch et al., 1994; Schotta et al., 2002), these individuals should be phenotypically equivalent to wm4, Su(var)3-9+/Su(var)3-9+ flies, which typically have variegating, unsuppressed eyes (Tartof et al., 1984; Grigliatti, 1991;  85  Reuter and Spierer, 1992, and references therein). Thus, the presence of a large proportion of strongly suppressed eyes in wm4/Y; Su(var)3-906/Su(var)3-9+, P[3-9egfp] individuals suggests that the ectopically inserted Su(var)3-9 transgene, P[3-9egfp], may not be functionally equivalent to the endogenous gene. The EGFP-tagged Su(var)3-9 may produce less protein product, and/or its product may function less efficiently than the wild-type SU(VAR)3-9. The highest proportion of strongly suppressed eyes was observed in flies carrying the Su(var)3-9311 allele, which suggests Su(var)3-9311 is the strongest suppressor of PEV in this genetic test. Individuals bearing the Su(var)3-9318 and Su(var)3-9330 alleles displayed approximately the same fraction of strongly suppressed eyes, indicating that Su(var)3-9318 and Su(var)3-9330 suppress PEV roughly to the same degree. Curiously, in this assay, Su(var)3-9309 resulted in the lowest proportion of strongly suppressed, and the highest proportion of unsuppressed eyes, suggesting Su(var)3-9309 is a weaker suppressor of PEV than Su(var)3-9330, Su(var)3-9318 or Su(var)3-9311. In summary, differences in the strength of PEV suppression were detected among the four Su(var)3-9 missense alleles. Using this particular genetic assay of function, Su(var)3-9311 was the strongest suppressor, while Su(var)3-9309 was the weakest, and also the only one to suppress PEV less efficiently than the «protein null» allele, Su(var)3-906. With the exception of Su(var)3-9309, these results correlate well with the levels of remaining HMTase activity of each mutant (see Discussion).  86  EGFP-tagged Su(var)3-9  a.  wild-type Su(var)3-9 X  mutant Su(var)3-9  b.  A  B  C  strongly sup  mildly sup  D  unsuppressed  enhanced 97.01  wm4/Y;  Su(var)3-9+ Su(var)3-9+,P[3-9egfp] (n=3; tot=410)  wm4/Y;  Su(var)3-906 Su(var)3-9+,P[3-9egfp] (n=3; tot=400)  wm4/Y;  Su(var)3-9309 Su(var)3-9+,P[3-9egfp] (n=3; tot=356)  wm4/Y;  Su(var)3-9330 Su(var)3-9+,P[3-9egfp] (n=3; tot=524)  wm4/Y;  Su(var)3-9318 Su(var)3-9+,P[3-9egfp] (n=3; tot=567)  wm4/Y;  Su(var)3-9311 Su(var)3-9+,P[3-9egfp] (n=3; tot=562)  100 50 0 100 50  100 0.30  +/- 1.96  0 100 50  37.71 +/- 0.07  0 100  50  +/- 0.42  53.51  65.68  100  0  50 0  100 50  29.29 +/- 2.38  100 66.76  +/-1.89  100  100 21.79 +/- 0.01  50  0 100  17.20  50 0  +/- 0.02  50  0  0  100  100  100  +/- 1.03  0  100 40.49  0  19.88  +/- 2.31  50  +/- 1.26  0  50  0  50  0  50  2.69  100  +/- 0.81  50  0  100  50 14.44  0  50  +/- 0.27  0  0  0  100  100  100  0  +/- 1.59  50 24.28  50  +/- 2.30  0  100 50  86.20 +/- 3.26  50  0  0  100  100  50 10.73  50  +/- 2.53  0  0  Figure 2.6.  87  0  8.95 +/- 0.73  50 0  0  100 2.90 +/- 0.89  50 0  0  Figure 2.6. Effect of several Su(var)3-9 alleles on PEV (here, wm4 variegation) in wm4/Y; Su(var)3-9+, P[3-9egfp]/Su(var)3-9mutant individuals. a) A schematic of the genetic configuration of the flies examined in this experiment. The EGFP-tagged Su(var)3-9 (also indicated as P[3-9egfp] for simplicity) represents the ectopic Su(var)3-9 insertion pP{GS[ry+,(10kb Su(var)3-9)EGFP} (Schotta and Reuter, 2000). This construct is inserted on the third chromosome, thus all flies harboring this insert also carry a wildtype copy of the endogenous Su(var)3-9 gene. b) Frequencies (%) of strongly suppressed, mildly suppressed, unsuppressed, and enhanced eyes in wm4/Y; Su(var)3-9+, P[3-9egfp]/Su(var)3-9mutant individuals carrying different Su(var)3-9 alleles. Pictures A-D show examples of strongly suppressed, mildly suppressed, unsuppressed and enhanced wm4 variegation, respectively. Genotypes are shown on the left, n=number of crosses analyzed, tot=total number of eyes scored. The bar graphs below each eye picture represent the fraction (%) of eyes displaying the depicted phenotype amongst the individuals of the genotype indicated (average of 3 independent crosses +/- S.E.M., rounded to the closest second decimal). Panels A and C show eyes of wm4/Y; Su(var)3-9309/Su(var)3-9+,P[3-9egfp] flies. The eye in panel B is from a wm4/Y; Su(var)3-906/Su(var)3-9+,P[3-9egfp] fly and the eye in D is from a wm4/Y; Su(var)3-9+/Su(var)3-9+,P[3-9egfp] individual.  88  2.3. Discussion 2.3.1. The Su(var)3-9 mutations are single base-pair substitutions clustered in the catalytic region. SU(VAR)3-9 has two highly conserved and functionally distinct regions: a chromodomain and a SET domain; the latter, together with the flanking pre- and postSET sequences, constitutes the HMTase activity of the protein. Both regions are present in several chromatin associated proteins and are highly conserved, and therefore it is reasonable to assume that the chromo and SET domains each have important roles in the function of SU(VAR)3-9. Other domains of the protein, as yet unidentified, may also be required for the function of SU(VAR)3-9. Indeed, we expected that the dominant suppressors of PEV would comprise mutations in the chromo and SET domains, and perhaps identify other regions of SU(VAR)3-9 that are required for the epigenetic gene silencing observed in PEV. Interestingly, the 9 confirmed EMS-induced Su(var)3-9 mutations altered 7 residues, with 2 residues hit twice, all of which are located in the catalytic region of SU(VAR)3-9; none of the Su(var)3-9 mutations occurred in or near the chromodomain of the protein. The concentration of missense mutations within the preSET/SET/postSET region of the protein suggests that the HMTase activity of SU(VAR)3-9 plays a crucial role in suppression of PEV. In contrast, single amino acid substitutions in the chromodomain are probably insufficient to cause a dominant suppression of PEV and thus affect the gene silencing function of SU(VAR)3-9. Alternatively, the absence of chromodomain mutants among the dominant Su(var)s recovered in the original screen may be due to the fact that the EMS mutagenesis failed to induce mutations in or around the chromodomain of Su(var)3-9. This latter hypothesis  89  seems unlikely, because Reuter and colleagues also failed to recover mutations in the chromodomain of SU(VAR)3-9 by screening for strong, dominant suppressors of PEV (Reuter and Wolff, 1981; Ebert et al., 2004). Mutations in the N-terminal region of the protein were indeed isolated in several screens using P element transposition or gamma rays as the mutagenic agent, but they represent insertions and deletions that produce either truncated proteins, or no protein at all (Tschiersch et al., 1994; Harrington, 2001; Schotta et al., 2002; Ebert et al., 2004, 2006). These mutants act as dominant Su(var)s because the Su(var)3-9 locus is dosage sensitive, that is hemizygosity for Su(var)3-9 suppresses PEV (Grigliatti, 1991; Reuter and Spierer, 1992; and references therein). Nevertheless, the observation that mobile genetic elements can insert into or near the chromodomain suggests that this region should be amenable to mutagenesis via EMS. Thus, the function of the chromodomain in SU(VAR)3-9 remains elusive, although it is more defined in other proteins, such as HP1, PC and MOF. In these proteins, the chromodomain appears to be required for chromatin binding (Platero et al., 1995; Messmer et al., 1992; Ma et al., 2001; Akhtar et al., 2000; Jacobs et al., 2001; Bannister et al., 2001; Lachner et al., 2001; Bouazoune et al., 2002; Fischle et al., 2003; Min et al., 2003; Pray-Grant et al., 2005). By analogy, a similar role was suggested for the SU(VAR)3-9 chromodomain (Schotta et al., 2002; 2003), but, to date, direct evidence is lacking. If the SU(VAR)3-9 chromodomain is involved in chromatin binding, then one would expect mutations within the chromodomain to cause mis-targeting, which should have a strong dominant Su(var) phenotype. The absence of chromodomain mutations among the respective collections of dominant Su(var)3-9 mutants (this paper and Ebert  90  et al., 2004; Donaldson et al., 2002), suggests that substitution of any of the ~40 amino acids that comprise the chromodomain is not enough to cause significant mis-targeting.  2.3.2. Reduction of H3K9me2 and HP1 in Su(var)3-9 mutants. At the cellular level, we found that all Su(var)3-9 missense mutants display a dramatic reduction in the levels of both H3K9me2 and HP1 associated with centric heterochromatin and with the HIS-C. Firstly, these in vivo observations corroborate the results obtained from the in vitro enzyme assay, namely that the amino acid substitutions present in the mutant alleles of SU(VAR)3-9 impair the protein’s HMTase function. Missense mutations in Su(var)3-9 also altered the abundance of HP1 binding, as measured by in situ immunofluorescence and ChIP analysis, which is consistent with the hypothesis that H3K9me2 constitutes a binding platform for HP1 (Bannister et al., 2001; Lachner et al., 2001; Nakayama et al., 2001; Ebert et al., 2006). Secondly, the observation that Su(var)3-9 mutants cause the same set of effects at the chromocentre and at the HIS-C, a euchromatic locus and natural target of SU(VAR)3-9, suggests that SU(VAR)3-9 may function as part of a silencing mechanism that affects numerous loci in the euchromatic as well as heterochromatic regions of the genome.  91  2.3.3. The SET mutants are hypomorphs Based on the crystal structures of the SU(VAR)3-9 homologues Clr4p and DIM-5 (Min et al., 2002; Zhang et al., 2003), both the HMTase active site, and the substratebinding cleft, are part of the SET domain. The mutant alleles Su(var)3-9311, Su(var)39325 and Su(var)3-9330 cause single amino acid substitutions in the SET domain. Each of the affected residues is predicted to lie within, or very near, the active site of the enzyme (Figure 2.7). Su(var)3-9330, which affects ASP536, is the most interesting of these SET mutants. ASP536 is conserved in all H3K9 and in some H3K4 methyltransferases (Aagard et al., 1999; Zhang et al., 2002; Mis et al., 2006). It is located in the portion of the cleft that is involved in stabilizing the enzyme-substrate complex; its side chain forms a hydrogen bond with the hydroxyl oxygen of SER10 of H3 (H3S10) (Min et al., 2002; Zhang et al., 2003). The HMTase activity of SU(VAR)3-9330, on full-length, unmodified histone H3, is reduced to about 31% of the wild-type. This partial loss of function is probably the result of an unstable enzyme-substrate interaction due to the replacement of ASP536 with ASN, which could disrupt the hydrogen bond formed between H3S10 and ASP536. Interestingly, the H3 peptides are much poorer substrates for SU(VAR)3-9330 compared with full length H3. The enzyme activity on unmodified, and H3K9me2 peptides is less than 5% of wild-type. Their smaller size, and the absence of backbone residues, which are present in full length H3, reduce the stability of binding of the peptides compared to the full length H3. Thus, the substitution of ASP536 probably has a more drastic effect on the peptides than it does on the full-length histones. Alternatively, the ASP536ASN substitution in SU(VAR)3-9330 may lead to an altered substrate specificity from H3K9 to another H3 lysine that is not present in the H3 tail  92  peptides, for example K27. Since K27 is absent in these peptides, the only activity detected would correspond to weak, residual methylation of K9. Since no appropriately modified peptides are available that include residues 1-27 of H3, this hypothesis cannot be tested without using mass spectrometry.  2.3.4. The preSET mutants are enzymatically inactive. There are nine key cysteine residues in the preSET region of SU(VAR)3-9. These residues are highly conserved (Aagaard et al., 1999; Min et al., 2002) and crystal structures of the Clr4p and DIM-5 proteins reveal that these cysteines coordinate 3 zinc ions that form a “zinc cluster”. This “cluster” has an important structural role as it holds together two random coils that form the bottom surface of the catalytic region of the protein (Min et al., 2002; Zhang et al., 2003). Although the preSET domain is not part of the enzyme active site, or the regions binding the substrate or cofactor per se, it is required for efficient H3 methylation (Rea et al., 2000). Four of our Su(var)3-9 missense alleles have mutations in preSET cysteine residues. We tested two of them, SU(VAR)3-9376 (CYS428TYR) and SU(VAR)3-9309/312 (CYS462TYR), for their in vitro HMTase activity, and in both cases the variant proteins were completely inactive. Given the role of these residues in protein structure, the complete loss of enzyme function is likely due to misfolding of the protein, which dramatically alters many aspects of substrate binding and enzyme function.  93  2.3.4. The postSET mutant lacks the ability to add a third methyl group to H3K9me2. Su(var)3-9318 and Su(var)3-9319 are two independently isolated mutations with the same SER616LEU mutation in the postSET region. Although this amino acid is not conserved in Clr4p, Suv39h1 and SUV39H1 (fig 1b), our results suggest that it is crucial for the addition of the third methyl group to H3K9. In addition, the fact that the corresponding residues are a GLN in Clr4p and an ASP in SUV39H1 and Suv39h1 (Fig.1b), and that the SER616LEU substitution in SU(VAR)3-9 causes a strong, dominant Su(var) phenotype, suggest that the presence of a polar residue at this position may be critical for SU(VAR)3-9 function. In the Clr4p structure the flexible postSET region is positioned near the active site where it acts as a “lid” and creates a solvent-secluded space for optimal methyl transfer (Min et al., 2002). The postSET domain of DIM-5 works in a similar manner and in the presence of H3 substrate it interacts directly with the enzyme active site, with the substrate, and with the cofactor AdoMet (Zhang et al., 2003). Hence, mutations in the postSET region could disrupt methyl transfer by altering the local architecture and exposing the active site to the solvent. Alternatively, the altered residue in the postSET may interfere with the normal positioning of the substrate or the cofactor in their respective binding pockets. As expected, SU(VAR)3-9318 has reduced HMTase function on unmodified substrates such as bulk histones, recombinant H3 and H3 tail peptide (120). However, SU(VAR)3-9318 fails to add a third methyl group to an H3 tail peptide already dimethylated at K9, suggesting that the mutation either interferes with the stability of the dimethylated peptide or the AdoMet in the catalytic cleft, or creates sufficient steric hindrance to impair methyl transfer.  94  95  1  .  .  .  B  3 4  5 6  C  .  324  7  .  8  9  .  10  .  311  330  309 312  C-terminus  318 319  4  5  9  B 10  D  330  7  N-terminus  zinc cluster  3  1  8  6 C  311  325 318 319  CLR4 399 QNYGDVSRFFNHSCSPNIAIYSAVRNHGFRTIYDLAFFAIKDIQPLEELTFDYAGAKDFSPVQSQKSQQN RISKLRRQCKCGSANCRGWLFG SU(VAR)3-9 550 ANYGNISHFINHSCDPNLAVFPCWIEHLNVALPHLVFFTLRPIKAGEELSFDY-IRADNEDVPYENLST- -A--VRVECRCGADNCRKVLF-  D  309 312  CLR4 303 VIYECNSFCSCSMECPNRVVQRGRTLPLEIFKTKE-KGWGVRSLRFAPAGTFITCYLGEVITSAEAAKRDKNYDDDGITYLFDLDMFD-DASEYTVDA SU(VAR)3-9 452 AIYECNSRCSCDSSCSNRLVQHGRQVPLVLFKTANGSGWGVRAATALRKGEFVCEYIGEIITSDEANERGKAYDDNGRTYLFDLDYNTAQDSEYTIDA  2  376  CLR4 220 --------------VTLVNEVDDEPCPSLDFQFISQYRLTQGVIPPDPNFQSGCNCSSLGG-CDLNNPSRCECLDDLDEPTHFAYDAQG-RVRADTGA SU(VAR)3-9 361 FEKRMNHVEKPSPPIRVENNIDLDTIDS-NFMYIHDNIIGKDVPKPEAGI-VGCKCTEDTEEC -TAST-KC-CARFAG--ELFAYERSTRRLRLRPGS  Figure 2.7.  b.  a.  490 635  398 549  302 451  Figure 2.7. Probable tertiary structure of SU(VAR)3-9 and relative positions of the mutated residues. a) Amino acid sequence alignment of the catalytic domain of Clr4p and SU(VAR)3-9. Identical residues are highlighted in black, similar residues are highlighted in grey. Red dots indicate the residues that are mutated in the different Su(var)3-9 alleles. Above the alignment is represented the secondary structure of Clr4p (aa 220-490), according to Min et al. (2002). The N-terminal region of the catalytic domain is shown in black, the pre- and postSET domains in green, and the SET domain in blue. The high level of homology between the two proteins at the primary sequence level suggests that their secondary and tertiary structures could also be very similar. b) A ribbon diagram representation of the Clr4 structure (residues 220-490). Under the assumption that the tertiary structure of SU(VAR)3-9 is very similar to Clr4’s, the red dots show the approximate positions of the amino acids that are mutated in SU(VAR)39309, SU(VAR)3-9330, SU(VAR)3-9318 and SU(VAR)3-9311. The diagram was generated partially using Raster3D (Kraulis, 1991) and MolScript (Merritt and Murphy, 1994).  96  2.3.5. Effect of Su(var)3-9 missense and null alleles on PEV in a strain that is hyperploid for Su(var)3-9. In our structure-function analysis of Su(var)3-9 we wanted to examine the morphological phenotype of PEV and attempt to correlate the morphological end point (eye colour pattern) with the cytological and molecular effects of the different mutations in Su(var)3-9. However, all Su(var)3-9 missense mutants suppress PEV very strongly. Therefore, we employed an assay for PEV, which examined the relative strength of the Su(var)3-9 mutations in individuals that carried three copies of Su(var)3-9. The three alleles were one wild-type, endogenous allele (Su(var)3-9+), one Su(var)3-9+-EGFP transgene, and one mutant Su(var)3-9 allele. At the morphological level (suppression of PEV) we found that all Su(var)3-9 mutant alleles were able to suppress PEV in at least a substantial fraction of wm4/Y; Su(var)3-9mutant/Su(var)3-9+, P[3-9egfp] individuals, but they differed in their strength of suppression. These differences may be due to differences in the residual HMTase activity of the mutant SU(VAR)3-9 proteins. However, it should be kept in mind that, unlike the HMTase assay, which provides a direct measure of enzyme activity under given conditions, or the immunohistochemical analyses, which identify the distribution patterns and can delineate the relative abundance of given proteins, PEV suppression is a tertiary phenotype involving numerous unknown variables, and may therefore not be directly indicative of SU(VAR)3-9 HMTase function. Flies carrying the Su(var)3-906 mutant allele (wm4/Y; Su(var)3-906/Su(var)3-9+, P[3-9egfp]) served as "baseline", as Su(var)3-906 does not produce any Su(var)3-9 mRNA or protein, and the products of the endogenous Su(var)3-9+ and the transgenic  97  Su(var)3-9+-EGFP must therefore account for the SU(VAR)3-9 function present in this strain. Individuals of this genotype showed strongly suppressed eyes at a frequency of about 53%. Curiously, the presence of missense Su(var)3-9 allele (wm4/Y; Su(var)39missense/Su(var)3-9+, P[3-9egfp]) resulted in either stronger (Su(var)3-9330, Su(var)3-9318, Su(var)3-9311)or weaker (Su(var)3-9309) suppression of PEV than with the Su(var)3-906 null allele (Fig. 6b). Su(var)3-9 alleles resulting in stronger suppression of PEV than Su(var)3-906 are likely antimorphs, while alleles that are weaker suppressors than Su(var)3-906, are probably hypomorphs. This logic suggests that Su(var)3-9311, Su(var)3-9318 and Su(var)3-9330 are antimorphs, and Su(var)3-9309 is a hypomorph. At a mechanistic level, the three antimorphic mutants possibly act as dominant negatives. The mutant SU(VAR)3-9 products may be incorporated into protein complexes like their wild-type counterparts, and these complexes would be correctly targeted, but would fail to efficiently methylate H3K9, interfering with the function of the wild-type SU(VAR)3-9. Indeed, the mutations present in Su(var)3-9311, Su(var)3-9318 and Su(var)39330 are located in the catalytic region of the protein, and do not affect its N-terminal protein-protein interaction domains. However, the possibility that amino acid substitutions in the SET