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A quest for function : identification of proteins that interact with the Drosophila melanogaster histone… McNamee, Jason Allan 2002

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A Q U E S T F O R F U N C T I O N : I D E N T I F I C A T I O N O F P R O T E I N S T H A T I N T E R A C T W I T H T H E Drosophila melanogaster H I S T O N E V A R I A N T H 2 A . V . D by Jason Allan John McNamee B.Sc , The University of Western Ontario 1999 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Zoology; Genetics Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A May 2002 © Jason Allan John McNamee UBC Rare Books and Special Collections - Thesis Authorisation Form Page 1 of 1 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of Z Q 0 I ° 3 J foeneKcS G r c a ^ f e . Progr^r~J K The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date http://www.library.ubc.ca/spcoll/thesauth.html 01/09/02 11 ABSTRACT Chromatin is a structure within the cell composed of D N A , histone and non-histone proteins. It packages the cells genome and thus regulates gene expression throughout development. Histones are an important component of chromatin, forming the nucleosome structure on which the D N A wraps in order to be condensed and packaged. Histone variants are variations of these histones that are encoded by single copy genes located elsewhere in the genome. Most of these variants are essential to the organism and cannot be compensated for by their core counter parts and therefore must have a defined role in the cell. One of the most studied classes of these variants is the H 2 A . Z class, a variant class of the major H 2 A core histone. Homo logs have been found in a variety of organisms from yeast to mammals all exhibiting very high sequence conservation. Here we attempted to determine function of one of these homologs of H 2 A . Z , Drosophila H 2 A . v D . We attempted to gain an understanding of protein function by identifying proteins with which H 2 A . v D interacts. We employed a Protein Interaction Trap direct test method followed by G S T pulldowns in order to confirm physical direct interactions. We identified the Drosophila gene CG5515 as a direct interactor o f H 2 A . v D in vitro. We used genetic analysis in an attempt to understand the in vivo significance of the interaction. We believe that the hypothetical protein product of CG5515 may work in a phosphorylation pathway for H 2 A . v D . This modification may either aid H 2 A . v D in generating alternate chromatin structure or possibly in its response to D N A damage. iii Table of Contents Abstract » Table of Contents iii List of Tables vii List of Figures viii Acknowledgements x Chapter 1 INTRODUCTION 1 1.1 Chromatin Structure 1 1.2 The Chromatin Fibre 2 (a) Histones 2 (b) The Nucleosome 3 1.3 Alterations to the Nucleosome 7 (a) Post-translational Modifications 7 (b) Chromatin Remodelling 10 1.4 Histone Variants 10 (a) Introduction 10 (b) Overview of Histone Variants and Variations 11 (c) Heteromorphic/ Minor Core Histone Variants 12 (d) Histone Variant H2A.Z 17 (e) Drosophila H2A.vD 22 1.5 Previous experiments 23 1.6 Thesis Goals and Hypothesis 26 iv (a) Goal/Hypothesis : 26 (b) Findings 27 Chapter 2 Materials and Methods 28 2.1 Cloning / Inserts 28 (a) Protein Interaction Trap / Protein Expression Vectors 28 (i) Reporter Vector 28 (ii) Bait Vector 28 (iii) Prey Vector 29 (iv) GST Fusion Vector 29 (v) Protein Expression Vector 31 (b) H2A.vD 3' Partial deletion PCR 31 (c) Cloning Strategy 33 2.2 Protein Interaction Trap Direct Interaction 36 (a) Theory 36 (b) Yeast Strain 37 (c) Media 38 (d) Yeast Transfrmations 38 2.3 P-Galactosidase Plate Assay 38 2.4 Liquid P-Galactosidase Quantification Assay 39 (a) Growth and Preparation of Cells 39 (b) Assay for P-Galactosidase Activity 40 2.5 GST Purification Pull-downs 40 (a) Theory 40 V (b) GST-Fusion Protein Expression 41 (c) GST Fusion Isolation / Solubility Protocol 41 (i) Isolation 41 (ii) Purification 42 (d) In vitro Transcription / Translation Reactions 42 (e) Co-purification / Pulldown Protocol 42 2.6 Database Searches 43 2.7 Southern Blot Analysis 44 2.8 Genomic PCR 45 2.9 Genetic Interaction Crosses 46 (a) Genetic Interactions 46 (b) Test for Fecundity / Fitness 47 2.10 Lethal Phase 47 Chapter 3 RESULTS 49 3.1 Protein Trap Direct Interactions 50 (a) Altered-H2A.vD / Putative Interacting Proteins 50 (b) H2A.vD / Putative Interacting Proteins 55 (c) H2A / Putative Interacting Proteins 59 (d) 3' Partial Deletion-H2A.vD / Putative interacting Proteins 59 3.2 P-Galactosidase Quantification 65 (a) Growh Times 65 (b) P-Galactosidase Quantification 67 vi 3.3 Confirmation of Physical Interactions / GST Pull-downs 69 3.4 Search for Putative Interacting Genes 71 (a) Search for Putative Interacting Protein Gene-II 71 (b) Search for Putative Interacting Protein Gene-III 73 3.5 Genetic Interactions 75 (a) Genetic Interaction Crosses 75 (b) Test for Fecundity / Fitness 76 3.6 Lethal Phase Assay 79 Chapter 4 DISCUSSION 81 4.1 Protein Interaction Trap: Interaction Specificity Between H2A.vD and the Putative Interacting Proteins 81 4.2 GST Pull-downs: Confirmation of the Direct Interaction 83 4.3 Putative Interacting Protein Identities 84 4.4 Role in Chromatin: Association with Post-translational Modifications 87 4.5 Discrepancies between aItered-H2A.vD and native H2A.vD 89 4.6 Other Possible Interactors 91 4.7 Conclusion 92 Bibliography 93 vii List of Tables Table 1 Cloning Strategy: Interaction Trap 34 Table 2 Cloning Strategy: GST Pulldown / Protein Expression 35 List of Figures Figure 1-1 X-Ray structure of nucleosome core particle 6 Figure 1-2 Phylogenetic tree of H2A sequences 13 Figure 1-3 Amino acid sequence comparison between different homo logs of H2A and H3 minor histone variants 14 Figure 1-4 Drosophila H2A, H2A.vD and altered-H2A.vD amino acid sequences 25 Figure 2-1 Plasmid vector constructs used in the Protein Interaction Trap assay 30 Figure 2-2 Plasmid vector constructs used in the the GST Protein Expression Assay 32 Figure 3-1 Altered-H2A.vD-putative Protein Interaction Trap direct test platings 52 Figure 3-2 Altered-H2A.vD-putative Protein Interaction Trap direct test platings 54 Figure 3-3 H2A.vD-putative Protein Interaction Trap direct test platings 56 Figure 3-4 H2A.vD-putative Protein Interaction Trap direct test platings 57 Figure 3-5 H2A-putative Protein Interaction Trap direct test platings 60 Figure 3-6 H2A-putative Protein Interaction Trap direct test platings 61 Figure 3-7 3' Deletion H2A.vD-putative Protein Interaction Trap direct test platings 63 Figure-3.8 3' Deletion H2A.vD-putative Protein Interaction Trap direct test platings 64 Figure 3-9 Yeast growth times 66 Figure 3-10 P-Galactosidase quantification 68 Figure 3-11 GST Pulldown 70 Figure 3-12 Southern Blot 72 Figure 3.13 Genomic PCR of putative II sequence 74 Figure 3.14 Analysis of genetic interactions 77 Figure 3.15 Test for fertility Figure 3.16 Lethal phase assay X Acknowledgments The perfect question is the idea that governs successful science. It enables one to follow a train of thought, and to pursue a goal in a logical and thoughtful manner. I would like to thank my supervisor, Dr. Thomas Grigliatti for teaching me this lesson. He has given me the freedom to discover and the guidance to do so successfully. I would like to thank the rest of the Grigliatti lab for their help and support throughout my studies, especially Randy Mottus who has been more of a mentor than a fellow student and Dr. Richard Sobel whose initial interest and intriguing questions allowed my project to take form. I would like to thank my parents, family and friends for their love and support. Without their encouragement many of my goals, both present and future, would not be possible. Lastly I would like to thank my beautiful wife Natasha, my companion, my inspiration and my soul. A tremendous journey has been undertaken and finally the light at the end of our tunnel is beginning to shine. 1 Chapter 1: I N T R O D U C T I O N 1.1 Chromatin Structure Considering the size and complexity of multi-cellular eukaryotic genomes the task of packaging the genome while allowing for gene regulation is an issue the cell must 7 8 overcome. For example genomes range in size from 1.2x10 bp in yeast to 1.8x10 bp in Drosophila melanogaster (Adams et al, 2000) to greater than 6x l0 9 in humans all of which must fit inside every single cell of ~ 0.01mm in diameter (Redon et al, 2002). Chromatin is the packaging structure cells use in order to accomplish this task. The chromatin model is described as follows: D N A folds around nucleosomes, nucleosomes then form lOnm fibres that in turn fold helically into 30nm chromatin fibres, termed solenoid fibres. These fibres then form prophase loops and organize radially from a prophase chromosome axis that coils to form the fully condensed metaphase chromosome (Alberts et al, 1994). It is clear that packaging D N A into chromatin is necessary for packaging the genome into the nucleus but the added complexity of D N A packaging also limits the accessibility of D N A to D N A binding factors, promoting an overall repressive state, and is functionally significant in terms of transcription, replication, repair, and recombination (Farkas et al, 2000). The chromatin structure is so essential in governing the transcriptional competency of a gene that in order to understand any regulated event in the nucleus it is necessary to define the chromatin structure within which the D N A is utilized (Wolffe, 1998). Chromatin is an overall structure composed of many components that function together to allow for 1) packaging and 2) regulation. The overall structure of the chromatin fibre can be dissected into linker D N A , non-histone proteins, 4 histones that comprise the nucleosome and histone HI which associates with 2 the internucleosome D N A and links adjacent nucleosomes. In order to fully understand this essential structure one must look first at the basic components of the chromatin fibre, the hi stones. 1.2 The Chromatin Fibre (a) Histones The histones themselves were originally thought to be genetic material. While this is not true, the histone proteins are an essential group of nuclear proteins involved in chromatin formation and structure. In Drosophila, the genes encoding HI and the four core histones H2A, H2B, H3, and H4, comprise a 5 kb long complex that is tandemly repeated -110 times and located at bands 39D3-E2 on the chromosome. These genes are intronless and their messenger RNA transcript is not polyadenylated. The histone proteins are all small, basic (11,000-16,000 kDa in weight) proteins and are composed mostly of arginine and lysine (Wolffe, 1998). A l l five histone proteins contain what is called a histone fold-domain at their carboxy terminal end of the protein. This is the site at which histone-DNA and histone-histone interactions take place. Tails, short stretches of amino acids that extend from the core itself, are located at the amino and carboxy terminal ends of H2A and just the amino terminal ends of the other core histones (Luger et al, 1997). The amino charged tails of the histones are the sites where most of the lysine residues are located (Arents et al, 1991b), and much of the post-translational modifications occur (Wolffe, 1998). A fifth histone known as the linker histone, termed HI (there is also a specialized linker termed H5 from chicken erythrocytes), is slightly larger than the core histones (>20,000 kDa), and is also basic and lysine rich. HI and its 3 counterparts have a central structural domain and charged regions at the carboxyl and amino terminal ends of the protein (Wolffe, 1998). These histones are essential proteins and are evolutionarily conserved across species (H4 is near perfect) and were previously thought to be among the most invariant proteins known (see Baxevanis and Landsman, 1994). However, not all eukaryotic cells contain them. Dinoflagellates use relatively small basic proteins, which do not resemble histones, to package their D N A (Vernet et al, 1990). Other lower eukaryotes are deficient in certain histones. Histones H3 and H4 are the most conserved while H2A and H2B are less so. This hints at a more defined structural role for H3 and H4. In most eukaryotes the histone proteins comprise approximately 50% of the total chromatin protein and the other 50% is composed of DNA, chromatin bound enzymes, high mobility group proteins, transcription factors, and scaffold proteins etc. The core histones accomplish their task of mediating the folding of D N A into chromatin (Wolffe, 1998) by forming a specialized structure along with D N A termed the "core particle" of the nucleosome (Kornberg and Lorch, 1999). (b) The Nucleosome The chromatin fibre itself can be seen as "beads on a string" due to discrete complexes of proteins and nucleic acid arrayed along a D N A backbone (Wolffe, 1998). These beads termed nucleosomes are the fundamental repeating unit of the chromatin fibre. The "core particle" of the nucleosome is composed of 2 of each of the 4 core histones (H2A, H2B, H3 and H4) arranged in a left-handed protein super helix matching 4 that of the -146 bp of D N A in a 1 % left handed superhelical turn that wraps around it (Arents etal, 1991a), (Wolffe, 1998). The histones are produced in strict stoichiometric ratio and the nucleosomes that they create incorporate 80% of the D N A in the nucleus (Noll, 1974). The attraction between D N A and the histones is electrostatic with H3 and H4 being more tightly associated than H2A and H2B. The association between the histones themselves is noteworthy with H2A and H2B forming a stable dimer (H2A/H2B) and H3 and H4 forming a stable tetramer ((H3/H4)2). The (H3/H4)2 tetramer organizes D N A into the nucleosome while the H2A/H2B protects either side of the D N A segment (periphery) (Wolffe, 1998). The histone fold domains of all four core histones are quite similar in structure: a main a-helical domain bordered by loop segments on each side followed by a shorter helix. Within each structure is an area of p-sheets. The main central helix is the interaction interface domain. The general shape of the histone octomer contains a central V shaped (H3/H4)2 tetramer flanked by two spherical like H2A/H2B dimers (Wolffe, 1998). This thesis focuses on a variant molecule of H2A. Subsequently, it is of particular interest to note its unique basic amino-terminal and carboxyl-terminal tails. The C-terminal tail of H2A binds to D N A around what is known as the dyad axis (Gushchin et al, 1991) and extends outside the nucleosome (Luger et al, 1997) and the N-terminal tails along with that of H2B binds D N A at the periphery of the nucleosome (Pruss and Wolfe, 1993), (Lee and Hayes, 1997). The phosphodiester backbone of D N A seems to interact electrostatically with the arginine residues found in the histone fold domains of the octomer. This is an important concept for organizing D N A in the nucleosome. The crystal structure of core particle, deduced in 1997 (Luger et al, 1997), shows the details of the protein-protein and protein D N A interactions (Fig. 1-1). There are two key points to note. The first is the twist of the D N A double helix associated with the core particle. A stretch containing 10 bp of altered twist is accommodated at various locations. This promotes the idea of structural "plasticity" and may underlie chromatin remodelling for gene regulation (Kornberg and Lorch, 1999). Secondly, there are sufficient gaps for the amino terminal tails of H2B and H3 to pass through to the outside of the core particle. H2A and H4 tails pass across the super helix on the flat faces of the particle to the outside as well. Along with the D N A that connects one nucleosome to the next in the fibre, the linker histone (HI or H5) binds near one end of the core D N A and is thought to promote coiling or folding of this fibre. The core histone tails that protrude from the core itself also seem to promote chromatin fibre formation possibly by associating with the neighbouring nucleosome or by influencing the configuration of the linker D N A (Kornberg and Lorch, 1999). These tails that extend out of the core also serve as the targets of histones, which may also influence chromatin formation and ultimately gene regulation. Three basic mechanisms influence or regulate chromatin structure to occur: covalent modifications of histones, nucleosome remodelling, or histone-histone interactions (Santisteban et al, 2000). Figure 1-1. X-Ray structure of nucleosome core particle. Ha l f the core particle with four histone molecules and 73 bp of D N A is shown. The histone tails are drawn in as dotted lines with circular headed sticks to indicate sites of acetylation. H 2 A shows both N-terminal (bottom) and C-terminal (top) protrusions, while the other core histones only have N-terminal protrusions.