Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
DSpace
UBCV
Shimizu, Jed Chih-Leh
Master of Science - MSc
University of British Columbia
The FACT (facilitates chromatin transactions) complex, a heterodimer comprised of Spt l6p and Pob3p, is involved in a number of processes involving DNA, including transcription initiation, elongation, and repression, DNA replication, and DNA repair. FACT is thought to facilitate these processes through its ability to bind histones and reversibly alter nucleosomal structure modulating DNA accessibility. Recent data has shown that the Sptl6p subunit of FACT interacts with Sas3p of the NuA3 HAT (histone acetyl transferase) complex. Interestingly, mutants of NuA3 share none of the many phenotypes observed in FACT mutants, suggesting that this putative interaction may be redundant to other unknown FACT interactions. In the first part of this study, we confirmed the interaction between FACT and NuA3, and proceeded to describe, for the first time, an interaction between FACT and the ADA HAT complex both in vivo and in vitro. The interaction of FACT with NuA3 and ADA was then mapped to the Nterminal 482 residues of the Sptl6Δp FACT subunit. However, attempts to implicate this interaction with known FACT functions were unsuccessful. Biochemical approaches similarly failed to demonstrate a role for FACT in NuA3 and ADA complex function. The second part of this study focused on the novel spt16Δ922 mutant, which expresses 182 residues more of the Sptl6Δp polypeptide than the sptl6ΔNTD allele, yet possesses a number of mutant phenotypes. Our results show that the spt16Δ922 mutation causes a loss in histone H3 acetylation, supporting the hypothesis that the protein encoded by spt16Δ922 is interfering with normal histone interactions. This loss in histone acetylation was shown to parallel the generation of an spt phenotype, suggesting a possible role for histone acetylation in preventing aberrant transcription. However, further work examining the role of histone acetylation with respect to FACT yielded conflicting results. We conclude that spt16Δ922 is interfering with histone modifications, although the molecular basis of this is as of yet unknown.
INVESTIGATING THE ROLE OF HISTONE ACETYLATION IN YEAST FACT COMPLEX FUNCTION by JED CHIH-LEH SHIMIZU B . S c , The University of British Columbia, 2003 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Biochemistry and Molecular Biology) T H E U N I V E R S I T Y OF BRIT ISH C O L U M B I A July 2006 © Jed Chih-Leh Shimizu, 2006 Abstract The F A C T (facilitates chromatin transactions) complex, a heterodimer comprised of Sptl6p and Pob3p, is involved in a number of processes involving D N A , including transcription initiation, elongation, and repression, D N A replication, and D N A repair. F A C T is thought to facilitate these processes through its ability to bind histones and reversibly alter nucleosomal structure modulating D N A accessibility. Recent data has shown that the Sptl6p subunit of F A C T interacts with Sas3p of the N u A 3 H A T (histone acetyl transferase) complex. Interestingly, mutants of N u A 3 share none of the many phenotypes observed in F A C T mutants, suggesting that this putative interaction may be redundant to other unknown F A C T interactions. In the first part of this study, we confirmed the interaction between F A C T and N u A 3 , and proceeded to describe, for the first time, an interaction between F A C T and the A D A H A T complex both in vivo and in vitro. The interaction of F A C T with N u A 3 and A D A was then mapped to the N -terminal 482 residues of the Sptl6p F A C T subunit. However, attempts to implicate this interaction with known F A C T functions were unsuccessful. Biochemical approaches similarly failed to demonstrate a role for F A C T in N u A 3 and A D A complex function. The second part of this study focused on the novel sptl6A922 mutant, which expresses 182 residues more of the Sptl6p polypeptide than the sptl6ANTD allele, yet