domain may play a role in the assembly, stability, or targeting of SU(VAR)3-9-containing complexes cannot be excluded. In this hyperploid genotype, Su(var)3-9311 is a stronger suppressor of PEV than Su(var)3-9330. This may be due to the fact that SU(VAR)3-9311 retains significantly less HMTase activity than SU(VAR)3-9330, as the in vitro enzyme assays indicate. In this genetic assay, Su(var)3-9311 is also a morphologically stronger PEV suppressor than Su(var)3-9318, but the in vitro enzyme activities of SU(VAR)3-9311 and SU(VAR)3-9318 do  98  not differ significantly. This may be explained by the peculiar biochemical phenotype of SU(VAR)3-9318. If, as suggested by the in vitro data (Figure 5), this mutant protein is an inefficient HMTase that is also completely unable to trimethylate histone H3, then its observed residual activity on unmodified substrates probably represents mono/dimethylation exclusively. Therefore, comparing the HMTase activity of SU(VAR)39318 and SU(VAR)3-9330 or SU(VAR)3-9311 based on the relative amount of radiolabeled methyl transferred onto unmodified H3 substrate, could be misleading. With respect to mono/dimethylation, SU(VAR)3-9318 may well retain as much activity as SU(VAR)3-9330 (i.e. significantly more than SU(VAR)3-9311) and, since H3K9me2 is sufficient for heterochromatic silencing in Drosophila (Fischle et al., 2003; Swaminathan et al., 2005), it would not be surprising that Su(var)3-9318 and Su(var)3-9330 may be equivalent in their ability to suppress PEV, and that neither is as strong as Su(var)3-9311. However, we cannot exclude the possibilities that 1) the in vitro HMTase activity of the mutant SU(VAR)3-9 proteins is not always a good indicator of their in vivo activity, and/or 2) the Su(var)3-9 missense mutations affect more than just the protein’s enzyme activity, and the enzyme activity of the different mutant SU(VAR)3-9 proteins is not the only factor determining the strength of the Su(var) phenotype. Interestingly Su(var)3-9309, which behaves as a hypomorph in the PEV assay employed here, results in a protein product with no in vitro HMTase activity at all. We propose that the amino acid substitution present in SU(VAR)3-9309 (CYS462TYR) causes the catalytic region of the protein to be misfolded since it affects a residue that likely plays an important structural role (Min et al., 2002). Thus, in vitro, the complete loss of enzyme function associated with this mutation is due to severe misfolding of the  99  polypeptide. Furthermore, we suggest that the catalytic domain of SU(VAR)3-9309 is misfolded in vivo. This misfolding not only renders it inactive, but also prevents it from being incorporated into SU(VAR)3-9-containing complexes, therefore not interfering with the function of the wild-type protein. Hence, Su(var)3-9309 is not an antimorph. We are left with the observation that Su(var)3-9309 is a weaker suppressor of PEV than the null allele, Su(var)3-906, indicating that Su(var)3-9309 is a hypomorph. One possibility is that, occasionally (i.e. at a low frequency, e.g. 10% of the time), SU(VAR)3-9309 is still incorporated into the SU(VAR)3-9-containing complex, and its incorporation into the complex stabilizes its tertiary structure. In such cases, SU(VAR)3-9309 is able to methylate histone H3 like its wild-type counterpart, since its active site is intact.  100  Table 2.2. Summary of the molecular, biochemical, and Su(var) phenotypes of five Su(var)3-9 mutant alleles examined in this study. The stick diagram of SU(VAR)3-9 shows the positions of the point mutations in Su(var)3-9309, Su(var)3-9311, Su(var)3-9330 and Su(var)3-9318 (vertical black bars). The chromodomain (red box), pre- and postSET domains (green boxes) and SET domain (blue box), and the N-terminal region that is in common with eIF2g are also indicated. For each allele, the result of the mutation (amino acid change), the relative amount of residual HMTase activity with 3 different substrates, and the percentage of strongly suppressed eyes in the PEV assay are shown. 309 eIF2g  chromo  Su(var)39 allele  Result of mutation  H3K9me2 in vivo  06  Reduced  309 311  No gene product C462Y G521D  330  D536N  Reduced  318  S616L  Reduced  Reduced Reduced  311 330  preSET  SET  318 postSET  HMTase in vitro (% of wild-type) H3 H3 tail H3K9me2 tail N/A N/A N/A  % strongly suppressed eyes 53.51+/-1.96  0 22.7+/1.2 31.3+/3.8 24.7+/1.7  0 14.3+/1.4 1.2+/-0.2  0 11.5+/-0.2  37.71+/-0.07 86.20+/-3.26  4.0+/-1.1  65.68+/-0.81  18.3+/0.3  0  66.76+/-1.59  101  2.4. Materials and Methods 2.4.1. Drosophila strains Unless otherwise specified, all fly strains were grown under standard conditions on glucose/yeast/cornmeal medium, with Tegosept (methyl-p-hydroxybenzoate) as a mold inhibitor.  2.4.2. Recombination mapping Allelism to Su(var)3-9 was determined by recombination analysis. Each putative EMS-induced Su(var)3-9 mutant (wm4;Su(var)X/Su(var)X) was first crossed to wm4;Su(var)3-9P25, which harbours a P-element insert in the first intron of the dual Su(var)3-9/eIF2g transcription units (Harrington, 2001; Ner et al., 2002). F1 females (wm4/wm4; Su(var)3-9P25/Su(var)X) were then crossed to wm4/Y;+/+ males, and in each case >2000 offspring were scored with respect to PEV suppression. Mutants that did not yield any wm4;+/+ recombinants, indicating that the distance between Su(var)3-9 and the Su(var)X mutation they carried was less than 0.1 cM, were further characterized by DNA sequence analysis. The maximum distance between Su(var)X and Su(var)3-9 was calculated as if the next fly to eclose would have been a wild-type recombinant; max distance = 2 [since the reciprocal event yields a double mutant, indistinguishable from the parentals]/(1+total number of flies scored).  102  2.4.3. Suppression of PEV in the presence of 2 wild-type copies of Su(var)3-9 Homozygous wm4; Su(var)3-9 females were crossed to wm4/Y; pP{GS[ry+,(10kb Su(var)3-9)EGFP]} homozygous males (Schotta and Reuter, 2000) and the eye colors of the offspring were scored. Only males were scored in order to avoid effects due to the presence of two copies of the white gene. All crosses were conducted in triplicate at 18 ºC. For each cross, a total of 356 to 580 eyes were examined. The phenotype of each eye was classified as “strongly suppressed”, “mildly suppressed”, “unsuppressed” (wm4-like), or enhanced. The reciprocal crosses gave similar results, but the number of offspring was much lower due to the low fecundity of wm4/ wm4; pP{GS[ry+,(10kb Su(var)3-9)EGFP]} homozygous females at 18 ºC. Crosses were set up in triplicates, and strength of PEV suppression was assessed based on the percentage of strongly suppressed eyes in the male offspring of each cross (average +/- S.E.M.). Student’s Ttests (p=0.05) were employed to determine whether differences between alleles were statistically significant.  2.4.4. Sequence analysis Genomic DNA was extracted from the following stocks: Oregon-R, wm4, wm4;Su(var)3-9309/Su(var)3-9309, wm4;Su(var)3-9312/Su(var)3-9312, wm4;Su(var)39311/Su(var)3-9311, wm4;Su(var)3-9317/TM3,Sb,Ser; wm4;Su(var)3-9318/Su(var)3-9318, wm4;Su(var)3-9324/TM3,Sb,Ser, wm4;Su(var)3-9325/TM3,Sb,Ser, wm4;Su(var)39327/TM3,Sb,Ser, Su(var)3-9319/TM3,Sb,Ser, and wm4;Su(var)3-9330/Su(var)3-9330. Segments of the Su(var)3-9 gene were amplified by PCR using the following primer  103  pairs: 39KYLE and 3SET, 3-95’ and 39-1, 39-2 and 3-95’, 5RI and 3RI, and 5SET and 3SET (all primer sequences are listed in supplementary table 2). The amplification conditions were: 94ºC, 5 min; (94ºC 45 sec, 57-60ºC, 30 sec, 72ºC, 1.5 min) for 30 cycles, 72ºC, 10 min. Each PCR reaction was performed three times and both strands of each product were sequenced twice.  2.4.5. RT-PCR Total RNA was prepared from wild-type and Su(var)3-906 homozygous flies by TRIzol extraction as recommended by the manufacturer (Invitrogen). 50 adults were used for each extraction. 2 µg of each RNA sample were reverse-transcribed with primers RP49rt (control) and 3906rt simultaneously, following standard procedures. Mock reactions (no reverse transcriptase) were carried out to ensure that no contaminating genomic DNA was present. cDNA (first strand) samples and mock reactions were amplified using primer pairs RP495-RP493, and 3906rt-3906pcr, separately. The amplification conditions were: 94°C, 5 min; (94°C, 30 sec; 52°C, 30 sec; 72°C, 30 sec) for 30 cycles; 72°C, 5 min.  2.4.6. Western blots Western blot analyses were performed according to standard procedure (Lacey et al., 1994). Embryo extracts were prepared from wild-type and mutant 12-16 hour old embryos as described below under (ChIP analysis). About 150 µg of each extract were used for the SU(VAR)3-9 and H3K9me analyses. The blots were probed with a polyclonal  104  anti- SU(VAR)3-9 antibody (a-3-9chr) (Ner et al., 2002) at 1:2000 dilution and subsequently the blots were reprobed with an anti-tubulin monoclonal antibody at 1:1000. To detect the methylation status of K9H3 we used commercial anti-H3K9me2 (Upstate Biotech #07-212) at 1:1000 and to detect total histone H3 the blots were probed with an anti-H3 monoclonal antibody at 1:30,000 dilution (Sauvé et al., 1999).  2.4.7. Immunostaining of polytene nuclei Salivary glands of wild-type and Su(var)3-9 mutant 3rd instar larvae were dissected in PBS, fixed in PBS + 2% formaldehyde for 15 min at room temperature, washed 3 times in PBS2+, and blocked in PBS2+ with 1% BSA for 60 min at room temperature (Cryderman et al., 1999). Protease inhibitors were added as required. The anti-H3K9me2 (Upstate Biotech #07-212) and anti-HP1 (C1A9) (James et al., 1989) were added at a final dilution of 1:250 each. The secondary antibodies were anti-rabbit Alexa488 and anti-mouse Alexa568 (Molecular Probes) at 1:1000 each.  2.4.8. ChIP analysis 12-16 hours old embryos were dechorionated in 50% bleach for 2 min and washed extensively with PBS+0.01% Triton-X 100. Cross-linking was achieved by incubation in 2% formaldehyde, 50mM Hepes pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA for 10 min at room temperature and then for 20 min at 4°C, and terminated by adding glycine at a final concentration of 250mM. The embryos were then washed twice with 10mM Tris-HCl, pH 8.0, 1mM EDTA, 0.5 mM EGTA and subjected to  105  sonication (9X15 sec at 30% output). Protease inhibitors were added as required. The soluble fraction of the lysate was adjusted to a final concentration of 3M urea and incubated on ice for 10 min. Nucleoprotein complexes were purified using a polyclonal anti-H3K9me2 (UPSTATE #02-441) or a polyclonal anti-HP1 (a-HP1 (Ner et al., in preparation)) antibodies. Mock reactions (no antibody) were included in each set of experiments. Immunoprecipitated DNA was purified as described by Nelson et al., (2006). Genomic sequences of interest (HIS-C) were detected by PCR using primer pair H3F/H3R. The amplification conditions were: 94°C, 5min; (94°C, 45 sec; 58°C, 30 sec; 72°C, 40 sec) for 26 cycles; 72°C, 5 min.  2.4.9. Expression and purification of active GST-SU(VAR)3-9 fusion proteins DNA fragments encoding amino acids 182 to 635 of wild-type SU(VAR)3-9, SU(VAR)3-9311, SU(VAR)3-9309, SU(VAR)3-9330, SU(VAR)3- SU(VAR)3-9318 and SU(VAR)39376, were amplified from genomic DNA of the corresponding strains using primers 39KYLE and 3SET. Each purified amplification product was cloned into the EcoRV site of pBluescript KS- and sequenced. An EcoRI/NotI fragment from the pBluescript Su(var)3-9 constructs was then cloned into pGEX 4T-1 resulting in an in-frame construct that produces GST-SU(VAR)3-9 polypeptides. The junctions and specific Su(var)3-9 mutation of each clone were verified by sequencing. The pGEX-Su(var)3-9 constructs were transformed into BL21(DE3)pLysS. For each construct, 500 ml of LB containing 100 µg/ml of ampicillin were inoculated with a single colony and incubated overnight in a 37º C shaker. The culture was induced by adding 150 ml of LB supplemented with 600 µg of ampicillin and 650 µl of 1M IPTG,  106  incubated for 5 hours at 37º C and then processed as described by Frangioni and Neel (1993) with the following modifications. The bacterial pellet was repeatedly frozen and thawed (four times) in liquid nitrogen and a 25º C water bath. The cells were resuspended in 36 ml of STE+ (10mM Tris, pH 8.0, 300 mM NaCl, 1mM EDTA) and lysozyme added to a final concentration of 1 mg/ml. After 20 minutes at room temperature the cells were adjusted to 5 mM DTT and 1.4% N-lauryl-sarcosine. The lysate was placed on ice for 5 min and then sonicated (Sonic 300 dismembrator, 6x45 sec at 40% power). Triton X-100 was added to a final concentration of 3.4%. The sonicate was then incubated for 5 min. at room temperature and spun for 6 min at 12000 rpm (bench top centrifuge) at 4º C. The supernatant was collected and the GSTSU(VAR)3-9 fusions were bound to glutathione-coupled matrix (Pharmacia Biotech) as recommended by the manufacturer. After binding, the matrix was washed 4 times with PBS, 0.1% NP40, 6 times with PBS, 500mM NaCl, 0.1% NP40, and twice with PBS alone. The matrix-bound fusion protein was stored at –80°C in 50% glycerol in PBS.  2.4.10. HMTase assays The recombinant variant GST- SU(VAR)3-9 polypeptides (1-10 µg) including the wild-type were incubated for 2 hours at room temperature in HMTase buffer (50mM Tris, pH 8.1, 20mM KCl, 10mM MgCl2, 10mM 2-mercaptoethanol, 250mM sucrose (Rea et al., 2000)) with 20 mg of bulk histones (Roche), or 2 µg of recombinant H3 (Upstate Biotech #14-411), or 2 µg of H3 tail peptide (Upstate Biotech #12-357, Upstate Biotech #12-430), and 0.125-0.25 µCi of S-adenosyl-methyl-methionine. The reactions were carried out in a final volume of 50 ml and stopped by adding 10 µl of 6X SDS loading  107  buffer. 30µl of each reaction was separated on a 13 or 15% polyacrylamide gel and stained with Coomassie. After drying, the gels were exposed and radioactive signal detected using a Phosphor Imager. The data were processed using the Image Quant software. The NIH Image software was used to quantify the amount of the various GSTSU(VAR)3-9 proteins in each Coomassie-stained band. The specific activity of each mutant was calculated as the ratio of the radioactive signal corresponding to the (methylated) substrate and relative amount of recombinant GST- SU(VAR)3-9 used as determined by the intensity of the Coomassie stained band. The relative activity of each mutant, expressed as a percentage, is the ratio between its specific activity and the specific activity of the wild-type recombinant protein (run on the same gel). Each experiment was performed three times using recombinant proteins from independent preparations. The results are expressed as the average of three independent trials +/- the S.E.M. Student’s T-tests (p=0.05) were used to determine whether differences between mutants were statistically significant.  108  Suppl. table 2.1. Enzymatic activity of selected SU(VAR)3-9 mutants on 4 different substrates (see Materials & Methods for details).  Percent activity (average +/- SEM) on the indicated substrates  GST SU(VAR)3-9wt SU(VAR)3-9309/312 SU(VAR)3-9311 SU(VAR)3-9318 SU(VAR)3-9330 SU(VAR)3-9376 1  Bulk histones 01 100 01 12.8 +/- 3.1 14.3 +/- 1.4 24.3 +/- 1.9 01  Recombinant H3 01 100 01 22.7 +/- 1.2 24.7 +/- 1.7 31.3 +/- 3.8 01  Values below 1% are listed as 0  109  H3 tail (1-20)  H3 K9me2 tail (1-21)  1  0 100 01 14.3 +/- 1.3 18.3 +/- 0.3 1.2 +/- 0.2 01  01 100 01 11.5 +/- 0.2 01 4.0 +/- 1.1 Not tested  Suppl. table 2.2. Sequences of the primers used in this study.  NAME 3SET 5SET 3-95’ 39-1 39-2 39KYLE 5RI 3RI RP49rt RP495 RP493 3906rt 3906pcr H3F H3R  SEQUENCE (5’ to 3’) TGTCTCAGGTGGGTAACGGCGTG GCCAACGGCAGCGGATGGGGGG CGGGATCCCGAATTCATGGCCACGGCTGAAGCC CTGCTGTCGCTGCTGCTTGGAGGT CAATACGCTCCACAACGTACTCTC TTCGCCAAACTGAAGCGTCG CGATATCGAGATTTGATGCCG TAGGGCACTACGGGGTTTAC CGCGCTCGATAATCTCC GCCCAAGATCGTGAAGAAGC CTGTTGTCGATACCCTTGGG TTTTTCGTCAAGCGTTC ATCCACGGTGGTCAAAG GCTCGTACCAAGCAAACT TGCCGTGTCAGCTTAAGCA  110  2.5. References Aagaard, L., Laible, G., Selenko, P., Schmid, M., Dorn, R., Schotta, G., Kuhfittig, S., Wolf, A., Lebersorger, A., Singh, P.B. et al.: Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromereassociated proteins which complex with the heterochromatin component M31. Embo J 18, 1923-1938 (1999) Akhtar, A., Zink, D., Becker, P.B.: Chromodomains are protein-RNA interaction modules. Nature 407, 405-409 (2000) Ayyanathan, K., Lechner, M.S., Bell, P., Maul, G.G., Schultz, D.C., Yamada, Y., Tanaka, K., Torigoe, K., Rauscher, F.J., 3rd: Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: a mammalian cell culture model of gene variegation. Genes Dev 17, 1855-1869 (2003) Bannister, A.J., Zegerman, P., Partridge, J.F., Miska, E.A., Thomas, J.O., Allshire, R.C., Kouzarides, T.: Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120-124 (2001) Berger, S.L.: Histone modifications in transcriptional regulation. Curr Opin Genet Dev 12, 142-148 (2002) Bouazoune, K., Mitterweger, A., Langst, G., Imhof, A., Akhtar, A., Becker, P.B., Brehm, A.: The dMi-2 chromodomains are DNA binding modules important for ATP-dependent nucleosome mobilization. Embo J 21, 2430-2440 (2002) Collins, R.E., Tachibana, M., Tamaru, H., Smith, K.M., Jia, D., Zhang, X., Selker, E.U., Shinkai, Y., Cheng, X.: In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases. J Biol Chem 280, 5563-5570 (2005) Cryderman, D.E., Morris, E.J., Biessmann, H., Elgin SC, Wallrath LL.: Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles. EMBO J 18, 3724-3735 (1999) Cryderman, D.E., Grade, S.K., Li,Y., Fanti, L., Pimpinelli, S., Wallrath, L.L.: Role of Drosophila HP1 in euchromatic gene expression. Dev Dyn 232, 767-74 (2005) Czermin, B., Schotta, G., Hulsmann, B.B., Brehm, A., Becker, P.B., Reuter, G., Imhof, A.: Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila. EMBO Rep 2, 915-919 (2001)  111  Donaldson, K.M., Lui, A., Karpen, G.H.: Modifiers of terminal deficiencyassociated position effect variegation in Drosophila. Genetics 160, 995-1009 (2002) Ebert, A., Schotta, G., Lein, S., Kubicek, S., Krauss, V., Jenuwein, T., Reuter, G.: Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev 18, 2973-2983 (2004) Ebert, A., Lein, S., Schotta, G., Reuter, G.: Histone modification and the control of heterochromatin gene silencing in Drosophila. Chromosome Research 14, 377-392 (2006) Eskeland, R., Czermin, B., Boeke, J., Bonaldi, T., Regula, J.T., Imhof, A.: The Nterminus of Drosophila SU(VAR)3-9 mediates dimerization and regulates its methyltransferase activity. Biochemistry 43, 3740-3749 (2004) Finch, J.T., Rhodes, D., Brown, R.S., Rushton, B., Levitt, M., Klug, A.: Structure of nucleosome core particles of chromatin. Nature 269, 29-36 (1977) Fischle, W., Wang, Y., Allis, C.D.:Histone and chromatin cross-talk. Curr Opin Cell Biol 15, 172-183 (2003) Fischle, W., Wang, Y., Jacobs, S.A., Kim, Y., Allis, C.D., Khorasanizadeh, S.: Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev 17, 1870-1881 (2003) Frangioni, J.V., Neel, B.G.: Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem 210, 179-187 (1993) Gatti, M., Pimpinelli, S.: Functional elements in Drosophila melanogaster heterochromatin. Annu Rev Genet 26, 239-275 (1992) Greil, F., van der Kraan, I., Delrow, J., Smothers, J.F., de Wit, E., Bussemaker, H.J., van Driel, R., Henikoff, S., van Steensel, B.: Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev 17, 2825-2838 (2003) Grigliatti, T.: Position-effect variegation--an assay for nonhistone chromosomal proteins and chromatin assembly and modifying factors. Methods Cell Biol 35, 587-627 (1991) Harrington, M.J.: Ph.D. Thesis The University of British Columbia (2001)  112  Ivanova, A.V., Bonaduce, M.J., Ivanov, S.V., Klar, A.J.: The chromo and SET domains of the Clr4 protein are essential for silencing in fission yeast. Nat Genet 19, 192-195 (1998) Jacobs SA, Taverna SD, Zhang Y, Briggs SD, Li J, Eissenberg JC, Allis CD, Khorasanizadeh S (2001) Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3. Embo J 20:5232-5241. Jenuwein, T., Allis, C.D.: Translating the histone code. Science 293, 1074-1080 (2001) Kraulis, P.J.: MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. Appl Cryst 24, 946-950 (1991) Krauss, V., Reuter, G.: Two genes become one: the genes encoding heterochromatin protein Su(var)3-9 and translation initiation factor subunit eIF2gamma are joined to a dicistronic unit in holometabolic insects. Genetics 156, 1157-67 (2000) Klug, A., Rhodes, D., Smith, J., Finch, J.T., Thomas, J.O.: A low resolution structure for the histone core of the nucleosome. Nature 287, 509-516 (1980) Krouwels, I.M., Wiesmeijer, K., Abraham, T.E., Molenaar, C., Verwoerd, N.P., Tanke, H.J., Dirks, R.W.: A glue for heterochromatin maintenance: stable SUV39H1 binding to heterochromatin is reinforced by the SET domain. J Cell Biol 170, 537-549 (2005) Lachner, M., O'Carroll, D., Rea, S., Mechtler, K., Jenuwein, T.: Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116-120 (2001) Lewis, E.B.: The phenomenon of position effect. Adv genet 3, 73-115 (1950) Litt, M.D., Simpson, M., Gaszner, M., Allis, C.D., Felsenfeld, G.: Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus. Science 293, 2453-2455 (2001) Liu, L.P., Ni, J.Q., Shi, Y.D., Oakeley, E.J., Sun, F.L.: Sex-specific role of Drosophila melanogaster HP1 in regulating chromatin structure and gene transcription. Nat Genet 37, 1361-1366 (2005) Lohe, A.R., Hilliker, A.J.: Return of the H-word (heterochromatin). Curr Opin Genet Dev 5, 746-755 (1995)  113  Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., Richmond, T.J.: Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251260 (1997) Ma, H., Baumann, C.T., Li, H., Strahl, B.D., Rice, R., Jelinek, M.A., Aswad, D.W., Allis, C.D., Hager, G.L., Stallcup, M.R.: Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter. Curr Biol 11, 1981-1985 (2001) Margueron, R., Trojer, P., Reinberg, D.: The key to development: interpreting the histone code? Curr Opin Genet Dev 15, 163-176 (2005) Merritt, E.A., Murphy, M.E.: Raster3D Version 2.0.: A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr 50:869-873 (1994) Messmer, S., Franke, A., Paro, R.: Analysis of the functional role of the Polycomb chromo domain in Drosophila melanogaster. Genes Dev 6, 1241-1254 (1992) Min, J., Zhang, X., Cheng, X., Grewal, S.I., Xu, R.M.: Structure of the SET domain histone lysine methyltransferase Clr4. Nat Struct Biol 9, 828-832 (2002) Min, J., Zhang, Y., Xu, R.M.: Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27. Genes Dev 17, 1823-1828 (2003) Mis, J., Ner, S.S., Grigliatti, T.A.: Identification of three histone methyltransferases in Drosophila: dG9a is a suppressor of PEV and is required for gene silencing. Mol Gen Genomics 275, 513-526 (2006) Muller, H.J.: Types of visible variations induced by X-rays in Drosophila. J Genet 22, 299-334 (1930) Nakayama, J., Rice, J.C., Strahl, B.D., Allis, C.D., Grewal, S.I.: Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110-113 (2001) Nelson, J.D., Denisenko, O., Sova, P., Bomsztyk, K.: Fast chromatin immunoprecipitation assay. Nucleic Acids Res 34, e2 (2006) Ner, S.S., Harrington, M.J., Grigliatti, T.A.: A role for the Drosophila SU(VAR)3-9 protein in chromatin organization at the histone gene cluster and in suppression of position-effect variegation. Genetics 162, 1763-1774 (2002)  114  Nielsen, S.J., Schneider, R., Bauer, U.M., Bannister, A.J., Morrison, A., O'Carroll, D., Firestein, R., Cleary, M., Jenuwein, T., Herrera, R.E. et al.: Rb targets histone H3 methylation and HP1 to promoters. Nature 412, 561-565 (2001) Peterson, C.L., Laniel, M.A.: Histones and histone modifications. Curr Biol 14, R546-551 (2004) Platero, J.S., Hartnett, T., Eissenberg, J.C.: Functional analysis of the chromo domain of HP1. Embo J 14, 3977-3986 (1995) Pray-Grant, M.G., Daniel, J.A., Schieltz, D., Yates, J.R., 3rd, Grant, P.A.: Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation. Nature 433, 434-438 (2005) Rea, S., Eisenhaber, F., O'Carrol, D., Strahl, B.D., Sun, Z-W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C.P., Allis, C.D. et al.: Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593-599 (2000) Reuter, G., Spierer, P.: Position effect variegation and chromatin proteins. Bioessays 14, 605-612 (1992) Reuter, G., Wolff, I.: Isolation of dominant suppressor mutations for positioneffect variegation in Drosophila melanogaster. Mol Gen Genet 182, 516-519 (1982) Rice, J.C., Allis, C.D.: Code of silence. Nature 414, 258-261 (2001) Schneider, R., Bannister, A.J., Kouzarides, T.: Unsafe SETs: histone lysine methyltransferases and cancer. Trends Biochem Sci 27, 396-402 (2002) Schotta, G., Ebert, A., Krauss, V., Fischer, A., Hoffmann, J., Rea, S., Dorn, R., Reuter, G.: Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. EMBO J 21, 1121-1131 (2002) Schotta, G., Ebert, A., Reuter, G.: SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing. Genetica 117, 149-158 (2003) Schotta, G., Reuter, G.: Controlled expression of tagged proteins in Drosophila using a new modular P-element vector system. Mol Gen Genet 262, 916-920 (2000) Seum, C., Reo, E., Peng, H., Rauscher, F.J. III, Spierer, P., Bontron, S.: Drosophila SETDB1 Is Required for Chromosome 4 Silencing. PLoS Genet 3, e76l (2007)  115  Sinclair, D.A.R., Mottus, R.C., Grigliatti, T.A.: Genes Which Suppress PositionEffect Variegation in Drosophila melanogaster are Clustered. Mol Gen Genet 191, 326-333 (1983) Strutt, H., Cavalli, G., Paro, R.: Co-localization of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression. Embo J 16, 3621-3632 (1997) Tachibana, M., Sugimoto, K., Nozaki, M., Ueda, J., Ohta, T., Ohki, M., Fukuda, M., Takeda, N., Niida, H., Kato, H. et al.: G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev 16, 1779-1791 (2002) Tschiersch, B., Hofmann, A., Krauss, V., Dorn, R., Korge, G., Reuter, G.: The protein encoded by the Drosophila position-effect variegation suppressor Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J 13, 3822-3831 (1994) Weiler, K.S., Wakimoto, B.T.: Heterochromatin and gene expression in Drosophila. Annu Rev Genet 29, 577-605 (1995) Xiao, B., Jing, C., Wilson, J.R., Walker, P.A., Vasisht, N., Kelly, G., Howell, S., Taylor, I.A., Blackburn, G.M., Gamblin, S.J.: Structure and catalytic mechanism of the human histone methyltransferase SET7/9. Nature 421, 652-656 (2003) Zhang, X., Tamaru, H., Khan, S.I., Horton, J.R., Keefe, L.J., Selker, E.U., Cheng, X.: Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase. Cell 111, 117-127 (2002) Zhang, X., Yang, Z., Khan, S.I., Horton, J.R., Tamaru, H., Selker, E.U., Cheng, X.: Structural basis for the product specificity of histone lysine methyltransferases. Mol Cell 12, 177-185 (2003)  116  3. THE ROLE OF SU(VAR)3-9 IN THE REGULATION OF DROSOPHILA’S HISTONE GENE CLUSTER (HIS-C)2.  3.1. Introduction In Drosophila, methylation of lysine 9 of histone H3 (H3K9me) is a hallmark of heterochromatin (Rea et al., 2000; Nakayama et al., 2001; Peters et al., 2001; Schotta et al., 2002; 2004; Ebert et al., 2004; 2006), and SU(VAR)3-9 is one of the major methyltransferases responsible for this modification (Rea et al., 2000; Schotta et al., 2002; 2003). SU(VAR)3-9 itself is associated with heterochromatic regions of the genome, and particularly with centromeric and pericentric heterochromatin, which in polytene chromosomes form the chromocentre. The formation of heterochromatin is thought to involve several steps, and a relatively detailed model has emerged, which describes the sequence of events and the role of SU(VAR)3-9 in this process (Nakayama et al., 2001; Czermin et al., 2001; Schotta et al., 2003; Swaminathan et al., 2005; Rudolph et al., 2007). In this model, chromatin compaction is initiated by the demethylation of H3K4 by the demethylase SU(VAR)3-3/dLSD1, followed by the deacetylation of H3K9 by the histone deacetylase HDAC1/RPD3 (Czermin et al., 2001; Rudolph et al., 2007). HP1 and SU(VAR)3-7, two other NHCPs, are then responsible for targeting and restricting SU(VAR)3-9 to the chromocentre, where it methylates H3K9, thus creating a binding site for the chromodomain of HP1 (Jaquet et al., 2002; Schotta et al., 2002; Delattre et al., 2004; Ebert et al., 2006; Jacobs et al., 2001; 2  A version of this chapter will be submitted for publication. Kalas, P. and Grigliatti, T.A. The role of SU(VAR)3-9 in the regulation of Drosophila’s Histone Gene cluster.  117  help of auxiliary factors, HP1 then binds to H3K9me, allowing the recruitment of more SU(VAR)3-9 and other factors, such as the H4K20 HMTase SUV4-20 (Schotta et al., 2004; Eskeland et al., 2007). However, SU(VAR)3-9 is also detected at a number of euchromatic loci, where it contributes to the regulation of gene expression (Nielsen et al., 2001; Ner et al., 2002; Greil et al., 2003; Koryakov et al., 2006). One of these euchromatic sites is the histone genes cluster (HIS-C), where SU(VAR)3-9 appears to affect gene expression by altering the chromatin structure of the locus (Ner et al., 2002). Several pieces of evidence seem to connect the function of SU(VAR)3-9 in euchromatin with silencing or down regulation of the target genes, but its mechanism of action is largely unknown (Nielsen et al., 2001; Ner et al., 2002; Macaluso et al., 2003). Moreover, it is becoming increasingly apparent that SU(VAR)3-9’s role and function may vary in a context-dependent manner. For instance, genome-wide localization studies showed that SU(VAR)3-9 is present at a large number of euchromatic loci, and it colocalizes with HP1 only at a subset of these loci; unlike in heterochromatin, where SU(VAR)3-9 and HP1 appear to overlap very broadly (Greil et al., 2003; Schotta et al., 2002). This suggests that SU(VAR)3-9 may have slightly different functions at different loci, and/or that it may elicit its function(s) through different mechanisms (e.g. in collaboration with HP1 or with some other non-histone chromatin protein). Particular attention has been devoted to the study of SU(VAR)3-9’s catalytic core, the preSET/SET/postSET domain (here referred to as "the SET domain" for simplicity). It has been demonstrated that the integrity of this domain is  118  necessary not only for the protein to carry out its enzymatic activity, which is believed to play a key role in heterochromatin formation, but also for its association with centric and pericentric heterochromatin (Schotta et al., 2002). FRAP-based studies in mammalian cell lines have also shown that the SET domain of SUV39H1 (the human ortholog of SU(VAR)3-9) contributes to its stable association to (hetero)chromatin, and that this function seems independent from its catalytic activity (Krouwels et al., 2005). Again, it is possible that the relative importance of each of SU(VAR)3-9’s multiple functions are context-dependent. Here, we use the HIS-C as a "model system" for investigating the function(s) of SU(VAR)3-9, and specifically those associated with its SET/preSET/postSET domain, in the regulation of a euchromatic locus. There are several reasons why the HIS-C was chosen. First, SU(VAR)3-9 has been shown to physically associate with the HIS-C, indicating that it probably plays a direct role in the chromatin architecture of this locus. Second, we know that the expression of at least two of the histone genes (H1 and H4) is altered in at least three Su(var)3-9 mutants, suggesting that SU(VAR)3-9 must play a role in their regulation (Ner et al., 2002). In addition, its mechanism of action seems to be chromatin-mediated, since the nucleosome spacing at the HIS-C appears altered in several Su(var)3-9 mutants (Ner et al., 2002). The simplest initial working model is that SU(VAR)3-9 acts at the HIS-C through the same mechanism as it does in heterochromatin. In particular, we hypothesize that methylation of H3K9 across the HIS-C is mainly dependent on SU(VAR)3-9,  119  and that the presence of SU(VAR)3-9 and that of H3K9me2 are necessary for proper localization of HP1 at this locus. In turn, the presence of HP1 at the HIS-C would be necessary for proper regulation of the histone genes expression. In order to test this hypothesis, we first need to obtain a reasonably detailed picture of the HIS-C "landscape" in terms of the distribution of SU(VAR)3-9, H3K9me2 and HP1 across the locus in a wild-type strain. Then, we take advantage of a set of well-characterized Su(var)3-9 mutants to investigate the functional relationships among SU(VAR)3-9, H3K9me2 and HP1 in the context of histone gene regulation. Specifically, we ask whether the distribution of SU(VAR)3-9, H3K9me2 and HP1 across the HIS-C, and the level of histone transcripts are altered in Su(var)3-9 missense mutants. We show that, in the three missense mutants analyzed, the Su(var)3-9 gene product is still present across the HIS-C. The levels of H3K9me2 and HP1 associated with the HIS-C are significantly reduced in all Su(var)3-9 mutants tested. These mutants also display an increase in the amount of histone H3 and histone H2A transcript levels, supporting the hypothesis that the enzymatic function of SU(VAR)3-9 is critical for regulation of the histone genes. Interestingly, in one Su(var)3-9 missense allele (Su(var)39330) the relative increase in the level of H3 transcript is much more pronounced than those of H2A and H2B, suggesting that the stoechiometry of these core nucleosome proteins may be disrupted in this particular strain. Finally, we show that the elevated levels of histone transcripts detected in Su(var)3-9 mutants are not due to an accumulation of abnormally high levels of histone mRNA synthesized outside S-phase. Hence, we conclude that the increased amount of  120  h2a and h3 transcripts is most likely a consequence of an increase in the number of templates transcribed/unit time, or an increase in the rate of transcription from each of an invariant number of templates, or a combination of these two factors.  121  122  1kb  histone H1  BamHI  Figure 3.1.  0kb  BglII  BglII  )  2kb  EcoRI  AflII  H1/H3 intergenic  (  histone H4  4kb  H2A  H3  3kb  H2A coding  StuI  histone H2B  H2B  H2A/H2B intergenic  histone H2A  H3 coding  H3/H4 intergenic  histone H3  5kb  BglII  BglII  Figure 3.1. A schematic of the histone unit. The histone genes cluster (HIS-C) is comprised of ~110 tandemly repeated histone units; the diagram shows the BglII genomic fragment representing the histone unit. The coding regions of histones H1 (yellow), H3 and H4 (red) and H2A and H2B (blue) are shown as plain boxes with an arrowhead pointing in the direction of transcription. The region delimited by the brackets represents the approximate extent and location of the deletion present in about 25% of the histone units. The blue lines labelled "H1/H3 intergenic", "H3/H4 intergenic", H2A/H2B intergenic", "H3 coding" and "H2A coding" indicate the sizes and positions of the fragments analyzed in the ChIP experiments. The dark red arrows show the positions and directions of the primers used to reverse-transcribe the histone RNAs, and the dark red lines labelled "H3", "H2A" and "H2B" indicate the extents of the amplified cDNAs produced. Below, the relative positions of four unique restriction sites are shown in relation to the two BglII sites defining the unit.  123  3.2. Results 3.2.1. SU(VAR)3-9 is associated with the HIS-C in wild-type and Su(var)3-9 missense mutants The Histone Gene Cluster (HIS-C) region of the second chromosome is comprised of about 100 tandemly reiterated copies of the histone unit (his unit). The his unit contains one copy of each of the five histone genes, with the core histone genes arranged into two gene pairs, the h3/h4 and the h2a/h2b couplets (Figure 3.1). The two members of each couplet share a regulatory region. There are two versions of the his unit: one, representing about 75% of the his units present in the HIS-C, is 5kb, and the other, less represented, is about 4.75kb in length (Lifton et al., 1977). The 250 bp difference is due to an indel located in the H1/H3 intergenic region (see parentheses in Figure 3.1.). SU(VAR)3-9 is associated with the HIS-C, where it is detected across the whole locus (Ner et al., 2002; Koryakov et al., 2006; Ner et al., in preparation). In addition, several tested Su(var)3-9 mutants, including the missense mutant Su(var)3-9330 display an alteration in the chromatin structure of the HIS-C and elevated levels of histone gene transcripts (Harrington, 2001; Ner et al., 2002). However, whether the physical presence of SU(VAR)3-9 is necessary and/or sufficient for proper regulation of the histone genes, and whether the single amino acid substitutions in its catalytic region affect its localization at the HIS-C, is unclear. In order to address these issues, we first used chromatin immunoprecipitation (ChIP) to determine the distribution of SU(VAR)3-9 at the HIS-C in wild-type and  124  Su(var)3-9 missense mutant embryos. Homozygous Su(var)3-906 individuals, which represent complete nulls producing no Su(var)3-9 mRNA or protein (Tschiersch et al., 1994; Schotta et al., 2002; this work, chapter 2) were also included in the analysis to serve as an internal standard/negative control. ChIP analysis of cross-linked extracts prepared from 12-16 hour old staged embryos was performed using an antibody raised against the N-terminal half of SU(VAR)3-9 (α-SU(VAR)3-9chr (Ner et al., 2002)), and the immunoprecipitated DNA was tested for the presence of 3 sequences belonging to the HIS-C: the H3 coding region ("H3 coding"), a fragment including the intergenic/regulatory region between the H2A and H2B genes ("H2A/H2B intergenic") and a section of the intergenic region between the H1 and H3 genes ("H1/H3 intergenic") (see also Figure 3.1). This last fragment spans a region of the HIS-C that, in ~25% of the 110 or so copies of the his unit, contains a ~250bp indel (Lifton et al., 1977). Thus, amplification with H1/H3 intergenic-specific primers gives rise to two different fragments: one representing the "longer" version of the his unit (5 kb) and one corresponding to the "shorter" one (4.75 kb). The sizes of these two fragments are roughly 600 bp and 350bp, respectively (Lifton et al., 1977; Samal et al., 1981; Worcel et al., 1983). As expected, the cross-linked material pulled down by the α-SU(VAR)3-9chr antibody from Su(var)3-9+/ Su(var)3-9+ extracts contains each of the three HIS-C sequences ("H3 coding", "H2 intergenic" and "H3/H1 intergenic"). In contrast, these fragments were not enriched in the material pulled down from Su(var)3-906 extracts (Figure 3.2.b), allowing us to conclude that the enrichment observed in  125  the wild-type strain is indeed due to the association of SU(VAR)3-9 with the HISC. In all the Su(var)3-9 missense mutants, fragments corresponding to the "H3 coding", "H2A/H2B intergenic" and "H1/H3 intergenic" regions were detected among the immunoprecipitated material (Figure 3.2). Accurate quantifications of one of the his unit fragments, "H3 coding", revealed that the relative enrichment obtained with α-SU(VAR)3-9chr is relatively small, although significant (Figure 3.2.c and appendix 2). Still, the data obtained allow us to conclude that that wildtype SU(VAR)3-9 protein (SU(VAR)3-9WT) is associated with the his unit. These data, together with those of Ner and colleagues suggest that SU(VAR)3-9WT is distributed throughout the his unit (Ner et al., 2002; Ner et al., in preparation). A similar set of ChIP analyses, performed on Su(var)3-9309, Su(var)3-9330 and Su(var)3-9318 12-16 hours old embryos, demonstrated that the SU(VAR)3-9309, SU(VAR)3-9330 and SU(VAR)3-9318 mutant proteins also associate with the his unit. The enrichment for the "H3 coding" fragment detected in the three missense mutants Su(var)3-9309, Su(var)3-9330 and Su(var)3-9318 was not significantly different from that of the wild-type strain (Figure 3.2.c and appendix 2). Hence, our results also suggest that the single amino acid substitutions present in the SU(VAR)3-9309, SU(VAR)3-9330 and SU(VAR)3-9318 variants do not prevent their association with the HIS-C.  126  a.  histone H3  histone H1  BglII  histone H2A  histone H4  H3 coding  H1/H3 intergenic  histone H2B BglII  H2A/H2B intergenic  b. H3 coding IN IP NO (1:100)  Ab  H2A/H2B intergenic IN IP NO  (3-9)  (1:100)  Ab  H1/H3 intergenic IN IP NO  (3-9)  (1:100)  wild-type  (3-9)  Ab  wild-type  Su(var)3-906 Su(var)3-906  Su(var)3-9309 Su(var)3-9330  Su(var)3-9309  Su(var)3-9318 1  2  3  4  5  6  Su(var)3-9330 Su(var)3-9318 7  d. Relative amounts of “H3 coding” fragment in the material immunoprecipitated from 12-16 hrs old embryos. 