(Adapted from Luger et al, 1997, taken from Kornberg and Lorch, 1999) 7 1.3 Alterations to the Nucleosome (a) Post-translational Modifications Chromatin is a dynamic structure exhibiting extensive morphological changes from one cell cycle to another. These alterations can be attributed, in part, to reversible chromosomal protein modifications. Core histones can undergo many different types of post-translational modifications including, but not limited to: acetylation, phosphorylation, methylation, ADP-ribosylation, and/or ubiquitination. These are not permanent changes and can be undone. These post-translational modifications are known to be involved in histone deposition, nucleosome assembly, chromosome condensation, and transcriptional regulation (Ren and Gorovsky, 2001). It has been suggested that these modifications could form a complex "histone code" that may specify unique chromatin functions (Strahl and Allis, 2000). Charge-altering modifications of the histone is another mechanism affecting chromatin structure. Initially many of these charge-altering modifications were described in Tetrahymena. Subsequently they have been documented in a variety of organisms. This is done by modifying the charge of a protein domain rather than specific residues (Dou and Gorovsky, 2000). Acetylation and phosphorylation are the best studied of these alterations. Acetylation is believed to play a key role in the transcriptional processes. This inference is based on the observation that some transcriptional activators have acetyltransferase activity (Brownell et al., 1996) and some transcriptional repressors have deacetylase function (Taunton et al, 1996). Acetylation occurs on all four core histones and in all higher eukaryotes, from animals to plants. The lysine residues at the amino terminal end of the four core histones are the primary targets for acetylation and each 8 acetate molecule neutralizes a negative charge at these sites of modification. Hyperacetylation is thought to have moderate effects on nucleosome conformation (Bode et al, 1983), (Oliva et al, 1990). The most dramatic affect on chromatin structure may come from protein-protein interactions. These interactions may occur among the histones themselves, between the histones and non-histone chromatin proteins; or between histones and chromatin remodelling complexes or transcription factors. Since the amino terminal tails protrude beyond the nucleosome core particle they are potential targets for interaction with DNA, linker histone, or non-histone chromatin proteins. Acetylation of the histone tails appears to be a necessary pre-requisite to transcription and may facilitate access of trans-acting factors to their respective target site. This suggests that modifications, such as acetylation, play a key role in the transcription process (Lee et al., 1993), (Vettese-Dadey et al, 1996). The correlation between histone acetylation and transcription is considerable. In S. cerevisiae most of the genome is transcriptionally active and contains acetylated histones; among the exceptions are areas such as the mating type locus and telomeres that are normally silenced, these regions contain hypoacetylated H4. Nucleosomes containing hyperacetylated histones are less stable and generally make the chromatin more soluble as a whole (Perry and Annunziato, 1989). Phosphorylation is another type of post-translational modification and is correlated with relaxing the chromatin for gene expression and condensing chromatin for cell division. Phosphorylation occurs on serine residues. In vivo phosphorylation of H2A occurs in the cytoplasm after synthesis (Dimitrov et al, 1994). This may target it to the molecular chaperones involved in nucleosome assembly at the replication fork (Wade et al, 1997). The variant H2A.X, aXenopus variant found in oocytes (Dimitrov et al, 9 1994), is phosphorylated and has a proposed role in nucleosome spacing during chromatin assembly (Kleinschmidt and Steinbeisser, 1991). A phosphorylated form of H2A.X gathers in Xenopus sperm chromatin, occupying 50% of the total H2A in the paternal pronucleus. The true reason for phosphorylation is still a mystery. Methylation is not just for DNA. Core histones are also methylated at their lysine residues, possibly to inhibit acetylation and therefore promote transcriptional repression. The histones H3 and H4 are preferentially involved in this type of modification. It has been suggested that ADP-ribosylation is involved in D N A repair and evidence suggests that ADP-ribosylation may lead to localized unfolding of chromatin possibly by partial disruption of nucleosomes (Wolffe, 1998). H2A and H2B can have the small peptide ubiquitin attached to their carboxyl terminal tails. Poly-ubiquitination has been shown to target proteins for degradation, where as mono-ubiquitination may play a'chaperone role. Ubiquitinated H2A is incorporated into the nucleosome without major changes to the core (Kleinschmidt and Martinson, 1981). Acetylation, phosphorylation and now methylation are at the forefront of post-translational modification research. Current evidence suggests that these modifications play a role in gene control. However, they are not the only mechanisms associated with nucleosome regulation in the nucleus. 10 (b) Chromatin Remodelling The ability to shift nucleosomes along the chromatin fibre allows for a different type of regulation to occur. Re-position the nucleosomes around promoters of certain genes allows promoters to be more or less accessible to the trans-acting factors responsible for transcriptional regulation of those genes. In Drosophila three such complexes that appear to shift nucleosome position have been discovered: nucleosome remodelling factor (NURF), chromatin accessibility complex (CHRAC) and ATP-utilizing chromatin assembly and remodelling factor (ACF). The protein products of brahma and kismet have ATPase domains similar to those from the yeast SWI/SNF complex and are thought to be involved in remodelling as well. The ideas presented thus far involve epigenetic changes or shifts to the nucleosome and D N A binding or position. We will now consider to effects of structural changes to the nucleosome core, or more accurately the histones themselves. 1.4 Histone Variants (a) Introduction Though histone modifications leading to a possible "histone code", charge patch or nucleosome shifting are popular ideas regarding chromatin and gene regulation, they are not the only mechanisms one must consider. Another aspect most relevant to this dissertation is the notion of histone variants. Changes in the actual chromatin proteins themselves may be an important component of the chromatin regulatory process. Although they have been less intensively studied than histone modifying enzymes, 11 histone variants may prove to have structural and functional consequences for chromatin structure and gene regulation. (b) Overview of Histone Variants and Variations There are numerous questions surrounding the structure of chromatin especially dealing with the nucleosome model. Are nucleosomes structurally equal? What are the differences within them that govern transcriptional competency from one developmental interval to the next? Core histone genes are present in multiple copies in most organisms, from two copies in yeast to several hundred copies of each histone gene in sea urchins (Wolffe, 1998). The multiple copies of a given histone gene (e.g. H2A) are identical in sequence. Interestingly most of the core histones have several non-allelic variants originally referred to as "orphan" loci because their role was undefined. These histone variants differ somewhat in amino acid composition and physiochemical and immunochemical properties. They can be grouped into two broad categories: homomorphous and heteromorphous (West and Bonner, 1980). Homomorphous variants are considered to be major variants due to their abundance in the cell. The have minor amino acid changes and are not known to have any differential function in the cell. Some examples are H2A.1, H2A.2, H2B.1, H2B.2, H3.1, H3.2 and H3.3. Interestingly H4 is the exception with no known species variants (though there are two in Tetrahymena) (Elgin, 1995). Of greater interest, heteromorphous variants are considered to be minor variants because of their rarity and apparent distinct function in the cell (Redon et al, 2002). These minor variants are distinct variations of the histones; they are transcribed at distinct periods in development and do not necessarily follow the cell cycle (unlike core 12 histone that are tightly linked to the S-phase of the cell cycle) (Poccia, 1986). These variant sequences are frequently highly conserved, sometimes they are more similar to each other than to their respective core counterparts (Ernst et al, 1987) (Fig. 1-2). These histone variant genes also differ from the genes encoding the core histones; the variants characteristically have introns and their mRNAs are polyadenylated (Brush et al, 1985); (Wells and Kedes, 1985). There are also non-nucleosomal histone variants or variations. The linker histone HI also shows pronounced variance. The H5 variation is believed to be involved in chromatin compaction and inactivation. Mammalian H i t is present in gametes. The subtypes Hies and Hlct are observed during embryonic development. H I 0 has been implicated in establishing and maintaining the terminally differentiated state (Elgin, 1995). Proteins such as TaflI40 resemble H3 and TAFII60 resemble H4. These are thought to create a nucleosome like structure around the T A T A box maintaining a semi-contacted state that is competent for transcription (Kokubo et al, 1993), (Nakatani et al, 1996), (Xie et al, 1996). These proteins are examples of transcriptional regulatory proteins that are extreme variations of histones. These differences can lead to differences in the way genes are regulated. (c) Heteromorphic / Minor Core Histone Variants The minor variants are extremely interesting because they are distinguishable from their major core histone counterparts. The best studied minor variants are CENP-A, H2A.X, macroH2A, H2A-Bdb, and H2A.F/Z (Fig. 1-3). Centromeric protein A (CENP-A) (Csep4 in yeast and Cid in Drosophila) is the most studied H3 variant. It is localized at the centromeres of eukaryotic chromosomes 13 TROUT T. PYRIFORMS DROSOPHILA SEA URCHIN XENOPUS HUMAN " CHICKEN ASPERGILLUS 5. CEREVISIAE •S. POMBEB S. POMBEA S. POMBE 2 H2A.vD ^ H2A.F/Z HVl H 2 A . Z Variants Major H 2 A Figure 1-2. Phylogenetic tree of H2A sequences. Bar length represents evolutionary time. The organism name represents the evolutionary decent of the major H2A sequences. The H2A.F/Z nomenclature represents the evolutionary decent of the H2A.Z class of minor H2A variants (S. Pombe 2, Drosophila H2A.vD, Sea Urchin H2A.F/Z , Chicken H2A.F, Mammal H2A.Z and Tetrahymena hvl) (adapted from van Daal, 1990). 14 Helix 1 H2A N J j ^ K ^ K g C ^ K A K A K A K - Uwt( /VGHYHKLLKKG ) 1RAERVGA Human H2A.Z N A G G X A - - L > S — K T - - V _ L J . a s f p r . " 3 - - H - K S K T ) . , . T S H G Drosophila H2A.Z N AGGKA- - D S - - - K - - - v„ i i a a [ ) — i - -H-HSRT) . . .TSH<3 Tetrahymena H2A2 N g-&-< J K - - - VOOAKW., 1 $ a a [} - - -1 - - F -RGKV )„,SAKN MaCrO H2A N -S—GKXKSTKTSRS S a a ( } * - - - H I . - V l K - - )..,„,K?KY I V Helix 2 Helix 3 H2A ()GAFVYK auV> rLEYLTAE IL5IJLGHAARDHK)z<TRri(J P R H L Q L A I RN3 Human H2A.Z ( ) T - A - - S - - I »• v-Drosophila H 2 A . 2 Q T - A - - S - - I -Tetrahymena H2A.Z Q T - A -Macro H2A fT~~ c --G--I— - S K - L - ) V R - - T Q - - — L - 0-Helix 1 Helix 2 H3 N " a . i . . . R .K . . , S aO-AB HK...2 6 J . - - [) TVA * .H E T R S ) K S T ELL I R (TKL P FQF. II VR E I A ) Q D F K T E L R F - - O S CENP-A N 6aa-.RK-.2aa-jmilR-29aa{j.y3t,r-K K L - ) - - - r t Q — - - S - - A C /VK-TRGVD-IW-A Helix 3 Helix 4 H3 (. )AAIGAI<:'EASE^YLV'5LF .^TOL£AT)EiAKRVTIM( )p)OIQLARRIg_)GERA C CENP-A (k-i-i- A—"-y- - A Y - L T X J - - G ; .F() V ) - L 5 S G L G G H 2 A . 1 H 2 A X H H 2 A H 2 A . 2 H 2 A . 1 H 2 A X B 8 2 A M 2 A . Z H 2 A . 1 H 2 A X t»B 2 A K 2 A . Z H 2 A . 1 H 2 A X » B 2 A H 2 A , £ JNPPW" — — — - — • .ATVgt^AP8430WOttQ*S5Sir j C l ' S E T I L 9 P P P E I W C R M T S < ^ « r R J - I U V A A F A T S I U » " S G j O - j t V -DIUSGTSNSTSEDGPCTGrTItSSKSLVIXiQKLSLTQSDISHIGSKMVXGIVHPTTAEIOLI^IGKAtEKAGGK ErLETVPLELRKSQG PLEVAEAAVSQBSOLAAXFVI BCHI PQWSSDKCEEQLEEYiKKCLSAAEDKKJJSSVAFPP r P S C R N C FPKgTAAQVT_KAI SAH FDDSSASSUCKVTr J X i r D S B S ICITVQEMA.KI.DAK Figure 1-3. Amino acid sequence comparison between different homologs of H 2 A and H3 minor histone variants. a) Cylindrical boxes indicate alpha-helices. Dashed lines represents identical amino acids between homologs. The C-terminal tail o f the H 2 A variants are represented by a zig zag line (taken from Wolfe, 1998). b) C-terminal amino acid sequences of H 2 A . 1 , H 2 A . X , macroH2A, and H 2 A . Z are aligned. The shaded boxes represent similarities between sequences. The dark grey box surrounding the amino acids S Q E at the C-terminal end of the H 2 A . X sequence represents a phosphorylation domain, (taken from Ausio & Abbott, 2002) 15 (Sullivan et al, 1994). At the carboxyl terminus, 139 amino acids contain the histone fold domain (Arents and Moudrianakis, 1995) and is 62% identical to its H3 counterpart (Sullivan et al, 1994). It has been speculated that the histone fold domain could participate in forming the specialized nucleosomes at the centromeres involved in folding on chromatin in that region (Basrai and Hieter, 1995). The N-terminal domain appears to be essential for function and is critical for interactions between this particular variant and other kinetichore proteins (Chen et al., 2000) This N-terminal region is also involved in higher order structure (Leuba et al, 1998). Neocentromeres observed in Drosophila (Williams et al, 1998) argue against the need for sequence specificity for targeting CENP-A containing nucleosomes. Rather than sequence specificity, targeting of CENP-A may be due to coupling time between the replication of the kinetichore D N A in late S phase and the synthesis of this variant in G2 (Shelby et al, 2000). Many of the H2B variants were initially found and described in Tetrahymena where they are developmentally regulated. An interesting variant of this class is the sea urchin variant which possesses an amino tail that is 21 amino acids longer than the major histone H2B, and is thought to interact with linker D N A , allowing for increased nucleosome stability in sperm (Hill and Thomas, 1990). H2 A is unique among histones in that it has a prominent carboxyl-terminal tail that extends beyond the histone fold (Arents and Moudrianakis, 1995). The variants of this protein are therefore very interesting and are presently becoming a focus of investigation. Many H2A variants have been characterized. H2A variants are uniquely classified by containing a common core protein sequence (AGLQFPVGR) termed the 16 H2A box (von Holt et al, 1979). They have long 3' untranslated regions, are polyadenylated and follow many of the rules of the other histone variants. One class of H2A variant is H2A.X (Mannironi et al, 1989). This variant has carboxyl-terminal tails that extend far past the histone fold domain. It contains a C-terminal D N A dependant protein kinase mutated consensus recognition sequence and is a phosphorylation substrate during double stranded break repair (Downs et al, 2000) and meiosis (Mahadevaiah et al, 2001). As an example, wheat H2A1 has a 19 amino acid extension that could theoretically protect 20 base pairs of D N A directly adjacent to the nucleosome (Lindsey et al, 1991). H2A-Bbd displays a truncated carboxyl-terminal tail and is enriched in active chromatin (Chadwick and Willard, 2001) and generally absent from the inactive X chromosome in mammals, and is thus associated with barr body deficient (Bbd) phenotype (Chadwick and Willard, 2001). This protein co-localizes with acetylated histone H4 suggesting a role in nucleosomes at actively transcribing genes. This variant cannot be ubiquitinated due to the truncation of both the n-terminal and c-terminal regions. Macro H2A has a large COOH non-histone region that is similar to that of viral proteins that interact with R N A binding proteins. Thus this is a specialized protein that works to transcriptionally silence chromatin (e.g. X chromosome inactivation in female vertebrates) (Pehrson and Fried, 1992), (Costanzi and Pehrson, 1998). 