possesses a number of mutant phenotypes. Our results show that the sptl6A922 mutation causes a loss in histone H3 acetylation, supporting the hypothesis that the protein encoded by sptl6A922 is interfering with normal histone interactions. This loss in histone acetylation was shown to parallel the generation of an spt phenotype, suggesting a possible role for histone acetylation in preventing aberrant transcription. However, further work examining the role of histone acetylation with respect to F A C T yielded conflicting results. We conclude that sptl6A922 is interfering with histone modifications, although the molecular basis of this is as of yet unknown. i i Table of Contents Abstract ii Table of Contents Hi List of Tables v List of Figures vi Nomenclature vii Acknowledgements viii Dedication ix CHAPTER 1 - Introduction 1 1.1 C h r o m a t i n - S t r u c t u r e a n d F u n c t i o n 1 1.1.1 The Nucleosome and Higher Order Structure 1 1.1.2 The Regulation of Chromatin Structure - Links to Gene Expression 2 1.1.3 The Histone Code Hypothesis 3 1.2 H i s t o n e A c e t y l a t i o n 4 1.2.1 Histone Acetylation 4 1.2.2 H A T and H D A C Complexes 6 1.3 S p t l 6 p a n d t h e Y e a s t F A C T C o m p l e x 9 1.3.1 The SPT Genes 9 1.3.2 The Yeast F A C T Complex - Sptl6p and Pob3p 10 1.3.3 FACT Functions in Processes Involving DNA 11 CHAPTER 2 - Materials and Methods 13 2.1 P r e p a r a t i o n o f Y e a s t S t r a i n s a n d P l a s m i d s 13 2.2 P C R T e c h n i q u e s 19 2.3 P l a s m i d S h u f f l e 19 2.4 P r e p a r a t i o n o f W h o l e C e l l E x t r a c t s f o r F L A G a n d T A P P u l l - d o w n s 2 0 2.5 P u l l - d o w n s o f F L A G - a n d T A P - T a g g e d P r o t e i n s 2 0 2.6 P r e p a r a t i o n o f W h o l e C e l l E x t r a c t s f o r C a l m o d u l i n P u l l - d o w n s 21 2.7 C a l m o d u l i n P u l l - d o w n P u r i f i c a t i o n s o f P r o t e i n C o m p l e x e s 21 2.8 P r e p a r a t i o n o f G S T - S p t l 6 p F u s i o n P r o t e i n 2 2 i i i 2.9 T h e S p t l 6 p P u l l - d o w n ( i n v i t r o a n a l y s i s ) 2 3 2.10 H i s t o n e H 3 A c e t y l a t i o n A n a l y s i s 2 3 CHAPTER 3 - Results 24 3.1 F A C T I n t e r a c t s w i t h t h e H A T C o m p l e x e s N u A 3 a n d A D A 2 4 3.1.1 NuA3 Interacts with FACT 25 3.1.2 Sptl6p Interacts Directly with both NuA3 and ADA 28 3.1.3 The ADA HAT Complex Interacts with FACT in vivo 31 3.1.4 Deleting the NTD of Sptl6p Causes a Loss of HAT Interaction 33 3.1.5 Phenotype Analysis Shows that Interaction with HATs is not Required for Known FACT Functions 36 3.1.6 NuA3 and ADA Mutants do not affect FACT Association with the Nucleosome 38 3.1.7 HAT Interactions with the Nucleosome are Unaffected in sptl6ANTD Mutants 41 3.1.8 Global Histone Acetylation is Unchanged in the sptl6ANTD Mutant 43 3.2 T h e s p t l 6 A 9 2 2 M u t a n t C a u s e s I n t e r f e r e n c e o f H i s t o n e A c e t y l a t i o n 4 6 3.2.1 FACT-Nucleosome Association is not Affected in the sptl6A922 Mutant 46 3.2.2 The sptl6A922 Mutant Shows a Loss in H3 Acetylation 47 3.2.3 Histone H3 Acetylation is Altered in Multiple sptl6 Mutants 51 3.2.4 The spt Phenotype of sptl 6 Mutants Correlates with the Loss in H3 Acetylation 53 3.2.5 The Association of HATs with the Nucleosomes is not Affected in sptl6A922 ....56 3.2.6 Deletion of the Histone H3 Specific HATs Does not Confer an spt Phenotype. 58 3.2.7 Genetic Interactions Suggest that the spt Phenotype of sptl6A922 is Due to Loss of Histone Acetylation 60 CHAPTER 4 - Discussion 65 4.1 T h e I n t e r a c t i o n o f F A C T w i t h N u A 3 a n d A D A 6 5 4.1.1 The Importance of the HAT Interaction Domain of Sptl 6p 65 4.1.2 The Relevance of NuA3 and ADA Interaction with FACT - Alternative Models 68 4.2 T h e s p t l 6 A 9 2 2 M u t a n t a n d t h e I n t e r f e r e n c e o f H i s t o n e A c e t y l a t i o n 7 0 4.2.1 Histone Acetylation and Cryptic Initiation 71 4.2.2 The Possibilities of an Interference Model 72 4.3 C o n c l u d i n g S t a t e m e n t . . . 