0.08 IP (SU(VAR)3-9) mock (no Ab) 0.06 0.04 0.02  9  Average amounts of IPed target (relative to the wild-type) 1  ratio to WT  0.8 0.6 0.4 0.2  0  Figure 3.2  127  8 31  318  0  330  33  309  T  06  W  WT  30 9  0  06  % of the input  c.  8  Figure 3.2. Relative levels of SU(VAR)3-9 associated with three regions of the histone unit in wild type and Su(var)3-9 mutant embryos. a) Schematic of the BglII fragment defining the histone unit. The five histone genes (H1, H3, H4, H2A and H2B) and the three regions analysed (H3 coding, H2A/H2B intergenic and H1/H3 intergenic) are shown. b) Chromatin immunoprecipitation of wild-type and Su(var)3-9 mutant embryo extracts. The recovered DNA was PCR amplified using primers specific for the “H3 coding” region (lanes 1-3), the “H2A/H2B intergenic region” (lanes 4-6) or the “H1/H3 intergenic” region (lanes 7-9). The template used in each PCR reaction is indicated above the corresponding lane; IN (1:100): input material diluted 100X, NO Ab: mock (no antibody) reaction, IP (α-3-9chr): immunoprecipitated material. The antibody used was raised against the N-terminal region (including the chromodomain) of SU(VAR)3-9 and has been previously described (Ner et al., 2002). c) Relative amounts of “H3 coding” fragment in the precipitated material as determined by real-time PCR (see materials and methods for details). The bar graphs represent the average of 3 independent experiments and the error bars span two standard deviations. d) Average amounts of immunoprecipitated “H3 coding” fragment (minus the average for the respective “mock” reaction) expressed as fractions of the wild type.  128  3.2.2. The level of H3K9me2 associated with the HIS-C is significantly reduced in Su(var)3-9 missense mutants H3K9me2 is enriched at the HIS-C (Figure 3.3 and Ner et al., 2002) and, since Su(var)3-9309, Su(var)3-9330 and Su(var)3-9318 have all been shown to produce SU(VAR)3-9 proteins with an impaired HMTase activity (chapter 2), we asked whether the levels and/or distribution of H3K9me2 across the HIS-C was altered in these Su(var)3-9 mutants. ChIP analyses of cross-linked embryo extracts from homozygous Su(var)3-9+, Su(var)3-906, and the three Su(var)3-9 missense mutant strains were performed using an antibody that specifically recognizes H3K9me2 (see material and methods for details). As shown previously (Ner et al., 2002), all three HIS-C fragments, "H3 coding", "H2A/H2B intergenic" and "H1/H3 intergenic" were detected in the material immunoprecipitated from the wild-type strain (Figure 3.3.a). Their enrichment was about 100-fold higher than that observed using nonspecific IgG (Supplementary Figure 1 and appendices 1 and 3), allowing us to conclude that H3K9me2 is indeed present at the HIS-C in the wild-type strain. As expected, the three HIS-C fragments analyzed were significantly less abundant in the material immunoprecipitated from Su(var)3-906 mutant extracts (Figure 3.3). The Su(var)3-906 mutant completely lacks the SU(VAR)3-9 protein. However, it is not surprising that a small amount of H3K9me2 associated with its HIS-C in this strain since other HMTases, capable of methylating H3K9, are present in the nucleus (see discussion). The target his unit fragments were also detected in the material immunoprecipitated from each of the three Su(var)3-9  129  missense mutants, but, as expected, their relative abundance was significantly lower than in the wild-type strain (Figure 3.3). The relative enrichment for one particular fragment, "H3 coding", was accurately quantified by real-time PCR. With regard to this fragment, Su(var)3-906 and the missense mutant Su(var)3-9330 displayed the lowest level of enrichment, less than 5% of the wild-type (Figures 3.3.c and d). Su(var)3-9309 and Su(var)3-9318 showed a slightly higher enrichment for this fragment, corresponding to about 10% and 13% of the wild-type, respectively (Figure 3.3 and appendix 3). We conclude that the levels of H3K9me2 associated with the three regions of the HIS-C, "H3 coding", "H2A/H2B intergenic" and "H3/H1 intergenic" are significantly lower in all Su(var)3-9 mutants tested than they are in the wild-type strain.  130  a.  histone H3  histone H1  BglII  histone H2A  histone H4  H2A/H2B intergenic  H3 coding  H1/H3 intergenic  histone H2B BglII  b. H3 coding IN IP NO (1:10)  Ab  (K9me2)  H1/H3 intergenic IN IP NO  H2A/H2B intergenic IN IP NO (1:10)  Ab  (1:10)  (K9me2)  wild-type  Ab  (K9me2)  wild-type  Su(var)3-906 Su(var)3-9309  Su(var)3-906  Su(var)3-9330 Su(var)3-9318  Su(var)3-9309 1  2  4  3  5  6 Su(var)3-9330 Su(var)3-9318 7  c. 12  9  Relative amounts of “H3 coding” fragment in the material immunoprecipitated from 12-16 hrs old embryos.  10  d.  Average amounts of IPed target (relative to the wild-type)  IP (H3K9me2) mock (no Ab)  8  1 6  ratio to WT  % of the input  8  4 2  0.8 0.6 0.4 0.2 0  WT  06  309  330  W T 06 30 9 33 0 31 8  0 318  Figure 3.3  131  Figure 3.3. Relative levels of H3K9me2 associated with three regions of the histone unit in wild type and Su(var)3-9 mutant embryos. a) Schematic of the BglII fragment defining the histone unit. The five histone genes (H1, H3, H4, H2A and H2B) and the three regions analysed (H3 coding, H2A/H2B intergenic and H1/H3 intergenic) are shown. b) Chromatin immunoprecipitation of wild-type and Su(var)3-9 mutant embryo extracts. The recovered DNA was PCR amplified using primers specific for the “H3 coding” region (lanes 1-3), the “H2A/H2B intergenic region” (lanes 4-6) or the “H1/H3 intergenic” region (lanes 7-9). The template used in each PCR reaction is indicated above the corresponding lane; IN (1:10): input material diluted 10X, NO Ab: mock (no antibody) reaction, IP (K9me2): immunoprecipitated material. The antibody used was an anti-H3K9me2 from UPSTATE (#07-441). c) Relative amounts of “H3 coding” fragment in the precipitated material as determined by real-time PCR (see materials and methods for details). The bar graphs represent the average of 3 independent experiments and the error bars span two standard deviations. d) Average amounts of immunoprecipitated “H3 coding” fragment (minus the average for the respective “mock” reaction) expressed as fractions of the wild type.  132  3.2.3. Association of HP1 with the HIS-C In heterochromatin, H3K9me is thought to represent a binding platform for HP1 (Lachner et al., 2001; Jacobs et al., 2002; Bannister et al., 2001; Nielsen et al., 2002). In addition, SU(VAR)3-9 is known to interact with HP1 physically and genetically (Schotta et al., 2002; 2003; our lab, unpublished data). Thus, not surprisingly, most models postulate an interaction among SU(VAR)3-9, HP1 and H3K9me as a central step in the formation of heterochromatin (Nakayama et al., 2001; Schotta et al., 2002; 2003; Ebert et al., 2006; Rudolph et al., 2007), and SU(VAR)3-9 and HP1 have been suggested to work together in the regulation of a subset of genes (Nielsen et al., 2001; Greil et al., 2003). Since HP1 has been detected at the HIS-C (Greil et al., 2003; Koryakov et al., 2006; chapter 2 of this work), we hypothesized that it may play a role in regulating the expression of the histone genes, as part of a SU(VAR)3-9-dependent mechanism. To test this hypothesis, we performed another set of ChIP analyses, in this case, with an anti-HP1 antibody (α−HP1 (Ner et al., in preparation)). The material immunoprecipitated with α−HP1 from wild-type embryo extracts contained significant, although not copious amounts, of his unit fragments (Figure 3.4 and appendix 4). This enrichment for his unit fragments was significantly higher than that obtained with pre-immune serum (Supplementary Figure 3.1 and Appendix 1), allowing us to conclude that HP1 is associated with the HIS-C in wild-type strains. In contrast, the material immunoprecipitated with α−HP1 from Su(var)3-9 mutant extracts displayed very low levels of enrichment for all HIS-C fragments analyzed (Figure 3.4.b). For the "H3 coding" and "H2A coding"  133  fragments, relative quantifications were performed by real-time PCR, and the Su(var)3-9 mutants showed a ~3 to 19-fold, and ~4 to 13-fold reduction in the enrichment for HP1, respectively, relative to the wild-type. We conclude that overall, in Su(var)3-9 mutants, the levels of HP1 associated with the HIS-C are significantly lower than in wild-type individuals.  134  histone H3  histone H1  BglII  a.  histone H2A  histone H4  H2A coding  H3 coding  H1/H3 intergenic  histone H2B BglII  H2A/H2B intergenic  b. H3 coding IN IP NO (1:100)  Ab  (HP1)  H2A/H2B intergenic IN IP NO (1:100)  Ab  H1/H3 intergenic IN NO IP  (HP1)  (1:100) Ab  wild-type Su(var)3-9 Su(var)3-9  wild-type  06  Su(var)3-906  309  Su(var)3-9309  Su(var)3-9330  Su(var)3-9330  Su(var)3-9318 1  2  3  4  5  Su(var)3-9318  6  7  c.  8  9  d.  0.3  Relative amounts of “H2A coding” fragment in the material immunoprecipitated from 12-16 hrs old embryos.  0.3  IP (HP1) mock (no Ab)  % of the input  % of the input  (HP1)  0.2  0.1  Relative amounts of “H3 coding” fragment in the material immunoprecipitated from 12-16 hrs old embryos. IP (HP1) mock (no Ab)  0.2  0.1  0  0  WT  06  309  330  318  WT  Figure 3.4  135  06  309  330  318  Figure 3.4. Relative levels of HP1 associated with four regions of the histone unit in wild-type and Su(var)3-9 mutant embryos. a) Schematic of the BglII fragment defining the histone unit. The five histone genes (H1, H3, H4, H2A and H2B) and the four regions analysed (H3 coding, H2A/H2B intergenic, H2A coding and H1/H3 intergenic) are shown. b) Chromatin immunoprecipitation of wild-type and Su(var)3-9 mutant embryo extracts. The recovered DNA was PCR amplified using primers specific for the “H3 coding” region (lanes 1-3), the “H2A/H2B intergenic region” (lanes 4-6) or the “H1/H3 intergenic” region (lanes 7-9). The material used as template is indicated above each lane; IN (1:100): input material diluted 100X, NO Ab: mock (no antibody) reaction, IP (HP1): immunoprecipitated material. See material and methods for details about the antibody. c) and d) Relative amounts of “H2A coding” and “H3 coding" fragments, respectively, in the precipitated material as determined by real-time PCR (see materials and methods for details). The bar graphs represent the average of 3 independent experiments and the error bars span two standard deviations.  136  3.2.4. The null, as well as the missense Su(var)3-9 mutants have elevated levels of H2A and H3 transcripts It was previously reported that the levels of H1 and H4 transcripts are elevated in three Su(var)3-9 mutants, including the missense mutant Su(var)3-9330 (Ner et al., 2002). To determine whether this is a common feature of Su(var)3-9 mutants, and its relationship to the abundance of H3K9me2, SU(VAR)3-9 and HP1 associated with the HIS-C, respectively, we quantified the relative amounts of two histone transcripts present in wild-type and several Su(var)3-9 mutants. We used 12-16 hour old embryos as our source of RNA, for two reasons. Firstly, at this stage of embryogenesis a significant proportion of cells are still going through the cell cycle (reviewed by Lee and Orr-Weaver, 2003) and therefore synthesizing copious amounts of histone mRNAs. Thus, if SU(VAR)3-9 participates in the regulation of the histone genes, the effect of Su(var)3-9 mutations on the levels of histone should be more pronounced at this stage of development than in adults. Secondly, we wanted to be able to relate the alterations (or lack thereof) in the relative levels of histone mRNA to the results of the ChIP analyses, which were performed on 12-16 hour old embryo extracts. For each strain (Su(var)3-9+, the various Su(var)3-9 missense mutants and Su(var)3-906), the relative levels of two histone genes transcripts, H2A and H3, were quantified by real time RT-PCR using rp49 as an internal standard (see materials and methods). Within the histone unit, H2A and H2B are transcribed in opposite directions and they share a promoter region, and the same is true for H3 and H4 (Figure 3.1). H2A and H3 were chosen for our analysis as  137  representative of each of the two "gene pairs", as we assumed that the two members of each pair would be co-regulated. The relative amount of H3 transcript detected in all Su(var)3-9 mutants was significantly higher than in the Su(var)3-9+ strain (Figure 3.5.b and 3.5.d, and Appendix 5). Su(var)3-906, Su(var)3-9330, and Su(var)3-9318 showed, on average a 6.46, 6.07 and 4.72 fold increase over the wild-type (n=3). For Su(var)3-9309 the increase was less substantial (1.56 fold relative to the wild-type), but still statistically significant (p=0.05). For Su(var)3-9309 and Su(var)3-9318, the relative increase in H2A was 1.86 and 5.60, respectively, which is comparable (not statistically different at p=0.05) to that observed for H3. In Su(var)3-906, the increase in H2A was only about 70% of that observed for H3 (see Appendix 6), but still significantly higher than what was observed both in the wild-type and in Su(var)3-9309 (Figure 3.5. and Appendix 6). Su(var)3-9330 was an exception in that the relative increase (over the wild-type) in its level of H2A transcript was much lower (<30%) than that observed for its H3 transcript. In fact, the relative abundance of H2A detected in Su(var)3-9330 was so low as not to be statistically different from that observed in the wild-type (Appendix 5). Despite the exceptional case of the H2A transcript in Su(var)3-9330, we conclude that elevated levels of histone transcripts are probably a common feature of those Su(var)3-9 mutations that suppress PEV, although the magnitude of the increase seems to be, at least in part, allele-dependent (see section 3.2.5 and discussion). In addition, the data obtained with Su(var)3-906, and especially with  138  Su(var)3-9330, suggest that the regulation of the H2A/H2B and the H3/H4 gene pairs may be uncoupled.  139  140  c.  2  4  5  6  7  8  9  Figure 3.5  309 330  318  2  3  4  5  6  7  13  -  +  309 - +  14 15 16  06  17  -  18  330 +  WT  06  *  309  *  330  *  318  *  Relative amounts of total H3 transcript in 12-16 hr old embryos (normalized to rp49)  11 12  WT - +  * Statistically different from wild type (at p=0.05)  0  06  d.  0  *  10  b. RTase  1 WT  *  *  Relative amounts of total H2A transcript in 12-16 hr old embryos (normalized to rp49)  3  318 - +  1  2  3  4  5  6  1  330 - + RP49  +  309 - +  RP49  -  06 H3 (1:10)  WT - +  H2A (1:10)  RTase  Ratio to wild-type  a.  Ratio to wild-type  318 +  19 20  -  Figure 3.5. Relative quantifications of total histone H3 and histone H2A transcript in wild-type and Su(var)3-9 mutant embryos. a) and b) End point RT-PCR reactions on total RNA extracted from 12-16 hours old wild-type (lanes 1,2, 11 and 12) or Su(var)3-9 mutant embryos (lanes 3-10 and 13-20). The transcripts amplified were H2A and H3, respectively, as well as RP49 as an internal standard. In each case, the allele number is indicated above the corresponding lanes. "-" signs denote mock reactions (no reverse transcriptase). c) and d) Relative quantifications of H2A and H3 transcripts by real-time PCR. For each reaction, the ratio of H2A or H3 between mutants and wild-type (standardized for RP49) was reported. The histograms represent the average of three independent reactions and the error bars span two standard deviations. See material and methods for additional details.  141  3.2.5. Histone genes expression in mutant Su(var)3-9330 Since the four core histones are required in equal amounts, and SU(VAR)3-9 is associated with both the H2A/H2B and the H3/H4 gene pairs, one would expect each Su(var)3-9 mutation to affect the expression of the core histone genes to the same degree. This was indeed the case for Su(var)3-9309 and Su(var)3-9318, but it was not the case in the Su(var)3-906 and Su(var)3-9330 strains. In the Su(var)3-906, the "protein null" strain, there was, on average, a 4.65 and 6.46 fold increase in H2A and H3, respectively. Statistically (p=0.05), the difference between these two values is only marginally significant. The difference between the increase in H2A and H3 was much more pronounced in the Su(var)3-9330 strain (1.76 fold and 6.07 fold, respectively). Since in Su(var)3-9330 there is such a dramatic difference in the increase of H2A versus H3 mRNAs, we decided to focus on this strain, and to ask whether the regulation of the histone genes can be misregulated in such a way that the expression of each of the four core histones, or each of the two usually co-regulated pairs, H2A/H2B and H3/H4 is decoupled. To examine this possibility, we measured the total H2B transcript present in embryo extracts from wild-type and Su(var)3-9330. We found that, as it is the case for H2A, the relative levels of H2B detected in Su(var)3-9330 embryo extracts are, on average, slightly less than 2 fold higher than in the wild-type (Figure 3.6). Similarly to what was observed for H2A, such difference is not statistically significant (at p=0.05), although it is probably significant biologically. We conclude that somehow, the amino acid substitution present in SU(VAR)3-9330 affects the relative abundance of H3 transcript, but not  142  that of H2A or H2B. This suggests that the H2A/H2B and the H3/H4 gene pairs may be independently regulated. To examine this further, we next asked whether, in Su(var)3-9330, the intergenic regions between the H3/H4 and H2A/H2B pairs show any differences in the level of H3K9me2 versus the wild-type. If H3K9me2 is responsible for the regulation of the histone genes, we would expect to see a substantial difference between the wild-type and Su(var)3-9330, with respect to such modification, within the H3/H4 region, and a less dramatic difference within the H2A/H2B region. A ChIP analysis was performed, and relative quantifications of the fragments of interest were carried out by real-time PCR. The results showed that the material immunoprecipitated from both the wild-type and the mutant extracts is enriched for "H3/H4 intergenic" and "H2A/H2B intergenic" fragments (Figure 3.7). However, as for all other regions tested, this enrichment is significantly higher in the wild-type than in Su(var)3-9330 (Figure 3.7. and Appendix 3). Intriguingly, it was the "H2A/H2B intergenic" region that showed the most substantial difference between Su(var)3-9330 and the wild-type (Figure 3.7. and Appendix 3), suggesting that there is no direct correlation between the levels of H2A, H2B and H3 transcripts and the amount of H3K9me2 associated with the genes’ regulatory regions.  143  a.  b. wild-type +  Su(var)3-9 +  H2B rp49 1  2  3  4  Ratio to wild-type  RTase  Relative amounts of total H2B transcript (standard=rp49)  330  2.5 2  1  0 WT  Figure 3.6  144  330  Figure 3.6. Comparison of H2B transcript present in wild-type vs. Su(var)39330 mutant embryos. a) and b) Relative abundance of total H2B transcript in 12-16 hours embryos, quantified by real-time RT-PCR following the same procedure as in Figure 3.5. "-" signs denote mock reactions (no reverse transcriptase).  145  H3/H4 intergenic  H2A/H2B intergenic a. WT  WT  330  330 5  6  7  8  Relative amounts of H2A/H2B intergenic” fragment in the material IPed from 12-16 hrs old embryos. 1.6 IP (H3K9me2)  % of the input  b.  