17 (d) Histone Variant H2A.Z The most studied minor H2A variant, and the focus of my research efforts, is the H2A.F/Z class (so named due to simultaneous discoveries in chicken and mammals). H2A.F/Z constitutes roughly 5-10% of total H2A in the chromatin of cells (Palmer et al., 1980), (West and Bonner, 1980). H2A variants have been identified in a wide variety of organisms including Drosophila H2A.vD (D2, H2A.2, His2A.vD, His2Av, and/or H2Av), chicken (H2A.F), Tetrahymena (hvl), Xenopus (H2A.Z), zebrafish (H2A.F/Z) budding yeast (S. cerevisiae) (HTA3 and/or HTZ1), fission yeast (S. pombe) homolog iphtl), mice (H2A.Z), and humans (H2A.Z). The breadth of organisms in which this H2A variant is found, as well as its conservation (Leach et al, 2000), suggests that incorporation of this class of variant into chromatin is not only longstanding, but H2A variants probably play a significant role in regulating chromatin structure and gene expression in one or more stages of development. For simplicity all homologs of H2A.F/Z will be referred to as H2A.Z except the Drosophila version, which will remain H2A.vD. The similarity between the H2A.Z variants themselves is striking. These variants are more similar to each other than the variants are to their core H2A counterparts. For example Drosophila H2A.vD and chicken H2A.Z are only 60% similar to their core counterparts (van Daal et al, 1988), (Harvey et al, 1983). Even more striking is the fact that at the amino acid level the similarity between the two variant proteins is 97%; there are only 5 amino acid changes among the total number of amino acids. The Tetrahymena version, is -84% similar to the chicken and sea urchin homologs at the amino acid level. Most of the divergence between the core histones and the variants occurs in the carboxyl tail, although there are slight differences in the amino 18 terminal region as well. The mammalian H2A.Z is identical across the human, calf, rat and mouse species at the amino acid level (94% - 97% similar in the coding region at the D N A level). The high protein similarity between this class of variants amongst such a wide range of species suggests a very important role of this class of protein. . H2A.Z is essential for survival in most organisms although its specific role in the cell remains enigmatic. A deletion in the Drosophila H2A.vD gene causes lethality (van Daal and Elgin, 1992). H2A.Z is also essential for viability in Tetrahymena (Liu et al, 1996b). In Tetrahymena, it been shown to associate with the actively transcribed chromatin in the macronucleus, but is not associated with genes in the inactive micronucleus (Allis et al, 1986). This observation suggests H2A.Z may be involved in differentiation between active and inactive genes (Stargell et al, 1993). In S. cerevisiae, deletion of H2A.Z results in lethality at 37°C but only slow growth and formamide sensitivity at 28°C (Santisteban et al, 2000). The mammalian counterpart has been shown to be essential for development, yet not essential for cell survival (Faast et al, 2001). This is similar to the phenotype in fission yeast, where cells deficient in H2A.Z show altered colony morphology, increased resistance to heat shock and a decrease in fidelity of segregation of an S. pombe mini chromosome (Carr et al, 1994). The specificity of the H2A.Z protein was shown when the Tetrahymena H2A.Z wild-type protein failed to compensate for the loss of the S. cerevisiae H2A genes (Liu et al, 1996a), and the major S. cerevisiae H2A failed to provide the function of S. cerevisiae H2A.Z and vice versa (Jackson and Gorovsky, 2000). Since H2A.Z has sequence differences compared to that of H2A, deletion/replacement studies have been done to determine the motifs that are essential for 19 function. In Drosophila the regions deemed necessary for survival have been determined. The a carboxy-terminal helix when replaced with the standard H2A amino acid causes lethality (Clarkson et al., 1999). This region has been demonstrated to reside deep within the nucleosome core (Luger et al, 1997), (Suto et al, 2000). In Tetrahymena the N-terminal region seems to be important. Acetylation of the amino terminal lysine residues is essential. When these lysine residues are replaced by similarly charged (basic) amino acids that cannot be neutralized by acetylation, the result is death. Interestingly when the N-terminal amino acids are deleted the cell is still viable (though deletion of the entire hvl is lethal) suggesting it is not specific residue neutralization that is necessary (Ren and Gorovsky, 2001). Since the amino terminal residues are most likely required for association with chromatin through modulation of a charge patch, it appears that modification of specific amino acids is less important than modifying the charge of a specific region of the H2 A.Z. When the area cannot be modulated, presumably gene activation is prevented (Redon et al, 2002). The localization of H2A.Z in the cell has been the focus of recent studies. H2A.vD was localized in polytene chromosomes by indirect immunofluoresence (van Daal and Elgin, 1992). The H2A.vD protein seems widely, but not uniformly distributed in Drosophila and is not limited to sites of active transcription. H2A.vD is present in thousands of euchromatic bands and at the heterochromatic chromocentre of the polytene salivary gland chromosomes. In order to help determine its precise binding sites, an anti H2A.vD antibody was developed. In chromatin immunoprecipitation (ChTP) experiments it precipitated transcribed and non-transcribed genes (Leach et al, 2000). 20 In S. cerevisiae, CMP studies also demonstrated a non-uniform pattern of distribution across the PH05 and GAL1 loci while the major H2A protein had a relatively uniform cross-linking pattern across the loci (Santisteban et al., 2000). Attempts have been made to define the structural and physical attributes of H2A.Z. A 2.6 A crystal structure of the Xenopus laevis nucleosome core particle was reported containing the H2A.Z variant (Suto et al, 2000). It showed very similar characteristics to the previously reported 2.8 A crystal structure containing the regular H2A (Luger et al, 1997). The presence of a metal ion is thought to change the overall surface presentation of the nucleosome possibly allowing for the association of specific nuclear protein association (Suto et al, 2000). The nucleosome, with the H2A.Z incorporated in place of the normal H2A, exhibits distinct localized charges which are hypothesized to destabilize the interaction between the (H2A.Z-H2B) dimer and the (H3-H4)2 tetramer. This destabilization would most likely affect protein-protein interactions within the nucleosome or between nucleosomes and would not interfere with D N A -protein interactions as most of the changes occur at the H4 docking area at the C-terminal end of the H2A.Z sequence. An interesting, though somewhat contradicting, note here is that separate studies have suggested that H2A.Z acts to globally decondense chromatin and that H2A.Z may cause the nucleosomes (i.e. interaction between the H2A.Z/H2B dimer and the H3/H4 tetramer) to be more stable (Fan et al, 2002). Fan et al. have gone on to suggest that the nucleosomal arrays are more stably folded which may in turn allow for higher order chromatin structure formation. It is obvious that the changes in the H2A.Z sequence affect the changes in the nucleosome. Whether those changes cause an overall stabilization or destabilization is debatable. Recently human H2A.Z was analysed 21 using recombinant H2A.Z and H2A.1 reconstituted onto 146 bp fragments obtained from chicken erythrocyte. The electrophoretic mobility and DNase I footprinting data suggested nucleosome properties that agree with the published crystal structure features found mXenopus (Suto et al, 2000). In a subsequent study Fan et al. have gone on to show that H2A.Z facilitates the intermolecular folding of nucleosomal arrays while at the same time inhibiting highly condensed structures (Fan et al, 2002). Either way it seems that H2A.Z may function to provide an alternate capability for nucleosome assembly or to generate alternate nucleosome structures and unique chromatin domains. The discovery of the S. cerevisiae homolog allowed, along with a powerful set of genetic tools, analysis of the H2A.Z class of variant in a relatively simple cell system. Loss of H2A.Z in S. cerevisiae affects recruitment of the R N A polymerase II transcriptional machinery to the GAL1-10 genes as well as their induction (Adam et al, 2001). Also, the H2A.Z C-terminus is sufficient to provide the variants the capability to function in positive regulation of gene expression (when tethered to chromatin appropriately). Most interestingly Adam et al showed that the C-terminal region is capable of interacting with transcriptional machinery, whether this association was direct or indirect could not be shown. The H2A.Z protein has been shown to regulate transcription positively by negating the need for chromatin remodelling complexes. Genetic studies in which H2A.Z was deleted suggests that H2A.Z is required for transcriptional activation (Santisteban et al, 2000). In contrast, ChIP analysis showed that H2A.Z preferentially associated with target promoters when their genes were repressed (Santisteban et al, 2000). Similarly H2A.Z is present at both GAL1 and GAL10 promoters prior to the addition of galactose, but upon the addition of galactose 22 the antibody signal at both promoters decreases with time (Adam et al, 2001). This suggests that H2A.Z may be important in establishing the state of active transcription (i.e. transcriptional competency) but may not be necessary for the maintenance of transcription. The association with chromatin under repressed conditions was investigated in a screen by Dhillon et al. H2A.Z was isolated independently when they looked for proteins that mediated silencing at the H M R locus. When over expressed the H2A.Z protein was able to over come the deletion of the Sirlp locus that is necessary for silencing. However, removal of H2A.Z gene only moderately de-represses the silenced loci, but increases silencing at the telomere (Dhillon and Kamakaka, 2000). The idea is that the H2A.Z protein leads to more stable binding of the Sir proteins through a specialized chromatic structure. Whether transcription or silencing is the major function in S. cerevisiae is still questionable (modifications of H4 have been shown to activate and repress transcription (Wyrick et al, 1999)). What we can see is that H2A.Z is necessary for chromatin integrity under certain physiological conditions (Adam et al, 2001). (e) Drosophila H2A.vD As mentioned previously the Drosophila variant is an excellent choice to study this class of variants. Drosophila melanogaster is an ideal choice as it is one of the most intensely studied organisms in biology. It serves as a model system for investigation of cellular and developmental processes than can be related to higher eukaryotes. The genome is substantially sequenced allowing for genomic comparison between organisms and allowing for the ease of nucleotide and amino acid searches. The Drosophila variant itself is interesting in its own regard as it is necessary and essential for viability of the fly. 23 It has been localized to both transcribed and non-transcribed genes. Its non-uniform distribution suggests a specific role in packaging and or regulation. Why is the H2A.vD variant essential? What does it do? With what proteins does it interact? These are the broad but essential questions surrounding these classes of variants. The H2A.vD is encoded by a single copy gene which is located within bands 97D1-9 on chromosome 3 and thus differs in position from the normal H2A gene which is reiterated approximately 110 times at bands 39E-F on chromosome 2. In addition H2A.vD is synthesized outside of the cell cycle and thus differs from the histone cluster in its regulation. The presence of a H2A box (protein sequence A G L Q F P V G R ) (von Holt et al, 1979) defines H2A.vD as an H2A class molecule (van Daal et al, 1988). The gene structure of H2A.vD differs from its core counterpart in that the coding sequence is interrupted by 3 introns. The 3 introns in H2A.vD are in the same position as the first 3 introns in the chicken H2A variant, and the mRNAs in both species are polyadenylated (van Daal et al, 1988). The H2A.vD protein appears to be essential. A 311 bp deletion that removes the second exon is lethal when homozygous and this exon does not appear to contain a "gene within a gene". Finally the recessive lethal phenotype was rescuable using P-element mediated transformation of a 4.1kb fragment containing the H2A.vD gene (van Daal and Elgin, 1992). 1.5 Previous Experiments The structure, expression patterns, its genomic position and lack of reiteration make H2A.vD an interesting protein and raise many questions about its role in gene expression and development. There were some previous experiments done in our 24 laboratory (just previous to my arrival) screening for proteins, and their genes, that interacted with H2A.vD. Dr. Richard Sobel performed a Protein Interaction Trap screen to identify putative proteins that interact with H2A.vD, and his data laid the groundwork for this dissertation. Dr. Sobel amplified an H2A.vD cDNA sequence by PCR and the amplified gene fragment was sub-cloned into the yeast bait construct (pEG 202). He used this "bait construct" expressing the Drosophila H2A.vD protein to screen a Drosophila cDNA library for proteins in a Protein Interaction Trap screen. The screen identified a number of cDNAs that encoded proteins that putatively interacted with H2A.vD in the Protein Interaction Trap screen. A set of cDNAs labelled A/C /E /H were represented 55 times (55 independent colonies) and cDNAs labelled D/F had 6 colonies, L had 44 colonies, and M had 53 colonies. As the H2A.vD clone used in the screen was subsequently found to be only approximately 70% accurate, due to a frame-shift alteration that added 7 novel amino acids and prematurely terminated the protein at the carboxyl-terminal end (sequencing done by G. Doheny), we termed it altered-H2A.vD (Fig. 1-4). For the sake of simplicity and since all the cDNAs were homologous, We have renamed A/C/E /H class as putative interacting protein-I (PIP-I), the D/F class as putative interacting protein-II (PIP-II), the L class as putative interacting protein-III (PIP-III), and the M class as putative interacting protein-IV (PIP-IV). These were all strong interactors with multiple colonies. The first goal was to determine whether the interactions detected between altered-H2A.vD and PIPs I through IV were true interactions. 25 (1) M a j o r H 2 A (2) H 2 A . v D O) A l t e re d-H2 A . V D (1) SG RGKGG K V K G _ K A KSRSNR (2) AGGKAGKDS GF . A K A K A V 3 R S A R (3) AGGKAGKDS G K A.KAKA V3 R S A R A G L Q F P Y G R I A G L Q F P V G R I AG LQF P V G R I H R L L R _ K G H Y A E HKELKS RTTSHG HRHLKSRTTSHG - / OTT—n m w — alpha-N helix alpha-1 helix loop 1 (1) R V G A G A F V Y L A A V M E Y L A A E V L E L A G H A A R D N K K T R I I P R H L Q L A I R N D (2) R VG A T A A V Y S A A ILi E Y L T A E V L E L AG W A SKD L K V K RIT P P B L Q LA. IR GD (3) R VGA T AAV Y S AA I L E Y L T A E V L E L AGT1A S KD L K VK RI TP PH L Q1A IR GD EELW EELD E E L D alpha-2 helix loop 2 alpha-3 helix (1) KL L S G VTIAQGG VL PWIQ A VL LPKKTEKE A (2) SLI KATIAGGGVIPHIEKSLIGKKEETYQDPQRKGNVILSQAY (3.) S L I KATIAGGGsfrtyts HSTKWRSSAKTAEPR (4) HGQQKTA HGSQKAT HGQQKTV h v l H 2 A . F H 2 A . F / Z H 2 A . Z W alpha-C helix Figure 1-4. Diagram of the Drosophila H2A, H2A.vD, and altered-H2A.vD amino acid sequences. The underlined letters represent the differences in amino acid sequence compared to the major H2A sequence. The lower case letters represent the novel C-terminal amino acids created by the alteration. The group of sequence marked by (4) denote the final terminal amino acid sequence of some other H2A.vD homologs (Tetrahymena, chicken, sea urchin and mammal respectively). The box denotes the H2A box. The brackets denote the histone fold motif. A schematic diagram of the secondary structure is represented below the sequence (modified from Clarkson et al. 1999) 26 1.6 Thesis Goals and Hypothesis (a) Goal/Hypothesis In order to help decipher the role of H2A.vD in chromatin and ultimately the cell, a helpful approach is to understand its interaction capabilities with other proteins and/or domains. The overall objective of my thesis is to gain an understanding of the role Drosophila H2A variant H2A.vD plays in the formation and structure of specific chromatin domains, and how that relates to gene expression. I would like to address questions regarding function. Specifically whether H2A.vD always associates with silencing or does it play a more positive role in determining transcriptional competency. One way of addressing these broad questions is to identify some or all of the other proteins with which a particular variant such as H2A.vD interacts. The specific goals of this thesis were: 1) to first confirm the interactions between the natural H2A.vD protein and one or more of the PIPs and 2) to identify the proteins with which it associates. This would allow me to infer function to H2A.vD by associating it with its interacting partners. By gaining knowledge of what a particular protein does can help one understand what cellular process the protein partners are involved in and what changes in chromatin structure and ultimately the cell they induce. The previous unpublished findings allow for an excellent starting point to begin addressing these goals. We will be able to get a glimpse into understanding H2A.vD's role and function in chromatin. We will hope to identify other proteins with which it interacts and which it might target to particular sites or aid in chromatin assembly. The PIPs elucidated from a previous global interaction trap screen (performed by R. Sobel) will act as the starting point for this research allowing for confirmation of protein-protein 27 interaction and further protein characterization (explained in chapter 2). The protein interaction trap has drawbacks in that it can give false positives, so a second technique to get around this problem was employed. The GST pull-down is a test of the ability of two proteins to physically interact directly. With this starting point (protein-protein interaction) well defined a combination of cell biology and genetics should provide the tools needed to address several of the larger issues concerning packaging and gene expression. This should elucidate the role H2A.vD has in gene expression, chromatin packaging and possibly development. (b) Findings This thesis will show that H2A.vD has the ability to interact with two of the proposed PIPs. One of these is a undefined Drosophila gene called CG5515 which may encode or recruit a protein kinase, the second identifies a sequence of unknown origin. Theses findings may contribute to the advancing knowledge of H2A.vD and histone variants in general. 