74 CHAPTER 5 - References 76 List of Tables T A B L E 1.1 S ITES OF H I S T O N E A C E T Y L A T I O N IN Y E A S T ( 3 4 , 4 2 , 4 3 ) 5 T A B L E 1.2 C O M P O N E N T S OF T H E G C N 5 P C O N T A I N I N G H A T C O M P L E X E S S A G A , S L I K / S A L S A , A N D A D A ( 4 2 ) A N D T H E N U A 3 C O M P L E X ( 6 2 , 6 3 ) 7 T A B L E 2.1 Y E A S T S T R A I N S U S E D IN TH I S S T U D Y 15 T A B L E 2 . 2 P L A S M I D S U S E D IN T H I S S T U D Y 1 8 v List of Figures F I G U R E 3.1 N u A 3 I NTERACTS WITH T H E F A C T C O M P L E X 26 F I G U R E 3.2 SPT16P INTERACTS WITH PURIF IED N U A 3 A N D A D A IN VITRO 29 F I G U R E 3.3 A D A INTERACTS WITH T H E F A C T C O M P L E X 32 F I G U R E 3.4 T H E N - T E R M I N A L D O M A I N OF SPT16P IS REQU IRED F O R N u A 3 A N D A D A I NTERACT ION 35 F I G U R E 3.5 SPT16ANTD SHOWS N O F A C T R E L A T E D M U T A N T P H E N O T Y P E S 37 F I G U R E 3.6 H A T M U T A N T S D O N O T A F F E C T T H E ASSOC IAT ION B E T W E E N F A C T A N D T H E N U C L E O S O M E 40 F I G U R E 3.7 Loss OF T H E SPT16P N T D DOES N O T A F F E C T N U A 3 OR A D A A SSOC IAT ION WITH T H E N U C L E O S O M E 42 F I G U R E 3.8 Loss OF T H E SPT16P N T D DOES N O T A F F E C T G L O B A L H3 H I S T O N E ACETYLAT ION . . . 45 F I G U R E 3.9 F A C T A S S O C I A T I O N WITH T H E N U C L E O S O M E IS N O T A L T E R E D IN T H E SPT16A922 M U T A N T 48 F I G U R E 3.10 SPT16A922 M U T A N T SHOWS A LOSS OF H3 A C E T Y L A T I O N 50 F I G U R E 3.11 SPT16 M U T A N T S D I S P L A Y A L T E R E D L E V E L S OF H I STONE H3 A C E T Y L A T I O N 52 F I G U R E 3.12 SPTI 6 M U T A N T S C O N F E R A N SPT P H E N O T Y P E 54 F I G U R E 3.13 Loss OF N T D DOES N O T A F F E C T N U A 3 A N D A D A A S S O C I A T I O N WITH T H E N U C L E O S O M E 57 F I G U R E 3.14 H A T M U T A N T S D O N O T C O N F E R A N SPT P H E N O T Y P E . 59 F I G U R E 3.15 D E L E T I O N OF RPD3 SUPPRESSES T H E SPT P H E N O T Y P E OF SPT16A922 61 F I G U R E 3.16 H I S T O N E H3 M U T A N T S C O N F E R A N SPT P H E N O T Y P E 63 Nomenclature The text follows the conventional method for designating genetic symbols and protein products. Dominant alleles of wi ld type genetic loci are designated by italicized upper case letters (e.g. SAS3) while mutant genes are designated with italicized lower case letters (e.g. sas3). Gene deletions are written as a mutation with a Greek 'delta' following the designation (e.g. sas3A indicates deletion of the SAS3 locus). Insertion mutations are indicated with the symbol ::. For example sas3::HISMX6 indicates that the HISMX6 gene is inserted within the SAS3 locus. Gene products are not italicized and only the first letter is capitalized followed by a 'p' postscript (e.g. Sas3p is the gene product of the SAS3 locus). v i i Acknowledgments There is no possible way this thesis could have been completed in the manner it was without the support and mentorship of all those around me during these last two years. First and foremost, I would like to thank my supervisor, Dr. LeAnn Howe, who took me on without a second thought, and, although absent for half of my time here, managed to be an exceptional mentor during the sometimes convoluted path my research took me on. I would also like to thank my committee members, Dr. Michel Roberge and Dr. Ivan Sadowski, for their input and suggestions over the course of my research. Thanks as well to all those that have come and gone (mostly gone) in the Howe Lab: David Martin for doing everything for the first time, Dan Grimes for keeping things interesting, Janet Dukowski for teaching me how to do things LeAnn did not, Julia Sheng for showing us how efficient protocols could be, Martin Wlodarski for his exceptionally witty and intelligent humour, and to V i c k i MacDonald to whom we pass the Howe Lab torch. As always, difficult times are overcome during any long-term project. Thank you to all the