IN (1:100)  NO IP Ab (anti-K9me2)  1.2  Mock (No Ab)  0.8 0.4 0  WT  9  Figure 3.7  146  10  1.2 0.8 0.4 0  330  NO IP (anti-K9me2) Ab  Relative amounts of H3/H4 intergenic” fragment in the material IPed from 12-16 hrs old embryos. 1.6  % of the input  IN (1:100)  WT  330  Figure 3.7. Relative levels of H3K9me2 associated with two intergenic regions of the histone unit in wild type and Su(var)3-9330 mutant embryos. a) Detection (end point PCR) and b) quantification (real-time PCR) of the "H2A/H2B intergenic" and "H3/H4 intergenic" fragments in the material immunoprecipitated with anti-H3K9me2 from wild-type and Su(var)3-9330 crosslinked embryonic extracts. In all cases the histograms represent the average of three independent experiments and the error bars span two standard deviations. See material and methods for additional details.  147  3.2.6. The elevated levels of histone transcripts in Su(var)3-9 mutants are not due to an increase in polyadenylated transcripts. The observed overproduction of histone H2A and histone H3 transcripts is an interesting phenotype, and its mechanism is unknown. There are three simple possibilities. First, in the Su(var)3-9 mutant strains there may be an increase in the number of histone templates transcribed at any point in time with respect to the wild-type, while the transcription rate remains constant. Second, Su(var)3-9 may result in an increase in the transcription rate of the histone genes, while the number of his templates that are transcribed within the HIS-C region remains constant. Finally, it is possible that neither the number of transcribed templates, nor the transcription rate are affected, but, in Su(var)3-9 mutants, expression of the histone genes may be uncoupled from the cell cycle (i.e. the histone genes may be transcribed outside, as well as during, S-phase). The latter hypothesis is easily tested. Histone transcripts synthesized during Sphase are not polyadenylated, while those synthesized outside S-phase are (Akhmanova et al., 1997). Therefore, histone transcripts produced outside Sphase (decoupled from DNA synthesis) can easily be detected and quantified using an appropriate primer (oligodT) for the reverse-transcription (RT) step. Taking this approach, we first measured the amount of polyadenylated H2A and H3 mRNAs in Su(var)3-9+/ Su(var)3-9+ embryos, in order to determine what proportion of the total histone transcripts are polyadenylated RNA. We found that the relative ratio of polyadenylated/total H2A and H3 was roughly 1/1000 (Figures 3.8.a and 3.8.b).  148  Hence, if the increase in the level of total H2A and H3 transcripts observed in the mutants is due, even only in part, to transcription outside S-phase, the Su(var)3-9 mutants should show a very prominent increase in the amount of polyadenylated H2A and H3 transcripts compared to the wild-type. This is not what we observed. Instead, we detected a small reduction in the levels of polyadenylated H2A and H3 in Su(var)3-906, Su(var)3-9309 and Su(var)3-9318, while Su(var)3-9330 showed a small reduction in H3 and an increase in H2A (Figures 3.8.c and 3.8.d). The differences in the levels of polyadenylated H2A and H3 between the wild-type and each of the Su(var)3-9 mutants are statistically significant. However, since the polyadenylated histone RNAs represent less than 1% of the total, these differences certainly do not account for the increase in the levels of total histone transcripts. Thus, we conclude that increased expression outside S-phase is not the mechanism responsible for the observed elevation in total histone transcripts.  149  Ratios of H2A/RP49 transcripts in 12-16 hr old wild-type embryos  b.  0.07  70  total +  polyA +  1  3  RTase  H2A/RP ratio  a.  H2A  1:10  RP49 2  4  50  0.05  30  0.03  10  0.01  0  d.  c.  -  WT  +  06 -  +  309  -  +  330 -  +  318  -  H2A H3 RP49 (1:20)  1  2  3  4  5  6  7  8  9  3  +  10  Ratio to wild-type  RTase  150  polyA  Relative amounts of polyadenylated H2A transcripts in 12-16 hr old embryos (normalized to RP49)  2  1  0  Figure 3.8.  0  total  WT  06  309  330  318  Figure 3.8. Relative quantifications of polyadenylated (polyA) histone H3 and histone H2A transcript in wild-type and Su(var)3-9 mutant embryos. a) Comparison between the relative amount of total and polyA H2A transcript in wild-type 12-16 hours old embryos. In lanes 1 and 2 the RNA was reversetranscribed with primers H2AR and RP49rt, and in lanes 3 and 4 with an oligodT primer. In both cases the reverse-transcribed material was then PCR amplified (separately) with primer pairs H2AFc/H2AR and RP495/RP493. Lanes 1 and 3 represent "mock" (no RTase) reactions. For the total H2A transcript sample, only 1/10 of the amplified product was loaded on the gel. b) Ratios of total H2A/total RP49, and polyadenylated H2A/polyadenylated RP49 transcripts, respectively, as determined by real-time PCR. The histograms represent the average of three independent experiments and the error bars span two standard deviations. c) RT-PCRs on total RNA extracted from 12-16 hours old wild-type and Su(var)3-9 mutant embryos. The extracts were reverse-transcribed with an oligodT primer and the material obtained amplified with primer pairs H2AFc/H2AR, H3F/H3R and RP495/RP493, respectively. "Mock" (no RTase) reactions are indicated with a "-" sign above the corresponding lanes. d) Relative quantifications of polyadenylated H2A and H3 transcripts by real-time PCR. For each reaction, the ratio of H2A or H3 between mutants and wild-type (standardized for RP49) was reported. The histograms represent the average of three independent experiments and the error bars span two standard deviations.  151  3.3 Discussion 3.3.1. Association of SU(VAR)3-9 with the HIS-C in 3-9 mutants The ChIP data demonstrate that SU(VAR)3-9 is present at the HIS-C in wild-type and in Su(var)3-9309, Su(var)3-9330 and Su(var)3-9318 embryos. This confirms previous observations (Ner et al., 2002; Koryakov et al., 2006) and suggests that in all three cases, single amino acid substitutions in the catalytic region of SU(VAR)3-9 do not substantially affect the protein’s ability to be recruited to, or associate with, the HIS-C. Since the three missense mutants tested carry distinct amino acid substitutions, and all of them are still targeted to, and associated with the HIS-C, we propose that their 3-dimensional structure, as well as their ability to interact with the other chromatin components, are mainly unaffected. If this were not the case, and one or more of the mutant gene products were misfolded and/or unable to properly interact with the customary partners of SU(VAR)3-9, we would expect them to fail to associate with their targets. The fact that no SU(VAR)3-9 was detected in Su(var)3-906 further validates this hypothesis. All Su(var)3-9 mutants tested, regardless of whether they were "protein nulls" (Su(var)3-906) or missense alleles (Su(var)3-9309, Su(var)3-9330 and Su(var)39318) display elevated levels of histone transcripts, suggesting that the physical presence of SU(VAR)3-9 at the HIS-C is not sufficient for normal regulation of the histone genes. It is certainly possible, at least in theory, that SU(VAR)3-9 has an essential structural role at the HIS-C, and that each one of the single amino acid substitutions present in the mutants analyzed impairs this function. We do not favour this possibility because, as discussed above, the mutant proteins still  152  associate with their target (although in some cases less efficiently), which suggests that their ability to interact with other chromatin proteins does not differ significantly from that of the wild-type SU(VAR)3-9. Although significant, the enrichment for HIS-C fragment detected in the material immunoprecipitated with α-SU(VAR)3-9chr was very weak, which may reflect the fact that the association of SU(VAR)3-9 with this locus is very dynamic, and the amount of protein physically associated with the HIS-C chromatin at any given time is very small. This would fit with the observation that SUV39H1, the human homolog of SU(VAR)3-9, can be found stably associated with heterochromatin, but not with euchromatin (Krouwels et al., 2005). It would also argue in favour of the hypothesis that SU(VAR)3-9 does not play a major structural role at the HISC. Alternatively, it is possible that the antibody was not very efficient in its recognition of SU(VAR)3-9 (possibly because the epitope against which it was raised is partially hidden by other chromatin proteins interacting with SU(VAR)39).  3.3.2. Reduced levels of H3K9me2 in Su(var)3-9 mutants Using ChIP analyses we were able to demonstrate conclusively that 1) at least three segments of the histone unit are enriched for H3K9me2, and 2) this enrichment is noticeably reduced in all four homozygous Su(var)3-9 mutants tested (and, for at least one fragment, "H3 coding" the reduction is as high as ~7 to 25-fold). The product of Su(var)3-9309 (SU(VAR)3-9309) is catalytically inactive in vitro, and those of Su(var)3-9330 (SU(VAR)3-9330) and Su(var)3-9318  153  (SU(VAR)3-9318) have dramatically reduced enzymatic activity (chapter 2). Nevertheless, all strains, including Su(var)3-906, display a statistically significant enrichment for H3K9me2 within at least one region of the his unit ("H3 coding", see also Appendix 3). Although weak cross-reactivity of the antibody with H3K9me1 or H3K27me2 cannot be completely excluded, we favour the hypothesis that the residual signal observed in Su(var)3-906 is due to the presence of other methyltransferases (MTases) that are able to methylate H3K9 at euchromatic loci, such as dG9a and DmSetDB1 (Mis et al., 2006; Stabell et al., 2006; Seum et al., 2007; Tzeng et al., 2007). This hypothesis could be tested by analysing cross-linked extracts from homozygous dG9a-; Su(var)3-906 and DmSetdb1-; Su(var)3-906 mutant embryos, respectively, and asking whether their level of H3K9me2 associated with the HIS-C is lower than that observed in their Su(var)3-906 counterparts. Unfortunately, this approach may not be simple. In the case of dSetdb1, homozygous dSetdb1- mutants survive until third instar larval stage (Seum et al., 2007), so dSetdb1-; Su(var)3-906 homozygous embryos could in principle be produced, although the viability of the double mutant strain is not known. In addition, it is likely that traces of H3K9me2, generated by the dG9a protein, would still be present in homozygous dSetdb-; Su(var)3-906 embryos . Since the levels of H3K9me2 associated with the HIS-C in Su(var)3-906 embryos are also very low, a comparison between dG9a-; Su(var)3-906 and Su(var)3-906 would likely involve working with trace amounts of material and trying to detect a very small difference, which would require extremely accurate and reliable quantification methods, as well as exceedingly specific antibodies. In a way, the  154  task could be simpler for dG9a, since the dG9aRG5 mutant line survives, as a homozygote, in combination with Su(var)3-906 (Seum et al., 2007). However, this particular dG9a mutant line does not have any particular phenotype and fails to show reduced levels of H3K9me or H3K9me27 at larval stages, so there is no guarantee that a difference in the levels of such modifications could be detected in homozygous dG9aRG5; Su(var)3-906 vs. dG9a+;Su(var)3-906 embryos. All Su(var)3-9 missense mutants tested showed a significant reduction in the levels of H3K9me2 associated with the HIS-C. In principle, this could be due to either a reduction in the catalytic function of the mutant SU(VAR)3-9 products, or a reduction in the amount of SU(VAR)3-9 protein present at the HIS-C. Since we have shown that the level of (mutant) SU(VAR)3-9 associated with the HIS-C in the Su(var)3-9 missense mutants are comparable to those observed in wild-type embryos (Figure 3.2), we conclude that the reduced levels of H3K9me2 are due to the altered enzymatic activity of the mutant products (SU(VAR)3-9309, SU(VAR)3-9330 and SU(VAR)3-9318, respectively). This was expected, as the catalytic activity of these mutant proteins is significantly impaired, at least in vitro (chapter 2). Curiously, although SU(VAR)3-9309, SU(VAR)3-9330 and SU(VAR)3-9318 differ in the strength of their catalytic phenotypes based on in vitro tests (chapter 2), we detected no significant differences in the levels of H3K9me2 that is associated with the HIS-C among the corresponding mutants. There are a number of possible reasons for this apparent discrepancy. The simplest explanation is that the conditions employed for the HMTase assay reported in chapter 2 do not  155  wholly simulate the nuclear environment in which SU(VAR)3-9 normally functions. For instance, in the in vitro assay the reactions were allowed to proceed uninterrupted for several hours, which is probably not the case in an in vivo context. More importantly, the in vitro reactions were carried out on free histones, in the absence of all the NHCPs and additional factors normally present in a cell nucleus, and the results obtained may not be representative of the enzyme’s activity on a chromatin template in an in vivo context. The dramatically reduced levels of HIS-C-associated H3K9me2 observed in the Su(var)3-9 mutants correlate with an overall upregulation of the histone genes, suggesting that dimethylation of H3K9 at the HIS-C is necessary to maintain normal levels of histone transcripts in the cell. However, this conclusion may be too simplistic since we don’t know what other functions SU(VAR)3-9 may have, and, if applicable, whether these other functions are affected in the Su(var)3-9 missense mutants. It has been suggested that the SET domain of SUV39H1, the human homolog of SU(VAR)3-9, may play an important structural role in the stable association of the protein with chromatin (Krouwels et al., 2005). We know that the residues that are affected in the Su(var)3-9 mutants tested here are not necessary for this process, since the association of these mutant forms of SU(VAR)3-9 with the HIS-C is not significantly affected (Figure 3.2). However, as discussed above, we can’t exclude the possibility that such residues are required for proper interaction with other chromatin components and/or regulators of the histone genes, and that the elevated levels of histone transcripts are partially due to the inability of the mutant SU(VAR)3-9 proteins to carry out such interactions.  156  3.3.3. Recruitment and role of HP1 at the HIS-C The ChIP results obtained show that HP1 is present at the HIS-C in wild-type embryos, at least within the three regions of the histone unit that we analyzed (Figure 3.4). This is in agreement with previous reports (Greil et al., 2003; Koryakov et al., 2006). We also detected HP1 association with the histone units in the Su(var)3-9 mutants, but its levels were drastically reduced in all mutants (Figure 3.4). We did not see a correlation between the magnitude of the reduction in the levels of H3K9me2 and the relative amount of HP1 associated with the different regions of the histone unit. This suggests that the presence of H3K9me2 is necessary for stable binding of HP1, but other factors, such as auxiliary proteins, are probably involved in the process, and these may influence the efficiency of HP1 binding to this chromatin domain. Interestingly, the amount of HP1 associated with the histone units in the Su(var)3-9 missense mutants was not significantly different from that found at the same locations in the "protein null" Su(var)3-906. Thus the presence of a SU(VAR)3-9 protein with a single amino acid substitution in its catalytic region is not sufficient to recruit and stabilize the association of HP1 with the HIS-C. There are at least two possible explanations for this. The first one is that each of the SU(VAR)3-9 residues that are mutated in the missense alleles is critical for the binding of HP1. The second one is that the physical presence of SU(VAR)3-9, whether wild-type or mutant, is not sufficient to recruit and/or to stabilize HP1 at the HIS-C.  157  Although a priori it is difficult to decide which hypothesis is most plausible, we tend to favour the second one, for the following reasons. Firstly, it has been shown that, at least in vitro, the presence of auxiliary factors is required for stable binding of HP1 to a chromatin template, even in the presence of H3K9me2,3 (Eskeland et al., 2007). Secondly, we showed that all the mutant SU(VAR)3-9 proteins, SU(VAR)3-9309, SU(VAR)3-9330 and SU(VAR)3-9318, localize at the HISC like their wild-type counterpart (Figure 3.2), suggesting that in vivo their structure and folding are relatively unaffected. Thus, it is likely that their ability to interact with other chromatin proteins does not differ significantly from that of wild-type SU(VAR)3-9. At this point, we propose that the most essential factor in the recruitment of HP1 at the HIS-C is the presence of a certain level of H3K9me2, and that this histone modification, in combination with the presence of NHCPs other than SU(VAR)39, are responsible for the stable association of HP1 with chromatin at this locus. Curiously, the relative enrichment for HP1 in Su(var)3-9330 is significantly lower than that observed in Su(var)3-906, suggesting that Su(var)3-9330 may be acting as an antimorph, effectively hindering the stable association of HP1 with the HISC. In chapter 2 we showed that, in vitro, the recombinant SU(VAR)3-9330 protein partially retains the ability to methylate histone H3, but can’t methylate a peptide representing the histone H3 tail alone. One of the hypotheses proposed to explain this result was that the amino acid substitution present in SU(VAR)3-9330 (D536N) might result in a change in specificity, causing SU(VAR)3-9330 to methylate a histone H3 residue other than K9. Several studies have shown that  158  chromodomains can show very high specificity with respect to modified histone residues; for example, the chromodomain of HP1 specifically binds H3K9me2,3, but not H3K27me, while the opposite is true for the chromodomain of POLYCOMB (Bannister et al., 2001; Nakayama et al., 2001; Muller et al., 2002; Jacobs and Khorasanizadeh, 2002; Nielsen et al., 2002; Cao et al., 2002; Min et al., 2003; Fischle et al., 2004; Pray-Grant et al., 2005; reviewed by Daniel et al., 2005). If the change in specificity hypothesized for SU(VAR)3-9330 occurs in vivo, then the methylation of this other H3 residue may create a binding platform that recruits or stabilizes the binding of a different chromodomain protein, and the binding of this inappropriate NHCP may preclude binding of HP1. Alternatively, it is conceivable that SU(VAR)3-9330 binds to histone H3, and may or may not methylate K9, but it remains tightly associated with the H3 tail, thus making it unavailable for the recruitment of HP1. We do not favour this possibility because, if SU(VAR)3-9330 remained tightly bound to the H3 tail, and thus to chromatin, we would expect to see higher levels of (mutant) SU(VAR)3-9 associated with the HIS-C in the ChIP experiments on Su(var)3-9330 extracts than in their wild-type counterparts. However, this is not the case (Figure 3.2).  3.3.4. Elevated levels of the histone transcripts in Su(var)3-9 mutants In a previous study, members of our lab (Ner et al., 2002) showed that the levels of histone H1 and H4 mRNA present in two Su(var)3-9 mutants (one missense allele, Su(var)3-9330, and one P element-induced allele, Su(var)3-9P25) is about two fold higher than in wild-type individuals. We confirmed and expanded this  159  observation. Our results show that the level of both the H2A and the H3 histone transcripts are elevated in three Su(var)3-9 missense mutants and the complete null allele Su(var)3-906. We find the difference between the mutant and wild-type strains to range between ~1.8 and 6.5-fold. The apparent discrepancies between these results and the data obtained in the 2002 study are likely due, at least in part, to differences in the experimental setup. In the former study the relative abundance of each histone transcript was quantified by northern blot analyses. In contrast, this study employed realtime/RT-PCR, a more accurate and reliable quantification system. Moreover, our mRNA samples are derived from staged embryos at a time when many of the cells are still undergoing mitosis (reviewed by Lee and Orr-Weaver, 2003). Since histone genes are expressed almost exclusively during S-phase, their misregulation would be more noticeable at this stage than in adult flies, which were used for the previous study (Ner et al., 2002).  