28 Chapter 2: M A T E R I A L S A N D M E T H O D S 2.1 Cloning / Inserts (a) Protein Interaction Trap /Protein Expression Vectors (see sect. 2.2/2.5 for theory) (i) Reporter Vector (Interaction Trap) The reporter gene is expressed if, and only if, there is a protein-protein interaction. The vector used to report on protein-protein interactions in the Protein Interaction Trap system is labelled pSH18-34 (10.3kb). It is a derivative from LR1A1 (West et al, 1984). It uses the LacZ reporter gene to produce the enzyme P-galactosidase, which is easily detectable due to blue colouration on X-Gal or yellow colouring with ONPG as a substrate, to confirm protein-protein interactions. It contains one LexA binding site inserted into the GAL1 promoter. It contains the URA3 selectable marker and 2 pm origin for selection and replication respectively, in yeast. The ampicillin resistance gene, and pBR origin allow for selection and replication in bacteria (Fig. 2-la). (ii) Bait Vector (Interaction Trap) pEG202 (10166 bp), a variation of Lex202 + PL (Ruden et al., 1991), is the vector used to create the bait fusion where the protein or domain of interest is fused to the LexA binding domain. The D N A inserts (in this case H2A.vD, altered H2A.vD, and H2A) were engineered into the EcoRI / Xhol (or Sail) sites of the MCS, fusing the sequence of interest to the D N A sequence of the D N A binding protein LexA. The A D H promoter drives expression of this fusion sequence ultimately producing a "protein of interest-LexA fusion protein". The plasmid contains the HIS3 selectable marker and 29 2um origin for selection and replication in yeast. The ampicillin resistance gene, and pBR origin allow for selection and replication in bacteria (Fig. 2-lb). (iii) Prey Vector (Interaction Trap) pJG4-5 (6449 bp) (Gyuris et al, 1993) is the vector used to generate the prey fusion protein where the sequences from a cDNA library (when conducting a screen) or a sequence of interest (when administering a direct test as in the experiment used here) is fused to a transcription activating or trans-activating domain. The D N A inserts (PIP D N A sequences I, II, III, or IV) were engineered into the EcoRI / Xhol M C S , fusing the sequences to a fusion cassette consisting of the SV40 nuclear localization sequence (NLS; P P K K K R K V A ) , the acid blob B42 domain (Ruden et al, 1991), and the hemagglutinin (HA) epitope tag ( Y P Y D V P D Y A ) capable of inducing transcription. Expression of the cassette is under the control of the GAL1 galactose inducible promoter. This allows for expression when galactose is in the media but repression when glucose is the sugar source. The plasmid contains the TRP1 selectable marker and 2um origin for selection and replication in yeast. The ampicillin resistance gene and the pUC origin allow for selection and replication in bacteria (Fig 2-lc). (iv) GST Fusion (Protein Expression) The vector used to fuse the glutathione S-transferase domain to the protein of interest is labelled pGEX4T- l (4900 bp). The insert (H2A.vD or H2A) was engineered into the EcoRI / Xhol (or Sail) sites of the MCS, fusing the H2A.vD or H2A sequence to the glutathione S-transferase (GST) sequence. When expressed the fusion protein will be c) Figure 2-1. Diagrammatic representation of the plasmid vector constructs used in the Protein Interaction Trap assay. Names in brackets represent names of sequences inserted into the M C S of the plasmids using EcoRI / X h o l restriction sites (EcoRI / Sal I for H2A). (a) Bait plasmid and (b) Prey plasmid. (c) is the lacZ reporter plasmid. 31 able to bind the Glutathione Sepharose resin and allow for purification of the GST fused proteins. The vector contains a tac promoter for high-level expression, an internal lac I q gene for E. coli. The ampicillin resistance gene allows for selection in bacteria. (Fig. 2-2a) (v) Protein Expression Vector (Protein Expression) The pET28a vector (5369 bp) is used for protein expression. The D N A inserts (PIP D N A sequences I, II, III, and IV) were cloned into the EcoRI /Xhol M C S and transcription occurred using the T7 R N A polymerase. 3 5 S was the radioisotope used in the in vitro transcription / translation reactions for expression and subsequent detection of the protein products. The Kanamyacin resistance gene allows for selection in bacteria (Fig. 2-2b). (b) H2A.vD 3' Partial Deletion PCR A 3' partial deletion was constructed to create a 3' truncation of the H2A.vD sequence (called H2A.vD-Tr). The truncated H2A.vD gene was made using PCR. The sequence was amplified out of the pEG202 construct using the pEG202F primer (2.2c) and the H2AZ3Tr primer (5 ' -CAC A T A C A C A A G T A G C T G A T C G G C CTC G A G -S'). The 3' primer is located from base pair 324 to base pair 334 this amplified product lacks the last 72 base pairs translating to 24 amino acids. Conditions for the PCR amplification was as follows: 1 pi of a 1:200 dilution of a bacterial mini-prep containing the H2A.vD insert in the pEG202 vector, 2.5 pi of lOpM of both 5' and 3' primers, 5.0pl of a lOpM stock of dNTPs, 5.0 pi of Pfu buffer (Fisher), l.Opl of Pfu enzyme (Fisher). a) b) H2A H2A.vD EcoRI Xhol P pGEX4T-1 \ GST-fusion" 4932 bp AMP r on III KAN r c f1 origin Putative Putative II Putative III EcoRI Putative IV pET28a "expression vector" 5369 bp H lad \ .,. on Figure 2-2. Diagrammatic representation of plasmid constructs used in protein expression for GST-pulldown experiments. Names in brackets represent sequences cloned into the M C S of the plasmids using EcoRI / Xhol restriction sites (EcoRI / Sal I for H2A). (a) GST fusion plasmid (b) protein expression plasmid used for in vitro transcription and translation. 33 The PCR reaction proceeded as follows using the PTC-100 Programmable Thermal Controller: 1) 45 seconds at 96°C, 2) 45 seconds at 60°C, 3) 2 minutes at 72°C, repeat 30 times, 10 minutes at 72°C, then held at 4°C. (c) Cloning Strategy The H2A and H2A.vD sequences were subcloned from the Bluescript vector pBKs. The inserts were removed using the restriction enzymes EcoRI and Xhol for H2A.vD and EcoRI and Sail for H2A and ligated into the bait vector (pEG202) in the EcoRI / Xhol and EcoRI / Sail sites within the MCS of the bait vector. The altered-H2A.vD-pEG202 construct and the four PIP-prey (pJG4-5) constructs were attained from the original Protein Interaction Trap screen. The chart below highlights the cloning strategy and the size of the inserts in each construct. 34 Table 1: Cloning Strategy: Protein Trap Interaction Insert (DNA sequences) Vector Enzymes used for insert & vector Digestion Insert Size H 2 A . v D pEG202 "Bai t" EcoRI X h o l 431 bp al tered-H2A.vD pEG202 "Bai t " EcoRI X h o l 429 bp H 2 A pEG202 "Bai t" EcoRI Sa i l 404 bp 3 "Deletion H 2 A . v D pEG202 "Bai t" " EcoRI X h o l 350 bp PIP-I pJG4-5 "Prey" EcoRI X h o l 838 bp PIP-II pJG4-5 "Prey" EcoRI X h o l 537 bp PIP-III pJG4-5 "Prey" EcoRI X h o l 393 bp P I P - I V pJG4-5 "Prey" EcoRI X h o l -1 .8 kb The H2A and H2A.vD sequences were subcloned from the Bluescript vector pBKs. The inserts were removed using the restriction enzymes EcoRI and Xhol for H2A.vD and EcoRI and Sail for H2A and ligated into the GST vector (pGEX4T-l) in the EcoRI / Xhol and EcoRI / Sail sites within the MCS of the GST vector. The four PIP D N A sequences were removed from the prey vector using an EcoRI / Xhol digestion and 35 ligated into the EcoRI / Xhol digested MCS of the pet28a expression vector. The chart below highlights the cloning strategy and the size of the inserts in each construct. Table 2: Cloning Strategy: GST Pull-down / Protein Expression Insert Vector Enzymes used for insert & vector Digestion Insert Size H2A.vD pGEX4T-l "GST" EcoRI Xhol 431 bp altered-H2A.vD pGEX4T-l "GST" EcoRI Xhol 429 bp H2A pGEX4T-l "GST" EcoRI Sail 404 bp PIP-I pET28a "expression" EcoRI Xhol 838 bp PIP-II pET28a "expression" EcoRI Xhol 537 bp PIP-III pET28a "expression" EcoRI Xhol 393 bp PIP-IV pET28a "expression" EcoRI Xhol -1.8 kb (c) Sequencing A l l of the vector and insert constructs were verified after construction but prior to use via D N A sequencing. Reactions were carried out using the "Protocol for automated sequencing on the 373 D N A sequencer" available from the Nucleic Acid-Protein Service (NAPS) Unit at the University of British Columbia. Upon completing reactions the 36 products were sent to the NAPS unit for sequencing. Primers used for sequencing specific for the bait were (p202F) 5'- CGT C A G C A G A G C TTC A C C ATT G-3' and (p202R) 5'-ACC TGA G A A A G C A A C CTG ACC-3' . Prey specific primers were (BC01) 5'-CCA GCC TCT TGC TGA GTG G A G ATG-3' and (BC02) 5'-GAC A A G C C G A C A A C C TTG ATT GGA-3'. Primers specific for the GST-fusion vectors were (GEX5 SQ) 5'-GGG CTG G C A A G C C A C GTT TGG TG-3' and (GEX3 SQ) 5'-CCG G G A GCT G C A TGT GTC A G A GG-3'. 2.2 Protein Interaction Trap Direct Interaction (a) Theory The Protein Interaction Trap (an independent version of the Yeast Di-hybrid system) developed by R. Brent and M . Ptashne, is a test for protein-protein interactions is based on re-constituting of two domains that when brought together are capable of transcription. The first is a D N A binding domain (i.e. the D N A binding domain from the E.coli repressor protein LexA) and the second is a transcription activating domain (i.e. B42 acidic domain from E. coli). The reconstitution is brought about by the interaction of two heterologous proteins one of which is fused to the D N A binding domain (bait) while the second heterologous protein is fused to the activation domain (prey). The bait plasmid (pEG202) is used to express the product of the LexA D N A binding domain fused to one of the H2A or H2A variants. The bait fusion product must not be able to activate the reporter construct on its own. This construct is then transformed into a yeast strain (EGY48) carrying a reporter plasmid (pSH18-34) and a chromosomal reporter gene. There are LexA binding sites located upstream of these two reporter genes. First is the 37 chromosomal LEU2 gene in which the normal activating sequences have been replaced. When this gene is activated the cells are able to complete the biosynthetic pathway for leucine and are viable on media lacking leucine. The second is the lacZ gene housed on the pSH18-34 plasmid. This gene contains a LexA binding site upstream and allows for selection based on colour due to the production of p-galactosidase in the presence of X -gal or another substrate. The activation of these genes only occurs when the B42 acid blob transactivating domain is brought to the site and thus induces transcription. When testing for interactors the interactor of interest is fused to the B42 domain in the pJG4-5 (prey) vector. This fusion is only expressed in the presence of galactose as it is controlled by the GAL1 promoter. If when the bait fusion is expressed the "bait" (in this case either H2A.vD, H2A or altered-H2A.vD) interacts with the "prey" (i.e. PIP-I, II, III, or IV fused to the transactivating domain), then the transactivating domain will be brought to the D N A by the D N A binding domain of LexA via the protein-protein interaction, and activation of the reporter genes will occur. If there is no interaction between the proteins of interest then the transactivating domain and the D N A binding domain never come in contact and transcription of the reporter genes never occurs, (for cloning and vectors see 2.1 i , ii,& iii) (b) Yeast strain EGY48 was the yeast strain used for the interactor hunt. It has the genotype of ura3, trpl, his3, lexA operator-LEU2 to allow for selection of the bait, prey, and reporter plasmids. 38 (c) Media For rich Y P D liquid media, which allows for healthy luxuriant growth of yeast cells, lOg yeast extract, 20g peptone, 20g dextrose was dissolved in 1 L of dti^O. For plates 20g agar and one pellet of NaOH were added. For complete minimal (CM) media, which allows for selection based on absent amino acids, 1.3g of dropout powder (missing amino acids needed for selection) 1.7g yeast nitrogen base without amino acids (YNB-A A ) , 5g (NH4 )2S04 , and 20g dextrose was dissolved in IL. For solid plate media 20g of agar and one pellet of NaOH was added. For liquid inducing media, which induces the expression of the prey construct driven by the GAL1 promoter, the sugar (dextrose) was replaced with 20g galactose and lOg raffinose. Cells were allowed to grow in this media in a 30°C incubator. Liquid cultures were kept shaking at a 45° angle at 200 rpm. (d) Yeast Transformations Yeast transformations were carried out as described in "The L i A c TRAFO Method" (Agatep et al, 1998). Each vector was transformed independently. Transformations were verified via yeast genomic mini-prep and subsequent PCR using bait and prey specific primers outlined in the sequencing section (2.1c). 2.3 P-Galactosidase Plate Assay The protocol was modified from (Duttweiler, 1996). Transformant colonies were allowed to grow for 2-5 days. The colonies were flooded with chloroform and allowed to sit for 5 minutes under the fumehood. The plates were overlaid with 1% low melt agarose containing 1 mg/ml X-Gal and lOOmM K P 0 4 p H 7.0 and cooled to 42°C. Once hardened the plate was inverted and incubated at 30°C for 24-48 hours. 39 2.4 Liquid P-Galactosidase Quantification Assay Assay was completed using modifications of the Assay for P-galactosidase in liquid cultures from Current Protocols in Molecular Biology supplement 39 (Wiley & Sons, Inc, 1997). (a) Growth and Preparation of Cells Five ml of complete minimal media (-uracil (U), -tryptophan (W), -histidine (H), -leucine (L) + Galactose and Raffmose (Gal/Raf)) was inoculated with a single yeast colony containing the H2A.vD-bait fusion plasmid, the PIP-prey fusion plasmid (containing either the group I, II ,111, IV PIP D N A insert) and pSH18-34, the lacZ reporter plasmid. This was done in duplicate for all 4 H2A.vD histone variant / PIP interaction tests. These cultures were grown to a density of 3.0x107 cells/ml at 30°C. This took differing amounts of time depending on the prey-fusion construct. Three ml of synthetic drop-out media (-U, -H, -W, -L, + Gal/Raf) was inoculated with 50ul of each culture. These cultures were grown to mid-late log phase (~2-5xl0 7 cells). Cells were centrifuged for 5 minutes at 2,100 rpm in a DAMON/IEC HN-S tabletop centrifuge. Cells were re-suspended in 1ml of Z buffer and placed on ice. Reaction tubes were set up containing 50ul of cells and 450ul of Z buffer. 15ul of 0.1% SDS and 30ul of chloroform were added to each sample. Tubes were vortexed and were set to equilibrate for 15minat30°C. 40 (b) Assay for B-galactosidase activity 200ul of 4 mg/ml ONPG was added to cells and then agitated for 5 seconds. The samples were then placed at 30°C and timing was started. When a medium yellow colour developed the reaction was stopped by adding 250pl of 1 M Na2C03 and the time was noted. The cells were centrifuged for 5 minutes at 11,000 x g. OD 4 2o and O D 5 5 0 of the supernatant was determined. The units were calculated using the following equation: U= 1000 x IYOD420HOD550)1 (t) x (v) x (OD600) 2.5 GST Purification Pull-downs (a) Theory GST pulldowns are a type of chromatography or co-immunoprecipitation in which one protein is able to associate with another protein and effectively "pull it down" out of solution. This association is a direct physical interaction between the two proteins and serves as an excellent reinforcement for experiments where protein-protein interactions are thought to have occurred, as in the Protein Interaction Trap method. This pulldown process is performed by creating a glutathione S-transferase (GST)- protein of interest fusion and subsequently purifying it using Glutathione Sepharose. An in vitro transcribed and translated product (i.e. PIP-I, II, III, or IV) containing radioactive S is then added to the GST fusion product. If there is an association directly between the GST-fusion protein and the radio labelled PIP, the two proteins will remain associated after washing. This physical interaction is detected by isolating the GST fusion protein using Glutathione Sepharose beads. The samples are then isolated on a SDS polyacrylamide gel; he gel is then exposed to allow for detection of the 3 5 S from the 41 interactor that was associated with the GST-fusion protein (for cloning and vectors see 2.1 i v & v ) . (b) GST-Fusion Protein Expression 3ml cultures of H2A.vD-GST, H2A-GST, GST construct containing E.coli were inoculated and allowed to grow overnight. 500ul of this culture was used to re-inoculate a 10ml culture. These were grown for 2-3 hours until cultures were thick and rich. Total volume was then used to inoculate 100ml. This was grown to an OD of 0.5 (X=600nrn). 10 ml of each culture were removed and labelled uninduced. 