In the present study, we were able to detect differences in the relative amounts of histone h2a and h3 transcripts between some of the mutants. In particular, Su(var)3-906 and Su(var)3-9318 have significantly higher levels of h2a and h3 than Su(var)3-9309. Since Su(var)3-906 is a "protein null" (no RNA or protein product detected, see chapter 2) we suggest that Su(var)3-9318 acts as an amorph (at least functionally) and Su(var)3-9309 behaves as a hypomorph, with respect to the regulation of h2a and h3 gene expression.  160  Once again, the Su(var)3-9330 mutant strain has a very curious phenotype. It shows dramatically elevated levels of h3 (~6 fold over the wild-type), but only a ~1.8 to 1.9 fold increase in the h2a and h2b transcripts. Thus, the Su(var)3-9330 mutation is unique in that, unlike the other alleles studied, it seems to decouple the h2a/h2b from the h3 expression levels. This mutant is therefore difficult to categorize, as it would be classified as a loss-of-function mutant (same phenotype as Su(var)3-906 and Su(var)3-9318) based on its h3 expression level, but not with respect to h2a and h2b.  3.3.5. Possible mechanism for the functions of SU(VAR)3-9 at the HIS-C In general, the results obtained in this study are consistent with the notion that the function of SU(VAR)3-9 at the HIS-C, a euchromatic locus, is very similar to its function in (pericentric) heterochromatin. In both cases SU(VAR)3-9 is required for downregulation or silencing, and its ability to methylate H3K9 is required to allow stable association of HP1 with chromatin. Our results suggest that the physical presence of SU(VAR)3-9 probably does not play a major role in the regulation of the histone transcripts levels. Su(var)3-9318, a mutant that produces a SU(VAR)3-9 protein with a single amino acid substitution, and which localizes at the HIS-C like its wild-type counterpart, shows the same h2a and h3 hyperexpression phenotype as Su(var)3-906, a mutant that does not produce any SU(VAR)3-9 protein at all (no statistical difference in a t test at p=0.05). In addition, the missense mutant Su(var)3-9330 also displays the same hyperexpression phenotype as Su(var)3-906 with respect  161  to the histone h3 gene. If the physical presence of SU(VAR)3-9 played a major role in the regulation of the histone genes expression, one would expect most missense mutants to have a milder overexpression phenotype than the "protein null" Su(var)3-906. All data presented point to the crucial role of SU(VAR)3-9’s enzymatic activity in the regulation of the levels of histone transcripts; all mutants have an impaired HMTase activity (chapter 2), all of them display a drastic reduction in the abundance of H3K9me2 associated with the HIS-C, and all of them show elevated levels of h2a and h3 transcripts. However, the relative levels of H3K9me2 associated with the various regions of the histone units are virtually indistinguishable in all Su(var)3-9 mutants, while the abundance of h3 and h2a transcripts may differ. The simplest explanation for this apparent disparity is that differences in the levels of H3K9me2 do exist among the mutants, but the resolution of the ChIP technique is not sensitive enough to detect them. It is also possible that the mutants do not differ significantly for H3K9me2, but they do with respect to another NHCP, or another histone modification, which could have a role in the fine-tuning of the regulation of histone genes expression. Interestingly, the levels of HP1 associated with the "H3 coding" region in the Su(var)3-906, Su(var)3-9318 and Su(var)3-9309 mutants correlates with their respective h3 transcript levels. For instance, the first two mutants show similar levels of HP1 associated with this region of the histone unit, and a similar increase in h3 transcript. The Su(var)3-9309 has a higher level of HP1 associated with the "H3 coding" region and a lower increase in its h3 transcript, which is  162  consistent with the view that the presence of HP1 is associated with silencing. As usual, Su(var)3-9330 represents an exception. In this case, its levels of "H3 coding"-associated HP1 are significantly lower than those observed in the other mutants, yet the increase in its h3 transcript level is similar to that of Su(var)3-906 and of Su(var)3-9318. In contrast, we did not detect a correlation between the level of HP1 associated with the "H2A coding" region and the relative increase in h2a transcript produced in the various mutants. The levels of HP1 associated with the "H2A coding" region did not differ significantly in Su(var)3-906, Su(var)3-9318 and Su(var)3-9309, but Su(var)3-9309 has a much weaker h2a hyperexpression phenotype than the other two strains. We have demonstrated that reduced levels of H3K9me2 and HP1 at the HIS-C correlate with an increase in the abundance of h2a and h3 transcripts produced. We propose that there is a causal relationship between the two observations, and that the presence of appropriate amounts of H3K9me2 and HP1 at the HIS locus is absolutely necessary for the production of wild-type levels of histone transcripts. Our data also suggest that high levels of H3K9me2 and/or of HP1 may not be sufficient, by themselves, to ensure a normal regulation of the histone genes expression. We propose that additional factors are involved in the process of fine-tuning the production of histone transcripts. In order to gain a thorough understanding of the mechanisms involved in the regulation of the HIS-C at the chromatin level, it will be necessary to identify the other factors associated with this locus, their respective functions and how they  163  interact with one another. So far, we know that the gene products of at least three other suppressors of PEV (abo, Bonus and Su(var)326/HDAC1) localize to the HIS-C (Berloco et al., 2001; Beackstead et al., 2005; Ner et al., in preparation). Investigations of their respective roles and functions in the modulation of the histone gene expression will likely help our understanding of this complex system.  3.3.6. Regulation of the histone gene expression Our data show that the amounts of h2a and h3 transcripts are higher in Su(var)39 mutants than in Su(var)3-9+ embryos and, as discussed previously, we think that this is most likely the result of overexpression of the histone genes. We have shown that the over production of histone transcripts is not due to decoupling of histone genes expression from the cell cycle (i.e. synthesis of histones outside S phase). Thus, we are left with two possibilities for the role of SU(VAR)3-9 in the regulation of the histone genes expression. In the first scenario, SU(VAR)3-9 determines the number of histone templates that are actively transcribed in a nucleus. In this model, mutations in SU(VAR)3-9 would cause an increase in the number of transcribed templates without affecting transcription rates. In a wild-type situation the HMTase activity of SU(VAR)3-9 may be mainly responsible for keeping a certain number of histone units inaccessible to transcription factors. In Su(var)3-9 mutant strains more units may be accessible and thus more histone transcripts are produced during S-phase while the transcription rate/template remains unchanged. This hypothesis is  164  supported by the fact that the chromatin structure of the HIS-C, as measured by the pattern of DNaseI and MNase hypersensitive sites, is altered in at least a subset of Su(var)3-9 mutants (Ner et al., 2002). The alternative model is that SU(VAR)3-9 modulates the rate of transcription of each histone template or unit. In this scenario, the same number of histone templates would be transcribed in wild-type and Su(var)3-9 mutants, but the mutants would have higher transcription rates. Of course, these two hypotheses are not mutually exclusive, and SU(VAR)3-9 could have several functions regulating template accessibility as well as modulating the transcription rate of each histone gene. Finally, it should be noted that the peculiar phenotype of the Su(var)3-9330 mutant, which shows a drastic increase in its levels of h3, but only a moderate elevation in h2a and h2b transcripts, suggests that the h2a/h2b and h3/h4 gene pairs may be regulated somewhat independently. In a wild-type situation, the regulation of these two gene pairs is usually co-ordinated. In two Su(var)3-9 mutants (Su(var)3-9309 and Su(var)3-9318) this co-ordinated regulation appears to be maintained, as their increase in h2a transcript is virtually identical to their increase in h3 transcript. Su(var)3-906 shows a slightly higher increase in h3 than h2a, and for Su(var)3-9330 this decoupling of h2a/h2b couplet from h3 is very dramatic. We do not have an explanation for these results, but it is possible that SU(VAR)3-9 is involved, directly or indirectly in the co-regulation of the h2a/h2b and h3/h4 pairs.  165  3.4.  Materials and methods  3.4.1 Drosophila strains All Drosophila strains used in this study are described in chapter 2, and were grown under standard conditions.  3.4.2. ChIP analysis ChIP analysis of the HIS-C was performed as described in chapter 2. The antibody to H3K9me2 was purchased from Upstate (#07-441) and the antiSU(VAR)3-9 antibody is described in chapter 2 and by Ner et al. (2002). The antibody to HP1 is a polyclonal and was raised in our lab (Ner et al., in preparation). The primers pairs used for amplification were H3F/H3R ("H3 coding" region), iH2AF/iH2BR ("H2A/H2B intergenic" region), H2AFc/H2BR ("H2A coding" region), iH3F/iH4R ("H3/H4 intergenic" region) and iH1Bf/iH3r ("H1/H3 intergenic" region) (see supplementary table 3.1 and Ner et al., 2002). The relative amounts of target DNA present in the immunoprecipitated material was quantified by real-time PCR. A dilution series (1:10-1:2000) of the input DNA was used to generate a standard curve, which was then used to estimate the amount of the fragment of interest present in the immunoprecipitated material (or in the mock reaction). In each case, the material pulled down in 3 independent experiments was individually analyzed for its enrichment in target (HIS-C) sequences, expressed as a % of the input for each IP (same for the mock IPs). The results obtained with each mutant strain were compared to those relative to the wild-type strain using Student’s T test. Differences were considered  166  significant if the absolute t value was above 2.78 (critical value for p=0.05, 4 df, in a two-tailed T test). The same test was also employed to determine whether, in each strain, the enrichment observed was above the background.  3.4.3. Quantifications of histone mRNAs Total RNA was isolated from 12-16 hours old embryos by TRIzol extraction as recommended by the manufacturer. Three independent extracts were prepared from each strain. For each extract, reverse-transcription reactions were performed as described in chapter 2. To reverse-transcribe total histone H2A, H3 and H2B mRNA, primers H2AR, H3R and dH2BR were used, each one in combination with RP49rt (internal control). A 17 nucleotide oligodT primer was employed to reverse-transcribe polyadenylated mRNAs. The relative amounts of histone cDNAs obtained from each RT reaction were quantified by real-time PCR amplification. For each sample, three reactions were run and, since the resulting Ct values were almost identical (<1% difference) among the three measurements, they were averaged, and referred to as "one measurement". The ratio between the histone and the rp49 measurements was calculated for three independent RNA extracts. In each case, the resulting value was then divided by the value obtained, by the same procedure, with the wild-type strain, giving rise to one "data point". For each strain, the results are presented as an average of three (independent) "data points" +/- the standard deviation. Differences among strains were considered significant if they yielded a |t value| >2.78 (critical value for p=0.05, 4df) in a two-tailed Student’s t-test.  167  168  H2Ac  H3c  b.  % of the input  IN  1  (1:10)  2  NO Ab  3  IgG Ab  4  (H3K9me2)  antiH3K9me2  0.08  0.12  0.16  0.2  0.24  0  IgG  IN  5  (1:100)  6  NO Ab  Ab  7  (HP1)  IN  8  (1:100)  9  preimmune  antiHP1  Mock  IP  10  NO P.I. Ab (pre-αHP1)  Relative amounts of “H2Acoding” fragment immunoprecipitated from 12-16hr old wild-type embryos  0  d.  integenic  0.04  Mock  IP  H3c H2A/H2B  c.  2  4  6  8  10  Relative amounts of “H3coding” fragment immunoprecipited from 12-16hr old wild-type embryos  integenic  H2A/H2B  a.  Supplementary figure 3.1  % of the input  Supplementary figure 3.1. Control ChIP reactions from wild-type 12-16 hours old embryo extracts using commercial, non-specific IgG and preimmune serum, respectively. a) Representative gels showing the PCR products obtained using the primers that amplify the "H3 coding" (H3c), "H2A coding" (H2Ac) and "H2A/H2B intergenic" fragments, respectively. The template was a 1:10 dilution of the ChIP input DNA (lane 1), the DNA obtained from a mock IP (no antibody, lane 2), or the DNA IPed with commercial non-specific IgG (lane 3) or with the anti-H3K9me2 antibody (lane 4). b) Relative abundance of the "H3 coding" fragment in the material IPed with IgG and with anti-H3K9me2 determined by real-time PCR (see materials and methods for details). c) Representative gels showing the PCR products obtained using the primers that amplify the "H2A coding" (H2Ac) and "H2A/H2B intergenic" fragments, respectively. The template was a 1:100 dilution of the ChIP input DNA (lanes 5 and 8), the DNA obtained from a mock IP (no antibody, lanes 5 and 9), or the DNA IPed with the anti-HP1 antibody (lane 7) or with pre-immune serum (lane 10). d) Relative abundance of the "H2A coding" fragment in the material IPed with IgG and with anti-H3K9me2 determined by real-time PCR (see materials and methods for details).  169  Supplementary table 3.1. List of primers used in this study. Primer name H2AR H2AFc H3R H3F RP495 RP493 RP49rt dH2BR dH2BF iH2BR iH2AF iH1Bf iH3R iH3F iH4R  Primer sequence (5’3’) AACGTTTAGGCCTTCTTCT TCTGGACGTGAAAAAGGTGG TGCCGTGTCAGCTTAAGCA GCTCGTACCAAGCAAACTG GCCCAAGATCGTGAAGAAGC CTGTTGTCGATACCCTTGGG CGCGCTCGATAATCTCC GTCCGCATTCGCAGGAG CCTCCGAAAACTAGTGGA ATGGCATAGCTCTCCTTCC TTCCGGAGCAAACGGTGA TCCGCAACAAAATTAGCCAA AAGCGCTAGCGTACTCTATAA GCGTGGCGCCTTTCCACCAGTC CGCTTGGCGCCACCCTTT  170  3.5.  References  Akhmanova, A., Miedema, K., Kremer, H., Hennig, W.: Two types of polyadenylated mRNAs and synthesized from Drosophila replication-dependent histone genes. Eur. J. Biochem. 244, 294-300 (1997) Bannister, A.J., Zegerman, P., Partridge, J.F., Miska, E.A., Thomas, J.O., Allshire, R.C., Kouzarides, T.: Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120-4 (2001) Beckstead, R.B., Ner, S.S., Hales, K.G., Grigliatti, T.A., Baker, B.S., Bellen, H.J.: Bonus, a Drosophila TIF1 homolog, is a chromatin-associated protein that acts as a modifier of position-effect variegation. Genetics 169, 783-94 (2005) Berloco, M., Fanti, L., Breiling, A., Orlando, V., Pimpinelli, S.: The maternal effect gene, abnormal oocyte (abo), of Drosophila melanogaster encodes a specific negative regulator of histones. Proc Natl Acad Sci U S A. 98, 12126-31 (2001) Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H., Tempst, P., Jones, R.S., Zhang, Y.: Role of histone H3 lysine 27 methylation in Polycombgroup silencing. Science 298, 1039-43 (2002) Czermin, B., Schotta, G., Hulsmann, B.B., Brehm, A., Becker, P.B., Reuter, G., Imhof, A.: Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila. EMBO Rep. 2, 915-9 (2001) Daniel, J.A., Pray-Grant, M.G., Grant, P.A.: Effector proteins for methylated histones: an expanding family. Cell Cycle 4, 919-26 (2005) Danzer, J.R., Wallrath, L.L.: Mechanisms of HP1-mediated gene silencing in Drosophila. Development 131, 3571-80 (2004) de Wit, E., Greil, F., van Steensel, B.: High-resolution mapping reveals links of HP1 with active and inactive chromatin components. PLoS Genet. 3, e38 (2007) Delattre, M., Spierer, A,. Jaquet, Y., Spierer, P.: Increased expression of Drosophila Su(var)3-7 triggers Su(var)3-9-dependent heterochromatin formation. J Cell Sci. 117, 6239-47 (2004) Dominski, Z., Marzluff, W.F.: Formation of the 3' end of histone mRNA: getting closer to the end. Gene 396, 373-390 (2007) Ebert, A., Schotta, G., Lein, S., Kubicek, S., Krauss, V., Jenuwein, T., Reuter, G.: Su(var) genes regulate the balance between euchromatin and heterochromatin in  171  Drosophila. Genes Dev. 18, 2973-83 (2004) Ebert, A., Lein, S., Schotta, G., Reuter, G.: Histone modification and the control of heterochromatic gene silencing in Drosophila. Chromosome Res. 14, 377-92 (2006) Eskeland, R., Czermin, B., Boeke, J., Bonaldi, T., Regula, J.T., Imhof, A.: The Nterminus of Drosophila SU(VAR)3-9 mediates dimerization and regulates its methyltransferase activity. Biochemistry 43, 3740-9 (2004) Eskeland, R., Eberharter, A., Imhof, A.: HP1 binding to chromatin methylated at H3K9 is enhanced by auxiliary factors. Mol Cell Biol. 27, 453-65 (2007) Fischle, W., Wang, Y., Jacobs, S.A., Kim, Y., Allis, C.D., Khorasanizadeh, S.: Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev. 17, 1870-81 (2003) Greil, F., van der Kraan, I., Delrow, J., Smothers, J.F., de Wit, E., Bussemaker, H.J., van Driel, R., Henikoff, S., van Steensel, B.: Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev. 17, 2825-38 (2003) Harrington, M.J.: Ph.D. Thesis. The University of British Columbia (2001) Jacobs, S.A., Taverna, S.D., Zhang, Y., Briggs, S.D., Li, J., Eissenberg, J.C., Allis, C.D., Khorasanizadeh, S.: Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3. EMBO J. 20, 5232-41 (2001) Jacobs, S.A., Khorasanizadeh, S.: Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail. Science 15, 2080-3 (2003) Jaquet, Y., Delattre, M., Spierer, A., Spierer, P.: Functional dissection of the Drosophila modifier of variegation Su(var)3-7. Development 129, 3975-82 (2002) Koryakov, D.E., Reuter, G., Dimitri, P., Zhimulev, I.F.: The SuUR gene influences the distribution of heterochromatic proteins HP1 and SU(VAR)3-9 on nurse cell polytene chromosomes of Drosophila melanogaster. Chromosoma 115, 296-31 (2006) Krouwels, I.M., Wiesmeijer, K., Abraham, T.E., Molenaar, C., Verwoerd, N.P., Tanke, H.J., Dirks, R.W.: A glue for heterochromatin maintenance: stable SUV39H1 binding to heterochromatin is reinforced by the SET domain. J Cell Biol.15, 537-49 (2005)  172  Lachner, M., O'Carroll, D., Rea, S., Mechtler, K., Jenuwein, T.: Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116-20 (2001) Lee, L.A., Orr-Weaver, T.L.: Regulation of cell cycles in Drosophila development: intrinsic and extrinsic cues. Annu. Rev. Genet. 37, 545-78 (2003) Lifton, R.P., Goldberg, M.L., Karp, R.W., Hogness, D.S.: The organization of the histone genes in Drosophila melanogaster: functional and evolutionary implications. Cold Spring Harb Symp Quant Biol. 42, 1047-51 (1978) Macaluso, M., Paggi, M.G., Giordano, A.: Genetic and epigenetic alterations as hallmarks of the intricate road to cancer. Oncogene 29, 6472-8 (2003) Min, J., Zhang, Y., Xu, R.M.: Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27. Genes Dev. 17, 1823-8 (2003) Mis, J., Ner, S.S., Grigliatti, T.A.: Identification of three histone methyltransferases in Drosophila: dG9a is a suppressor of PEV and is required for gene silencing. Mol Genet Genomics 275, 513-26 (2006) Müller, J., Hart, C.M., Francis, N.J., Vargas, M.L., Sengupta, A., Wild, B., Miller, E.L., O'Connor, M.B., Kingston, R.E., Simon, J.A.: Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 111, 197-208 (2002) Nakayama, J., Rice, J.C., Strahl, B.D., Allis, C.D., Grewal, S.I.: Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110-3 (2001) Ner, S.S., Harrington, M.J., Grigliatti, T.A.: A role for the Drosophila SU(VAR)3-9 protein in chromatin organization at the histone gene cluster and in suppression of position-effect variegation. Genetics 162, 1763-74 (2002) Nielsen, S.J., Schneider, R., Bauer, U.M., Bannister, A.J., Morrison, A., O'Carroll, D., Firestein, R., Cleary, M., Jenuwein, T., Herrera, R.E., Kouzarides, T.: Rb targets histone H3 methylation and HP1 to promoters. Nature 412, 561-5 (2001) Nielsen, P.R., Nietlispach, D., Mott, H.R., Callaghan, J., Bannister, A., Kouzarides, T., Murzin, A.G., Murzina, N.V., Laue, E.D.: Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature 416, 03-7 (2002)  173  Peters, A.H., O'Carroll, D., Scherthan, H., Mechtler, K., Sauer, S., Schofer, C., Weipoltshammer, K., Pagani, M., Lachner, M., Kohlmaier, A., Opravil, S., Doyle, M., Sibilia, M., Jenuwein, T.: Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107, 323-37 (2001) Peters, A.H., Mermoud, J.E., O'Carroll, D., Pagani, M., Schweizer, D., Brockdorff, N., Jenuwein, T.: Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin. Nat Genet. 30, 77-80 (2002) Pray-Grant, M.G., Daniel, J.A., Schieltz, D., Yates, J.R. 3rd, Grant, P.A.: Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation. Nature 433, 434-8 (2005) Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B.D., Sun, Z.W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C.P., Allis, C.D., Jenuwein, T.: Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593-9 (2000) Rudolph, T., Yonezawa, M., Lein, S., Heidrich, K., Kubicek, S., Schafer, C., Phalke, S., Walther, M., Schmidt, A., Jenuwein, T., Reuter, G.: Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3. Mol Cell. 26, 103-15 (2007) Samal, B., Worcel, A., Louis, C., Schedl, P.: Chromatin structure of the histone genes of D. melanogaster. Cell 23, 401-9 (1981) Schotta, G., Ebert, A., Krauss, V., Fischer, A., Hoffmann, J., Rea, S., Jenuwein, T., Dorn, R., Reuter, G.: Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. EMBO J. 21, 1121-31 (2002) Schotta, G., Ebert, A., Reuter, G.: SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing. Genetica 117, 149-58 (2003) Schotta, G., Lachner, M., Sarma, K., Ebert, A., Sengupta, R., Reuter, G., Reinberg, D., Jenuwein, T.: A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. Genes Dev. 18, 1251-62 (2004) Seum, C., Reo, E., Peng, H., Rauscher, F.J. 3rd, Spierer, P., Bontron, S.: Drosophila SETDB1 is required for chromosome 4 silencing. PLoS Genet. 3, e76 (2007) Seum, C., Bontron, S., Reo, E., Delattre, M., Spierer, P.: Drosophila G9a is a nonessential gene. Genetics 177, 1955-7 (2007) Stabell, M., Eskeland, R., Bjorkmo, M., Larsson, J., Aalen, R.B., Imhof, A.,  174  Lambertsson, A.: The Drosophila G9a gene encodes a multi-catalytic histone methyltransferase required for normal development. Nucleic Acids Res. 34, 4609-21 (2006) Swaminathan, J., Baxter, E.M., Corces, V.G.: The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin. Genes Dev. 19, 65-76 (2005) Tschiersch, B., Hofmann, A., Krauss, V., Dorn, R., Korge, G., Reuter, G.: The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J. 13, 3822-31 (1994) Tzeng, T.Y., Lee, C.H., Chan, L.W., Shen, C.K.: Epigenetic regulation of the Drosophila chromosome 4 by the histone H3K9 methyltransferase dSETDB1. Proc Natl Acad Sci U S A. 104, 12691-6 (2007) Worcel, A., Gargiulo, G., Jessee, B., Udvardy, A., Louis, C., Schedl, P.: Chromatin fine structure of the histone gene complex of Drosophila melanogaster. Nucleic Acids Res. 11, 421-39 (1983)  175  4. GENERAL SUMMARY AND DISCUSSION 4.1. Summary of results In chapter 2 we characterized a subset of EMS-induced Su(var)3-9 missense alleles with respect to a variety of phenotypes, including the relative strength with which they suppress PEV, and the levels of residual HMTase activity of their respective gene products. Based on sequence information, and on the crystal structure of Clr4 (the S. pombe homolog of SU(VAR)3-9) (Min et al., 2002) we were able to carry out a structure/function study of SU(VAR)3-9 and to rationalize the effects of each amino acid substitution on the various phenotypes analyzed. The nine mutants characterized were originally generated in a screen for strong, dominant modifiers of PEV (Sinclair et al., 1983) but, remarkably, all of them have single amino acid substitutions only in the catalytic region of the protein and show significantly impaired HMTase activity. This result strongly suggests that the enzymatic activity of SU(VAR)3-9 is crucial for its silencing function and, perhaps more interestingly, that single amino acid substitutions elsewhere in the protein have no discernable effect on silencing, at least in this particular assay. The data presented in chapter 3 shows the effects of one "protein null", and three of the Su(var)3-9 missense mutants, on the regulation of a euchromatic locus, the HIS-C. Again, our results suggest that the HMTase activity of SU(VAR)3-9 is essential for the recruitment and/or binding of HP1 to the his units that comprise the HIS-C, and in the (down)regulation/silencing of the locus. Taken together, these two data sets strongly suggest that SU(VAR)3-9 likely acts in similar ways at the HIS-C and within the wm4 variegating  176  rearrangement and, by extension, heterochromatin. The results presented in chapter 3 strongly suggest that it is the catalytic activity of SU(VAR)3-9, rather than its physical association with the HIS-C locus, that plays a major role in the regulation of the histone gene transcription or in the association of HP1 with this locus. Overall, we compared between 4 and 7 Su(var)3-9 mutants with the wildtype strain (and with each other) in a number of biochemical, molecular, cytological and morphological assays. When trying to integrate the data from the different types of assays, in most cases it was possible to detect some general trends and to rationalize the results obtained. However, no two assays gave results that were completely consistent with each other for every mutant (Table 4.1 and Figure 4.1). I suggest that these slight variations may derive mostly from the nature and limitations of the assays employed. However, slight differences in the role(s) of SU(VAR)3-9 in the regulation of the HIS-C and in PEV cannot be ruled out. The presence of these apparent "inconsistencies" between data sets from different assays highlights a third, very important point, namely, the advantage of testing mutants for a variety of parameters, rather than solely focusing on one specific aspect of their phenotypes. In this specific case, such a multi-assay approach has allowed us to uncover some subtle, and yet informative, differences among the Su(var)3-9 mutants. Indeed, this thesis represents one of a few cases where the function of mutant alleles of a gene have been assayed in a wide variety of contexts. The results presented indicate that in many cases, the  177  effect(s) of a mutation on one particular phenotype do not accurately predict its effect(s) on a different phenotype. This may be true not only for Su(var)3-9, but also for other genes encoding chromatin and, most likely, other proteins. I suggest that this may be the case especially for those proteins that function as components of one or more multimeric complexes. Hence, it should be kept in mind that studies relying on only one or two phenotypic assays may give a biased, and possibly misleading idea of the function of the gene/gene product in question.  4.2. The function(s) of SU(VAR)3-9 in PEV and in the regulation of the HIS-C. In this and the following sections I will try to briefly relate the role(s) and function(s) of SU(VAR)3-9 in the regulation of the HIS-C and in PEV, as well as some of the limitations of our experimental setup. 4.2.1. The white gene in the wm4 variegating rearrangement and the HIS-C. In chapter 3 we set out to address the role of SU(VAR)3-9 in the regulation of one of its natural targets, the histone genes, and to compare it to what we know about its functions in heterochromatin, represented here by the wm4 variegating rearrangement. There is a valid rationale behind the choice of these two loci, but one should also keep in mind that the HIS-C and the white gene in the wm4 strain are only single examples of a euchromatic locus and a heterochromatin-induced variegating rearrangement, respectively, and as such may not be representative of what typically occurs in euchromatin and heterochromatin, respectively. The white locus in the wm4 variegating rearrangement has many features in common  178  with heterochromatin, for instance, it is enriched in heterochromatin marks such as H3K9me2, HP1 and HDAC1 (Rudolph et al., 2007; Mottus, personal communication). However, the very fact that it shows variegated expression suggests that, at least in some nuclei, it is transcriptionally competent, and therefore presumably has an "open" chromatin structure. Moreover, in PEV the variegating gene does not reside in heterochromatin per se, but rather in close proximity to a disrupted heterochromatic region, and this distance varies depending on the white+ rearrangement in question (Tartof et al., 1989). Hence, one should not automatically extrapolate all that applies to the role of SU(VAR)39 in the context of the white gene in the wm4 rearrangement to heterochromatin in general. The case of the HIS-C is the opposite in that, while it is definitely a euchromatic locus, the HIS-C also has several characteristics that are perhaps more typical of heterochromatin: it is a reiterated locus, somewhat latereplicating, mildly underreplicated in polytene chromosomes, and it is organized into a higher order chromatin structure (Samal et al., 1981; Ner et al., 2002; Zhimulev and Belyaeva, 2003). For these reasons the HIS-C, albeit euchromatic, may not be representative of all euchromatic genes, especially single copy genes, and it would not be surprising if SU(VAR)3-9 acted through slightly different mechanisms and the recruitment of slightly distinct factors, in the regulation of other euchromatic loci.  179  4.2.2. Considerations regarding the assays used to assess the effect of Su(var)39 mutants on gene expression. For our purposes, the relative proportion of pigmented (red) vs. white pigment cells in flies carrying the wm4 variegating rearrangement can be considered as the readout of an assay for gene expression. In this sense, the PEV assay employed in chapter 2 is not substantially different from the RT-PCR approach used to determine the relative levels of histone transcripts in chapter 3. However, each of the two assays has its advantages and disadvantages. The RT-PCR approach is a direct assay of transcript accumulation, as it allows us to measure the relative amounts of transcripts of interest. Obviously, infer that an elevated transcript level reflects an increase in expression. However, we have not ruled out the possibility that the elevated transcript levels result from an increase in RNA stability, or a decrease in RNA turnover. In contrast, the PEV assay is based on a tertiary phenotype, eye pigmentation, which is the result of the coordinated action of several factors, including events and proteins that are not related to the expression of the white gene per se. This assay has, however, one big advantage over the RT-PCR approach. Since the phenotype of each eye is individually recorded, it is possible to observe and document the phenotypic variability within each strain. This allows for a more accurate and, possibly, more relevant comparison among strains. For example, we were able to notice that the presence of the Su(var)3906 mutant allele in a strain that is hyperploid for Su(var)3-9 (2 wild-type copies of Su(var)3-9, plus the mutant allele Su(var)3-906) results in a surprisingly high  180  frequency of "mildly suppressed" eyes (chapter 2, Figure 2.6.b), and that the presence of Su(var)3-9318, instead of Su(var)3-906, in the same hyperploid strain often causes the eyes to be "sectored" (large, well-defined regions of red and large, well-defined regions of white). Such observations would be impossible with the approach employed to assess the levels of histone transcripts, since each RNA preparation used was derived from a population of embryos, which invariably contain cells that are in different stages of the cell cycle. Hence, it is formally possible, although probably highly unlikely, that embryos of a certain genotype could have a great variation in their levels of histone transcripts, with some showing strong overexpression and others being indistinguishable from the wild type, while those of a different genotype could all be overexpressing the histone genes. In both cases, the RT-PCR assay would show a moderate increase in the transcripts levels.  4.2.3. Correlations between the effects of the Su(var)3-9 mutants on PEV and on the levels of histone transcripts. Keeping in mind the respective limitations of the techniques used, if the function of SU(VAR)3-9 within the HIS-C is the same as in the wm4 variegating rearrangement, and if it acts through the same mechanism at both loci, then any given mutation in Su(var)3-9 should affect the expression of the white gene in wm4, and the histone genes transcription similarly. Thus, some general predictions can be made. Firstly, a screen for dominant mutants that result in overexpression of the histone genes should lead to the isolation of the same  181  Su(var)3-9 alleles that were recovered in the original screen for strong, dominant suppressors of PEV, as well as mutations in other loci. Secondly, all Su(var)3-9 alleles that strongly suppress PEV should also show elevated levels of histone transcripts. Finally, mutations in Su(var)3-9 that do not cause suppression of PEV should not result in elevated levels of histone transcripts. An extension of this hypothesis predicts that, for each Su(var)3-9 mutation, there is a correlation between overproduction of histone transcripts and the strength of Su(var) phenotype. That is, an allele that shows a very high increase in the level of H2A and H3 transcripts should also be a very strong suppressor of PEV. Conversely, alleles that are weaker suppressors of PEV are expected to show a smaller elevation in the abundance of H2A and H3 RNAs. In the present case, Su(var)3-9309 was the weakest suppressor in our PEV assay (chapter 2), and it also showed the most limited increase in the levels of H2A and H3 transcripts (only ~1.6-1.8 fold higher than the wild-type, see chapter 3). The H2A and H3 transcripts are significantly more abundant in Su(var)3906 and Su(var)3-9318 embryos (~4.5 to 6-fold higher than wild-type, see chapter 3), and both Su(var)3-906 and Su(var)3-9318 displayed a stronger Su(var) phenotype than Su(var)3-9309. However, while no significant differences are detectable between Su(var)3-906 and Su(var)3-9318 at the level of histone transcripts, Su(var)3-9318 is a stronger Su(var) than Su(var)3-906 (chapters 2 and 3, respectively). This apparent discrepancy may be a result of the experimental systems used (discussed above), or may reflect a difference in the role of SU(VAR)3-9 in the regulation of the two loci, or in their respective sensitivity to  182  the presence of a malfunctioning SU(VAR)3-9 (as in Su(var)3-9318) versus no SU(VAR)3-9 at all (as in Su(var)3-906).  4.2.4. The Su(var)3-9330 mutation appears to be a special case. The Su(var)3-9330 mutant shows peculiar phenotypes in nearly every assay that we employed. In the in vitro enzyme assay this strain showed a unique characteristic. Its HMTase activity on full length histone H3 is higher than that of any other mutant SU(VAR)3-9 protein tested (>30% of wild-type), but it fails to function if a peptide representing the H3 N-terminal tail (20 amino acids) is used as the substrate. Su(var)3-9330 is also peculiar in that the levels of H3K9me2 associated with the histone unit, as determined by ChIP, are comparable to those observed in Su(var)3-906, yet the relative amount of HP1 associated with the same region is significantly lower than in Su(var)3-906. In fact, Su(var)3-9330 has the lowest level of his unit-associated HP1 among the mutants tested, suggesting that it may be acting as an antimorph. With respect to suppression of PEV, the strength of suppression of the Su(var)3-9330 mutant is similar to that of Su(var)39318 (although Su(var)3-9330 individuals do not show "sectored" eyes). The most interesting feature of this mutant, however, is that the expression of its h2a/h2b and h3 histone genes appears to be dramatically decoupled, to the point where the relative abundance of the h3 transcript is 6 fold higher than in wild-type (one of the strongest hyperexpression phenotypes observed), while the levels of h2a and h2b are only about 1.7-1.8 fold above the wild-type (Table 4.1). This is a very unique, and intriguing feature. It may be  183  worth pointing out that there are presently no data formally demonstrating that the H2A/H2B, and the H3/H4 transcripts, are expressed in equal amounts in a wild type Drosophila (we are currently examining this). The data obtained with the Su(var)3-9330 mutant strain suggest that there is some degree of independence between the regulation of h2a/h2b and h3 expression, and that there may be a mechanism in place to ensure co-ordinated expression.  184  Wild-type 100  HMTase activity  80  HP1 recruitment  60  Gene expression  40 20 0 H3  tail  K9 H3K9 HP1 wm4 me2 me2  In vitro assay  ChIP  PEV  h3  h2a  RT-PCR  Su(var)3-906  Su(var)3-9309  100  100  80  80  60  60  40  40  20  20  0 H3  tail  K9 H3K9 HP1 wm4 me2 me2  In vitro assay  ChIP  PEV  0 h3  h2a  H3  RT-PCR  100  80  80  60  60  40  40  20  20 tail  K9 H3K9 HP1 wm4 me2 me2  In vitro assay  ChIP  ChIP  PEV  h3  h2a  RT-PCR  Su(var)3-9318  100  H3  K9 H3K9 HP1 wm4 me2 me2  In vitro assay  Su(var)3-9330  0  tail  PEV  0 h3  H3  h2a  tail  K9 H3K9 HP1 wm4 me2 me2  In vitro assay  RT-PCR  ChIP  PEV  h3  h2a  RT-PCR  Figure 4.1. A semi-quantitative summary of the phenotypes of four Su(var)3-9 mutants analyzed. 185  Figure 4.1. A semi-quantitative summary of the phenotypes of four Su(var)3-9 mutants analyzed. The results presented in chapters 2 and 3 relative to in vitro HMTase activities of recombinant SU(VAR)3-9 proteins, association of H3K9me2 and HP1 with the HIS-C, suppression of PEV in Su(var)3-9 hyperploid strains and abundance of h3 and h2a transcripts were simplified and compiled in order to give a general "overview" of each mutant’s phenotype. For the in vitro enzyme assay, only the averages of 3 independent trials are indicated. The same is true for the ChIP data reported. Such data refer to the enrichment for "H3 coding" fragment in the material IPed with antiH3K9me2 and anti-HP1, respectively. Note that no data regarding the relative enrichment for H3K9me2 or HP1 within the chromocentre is reported here. For the abundance of h3 and h2a transcripts, the wild-type strain was arbitrarily assigned the value of 10, and the mutants are expressed as proportions of the wild type. For the in vitro assays, the activity of the wild-type strain was always arbitrarily assigned a value of 100. The ChIP data is expressed in each case as % of the input, and the PEV value represent the % of fly eyes showing a strong Su(var) phenotype in the assay described in chapter 2.  