100ml cultures were induced with IPTG (Sigma) (ImM final concentration). Induction was allowed to proceed for 20-24 hrs. Cultures were then spun down using DAMON/IEC HN-S tabletop centrifuge at maximum speed and supernatant removed. The pellets were stored at -70°C. (c) GST Fusion Isolation/Solubility Protocol (i) Isolation Cells were re-suspended in 5-10 ml PBS + protease inhibitors (Apoprotinin 2pg/ml, Leupeptin, 2ug/ml, Pepstatin A, 2tig/ml, PMSF, 2pg/ml). Glass beads were added. Suspension was sonicated at 15-second intervals 3-4 times, with 30-second incubation times, on ice, between pulses. Cells were spun at 14K at 4°C for 20 minutes. Separately the supernatant and pellet were stored at 4°C. Supernatant was then run along side a dilution series of BSA (10,5,2,1, 0.5 ng) to determine concentration of the soluble fraction. 42 (ii) Purification To 1ml PBS/0.1%NP40 5ul of soluble GST and 20ul of H2A.vD and H2A fusion were added. 20ul of a 50% slurry of glutathione resin and lul of PMSF in DMSO were added. This was mixed on a platform rocker for 30 minutes. Wash using spins of 14K for 10 minutes. Rinse I X with PBS/0.1% NP40, I X with PBS/0.l%NP40/500mM NaCl, and I X with PBS. (d) In vitro Transcription / Translation Reactions In order to generate labelled PIP proteins in vitro transcription / translation methods were employed using 3 5 S to radioactively label the PIP translation products. The four reactions (one for each of the PIPs) were performed according to the "General Protocol for TNT® Quick Coupled Transcription/Translation Reactions" found in the Promega Technical Manual for TNT® Quick Coupled Transcription/Translation Systems. (e) Co-purification / Pulldown Protocol One microphage tube of H2A.vD-GST fusion protein and two tubes of GST protein alone were used for each of the four interaction tests. To the tubes lOOOul of PBS, 0.1% NP40, - l u g of H2A.vD fusion protein (or GST alone), l u l of 0.5M PMSF in DMSO and 20ul of 50% slurry of glutathione agarose beads (Amersham pharmacia) were added. The tubes were then placed on a rocking platform at 4°C for 30 minutes, then washed once with PBS/0.1% NP40 and once with PBS/0. l%NP40/500mM NaCl, and once with PBS alone. For each of the four in vitro translated products GST + 43 glutathione agarose beads, 450ul PBS, 50ul in vitro translation reaction, and 1 pi PMSF in DMSO were added together in a microfuge tube. These reactions were then placed on a rocking platform for 30 minutes. After 30 minutes the tubes were spun and the in vitro translation dilution was drawn off and saved to another tube. The blocking reaction contained H2A.vD-GST fusion absorbed to the glutathione beads (or GST alone absorbed to beads), 1ml of PBS, 2% BSA, l p l PMSF in DMSO (0.5M). This was placed on a rocking platform for 1 hour. NP40 was added to the blocked beads in solution at a concentration of 0.1%. An excess volume of precleared in vitro translation reaction was then added. This was placed on a rocking platform at 4°C for 90 minutes. The binding reaction was then washed twice with PBS/0.1% NP40, twice with PBS/0.l%Np40/500mM NaCl, and twice with PBS alone. After washing 15pl of 2x SDS loading buffer was added. This was heated at 65°C for 10 minutes (along with prestained marker) and run on a 10% polyacrylamide gel was then dried with the gel dryer (Bio Rad) for 1.5 hours at 60°C, then developed using Storm Imager for 4 hours at room temperature and visualized using ImageQuant. 2.6 Database Searches The sequences of the PIPs attained from the Protein Interaction Trap screen were submitted to the B L A S T search engine at the National Centre for Biotechnology website (NCBI http://www.ncbi.nlm.nih.gov/). The searches used were the blastn (nucleotide BLAST), blastp (protein blastp), blastx (converts a nucleotide query sequence into protein sequences in all 6 reading frames, the translated protein products are then compared against the NCBI protein databases), tblastn (takes a protein query sequence and compares it against an NCBI nucleotide database which has been translated in all six reading frames), tblastx (converts a nucleotide query sequence into protein sequences in all 6 reading frames and then compares this to an NCBI nucleotide database which has been translated in all six reading frames), CD-search compares a protein sequence against the Conserved Domain Database with the RPS-BLAST program (this database currently contains domains derived from two popular collections, Smart and Pfam, plus contributions from colleagues at NCBI), and PSI B L A S T (Position specific Iterated B L A S T used for iterative searches in subsequent rounds). Generated sequences were then used for putative gene searches using the FlyBase database (http://flybase.bio.indiana.edu). 2.7 Southern Blot Analysis Approximately lOug of each sample (D. melanogaster, SF9, S. cerevisiae, E.coli, and murine genomic D N A digested with Eco RI overnight) were electrophoresed in a 0.8% agarose gel in a 11x14 Horizon gel apparatus for 3hrs at 50V. Blotting and washing procedures were completed as described in Amersham LIFE SCIENCE Hybond™-N + nylon membrane handbook with the following revisions. A l l washes were done with SSC. The Blot probed with the PIP-II D N A sequence had 2 washes with 2x SSC, 0.1% SDS for 10 minutes at room temperature. The probe was developed using Boehringer Mannheim Biochemica Random Primed D N A Labelling Kit. The PrP-U D N A sequence of 544 bp. was used as the template D N A for the reaction. A PCR product of Labelling was completed as described in the Random Primed D N A labelling kit protocol. The likely hood homologous sequence detection was governed by the fact that the Tmof a double stranded D N A decreases by 1-1.5°C with every 1% decrease in homology (Bonner et al, 1973). 2.8 Genomic P C R 1 ng of Drosophila genomic D N A was used as the substrate for a genomic P C R reaction. Primers specific for the PIP-II D N A sequence, (DFfr) 5'- G G C A C G A G G C G T G A G C G T C A G - 3 ' and (DFrv) 5'- C G G G C C G T G C A A A C T C T T - 3 ' were used to attempt to amplify the genomic sequence from the sample. A s a positive control H D A C 1 (histone deacetylase 1), a single copy gene, was amplified from the genomic D N A sample. Primers for the P C R reaction used to amplify H D A C 1 , were (RPD3-5) 5'-C G G T T A G G C T G C T T C A A T C T - 3 ' and (RPD3-3) 5 ' - T A T T G G T A T T A G A C G C C G T C G ) - 3 \ The reaction conditions were as follows: l p l of lng /p l of a genomic prep of Drosophila melanogaster D N A , 2.5 pi of l O p M of both 5' and 3" primers, 5.0pl of a l O p M stock of dNTPs, 5.0 pi of Pfu buffer (Fisher), l .Opl of Pfu enzyme (Fisher). The P C R reaction, using the PTC-100 Programmable Thermal Controller, proceeded as follows: 1) 45 seconds at 96°C, 2) 45 seconds at 60°C, 3) 2 minutes at 72°C, repeat 30 times, 10 minutes at 72°C, then held at 4°C. As a positive control for the PIP-II D N A sequence primers, a separate P C R reaction was run using l p l of a 1:200 dilution of a plasmid mini-prep containing the PIP-II D N A sequence in the pJG4-5 (prey) vector as the source D N A . 46 2.9 Genetic Interaction Crosses Genetic interaction is often used as one of the criteria for establishing protein-protein interactions in vivo. Hence, we decided to look for interactions in or encompassing H 2 A . v D and CG55515. The following crosses were done to test for lethal, sterile, or morphological aberrancies. The phenotypic markers used were stubble (sb), a dominant allele (homozygous lethal) in which the Drosophila bristles are less than one half the normal length and thicker than wild-type; ebony (e), a recessive allele when in the homozygous state causes body colour to vary from shining black to slightly darker than wild-type; scarlet (st), when in the homozygous state causes eyes to be bright vermillion, darkening with age; and beaded-serrate (bds)(ser), a dominant allele in which Drosophila wings are notched at the tip, homozygousity produces extreme incision of wing margins. Balancer chromosomes are used to maintain a homologous chromosome in a stock or cross by containing multiple inversions. They are usually homozygous lethal, and are dominantly marked. The balancer chromosome used to prevent recombination in these experiments were T M 3 marked with sb and ser separately, which effectively balances chromosome 3. (a) Genetic Interactions. The deficiencies Df(3R)crb87-4 stl, el (breakpoints spanning 95D11-E2; 96A2 determined cytologically) and Df(3R)crb-F89-4 stl, el (breakpoints spanning 95D7-11; 95F15 determined cytologically) should encompass the hypothetical gene CG5515 ( bands 95E4-5) (Bloomington stock BL-2362 and BL-4432 respectively). Flies heterozygous for these deficiencies were crossed separately to flies heterozygous for the 1(3)810 H 2 A . v D mutation. The 1(3)810 mutation removes exon 2 of H 2 A . v D and the 47 surrounding region but keeps the rest of the protein in frame. It is homozygous lethal but viable as a heterozygote. Reciprocal crosses were done. In each cross, vials contained 10 females and 5 males and a minimum 600 progeny were examined from each cross. A l l crosses were carried out at room temperature. (b) Test for Fecundity/ Fitness These crosses were done to determine if there were any genetic effects on the fecundity of the F l generation (trans-heterozygotes) produced from the crosses between flies with the H2A.vD mutation and flies with the deficiency mutations. The fecundity was checked by self crossing Df(3R)crb87-4/l(3)810, Df(3R)crb-F89-4/l(3)810, 1(3)810/TM3, Df(3R)crb87-4/TM3 ser, and Df(3R)crb-F89-4. Vials contained 10 females and 5 males. Files were scored until 250 flies from the control cross (l(3)810/TM3 sb) were scored. Crosses were carried out at room temperature. The number of next generation flies were compared in order to determine i f there was a loss of fecundity/fitness (i.e. were there less offspring produced) in the original F l parents (Df(3R)crb87-4/l(3)810 and Df(3R)crb-F89-4/l(3)810). 2.10 Lethal Phase 1(3)810/1(3)810 flies were allowed to mate in the dark for 48 hours at room temperature. The mated adults were then placed into empty glass bottles which were fitted with adapters attached to egg lay plates, composed of 2% agar which had a small smear of active yeast paste in the centre, to capture egg deposits. Embryos were collected after 24 hours and placed on dampened construction paper (100 embryos per piece). These were inserted into fly food contained in vials, one piece per vial. Embryos underwent development at room temperature for 72 hours. Those left behind were counted as either unfertilized, broken, or dead. Larval lethality was determined by subtracting the number of pupae from the number of hatched eggs (larvae). 49 Chapter 3: R E S U L T S There is increased interest in histone variants including The Drosophila histone variant H 2 A . v D , since these paralogous histones provide a possible mechanism for governing chromatin regulation and gene control in development. The role of these variants in chromatin assembly at specific developmental stages or tissue types, the type of post-translation modifications they undergo, and the proteins they interact with, are the main questions that need to be addressed. The results presented here attempt to add information to this growing body of knowledge on the H 2 A variants, specifically H 2 A . v D . The main focus of my efforts was to identify genes whose products interact with H 2 A . v D . This should lay the groundwork for further characterizing the newly discovered interactions themselves. The over all research goal was to determine the possible function of the Drosophila H 2 A histone variant H 2 A . v D . This was to be done by determining the authenticity of four previously determined H 2 A . v D interactors (R. Sobel, unpublished) by multiple methods, namely the Protein Interaction Trap direct test followed by G S T Pulldowns for conformation. This was not simply a validation of previous results. There was mutation in the H 2 A . v D sequence that was used as the "bait" in the initial Protein Interaction Trap screen. The mutation was discovered subsequent to the screen was initially carried out. The mutation caused a frameshift that created a stop codon at amino acid 116 (truncating the final 25 amino acids and adding 7 novel amino acids before the truncation). Since this mutation altered the C-terminal tail o f H 2 A . v D the first question We needed to address was whether any of the PIPs actually interact with H 2 A . v D or H 2 A or are these interactions specific to the errant version of the H 2 A . v D protein. 3.1 Protein Trap Direct Interactions The original Protein Interaction Trap screen identified four putative interacting proteins (PIPs), using the above mentioned altered form of the H2A.vD protein. To determine whether any or all of these interactions were true. We employed a direct test for interaction between H2A.vD and the four PIPs. We used the Protein Interaction Trap system except the cDNA for each individual PTP was cloned into the prey vector substituting for the prey-library that was used in the initial screen. The interactors were then retested directly with this method (i.e. a direct test betweenH2A.vD "bait" construct and, separately, each PIP "prey" construct) in order to substantiate or refute the previous unpublished results. (a) Alter•ed-H2A.vD /Putative Interacting Proteins Initially the unpublished data presented four key putative interacting proteins (PIPs) with H2A.vD. These results were achieved with an altered form of the H2A.vD protein (altered-H2A.vD) (Fig 1-4). Confirmation of previous the previous results were necessary in order to ensure the technique was repeatable. The above-mentioned direct test was done in order to retest the interaction capability of altered-H2A.vD with each of the four PIPs. The Su(Var)3-9 / HP-1 interaction was used as a positive control for the interaction (Suppressor of variegation 3-9 and Heterochromatic Protein 1 respectively) as they have been shown to physically interact in vitro and give a strong positive signal in the protein interaction trap system using either the Leu2 or LacZ reporters. (Schotta et al., 2002). The question is whether altered-H2A.vD has the ability to physically interact with any of the four PIPs in the Protein Interaction Trap direct test. The Figure 3-la result shows that all required constructs are in the yeast cell and functioning properly. 51 Specifically it shows all four of the yeast altered-H2A.vD / PEP transformants (and the positive control) exhibiting luxuriant growth on media containing - U , -W, -H, +Dextrose (i.e. media lacking uracil, tryptophan, histidine, with Dextrose as the sugar source), after a 48 hour incubation period at 30°C. Each colony (labelled i , i i , i i i , and iv), represent separate independent isolates from the original yeast transformation in which the "bait", "prey" and reporter plasmids were transformed into the EGY48 yeast strain. This plating is described as the master plate. Here selection occurred strictly based on the resistance conferred by the transformed plasmids (bait-pEG202 encoding HIS3, prey-pJG4-5 encoding TRP1, and LacZ reporter pSH18-34 encoding URA3). The result shows that all of the vectors are intact and capable of producing the required proteins to allow the yeast cell to survive on the selection media but does not give information regarding the possible interactions. Figure 3-lb is a test for self-activation or contamination. This figure shows the inability of the altered-H2A.vD / PIP yeast transformants to grow on the negative control plate, containing the - U , -H, -W, -L, +Dextrose media, after a 96 hour incubation period at 30°C. The absence of leucine in the media does not allow for the cells to grow (none of the plasmids have selectable markers enabling the cells to grow without leucine in the media). In this instance the only way for cells to grow would be i f the H2A.vD-Lex A fusion (i.e. bait) were to act as a trans-activation factor itself and essentially "self activate" the chromosomal leucine reporter gene in the EGY48 yeast strain or that the growth seen on the master plate was due to some form of contamination able to grow on the master plate selection media. The prey-PIP fusions, containing the 52 a) Master Media: -U.-H.-W, + Dex b) Negative Control Media -U.-H.-W.-L + Dex Altered H2A.vD - putative I r w i i T i i ii iii iv (+) i ii iii iv (+) Altered H2A.vD - putative II i ii iii iv (+) i ii iii iv (+) Altered H2A.vD - putative III © Q Q 9 w i ii iii iv (+) i ii iii iv (+) Altered H2A.vD - putative IV i ii iii iv (+) i ii iii iv (+) Figure 3-1. Altered-H2A.vD-putative Protein Interaction Trap direct test platings, (a) Master Plate displaying four separate isolates from original transformation plate (containing bait, prey and reporter plasmid constructs) plated on media selecting for the plasmids. (b) Negative control. Similar to (a) with the exception of leucine being absent in the media. 53 see "self activation" or contamination in this result. Figure 3-2a shows the results of the test for interaction ability using the Leu2 reporter gene. Specifically it shows the ability of the yeast colonies containing the altered-H2A.vD and PIP fusion constructs along with the reporter plasmid to allow for growth on the experimentation media containing - U , -H, -W, -L , +Galactose. This media is known as selection/induction media as the missing amino acids constitute the "selection" while the galactose is responsible for "induction" of the "prey" fusion construct. With the prey-fusion plasmids induced by the galactose they allow for the protein-protein interaction to take place. If a successful interaction takes place the chromosomal Leucine reporter gene is activated due to the "bait" fusion interacting with the "prey" fusion which can then initiate transcription of the Leu2 gene allowing for growth on media lacking leucine. These results presented here agree, and therefore confirm repeatability, with the previous unpublished findings (R. Sobel) that the altered-H2A.vD has the ability to interact with the four PIP fusions in the Protein Interaction Trap system. A confirmation of physical interaction in the Protein Trap system is the expression of the extra-chromosomal reporter LacZ located on the plasmid pSH18-34. This promoter also contains a Lex-A binding site. With the protein-protein interaction taking place the trans-activating domain is brought to the promoter and activation occurs producing P-galactosidase. p-galactosidase produces a blue colouration in the presence of X-gal. The amount and intensity of the colouring indicates the strength and or duration of the interaction. Figure 3-2b shows the test for interaction ability using the LacZ reporter gene. A l l of the alterd-H2A.vD / PIP transformants produced the blue 54 a) Galactose Induction b) (3-Galactosidase Assay Media: -U,-H,-W,-L + Gal/Raf Media -U,-H,-W,-L+ Gal/Raf + X-Gal Altered H2A.vD - putative 1 i ii iii iv (+) I f ','.[% ***** • i ii iii iv (+) Altered H2A.vD - putative II i ii iii iv (+) i ii iii iv (+) Altered H2A.vD - putative III 19 0 0 © £ ^^^^H - 1 i ii iii iv (+) i ii iii iv (+) Altered H2A.vD - putative IV L U L I i ii iii iv (+) i ii iii iv (+) Figure 3-2. Altered-H2A.vD-putative Protein Interaction Trap direct test platings, (a) Four separate isolates from original transformation plate (containing bait, prey and reporter plasmid constructs) plated on media selecting for the plasmids and protein-protein interactions, (b) Similar to (a) with X - G a l laid over the transformant colonies. 55 W, -L, +Galactose) (Fig. 3-2b). Qualitatively, the altered-H2A.vD / PIP-I and PIP-H transformants seem to show the greatest amount/intensity of colouration. This was consistent with the previous unpublished findings. (b) H2A.vD /Putative Interacting Proteins Whether or not the proper version of H2A.vD had the ability to interact with the four PIPs was the next question that needed to be addressed. A repeat of the preceding experiment using the native H2A.vD in place of the altered-H2A.vD construct was performed. The H2A.vD / PIP yeast transformants as well as the positive control also show the ability to grow on the master plate media (-U, -H , -W, +Dextrose) confirming that the plasmids are intact and expressing the nutrient gene products (Fig. 3-3a). Figure 3-3b shows the inability of the H2A.vD / PIP yeast strains to grow on the negative control media (-u, -H , -W, - l , +Dextrose) demonstrating that the H2A.vD-LexA fusion does not "self activate" and that the colonies seen on the master plate are not contaminants. The results in figure 3-4a show whether the initial unpublished findings using the altered-H2A.vD construct were comparable to the physical interaction capability of H2A.vD with the four P IP-fusion products. Here we ask whether H2A.vD can interact with of the four PIPs. As we can see in Figure 3-4a, the H2A.vD / PIP-I transformants were unable to grow on the inducing/selection media (-U, -H , -W, -L , +Gal) suggesting the inability for the H2A.vD-LexA fusion to interact with the PIP-I fusion protein. The H2A.vD / PIP-II and PIP-III strains exhibit growth on the 56 a ) Master b) Negative Control Media: -U.-H.-W, + Dex Media -U,-H,-W,-L + Dex H2A.vD - putative I i ii iii iv (+) i ii iii iv (+) H 2 A . V D - putative II i ii iii iv (+) i ii iii iv (+) H 2 A . V D -- putative III W W W W w | i ii iii iv (+) i ii iii iv (+) H 2 A . V D - putative IV L M L M W I J i ii iii iv (+) i ii iii iv (+) Figure 3-3. H2A.vD-putative Protein Interaction Trap direct test platings, (a) Master Plate. Four separate isolates from original transformation plate (containing bait, prey and reporter plasmid constructs) plated on media selecting for the plasmids. (b) Negative control. Similar to (a) with the exception of leucine being absent in the media. 57 a) Galactose Induction b) p-Galactosidase Assay Media: -U,-H,-W,-L + Gal/Raf Media -U)-H,-W,-L+ Gal/Raf + X-Gal H2A.vD - putative I JHHLk^l 9 B S B i ii iii iv (+) i ii iii iv (+) H2A .VD - putative II l # • • • 1 - i - N />«»»» 1 ^ f # % 1 * %W ^^^^^Hi • * i \—/ \ / V ;' V. i ii iii iv (+) i ii iii iv (+) H2A .VD - putative III A jfc A Jfc A W «W • • • • w w w x - • 1 ^•r ^HF w • • i ii iii iv (+) i ii iii iv (+) H2A.VD - putative IV i ii iii iv (+) i ii iii iv (+) Figure 3-4. H2A.vD-putative Protein Interaction Trap direct test platings, (a) Four separate isolates from original transformation plate (containing bait, prey and reporter plasmid constructs) plated on media selecting for the plasmids and protein-protein interactions, (b) Similar to (a) with X - G a l laid over the transformant colonies. 58 H2A.vD / PrP-IV transformants also were not able to survive on the selection/induction media (-U, -H , -W, -L , +Gal), suggesting there was an inability for the H2A.vD-LexA fusion protein and the PIP-IV fusion proteins to interact. To confirm the possible interaction between the "bait" H2A.vD and PIP-II and III, the second confirmation method, consisting of LacZ expression, was again used. The colonies were covered with the low melt agarose containing the X-Gal in order to look for the blue colouration, the sign of an interaction (Fig. 3-4b). The H2A.vD / PIP-I yeast transformants show very little blue colouration after the 24 hour incubation period when compared to the positive control substantiating the claim that there is not a substantial interaction taking place between the H2A.vD-LexA fusion and the PIP-I fusion proteins. The H2A.vD / PIP-II and PLP-III transformant strains show comparable colouration to the positive control. This suggests a physical interaction has taken place. The H2A.vD / PEP-IV transformant strain does not exhibit any significant colouration, correlating with the inability to grow seen on the selection/induction media (-U, -H, -W -L, +Gal) (Fig. 3-4a) These results indicate that there are possible interactions between H2A.vD and PIP-II, as well as H2A.vD and PIP-III. Also the results are not identical between the native H2A.vD and the altered form of H2A.vD. A l l four altered H2A.vD / PIP strains were able to show the signs of a physical interaction in yeast. When the native version of H2A.vD was used only the H2A.vD / PIP-II and PtP-UI strains were able to demonstrate a physical interaction when shown by the protein trap direct interaction test. 59 (c) H2A / Putative Interacting Proteins We then asked whether the interactions we saw between H2A.vD and PIPs II and III were specific to the H2A.vD variant. To decipher whether the interactions were specific to H2A.vD the experiment was performed using the major H2A sequence in the "bait" vector. The H2A / PIP yeast transformants as well as the positive control also show the ability to grow on the master plate media (-U, -H, -W, +Dextrose) confirming that the plasmids are intact and expressing the nutrient gene products (Fig. 3-5a). Figure 3-5b shows the inability of the H2A / PIP yeast strains to grow on the negative control media (-U, -H, -W, -L , +Dextrose) displaying that H2A-LexA fusion does not "self activate" and that the colonies visible on the master plate are not due to contamination. As we can see in figure 3-6a the H2A / PIP transformants were unable to grow on the inducing/selection media (-U, -H, -W, -L, +Gal) suggesting the inability for the H2A -LexA "bait" fusion to interact with the PIP "prey" fusion proteins. To confirm this negative result the colonies were covered with the low melt agarose containing the X-Gal in order to look for the blue colouration, the sign of an interaction (Fig 3-6b). There was very little to no colouration in all transformant strains. These results indicate that H2A does not physically interact with any of the PTP fusions in the Protein Interaction Trap system. (d) 3' Partial Deletion-H2A.vD/Putative Interacting Proteins The question arose as to why did the altered-H2A.vD have the ability to interact with all four PIPs in the Protein Interaction Trap system while the native form only 6 0 a) Master Media: -U.-H.-W, + Dex b) Negative Control Media -U,-H,-W,-L + Dex Truncated-H2A.vD - putative I i ii iii iv (+) i ii iii iv (+) Truncated-H2A.vD - putative i ii iii iv (+) i ii iii iv (+) Truncated-H2A.vD - putative III i ii iii iv (+) i ii iii iv (+) Truncated-H2A.vD - putative IV • • • i ii iii iv (+) i ii iii iv (+) Figure 3-7. Truncated H 2 A . v D (3' Deletion) - putative Protein Interaction Trap direct test platings, (a) Master Plate. Four separate isolates from original transformation plate (containing bait, prey and reporter plasmid constructs) plated on media selecting for the plasmids. (b) Negative control. Similar to (a) with the exception of leucine being absent in the media. 61 a) Galactose Induction Media: -U,-H,-W,-L + Gal/Raf b) p-Galactosidase Assay Media -U,-H,-W,-L+ Gal/Raf + X-Gall 3^  Deletion H2A.vD - putative I i ii iii iv (+) i i i i i i iv (+) i ii iii iv (+) • i ii iii iv (+) i ii iii iv (+) 3 - putative II i ii iii iv (+) - putative III i ii iii iv (+) ) - putative IV i ii iii iv (+) Figure 3-8. 3' Deletion H 2 A . v D - putative Protein Interaction Trap direct test platings, (a) Four separate isolates from original transformation plate (containing bait, prey and reporter plasmid constructs) plated on media selecting for the plasmids and protein-protein interactions, (b) Similar to (a) with X - G a l laid over the transformant colonies. 62 demonstrated that it had the ability to interact with PIPs II and III. To determine whether the ability of altered-H2A.vD to physically interact was due to the loss of amino acids or was due to a novel set of amino acids (Fig. 1-4) at stop codon was engineered at the 3' end of H2A.vD ORF using PCR. This D N A sequence was then engineered in to bait vector and upon expression generated a "truncated" protein product (H2A.vD-Tr) that was an identical length to the altered version of H2A.vD the difference being that there was not any novel amino acid generation. The construct was transformed into the EGY48 yeast strain along with the PIP "prey" fusion and reporter vectors. The truncated-H2A.vD / PIP yeast transformants as well as the positive control also show the ability to grow on the master plate media (-U, -H, -W, +Dextrose) confirming that the plasmids are intact and expressing the nutrient gene products (Fig 3-7a). Figure 3-7b shows the inability of the H2A.vD / PIP yeast strains to grow on the negative control media ( - U , -H, -W, -L , +Dextrose) displaying that H2A.vD-LexA fusion does not "self activate" and that the colonies seen on the master plate selection media are not contaminants. Figure 3-8 a shows the ability of the yeast colonies, containing the truncated-H2A.vD and PIP fusion constructs along with the reporter plasmid, to grow on the selection/induction media (containing - U , -H, -W, - L , +Galactose). These results coincide with the results found for the altered-H2A.vD. To substantiate this the (3-galactosidase test, using the LacZ reporter gene, for the truncated-H2A.vD / PIP interactions showed similar results to the altered-H2A.vD results exhibiting similar blue colouration when immersed in low melt agarose containing X-Gal and incubated for 24 hours after being allowed to grow for 96 hours at 30°C on the selection media (-U, -H, -W, -L, +Galactose) (Fig. 3-8b). 63 a) Master b) Negative Control Media: -U,-H,-W, + Dex Media -U,-H,-W,-L + Dex H 2 A - putative I • •••• i i ii iii iv (+) i ii iii iv (+) H 2 A - putative II L X T T T J i ii iii iv (+) H 2 A - putative i ii III iii iv (+) i TT I I J I i ii iii iv (+) i ii iii iv (+) H 2 A - putative IV i ii iii iv (+) i ii iii iv (+) Figure 3-5. H 2 A - putative Protein Interaction Trap direct test platings, (a) Master Plate. Four separate isolates from original transformation plate (containing bait, prey and reporter plasmid constructs) plated on media selecting for the plasmids. (b) Negative control. Similar to (a) with the exception o f leucine being absent in the media. 64 a) Galactose Induction b) p-Galactosidase Assay Media: -UrH,-W,-L + Gal/Raf Media -U>-H,-W>-L+ Gal/Raf + X-Gal H 2 A - putative I • 1 i ii iii iv (+) H 2 A -i putative II ii iii iv (+) i ii iii iv (+) i ii iii iv (+) H 2 A - - putative III \_J: 1 ii iii iv (+) H 2 A -i putative IV ii iii iv (+) i i ii iii iv (+) i ii iii iv (+) Figure 3-6. H 2 A - putative Protein Interaction Trap direct test platings, (a) Four separate isolates from original transformation plate (containing bait, prey and reporter plasmid constructs) plated on media selecting for the plasmids and protein-protein interactions, (b) Similar to (a) with X - G a l laid over the transformant colonies. 65 3.2 p-Galactosidase Quantification In order to determine the strength or duration of the interaction a method of quantification is necessary. The interaction system is designed with this in mind. As the interaction occurs the transcription "unit" activates the lacZ gene on the reporter plasmid allowing for the yellow colouration, in a certain amount of time, when O N P G (the substrate) is added to the medium. This can then be translated into an O.D. reading and ultimately P-Gal units. (a) Growth Times In order to perform the P-Galactosidase Quantification experiment the cells must reach a certain density, in this case 3 .0x l0 7 cells/ml in the selection /induction media (-U, - H , -W, +Gal/Raf). Leucine was kept in the media as not to select for growth differences between the transformant strains. In other words, how well the cells produce leucine due to the interactions between the H 2 A . v D fusion and the PIP fusions would not be an issue as the ability to produce leucine is not part of the selection in this experiment. The interaction w i l l produce P-galactosidase from the reporter plasmid. We wanted to know how long (i.e. how many hours), it took for each strain to reach the desired density so that following experiments could be initiated at the same time. We were not interested in the growth patterns, but rather a start and finish time for the transformant strains. Each transformant strain took a different amount of time to reach this desired density (Fig. 3-9). This chart is not a growth curve but rather a graphical representation of the time in hours the individual strains needed in order to reach the desired cell density. In order to determine the reason for this time differential between the transformant strains, the same 66 Yeast Growth Times y . ' • / / / / / y / If / ' / / / / / / / / / / 11/ I ^ if /A / V / ^/ I 1/ ///// / / // / f / /. y •' s 1 // v / 1 r / if / / / '/ / / / / / / / / If/ 0 24 48 72 U 120 ! 144 168 192 •Hp-1 /Su(Var3-9) H2AvD - Putative 1 (ACEH) H2AvD - Putative 2 (DF) H2A.vD/Putative 3 (L) • H2AvD - Putative 4 (M) • 0 A O 0 Dashed lines represent putative-prey fusions + reporter plasmids time (hrs) Figure 3-9. Diagrammatic representation of growth times for each transformant strain. The diagram indicates the time each strain took to reach the desired cell density of 3.0x10 7 cells in selection/induction media ( -U,-H,-W,+Gal/Raf). Diagram represents total time of growth, and is not a growth curve. Dashed lines represent the transformant strains minus the H 2 A . v D - L e x A Bait fusion construct. 67 strains minus the H2A.vD "bait" fusion construct were also grown to a density of 3.0x10 cells/ml and the time noted (Fig. 3-9). There was a slight decrease in the amount of time (6-12 hrs) required for each strain to reach the desired density when the H2A.vD-LexA bait fusion was absent, though the overall pattern did not change. The Su(Var)3-9 / HP1 strain reached the desired density first (approximately 48 hours after inoculation), the Ff.2A.vD/ PIP-III reached second (approximately 72 hrs after inoculation) , the H2A.vD / PIP-II reached third (approximately 120 hrs after inoculation), the H2A.vD / PIP-I reached fourth (approximately 192 hrs after inoculation), H2A.vD / PTP-IV never reached a suitable cell density (Fig. 3-9). We can conclude that it is not the "bait" fusion product that is responsible for the growth differences, but more likely the "prey" fusion products that are responsible; maybe due to some cellular toxicity attributed to some of the PIP "prey" fusion products. (b)P-Galactosidase Quantification Once the desired cell density was reached the assay was done in triplicate. The positive control (Su(Var)3-9 / HP1 interaction) gave a value of 22.3 P-Gal U/10 cells/ml (Std Dev = 3.1). This was used as the standard. The H2A.vD / PIP-I gave a value reaching only 2.0 p-Gal U/10 7 cells/ml (Std Dev = 2.0). H2A.vD / PIP-II produced a value of 44.0 P-Gal U/10 7 cells/ml (Std Dev = 7.8). The H2A.vD / PIP-HI produced a value of 20 p-Gal U/10 7 cells/ml (Std Dev = 2.0). The H2A.vD / PTP-IV gave a value of 0 p-Gal U/10 7 cells/ml (Std Dev = 0) (Fig 3-10). p-Galactosidase Quantification 601 50 1 Figure 3-10: Graphical representation of P-Gal units from each of the transformant lines. Cells were grown in the selection/induction media ( - U , - H , - W , +Gal/Raf) to a density of 3.0x107 cells/ml. Error bars represent standard deviation where n = 3. 