186  4.2.5. A possible mode of action for SU(VAR)3-9 The genetic screens for dominant Su(var)s were originally designed as a way to identify non-histone chromatin proteins (NHCPs). The term NHCP generally describes proteins that are either relatively stable components of chromatin, or that act as chromatin-modifying enzymes. There are of course proteins that fit both descriptions: SU(VAR)3-9, for example, is both a chromatin-modifier and a chromatin-associated protein (Rea et al., 2000; Nakayama et al., 2001; Schotta et al., 2002; Ebert et al., 2004; 2006). Given these two functions, it is easy to hypothesize how SU(VAR)3-9 is involved in the variegated, epigenetic silencing of the white gene in the wm4 rearrangement: it is recruited to centromeric and pericentric chromatin, where it methylates H3K9, thus allowing the recruitment and binding of HP1, a crucial step in the "heterochromatinisation" of the region. Therefore, the absence of functional SU(VAR)3-9 will result in a drastic reduction on H3K9me2 within pericentric chromatin; consequently low levels of HP1 are recruited and/or bound to the region (Rudolph et al., 2007; Mottus, personal communication). This will prevent or reduce the rate at which heterochromatin is assembled in most cells, leading to the Su(var) phenotype. However, we now know that Su(var)3-9 mutations that suppress PEV cause increased levels of histone transcripts (chapter 3 and Ner et al., 2002). We also know that strains that are hemizygous for the HIS-C have a Su(var) phenotype (Moore et al., 1979; 1983) and surprisingly these strains also produce increased levels of histone transcripts (Ner et al., 2002). Therefore, it is possible that the Su(var) phenotype observed in wm4; Su(var)3-9 mutant individuals is due  187  to the combination of a lack of methylation of H3K9 within pericentric chromatin, where the variegating white gene is located, and an increase in the amount of histone transcripts produced. It has previously been shown that individuals with elevated levels of histone gene transcripts also have an increased level of nucleus-associated histone proteins (Ner et al., 2002). We hypothesize that, since the euchromatic portion of the genome generally replicates earlier than the heterochromatic regions, the increased availability of histone proteins may give the euchromatic portion of the genome a competitive advantage over the later replicating heterochromatin. Therefore, in Su(var)3-9 mutants the formation of heterochromatin would be impeded in two ways. Firstly, by the failure to methylate H3K9 and secondly, by an excess of histone proteins that may allow the euchromatin to replicate slightly more rapidly or more completely, thus interfering with heterochromatin assembly. It will be interesting to investigate whether other Su(var)s also have elevated levels of histone transcripts, and how their levels of histone transcripts correlate with the strength of their Su(var) phenotype, and with that of individuals that are hemizygous for the HIS-C. Our hypothesis would predict that 1) all Su(var) mutations that result in increased levels of histone transcripts should be stronger suppressors of PEV than the lack of one copy of the HIS-C, and 2) hemizygosity for the HIS-C and Su(var) mutations should have an additive effect on the Su(var) phenotype, particularly if the Su(var) mutation in question does not result in increased levels of histone gene products.  188  189  Result of mutation  N/A No product C462Y D536N S616L G521D  Allele  + 06 309 330 318 311  100% N/A 0% 31.3% 24.7% 22.7%  100% N/A 0% 1.2% 18.3% 14.3%  100% N/A 0% 4.0% 0% 11.5%  strong reduced reduced reduced reduced reduced  100% 3.9% 10.4% 4.0% 13.3% NT  HMTase function In vitro (enzyme assays) In vivo (H3K9me2) rH3 H3 tail K9me ChromoHIS-C (H3 coding) tail centre  strong reduced reduced reduced reduced reduced  100% 12.7% 35.9% 5.3% 13.7% NT  HP1 recruitment/ association ChromoHIS-C (H3 coding) centre  <1% 53.5% 37.7% 65.68% 66.76% 86.20%  1 6.46 1.56 6.07 4.72 NT  1 4.56 1.86 1.76 5.60 NT  Loss of silencing/ overexpression white in histone RNAs wm4 h3 h2a  Table 4.1. Summary of the characteristics of the Su(var)3-9 mutants analyzed. All numerical data reported represent the average of 3 independent experiments (for details, see corresponding sections in chapters 2 and 3). The detection of H3K9me2 and HP1 associated with the chromocentre was carried out by immunofluorescence on polytene nuclei, which only provided qualitative data (expressed as "strong" vs. "reduced"). No HMTase assays were run for mutant Su(var)3-906 because such allele results in no gene product (see chapter 2). Su(var)3-9311 was not chosen for the HIS-C and histone transcripts analysis (NT=not tested).  4.3. Drosophila SU(VAR)3-9 has a unique N-terminal region Like many Su(var) genes, Su(var)3-9 is highly conserved from yeast to mammals. Therefore, one might infer that the basic function of the protein and our findings in Drosophila are transferable to other organisms. However, Drosophila’s SU(VAR)3-9 is larger than its homologs, and it contains a "unique" N-terminal region. This region is comprised of the 82 amino acids that are in common with (eIF2γ common domain) and a region, sometimes called "region 2" (residues 83-219), that is required for the dimerization and full enzymatic function of SU(VAR)3-9, as well as for the interactions with SU(VAR)3-7 and HP1 (Krauss and Reuter, 2000; Schotta et al., 2002; Eskeland et al., 2004; Krauss et al., 2006). The lack of these N-terminal domains in the yeast and vertebrate homologs of SU(VAR)3-9 suggest that they may function slightly differently from the Drosophila protein. Neither the vertebrate nor the yeast homologs have the regions required for dimerisation, implying that they may not need to dimerize in order to be fully functional. The fact that they also lack the region of interaction with SU(VAR)3-7 should not be surprising, since SU(VAR)3-7 does not exist outside the Drosophila genus (Jaquet et al., 2006), where it is required for proper localization of SU(VAR)3-9 (Delattre et al., 2004). In the fission yeast this function is carried out, at least in part, by an RNAi-based mechanism (Bühler et al., 2006; Zhang et al., 2008). In spite of its lack of region 2, the mammalian homolog of SU(VAR)3-9 can interact with HP1 (Aagaard et al., 1999; Melcher et  190  al., 2000). This interaction occurs through the first 44 amino acids of Suv39h1 (Melcher et al., 2000), which correspond to the last 44 residues of SU(VAR)3-9’s region 2. In addition, SUV39H1 (the human homolog) can partially rescue the dominant Su(var) phenotype of Su(var)3-9 mutants (Schotta et al., 2002). A physical interaction between S. pombe’s Clr4 and Swi6 (the homolog of HP1) has not been formally demonstrated, and in any case it would be unlikely to occur through the N-terminal region of Clr4, since this portion of the protein is only 7 amino acids long. Curiously, although the N-terminal region of SU(VAR)3-9 has such important functions, no single amino acid substitutions within this portion of the protein have been isolated in the screens for dominant suppressors of PEV (Sinclair et al., 1983; Tschiersch et al., 1994; Ebert et al., 2004). As in the case of the chromodomain (see discussion in chapter 2), this suggests that mutating any single amino acid may not be sufficient to cause a dominant Su(var) phenotype. In order to dissect the function of region 2, it would be interesting to create transgenic Drosophila lines that express tagged SU(VAR)3-9 proteins with single and multiple amino acid substitutions within this region, and investigate the targeting and chromatin association of the mutant proteins, as well as their effects on the targeting of the endogenous (wild-type) SU(VAR)3-9 and some of its known partners, HP1 and SU(VAR)3-7. We would not expect the single amino acid substitutions to have any effects on the behaviour of the endogenous protein. The same experiment, carried out in a Su(var)3-9 null background, would allow us to determine whether any missense mutations in region 2 result in a  191  recessive suppression of PEV phenotype, and what their effect on in vivo H3K9me2,3 is.  192  4.4. References Delattre, M., Spierer, A., Jaquet, Y., Spierer, P.: Increased expression of Drosophila Su(var)3-7 triggers Su(var)3-9-dependent heterochromatin formation. J Cell Sci 117, 6239-47 (2004) Ebert, A., Schotta, G., Lein, S., Kubicek, S., Krauss, V., Jenuwein, T., Reuter, G.: Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev. 18, 2973-83 (2004) Ebert, A., Lein, S., Schotta, G., Reuter, G.: Histone modification and the control of heterochromatic gene silencing in Drosophila. Chromosome Res. 14, 377-92 (2006) Greil, F., van der Kraan, I., Delrow, J., Smothers, J.F., de Wit, E., Bussemaker, H.J., van Driel, R., Henikoff, S., van Steensel, B.: Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev. 17, 2825-38 (2003) Jaquet, Y., Delattre, M., Montoya-Burgos, J., Spierer, A., Spierer, P.: Conserved domains control heterochromatin localization and silencing properties of SU(VAR)3-7. Chromosoma 115, :139-50 (2006) Krauss, V., Reuter, G.: Two genes become one: the genes encoding heterochromatin protein Su(var)3-9 and translation initiation factor subunit eIF2gamma are joined to a dicistronic unit in holometabolic insects. Genetics 156,1157-67 (2000) Krauss V, Fassl A, Fiebig P, Patties I, Sass H.: The evolution of the histone methyltransferase gene Su(var)3-9 in metazoans includes a fusion with and a refission from a functionally unrelated gene. BMC Evol Biol 6/18 (2006) Min, J., Zhang, X., Cheng, X., Grewal, S.I., Xu, R.M.: Structure of the SET domain histone lysine methyltransferase Clr4. Nat Struct Biol. 9, 828-32 (2002) Moore, G.D., Procunier, J.D., Cross, D.P., Grigliatti, T.A.: Histone gene deficiencies and position-effect variegation in Drosophila. Nature 282, 312-4 (1979) Moore, G.D., Sinclair, D.A., Grigliatti, T.A.: Histone gene multiplicity and position effect variegation in Drosophila melanogaster. Genetics 105, 327-44 (1983) Nakayama, J., Rice, J.C., Strahl, B.D., Allis, C.D., Grewal, S.I.: Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110-3 (2001)  193  Ner, S.S., Harrington, M.J., Grigliatti, T.A.: A role for the Drosophila SU(VAR)3-9 protein in chromatin organization at the histone gene cluster and in suppression of position-effect variegation. Genetics 162, 1763-74 (2002) Nielsen, S.J., Schneider, R., Bauer, U.M., Bannister, A.J., Morrison, A., O'Carroll, D., Firestein, R., Cleary, M., Jenuwein, T., Herrera, R.E., Kouzarides, T.: Rb targets histone H3 methylation and HP1 to promoters. Nature 412, 561-5 (2001) Rea, S., Eisenhaber, F., O'Carroll, D., Strahl, B.D., Sun, Z.W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C.P., Allis, C.D., Jenuwein, T.: Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593-9 (2000) Rudolph, T., Yonezawa, M., Lein, S., Heidrich, K., Kubicek, S., Schafer, C., Phalke, S., Walther, M., Schmidt, A., Jenuwein, T., Reuter, G.: Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3. Mol Cell. 26, 103-15 (2007) Samal, B., Worcel, A., Louis, C., Schedl, P.: Chromatin structure of the histone genes of D. melanogaster. Cell 23, 401-9 (1981) Schotta, G., Ebert, A., Krauss, V., Fischer, A., Hoffmann, J., Rea, S., Jenuwein, T., Dorn, R., Reuter, G.: Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. EMBO J. 21, 1121-31 (2002) Sinclair, D.A.R., Mottus, R.C., Grigliatti, T.A.: Genes which suppress position effect variegation in Drosophila melanogaster are clustered. Molec. gen. Genet. 191, 326-33 (1983) Tartof, K.D., Bishop C, Jones M, Hobbs CA, Locke J.: Towards an understanding of position effect variegation. Dev. Genet. 10, 162-76 (1989) Tschiersch, B., Hofmann, A., Krauss, V., Dorn, R., Korge, G., Reuter, G.: The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J. 13, 3822-31 (1994) Zhimulev, I.F., Belyaeva, E.S.: Intercalary heterochromatin and genetic silencing. Bioessays 25, :1040-51 (2003)  194  Appendix 1: ChIP with commercial non-specific IgG, and "SN1 preimmune" (Ner et al., in preparation) serum. (|Critical value| for a two-sided, unpaired T-test at p=0.05: 2.78)  Strain: WT Region: H3c Antibody: non-specific IgG (CALBIOCHEM NI01, control for H3K9me2 IPs) IP# Ab (%IN) Avg SD NO (%IN) Avg SD t 1 0.102 0.101 0.004 0.0367 0.026 0.0136 9.20 2 0.105 0.0306 3 0.097 0.0107 Strain: WT Region: H2Ac Antibody: "SN1 pre-immune serum" (control for HP1 IPs) IP# Ab (%IN) Avg SD NO (%IN) Avg 1 0.1642 0.139 0.022 0.0779 0.069 2 0.1306 0.0742 3 0.1343 0.0550  SD 0.012  t 4.85  Comparison between "control IPs" and corresponding "specific IPs"  H3K9me2 HP1  Control Ab-NO Ab (average)  Specific Ab-NO Ab (average, in % input)  Significantly different?  0.097 0.142  9.154 0.205  t=9.67; YES at p=0.05 t=4.72; YES at p=0.05  195  Appendix 2: ChIP with anti-SU(VAR)3-9 (α-SU(VAR)3-9chr; Ner et al., 2002). (|Critical value| for a two-sided, unpaired T-test at p=0.05: 2.78)  Strain: WT Region: H3c IP# Ab (%IN) 1 0.0652 2 0.0413 3 0.0449 Strain: 06 Region: H3c IP# Ab(%IN) 1 0.0174 2 0.0160 3 0.0198  Avg  SD  0.0504 0.0128  Avg  SD  0.0177  0.0019  Strain: 309 Region: H3c IP# Ab (%IN) Avg 1 0.0311 0.0345 2 0.0405 3 0.0320 Strain: 330 Region: H3c IP# Ab (%IN) Avg 1 0.0383 0.0358 2 0.0254 3 0.0439 Strain: 318 Region: H3c IP# Ab (%IN) Avg 1 0.0874 0.0653 2 0.0530 3 0.0556  SD 0.0051  SD 0.0094  SD 0.0191  NO (%IN) 0.0104 0.0079 0.0126  Avg  SD  t  0.0103  0.0023  5.31  NO (%IN) Avg 0.0232 0.0173 0.0138 0.0151  SD  t  0.0051  0.117  NO (%IN) 0.0128 0.0118 0.0095  Avg  SD  t  0.0113  0.0017  7.35  NO (%IN) 0.0127 0.0100 0.0136  Avg  SD  t  0.0121  0.0018  4.26  NO (%IN) 0.0244 0.0251 0.0246  Avg  SD  t  0.0247  0.0004  3.67  Comparison of mutants to WT Mutant Avg-avg NO Ratio to WT 06 0.0004 (%) 0.007 309 0.0232 (%) 0.578 330 0.0237 (%) 0.591 318 0.0406 (%) 1.012  Significantly different from WT? t= 4.99; YES at p=0.05 t= 2.15; NO at p=0.05 t= 1.92; NO at p=0.05 t= -0.035; NO at p=0.05  196  Appendix 3: ChIP with anti-H3K9me2 (UPSTATE#07-441) (|Critical value| for a two-sided, unpaired T-test at p=0.05: 2.78)  Strain: WT Region: H3c IP# Ab (%IN) Avg 1 7.474 9.232 2 10.71 3 9.512 Strain: 06 Region: H3c IP# Ab (%IN) Avg 1 0.323 0.378 2 0.343 3 0.470  SD 1.63  NO (%IN) 0.058 0.130 0.046  SD NO (%IN) 0.079 0.030 0.002 0.007  Avg 0.078  SD 0.045  Avg 0.013  SD 0.015  t 9.67  t 7.81  Strain: 309 Region: H3c IP# Ab (%IN) Avg 1 1.707 1.707 2 1.660 3 1.755  SD 0.047  NO (%IN) 1.052 0.878 0.333  Avg 0.754  SD 0.375  t 4.37  Strain: 330 Region: H3c IP# Ab (%IN) Avg 1 0.630 0.480 2 0.283 3 0.526  SD 0.178  NO (%IN) 0.114 0.128 0.075  Avg 0.106  SD 0.027  t 3.60  Strain: 318 Region: H3c IP# Ab (%IN) Avg 1 1.615 1.21 2 1.224 3 0.796  SD 0.410  NO (%IN) 0.049 0.039 0.177  Avg 0.088  SD 0.076  t 4.67  Comparison of mutants to WT Mutant Avg-avg NO Compared to WT 06 0.365 (%) 0.039 309 0.953 (%) 0.104 330 0.374 (%) 0.040 318 1.122 (%) 0.133  197  Significantly different from WT? t= 9.36; YES at p=0.05 t= 7.96; YES at p=0.05 t= 9.21; YES at p=0.05 t= 8.24; YES at p=0.05  Strain: WT Region: H3/H4 intergenic IP# Ab (%IN) Avg SD 1 1.208 1.179 0.065 2 1.226 3 1.104  NO (%IN) 0.038 0.048 0.066  Avg  SD  t  0.050  0.014  29.0  Strain: 330 Region: H3/H4 intergenic IP# Ab (%IN) Avg SD 1 0.922 0.871 0.045 2 0.836 3 0.854  NO (%IN) 0.008 0.010 0.011  Avg  SD  t  0.009  0.001  32.9  Strain: WT Region: H2A/H2B intergenic IP# Ab (%IN) Avg SD 1 1.640 1.350 0.261 2 1.134 3 1.276  NO (%IN) 0.012 0.006 0.002  Avg  SD  t  0.006  0.005  8.91  Strain: 330 Region: H2A/H2B intergenic IP# Ab (%IN) Avg SD 1 0.314 0.315 0.038 2 0.354 3 0.278  NO (%IN) 0.024 0.006 0.011  Avg  SD  t  0.013  0.009  13.4  Comparison of 330 to WT for H3/H4 intergenic region Strain Avg-avg NO Compared to WT Significantly different from WT? WT 1.13 (%) N/A N/A 330 0.862 (%) 0.762 t= 5.07; YES at p=0.05 Comparison of 330 to WT for H2A/H2B intergenic region Strain Avg-avg NO Compared to WT Significantly different from WT? WT 1.344 (%) N/A N/A 330 0.304 (%) 0.226 t= 6.93; YES at p=0.05  198  Appendix 4: ChIP with anti-HP1 (α-HP1; Ner et al., in preparation). (|Critical value| for a two-sided, unpaired T-test at p=0.05: 2.78)  Strain: WT Region: H2Ac IP# Ab (%IN) Avg 1 0.2179 0.234 2 0.2194 3 0.2646 Strain: 06 Region: H2Ac IP# Ab (%IN) Avg 1 0.0942 0.078 2 0.0652 3 0.0754  SD 0.0548  NO (%IN) 0.0461 0.0137 0.0252  Avg 0.0282  SD 0.0164  t 11.4  SD NO (%IN) 0.0146 0.0207 0.0136 0.0149  Avg 0.0162  SD 0.0040  t 7.06  Strain: 309 Region: H2Ac IP# Ab (%IN) Avg 1 0.0553 0.066 2 0.0568 3 0.0862  SD 0.0174  NO (%IN) 0.0082 0.0088 0.0154  Avg 0.011  SD 0.0040  t 5.36  Strain: 330 Region: H2Ac IP# Ab (%IN) Avg 1 0.0230 0.020 2 0.0156 3 0.0224  SD 0.0040  NO (%IN) 0.0084 0.0018 0.0040  Avg 0.0048  SD 0.0032  t 5.10  Strain: 318 Region: H2Ac IP# Ab (%IN) Avg 1 0.0781 0.0804 2 0.0824 3 0.0808  SD 0.0024  NO (%IN) 0.0342 0.0207 0.0178  Avg 0.0242  SD 0.0086  t 10.8  Comparison of mutants to WT Mutant Avg-avg NO Ratio to WT 06 0.0618 0.3002 309 0.0550 0.2682 330 0.0152 0.0758 318 0.0562 0.2730  Significantly different from WT? t= 8.89; YES at p=0.05 t= 9.16; YES at p=0.05 t= 13.8; YES at p=0.05 t= 9.99; YES at p=0.05  199  Strain: WT Region: H3c IP# Ab (%IN) Avg SD 1 0.3238 0.2750 0.0472 2 0.2705 3 0.2295  NO (%IN) 0.0061 0.0352 0.0352  Avg 0.0255  SD 0.0168  Strain: 06 Region: H3c IP# Ab (%IN) Avg 1 0.0426 0.0351 2 0.0339 3 0.0292  SD 0.0066  NO (%IN) Avg 0.0089 0.0061 0.0039 0.0055  SD 0.0025  Strain: 309 Region: H3c IP# Ab (%IN) Avg 1 0.1098 0.1150 2 0.1238 3 0.1123  SD 0.0074  NO (%IN) 0.0230 0.0244 0.0286  Avg 0.0253  SD 0.0029  t 19.4  Strain: 330 Region: H3c IP# Ab (%IN) Avg 1 0.0208 0.0187 2 0.0153 3 0.0200  SD 0.0030  NO (%IN) 0.0084 0.0070 0.0045  Avg 0.0066  SD 0.0020  t 5.86  Strain: 318 Region: H3c IP# Ab (%IN) Avg 1 0.0473 0.0487 2 0.0500 3 0.0489  SD 0.0013  NO (%IN) 0.0204 0.0123 0.0105  Avg 0.0144  SD 0.0052  t 10.9  Comparison of mutants to WT Mutant Avg-avg NO Ratio to WT 06 0.0290 0.1273 309 0.0897 0.3595 330 0.0121 0.0531 318 0.0343 0.137  t 8.60  t 7.03  Significantly different from WT? t= 6.04; YES at p=0.05 t= 4.35; YES at p=0.05 t= 6.52; YES at p=0.05 t= 18.0; YES at p=0.05  200  Appendix 5: relative quantifications of histone transcripts by RT-PCR. Comparison between the relative abundance of total h2a and h3 transcripts in wild-type vs. Su(var)3-9 mutant embryo extracts. (|Critical value| for a two-sided, unpaired T-test at p=0.05: 2.78)  Exp #  Strain  h2a/rp49 in mut.  Avg  St. dev.  t* (mutant/WT)  (1)  N/A  N/A  4.56  0.51  -12.0 √  1.86  0.53  -2.82 √  1.76  0.52  -2.56 X  5.60  0.72  -11.0 √  Avg  St. dev.  t*  (1)  N/A  N/A  6.46  0.53  -17.9 √  1.56  0.15  -6.25 √  6.07  0.50  -17.7 √  4.72  1.38  -4.67 √  h2a/rp49 in WT 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Exp #  WT 06 309 330 318  Strain  (1) (1) (1) 5.01 4.00 4.67 1.53 1.58 2.47 2.21 1.87 1.20 5.17 5.22 6.43 h3/rp49 in mut. h3/rp49 in WT  1 2 3 1 2 3 1 2 3 1 2 3 1 2 3  WT 06 309 330 318  (1) (1) (1) 6.70 5.85 6.82 1.71 1.40 1.57 6.03 6.58 5.59 6.24 3.56 4.35  201  Appendix 6. Comparison between the relative increase in h2a and h3 transcripts detected in the Su(var)3-9 mutants. The data presented in appendix 5 were compared using a Student’s T test. (|Critical value| for a two-sided, unpaired T-test at p=0.05: 2.78)  Strain 06 309 330 318  Relative increase h2a 4.56 +/- 0.51 1.86 +/- 0.53 1.76 +/- 0.52 5.60 +/- 0.72  Relative increase h3 6.46 +/- 0.53 1.56 +/- 0.15 6.07 +/- 0.50 4.72 +/- 1.38  Statistically different? YES at p=0.05; t=-4.46 NO at p=0.05; t=0.943 YES at p=0.05; t=-10.5 NO at p=0.05; t=0.994  Appendix 7. Comparison between the relative abundance of total h2b transcript in wild-type vs. Su(var)3-9330 embryo extracts. The indicated t* refers to the result of an unpaired T-test in which the h2b/rp49 ratio of each mutant was compared to that of the wild-type strain. (|Critical value| for a two-sided, unpaired T-test at p=0.05: 2.78) h2b/rp49 in mut. Exp #  1 2 3 1 2 3  Strain  WT 330  h2b/rp49 in WT  (1) (1) (1) 2.77 1.32 1.76  Avg  St. dev.  t*  (1)  N/A  N/A  1.95  0.74  -2.67 X  202  

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