69 3.3 Confirmation of Physical Interactions / GST Pulldowns In order to confirm that the interactions in the Protein Interaction Trap direct test were valid a confirmational experiment or "test" had to be done. The reason for this lies behind the faults in the mechanism of the Protein Interaction Trap screen (to be discussed in Chapter 4). The GST Pulldown, a form of co-immunoprecipitation is a suitable assay for this situation. Rather than co-immunoprecipitating the prey proteins out of total cell lysate and then confirming with an anti-body (which may or may not be possible depending on the identity of the proteins in question), the question we posed was: do the two proteins physically interact with one another (i.e. H2A.vD with either PIP-II or PEP-III)? The method used here uses only the two proteins in question, at a time, the H2A.vD fused to Glutathione-S transferase (GST) and the PIP D N A sequence in vitro transcribed and translated with 3 5 S to detect the presence of the translation product in an SDS gel. If these two proteins associate and can be detected, then a physical interaction has been determined. H2A.vD and H2A were fused to GST in order to purify them along with their interacting proteins. We asked whether H2A.vD-GST was able to interact directly with any of the in vitro translated PIPs. GST alone was used to determine i f there were any unspecific associations of the PIP in vitro translation products with GST. H2A.vD-GST was not able to pull-down PIP-I or PIP-IV (Fig. 3-1 l a lanes 2& 11). H2A.vD-GST was able to pull-down PIPs-II and III showing at least 2-3x more recovery than the control GST lanes (Fig 3-1 l a lanes 5 & 8). GST alone exhibited some background and therefore the results suggest that there is a slight ability for the GST domain to associate with the translated PIP sequences but this is strictly at a background level and therefore confirms 70 a) kDa 175 83 62 47.5 32.5 25 16.5 b) kDa 175 83 62 47.5 32.5 25 16.5 A. A. CO CO r% £ & & P v i 5 ' >*» _v A, v' <o & -CP <o <V Co ^ / ^ / ^ # * dr # *t <r (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11)(12) ^ > C o - A> . O T I ? ^> "V? ^ A A A / / £ / / <* / / # / / o * (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) Figure 3-11. G S T Pulldown. In vitro transcribed and translated products containing 3 5 S . Lanes 1,4,7,10 contain the in vitro translated product. Lanes 2,4,8,11 contain the H 2 A . v D GST (a) or H 2 A G S T (b) fusions mixed with in vitro translated product and subsequently washed Lanes 3,6,9, and 12 contain the control G S T alone. 71 the above conclusion that PIPII has the ability to interact with the H2A.vD protein and not with the GST domain. Major H2A was used in order to confirm that the interaction was specific to H2A.vD. Thus we asked whether major H2A was able to interact directly with any of the PIP in vitro translation products. H2A was not able to pulldown any of the four PIP translation products (Fig 1 lb lanes 2,5,8, &11). This suggests that the physical interactions between H2A.vD and PIPs II and III specifically are confirmed in vitro. This second assay confirms the results found in the Protein Interaction Trap direct test and (3-galactosidase assays that showed a similar result (Fig 3-2). Specificity has been demonstrated, i.e. the ability to interact with H2A.vD alone, as the second method of detecting interactions is in agreement with the original Protein Interaction Trap direct test result. 3.4 Search for Putative Interacting Protein Genes (a) Search for Putative Interacting Protein Gene-II Upon determining which PIP could physically interact with H2A.vD we investigated the identities of PIP-II and PIP-III. The D N A sequence for PIP-II obtained from the original Interaction Trap experiment was submitted to the database at NCBI and F L Y B A S E . This resulted in no significant sequence matches being obtained. In order to help determine the origin of the PIP-II D N A sequence, Southern Blot Analysis was done. Genomic samples from Drosophila, SF9 cells, S. cerevisiae, E. coli, and Mouse were used (Fig. 3-12, lanes 6-10). The blot was probed with a probe specific for the Drosophila HDAC1 sequence. Two bands were visible in the positive control lane (Fig. 3-12a lane 1). One corresponding to the 1.8 kb HDAC1 PCR product, the second is 72 //o ^ A . i m r> CN O CN in o X i o u-cu ' I I U H & § E | I I 3 <D O <*> CO cN C3 0> > s s Q I E o c u -o 60 C & I £ S S .S3 o c +-» . M C ' w o o 5 c° a o JC 'o <D <D W O £ a a o 2 o £ , o JO <C 5 u 6 i £ M m B o - a 5? ° E P ^ 3 ffl o I g 0 g . £ CN M m "a-1 -r ° o 60 3 o ^ Cu & V—' ' C . —^» Q a E s I - o 1 i C/3 ( U most likely a PCR artefact. There were two bands visible in the Drosophila lane (Fig. 3-12a lane 6) corresponding to the genomic HDAC1 sequence. Two bands are visible as the genomic D N A was digested with EcoRI and the genomic sequence contains an internal EcoRI sequence. No other bands are visible in any of the lanes. Homologous sequences in other organisms are not detected due to the high stringency conditions of the protocol. After stripping, the blot was re-probed with a probe specific for the PIP-II D N A sequence. A band is visible in each of the positive control lanes containing dilutions of the PIP-II PCR product (Fig. 3-12b, lanes 2-4). There are no bands visible in any of the other lanes (5-10). Lanes 1 and 6 (Fig 3-12b) show residual banding due to inefficient stripping of the HDAC1 signal. In order to help verify or refute the Southern blot results, a genomic PCR was performed from genomic Drosophila D N A using primers specific for HDAC1 and a separate pair designed for the PIP-II D N A sequence. A band is visible in Lane 2 (Fig 3-13) corresponding to the size of the HDAC1 genomic PCR fragment (740 bp). No band is detected in the PIP-II lane at (lane 3) (Fig. 3-13). A product is visible in the control lane 4 (~400bp) (positive control for PIP-II primers). (b) Search for Putative Interacting Protein Gene-Ill The PIP-III sequence (nucleotide and amino acid) derived from the original Interaction Trap experiment (R. Sobel) was also submitted to the databases at NCBI and F L Y B A S E for sequence similarity matches. The result produced a score of 716 bits with an E value of 0.0 (99% identity) for the nucleotide search and a score of 142 with an E value of le-34 (76% identity) for amino acid. The resulting gene was identified as 74 (1) (2) (3) (4) - 740 bp - 420 bp Figure 3-13. Genomic P C R of putative II sequence. Lane (1) contains 250ng of N E B lkb ladder. Lane (2) is the genomic H D A C 1 P C R product, positive control for the genomic P C R Lane (3) contains the putative II genomic P C R product. Lane (4) contains the putative II plasmid P C R product, a positive control for the primers. 75 CG5515, an open reading frame with a putative protein of unknown function. The representative D N A sequence is 218077 bp and the representative protein sequence is 244 amino acids. The gene resides between bands 95E4—5 in the Drosophila genome. Upon original discovery in the database the function of this protein is unknown but similar sequences have been found in Homo sapiens (CGI-24 protein), S. cerevisiae (hypothetical protein YDR152w), and S. pomhe (hypothetical protein). As these also are hypothetical proteins, their function is unknown. Domain analyses depict it as having two domains that are shared with its similar sequence partners. The first domain encompasses amino acids 4 to 82, being characterized simply as a domain similar to a protein found in the Homo sapiens protein which in itself is similar to the musculus RING finger protein A 0 7 (ProDom identifier: 119917). The second extends from amino acid 97 -175 characterized as sharing similarity to the Homo sapien protein CGI-24 (ProDom identifier: 29695). 3.5 Genetic Interactions In order to determine whether H2A.vD and the putative gene CG5515 interact act a genetic level as well as a physical level, genetic crosses were employed. (a) Genetic Interaction Crosses Here we asked whether H2A.vD and the gene CG5515 interact at a genetic level in order to attain in vivo significance. The crosses were set up using the 1(3)810 mini-deletion strain used to determine the essentiality of H2A.vD (van Daal and Elgin, 1992) which has a deletion encompassing the second exon of the gene. This was crossed with 76 two deficiency strains, Df(3R)crb87-4 and Df(3R)crb-F89-4 thought to contain CG5515 the putative gene. The crosses were set up in duplicate with one set having the male carry the 1(3)810 mutation and the second set having the female carrying the 1(3)810 H2A.vD mutation. This was done to determine if the maternal product (H2A.vD protein deposited by the mother) had any influence (van Daal and Elgin, 1992). The results yielded no difference. This is obvious when comparing the progeny from the deficiency crosses. The 1(3)810/Df(3R)crb87-4 and the 1(3)810/Df(3R)crb-F89-4 progeny are not substantially lower than the Df(3R)crb87-4/ TM3 sb or Df(3R)crb-F89-4/ TM3 sb (Fig. 3-14 line 2&3 and 6&7). Interestingly it seems when the mother has the 1(3)810 mutation there was less scorable progeny with identical set-up in experimentation between the two crosses, this result was not proven to be significant. (b) Test for Fecundity / Fitness We next asked whether the F l generation (1(3)810 / Df(3)crb 87-4 or 1(3)810 / Df(3)crb-F-89-4) from the previous genetic crosses were less fit than the control flies (1(3)810/TM3 sb or Df / TM3 ser)) (i.e. produced less offspring in a similar amount of time), another indicator of possible genetic interaction. None of the F l x F l crosses displayed greatly reduced number of offspring a when compared to the progeny of either of the control crosses (other than the expected lethality of mutation / mutation or balancer / balancer as both are homozygous lethal) (Fig. 3-15). CO CO o CO CO CO O o O) m i o loo o CO .CM (N o 1 T— 00 CO CN CO CO CNI • j in c> \ l-O CD 10 CD ! (0 0) CO 05 !•§ co CO O O \ ^ + + 0 0 + ss, + !'— '. £ 5 , o o sb se O o sb se 5 CO CO CO CO [CO CO TM CH- CO CO, 1^  I O oo CO 0) CM CO ^J" Ifl CD N 00 oo fc -e Q o oo m C/5 m O) O") (J) CD O) CO CM CM T - T -hrr8 1 v . i? ^ co £2.'" C N co O f O T - : 1^ CN 2 » 8 . 2 . 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N O -4—» 43 O 2 fl Lfl 13 60 • M s> Cu o \ o 0O |_ r ' 1> P H 43 •e I C 03 03 C O 03 cm c 03 03 "5 43 1/3 13 C O C O O M o 03 o >> fl 03 60 -4-> O nd pr 03 o 43 13 » fl o o fl r M C M o ' - M 03 S3 .fl g 13 +-» -*-> 03 13 "O Q co in i c n '-fl ure unc 60 ec •a t oo o ^ & ° Q ° O -t-» 1 8 13 U •3 s « p 13 03 ai c n fl 5 60 H N 2 2 00 03  43 13 C M O M 03 - D S fl c 13 43 o 43 co C O 03 . f l 03 03 43 H C O 13 C O C O O M o "o M M fl o o 03 43 • —H " M C M ^ O O ^ C O 13 03 C O C O O M 43 O ° 5 -*-» *x3 X ) «2 13 o iS 13 2 CL CL S g o o u 13 M > s 43 O O 00 cn 5 C fl T 3 03 C O O M o 13 c 13 B fl 03 60 O M CL 03 j> 2 CL o X O 03 C O 79 3.6 Lethal Phase Assay We know that the 1(3)810 mutation is homozygous lethal (van Daal and Elgin , 1992). We asked at what stage in development does the 1(3)810 mutation exhibit its lethality. In order to determine the phase at which the 1(3)810 mutation is lethal we set up a lethal phase experiment. Control Cross 1 was done in order to determine the lethality of the TM3/+ genotype. Control Cross 2 was done in order to determine the lethality of the 1(3)810/+ genotype (minus the lethality caused by the TM3/+) (Fig. 3-16). There were no more lethal embryos in the experimental cross (1(3)810/TM3) x 1(3)810) than in Control Cross 2. Those embryos broken were similar between the two crosses as were those eggs unfertilized. This results in similar numbers making it to larval phase (Fig. 3-16). The number of pupae was different however. There were roughly 28% less flies surviving to the pupal stage than in the experimental cross than the control cross (Control Cross 2). This suggests that the mutation is larval lethal which aggress with a similar study done using T M 6 Tb as the balancer chromosome (Clarkson et al., 1999). 80 Experimental cross Control Cross (1) Control Cross (2) 1(3)810 x 1(3)8101 TM3 sb + 1(3)810 1(3)810 1(3)810 TM3 sb 1(3)810 + TM3 sb TM3 sb TM3 1(3)810 x _+_ TM3 sb + U3)810 + TM3 EXPERIMENTAL CROSS (1) CONTROL CROSS (1)1 CONTROL CROSS (2)1 1 Total 500 Total j 500 Total 500 broken 10 broken 2| broken 29 lethal embryos 22 lethal embryos 5 I lethal embryos 31 unfertilized 77 unfertilized 34 (unfertilized 129 made to larval phase 391 made to larval phase 459 made to larval phase 311 j pupae 280j pupae | 430 i pupae j 298 Figure 3-16. Lethal phase determination of homozygous 1(3)810 flies. The lethality of +/TM3 is determined by control cross (1). The lethality of 1(3)810/+ is determined by control cross (2). 81 Chapter 4: DISCUSSION H2A.Z has been studied in a variety of organisms. The research is becoming more intense and findings more frequent. H2A.Z in Drosophila melanogaster (H2A.vD) has been studied via localization experiments, domain alterations, and genetic studies in hopes of contributing to the understanding of function of these H2A.Z variants. In this chapter we infer function by analyzing the relationship between H2A.vD and its protein partners. We suggest possible roles for these variant proteins based not only on these findings but also on incorporation of previous results from the literature. This information adds to the larger picture and solidifies our knowledge of H2A.vD and its role in chromatin formation, structure, and function. 4.1 Protein Interaction Trap: Interaction Specificity between H2A.vD and the Putative Interacting Proteins Upon completion of the Protein Interaction Trap direct test experiment we saw dramatic differences between results obtained with the altered version of H2A.vD and the native form of H2A.vD. A l l four PIPs displayed the ability to interact in the Interaction Trap direct test with the altered form, while only two of the four PIPs (PIPs II and III) showed interaction capabilities in the direct test with the full length H2A.vD (Fig. 3-2 & 3-4). The possible reasons for this will be discussed later in this chapter. The results obtained from the direct test suggest that there is a physical interaction between two previously detected proteins from the original screen, PIP-II and PIP-HI. Since PIPs I and IV did not pass the growth test on the selection/induction media (media lacking 82 Leucine +GAL/RAF) or the P-galactosidase test, they were dismissed as potential interactors though further analysis was continued for completeness. The question surrounding the protein interaction capability of H2A.vD with the PIPs essentially had two parts. Firstly, do any of the PIPs interact with H2A.vD and secondly, is the interaction specific? The possibility for general associations exists. In other words associations based on general structure as opposed to a specific protein structure and sequence. To determine if this was the case the major histone H2A was used as a "bait" fusion. Under identical testing conditions as those done with H2A.vD, there was no evidence of an interaction between major H2A and any of the four PIPs (Fig 3-6a and b). This initially suggested that the results obtained in the Interaction Trap experiment between H2A.vD and PIP-II and PIP-III are specific to H2A.vD. Therefore the interaction was not due to a general association between these PIPs and core histone proteins or proteins with a histone fold motif for example. This has been known to happen with certain domains (e.g. leucine zippers when expressed in the uM range (Phizicky and Fields, 1995). A common procedure to overcome this problem is to test the LexA-fusions that contain a bait protein from within a family. This was done here, using the major H2A. To determine the strength of the interactors, conformational analysis was performed. The strength of colouration shown on the plates is strictly qualitative. In order to determine whether the interaction results were reliable we deemed quantified the results. In figure 3-10 we see that the PIPs that interacted with H2A.vD on the plate assays exhibit the greatest interaction capability as measured in P-Gal units per number of cells per ml. The strength of the interaction with PIP-II was comparable to that of the 83 positive control (Su(var)3-9 / HP1). Even more interestingly was the result obtained from the interaction of H2A.vD with PIP-III that seemed stronger than that of the positive control, though statistical significance could not be determined. The assay for each H2A.vD / PEP interaction was repeated multiple times (n=3) allowing us to generate reliable and repeatable results. Due to the standard deviation we can only conclude that the interaction capability is at a considerable level for H2A.vD with PIP-II and PIP-ni, a level comparable to that of the positive control. We are unable to distinguish the degree at which the interactions between H2A.vD / PIPII, H2A.vD / PIP-III, and the positive control differ due to the standard deviation. Though we can conclude a strong interaction has taken place when comparing the H2A.vD / PIP II and PIP III results to the positive control. 4.2 GST Pulldowns: Confirmation of the Direct Interaction In order to confirm that that these interactions were valid, a conformational experiment or "test" was done. This is standard practice for any screen as there are faults in the mechanism of the Protein Interaction Trap screen. Self activation in which the bait fusion protein can activate transcription on its own, mutations leading to EGY48 yeast strains that grow favourably on galactose media, proteins that interact with the LexA binding domain, or proteins that are generally sticky, are possible false positives that can be detected. Most can be eliminated using control experiments to validate the results obtained from the Protein Trap method. Other types of false positives however cannot be accounted for in the context of the Protein Trap method. As an example, the type of association is an issue. Since the 84 yeast cell is essentially used as a biological test tube, the conservation of proteins among organisms is of concern. Yeast proteins may act as intermediates in the association of the proteins in question. This gives a clouded view as to what is happening at the molecular level. The results from the Protein Interaction Trap experiment may be able to tell the investigator that the two proteins of interest are associated in a complex. It does not however, describe the physical relationship between the proteins in question. The strength of the interaction will not be accurate due to over expression of the bait and prey fusions. As well, sequestering of these proteins by native yeast proteins may not allow for weaker or less frequent interactions to be discovered. In order to compensate for the possibility that endogenous yeast proteins facilitate the interaction, a method was needed that isolates the proteins and determines whether the proteins in question interact with each other directly. For this reason the GST pulldown was chosen. The technique was used to isolate the two proteins of interest and to look for direct interaction as described earlier in chapter 3. Upon analysis of the results it was shown that H2A.vD is able to "pulldown" two PIP translation products (PIPs II and III) showing a direct physical interaction between the two. Upon confirmation of the interaction we had to determine the function of these interactors in order to hypothesize a function for H2A.vD. 4.3 Putative Interacting Protein Identities PIP-II has no known sequence identity. We were unable to discover an identity through database searching or via molecular techniques. The weak stringency conditions used for the PIP-II Southern blotting procedure allowed for a 30% decrease in identity 85 (Bonner et al, 1973), therefore any homologous sequences in any of the other organisms should have been identified in the Southern Blot, i f they were at least 30% similar. It has been suggested that heterochromatic sequences have difficulty transferring through the gel and onto the membrane during Southern blotting procedures (Glaser and Spradling, 1994). This idea was supported when restriction fragments spanning euchromatic-heterochromatic junctions that extended into peri-centromeric regions had the unusual property of being selectively resistant to transfer from agarose during blotting procedures (Glaser and Spradling, 1994). This could explain why there was no detection of the sequence in the Southern blot experiment (Fig 3-12). This implies that the PIP-n D N A sequence may heterochromatic. It is understood that there are small islands of unique sequence embedded within heterochromatin, as in the case of the Drosophila rolled gene, which is flanked on each side by at least 3 M B of heterochromatin (Adams et al, 2000). Heterochromatic regions, especially those surrounding the centromeres, are undetectable to current sequencing methods (Adams et al, 2000). This could be why there is not a corresponding sequence to PIP-II in the Drosophila database. The follow up experiment, genomic PCR, was designed to address this. The genomic PCR experiment also could not detect a sequence matching that of PIP-II. This could be due to one of two reasons. Firstly, the primers were designed to amplify a region of ~400bp according to a cDNA sequence. As this was a genomic PCR the possibility exists that the primers were designed in separate exonic regions far from one another extending beyond the -600 bp limit of the polymerase, therefore producing no product. The second possibility is to suggest that the PIP-II sequence is an artefact of the Protein Interaction Trap screen, which unfortunately has the ability to interact with H2A.vD. 86 We were able to find an identity for PIP-III. The Drosophila CG5515 gene is real but has no known function. It was originally proposed to be a protein kinase by the FlyBase database but that designation has since been recalled. This association still has some very interesting connotations. The first domain encompasses amino acids 4 to 82. It is characterized simply as a domain similar to a domain found in a Homo sapiens protein which in itself is similar to the musculus RING finger protein A 0 7 . The second domain has a characterization of being similar the human CGI-24 protein as well as the other similar sequences mentioned above from amino acids 97 to 175. The functions of these similar sequences are also unknown leading one to hypothesize about the function of this putative protein product. By gaining information on other proteins that share these domains one may extrapolate a function for the original protein of interest. Upon domain matching the most common proteins containing a recognizable domain were those in which the domain was a protein serine/threonine kinase, for example the human protein with similarity to the musculus A 0 7 protein. We cannot state for certain that the protein generated from CG5515 is a protein kinase but we can suggest that it associates with the histones, as a protein kinase would, and may play a role in histone modification by possibly recruiting a kinase that phosphorylates H2A.vD. As mentioned in chapter one, protein modification affects histones by altering amino acids at the N - and C-terminal region to allow for regulation and other processes within chromatin. Histone variants have also been implicated in the chromatin remodelling process, substituting for the core histones and altering chromatin structure allowing for gene regulation. The results and ideas presented here bring about the notion 87 of histone modification and histone variants working in tandem to affect the overall structure of chromatin and its ability to package and regulate the genome. 4.4 Role in Chromatin: Association with Post-Translational Modifications Our current knowledge about histone variants and their post-translational modification is limited when compared to our knowledge of their functional relevance. Unfortunately this relevance has in turn provided little information about their molecular mechanisms. We can anticipate that functional correlations between histone variants and post-translational modifications will become clearer, and the idea that they act synergistically may become apparent. There are a few examples of such collaborations between modifications acting together. The coupling between H3 phosphorylation (at serine 10) and acetylation (at lysine 14) (Cheung et al, 2000), (Lo et al, 2000) or the coupling between H2A.X and its phosphorylation (at serine 139) (Rogakou et al, 2000) are under current scrutinization. It is known that the C-terminal tail of H2A is located in the region where D N A enters and exits the nucleosome (Luger et al, 1997). H2A.X has been implicated in double strand break repair (Rogakou et al, 1998), meiotic recombination preceding synaptic cross over(Mahadevaiah et al, 2001), and apoptotic digestion following caspase-activated DNase activity (Rogakou et al, 1998). It may also be involved in destabilization of the chromatin fibre (Ausio et al, 2001). If H2A.X was to replace major H2A in the nucleosome then phosphorylation of this region could alter the trajectory of linker D N A in this region (serine 139 in mammals) (Ausio et al, 2001). This is substantiated by the fact that yeast H2A, which has very high homology to the C-terminal two thirds of 88 H2A.X, allows for MNase accessibility when its serine residues are replaced by glutamic acid (which mimics serine phosphorylation), suggesting susceptibility of internucleosomal D N A digestion (Downs et al, 2000). It would then be reasonable to assume that Drosophila H2A.vD, which is somewhat longer than the rest of the H2A.Z molecules (Fig. 1-3), could have a similar role in altering the structure of the nucleosome. If in fact the role of H2A.vD is to destabilize the nucleosome, as suggested by the research of its homologs (Suto et al, 2000), (Abbott et al, 2001), then phosphorylation of the C-terminal tail could add to this phenomenon. Upon analysis of the amino acid sequence we find that the essential phosphorylation domain of H2A.X is the SQ(E,A,D)(I,L,F,Y) motif. This acts as the substrate for phosphorylation on the serine residue. Upon closer examination of the Drosophila H2A.vD amino acid sequence we see that it also has this domain. According to recent studies it is phosphorylated at the C-terminal serine (Rogakou et al, 1998), though in this study the H2A variant protein is referred to as an H2A.X homolog. When comparing between H2A.Z homologs, which are extremely conserved, we do not see another member with this phosphorylation domain (Fig. 1-4). We hypothesize then that H2A.vD may be a fusion of the H2A.Z and H2A.X domains and that H2A.vD has a dual role in the Drosophila cell. It has a specific H2A.Z function, perhaps stabilization or destabilization of the nucleosome, to promote higher order chromatin structure. As well it may function similarly to that of H2A.X proteins in mammals and Xenopus, such as involvement in meiotic recombination and response to double stranded break repair among others. It is hypothesized that double strand break repair requires the chromatin to unfold allowing access to modulating enzymes as well as serine phosphosignalling which in turn recruits down stream proteins 89 to site of D N A breakage (Ausio and Abbott, 2002). H2A.vD could aid in destabilizing the nucleosome and therefore allow this process to happen more efficiently by altering the chromatin formation at this site. If phosphorylation is indeed the result of the interaction discovered here, it will be very interesting to see which kinases are involved. Players are not monogamous in H2A.X phosphorylation, as 3 distinct kinases have been shown to phosphorylate H2A.X (Ausio and Abbott, 2002). We know that modifications can be essential in the H2A.Z variant class. Tetrahymena acetylation is necessary at its N -terminus where replacing 6 essential lysines with arginines causes lethality (Ren and Gorovsky, 2001). It is obvious that post-translational modifications and histone variants can work together to alter the structure of chromatin. Now, even more importantly it seems that modifications to the variants themselves will prove to play an integral role in manipulating chromatin structure. This manipulation may be essential to form specialized domains necessary for transcriptional organization and genome function. 4.5 Discrepancies between altered-H2A.vD and native H2A.vD As mentioned earlier in the chapter there were discrepancies between the results found during this research and those obtained from the original library screen. This was thought to be due to the differences in amino acid composition between the two sequences and therefore structural differences between the two versions of the protein (Fig. 1-4). The results suggest (Fig. 3-8) that it is in fact the absence of the twenty-five C-terminal amino acids that allows for the seemingly stronger interaction between the altered-H2A.vD protein and the putative fusion proteins rather than a "new domain" formed from the novel seven amino acids at the C-terminal end of this altered form. This 90 is because the results between the altered-H2A.vD / PIP interactions and the H2A.vD-Tr / PIP interactions are quite similar (Fig. 3-2 & 3-8). It would then suggest that the C-terminal tail of H2A.vD in some manner inhibits possible interactors, possibly non-specific interactions that may occur with this altered and/or truncated form. The results would seem to indicate that the tail serves an inhibition purpose, in some regard inhibiting binding. The major histone H2A protein was used as a control to determine the specificity of the interaction of the PIPs with the native H2A.vD. The H2A direct test shows the inability to interact with any of the four PIPs tested here. This would allow one to hypothesize that and extended tail does prevent association between core histones (H2A). As described earlier the major difference between major H2A and it's variant H2A.vD is in the C-terminal region. This would enable the idea to be put forth that these differences in the C-terminal region either promote or allow for the association between H2A.vD and PIPs II and III. Whereas the extended tail, when compared to either altered-H2A.vD or H2A.vd-Tr, may inhibit the interaction with PIPs I and IV. The reported core structure containing the H2A.Z histone (Suto et al., 2000) displays subtle changes in the nucleosome architecture and altered surface on the H2A/H2B dimer that results in an altered surface (Suto et al., 2000). Suto et al. have gone on to suggest that these changes create an uninterrupted acidic surface across the face of the H2A.Z-containing histone octomer. This in turn creates a site for protein-protein interactions. It seems to be subtle amino acid changes in the variant that allow for interactions between H2A.vD and the PIPs. This is a possible reason that interactions between the major H2A and PIPs do not occur. Along with the fact that the tail of H2A.vD seems to have some inhibitory function, it is interesting to note that while the C-terminal region of the H2A.vD is 91 important, it is not the terminal 14 amino acids (that extend past the normal H2As length) that are required for viability (Clarkson et al, 1999). Only the C-terminal region encompassing the ccC helix (i.e. the final a helix of the protein) and the amino acid stretch immediately following this helix is important (all of which are altered or absent in the altered form of H2A.vD). Interestingly, Clarkson et al. also found that removal of the C-terminal 14 amino acids actually increased the percentage rescue of Drosophila reaching adulthood at 29°C (an elevated stress temperature). It would seem then that the 14 amino acids are in some manner inhibiting the ability of. Drosophila to survive under stress conditions. The removal of the amino acids in question may enhance the ability to interact, i.e. possessing the terminal 14 amino acids inhibits the ability to interact as seen when comparing the results of the altered-H2A.vD and H2A.vD's interaction capabilities with the PIPs (I, II, III, & IV) (Fig 3-2 & 3-4). The removal of the 14a. a. mimics the altered-H2A.vD (premature truncation). We noted that premature truncation allows for a greater number of interactions (PIPs I through IV whereas only PIPs II and III interacted with native H2 A.vD). This may help suggest as to why Drosophila is able to survive the stress conditions when a portion of H2A.vD protein is truncates. These novel interactions may bring about or be involved in a mechanism enabling Drosophila to survive the stress conditions. 4.6 Other Possible Interactors Since H2A.Z is believed to be involved in transcriptional regulation it would make sense that it would have the ability to interact with some transcriptional machinery. In S. cerevisiae it has been shown that H2A.Z interacts with transcriptional proteins through 92 its C-terminal region, specifically R N A polymerase II and T A T A binding protein (TBP). If H2A.Z is involved in either transcription or repression, one would expect its involvement with transcriptional machinery, whether for recruitment, or inhibition. Due to the differences on the face of the nucleosome when H2A.Z is incorporated (Suto et al, 2000) one would expect these interactions to be specific to the H2A.Z incorporated nucleosome. Specific interactions allow for a specific function that cannot be compensated for by other related proteins. 4.7 Conclusions H2A.vD and other histone variants are gaining increasing notoriety as a modifiers and manipulators of chromatin structure and ultimately function. The possibilitity exists that H2A.vD has dual roles in the cell allowing for altered chromatin domains that in turn will govern chromatin function as well the possibility exists that it has a role in D N A repair or other such mechanisms. The specific molecular process and exact cellular pathways in which histone variants act will provide exciting biological questions. These variants are necessary for the organism, implying defined roles in nucleosome structure, chromatin packaging, and transcriptional regulation and ultimately global cellular function. 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