friends (many of whom have already been mentioned) and family that have always listened, despite often not completely understanding what it was I was talking about. v i i i Dedication This thesis is dedicated to my parents and grandparents. Chapter 1 - Introduction 1.1 Chromatin - Structure and Function 1.1.1 The Nucleosome and Higher Order Structure The basic repeating subunit of chromatin is the nucleosome core particle. It is made up of 147 base pairs (bp) of D N A wound 1.7 times around a histone octamer (1). Separating each core particle is a 10-60 bp stretch of linker D N A , the length of which varies depending on a number of factors including the presence of D N A binding proteins (2). This repeating structure of the nucleosome core and linker D N A forms the beads-on-a-string model and is the foundation for all higher order chromatin structure. The histone octamer is composed of two copies each of four highly conserved histone proteins. The central tetramer is made up of two copies each of histones H3 and H4. Two H 2 A -H2B dimers flank the central (H3)2(H4)2 tetramer and complete the histone octamer (3-5). The interactions of histone proteins with other histones and with D N A are the result of a common core histone motif termed the histone fold (6). The association with D N A is through the phophodiester backbone, forming a coiled structure around the histone core of 1.7 left-handed superhelical turns (2). Sticking out of the histone core are the largely unstructured, highly basic, N-terminal tails of each histone protein (8). These tails are thought to modulate nucleosomal folding and higher order chromatin structure through a variety of mechanisms, including protein-protein interactions, and charge neutralization of D N A (9). The next order of chromatin structure believed to form in vivo is the 30 nm fibre (1). This structure and further fibre-fibre interactions are modulated by both histone tails and the 1 actions of linker histones. These linker histones are non-core histone proteins, and associate at the periphery of the nucleosome. They are believed to impart stability to the core complex and play a role in organizing greater oligomeric interactions between nucleosomes. In vertebrates, linker histones H I and H5 are essential, and associate at a 1:1 ratio with the nucleosome core (9). In yeast, the linker histone H h o l p has not been shown to have any clear ratio (10). In fact, linker histones are not essential in yeast or lower organisms (11, 12), suggesting that histone tails may play a more fundamental role in chromatin condensation (1). Additional structural organization beyond the 30 nm fibre also involves non-histone proteins. These include Sir3p and Tuplp , two yeast silencing proteins that function through interactions with histone tails (13, 14). These proteins function by cross-linking chromatin fibres through the interaction of other silencing proteins. This causes an overall coming together of nucleosomes over long distances, resulting in the formation of a highly ordered structure (9). These highly condensed and tightly packaged regions of chromatin are referred to as heterochromatin, and generally have a lower gene density. Conversely, less ordered chromatin, or euchromatin, has a higher gene density (15). 1.1.2 The Regulation of Chromatin Structure - Links to Gene Expression The structure of chromatin inherently restricts the access of D N A by cellular machinery for processes like transcription or replication (16, 17). The regulation of chromatin structure, then, has an important role in the control of gene expression (18-20). The repressive effects of chromatin structure are thought to be mediated by two sets of interactions, those between histones and D N A , and those involving the histone tails. Mechanisms that modulate these 2 interactions include the incorporation of histone variants, the action of ATP-dependent chromatin remodelers, and post-translational modification of histones (2). In yeast, the histone variant H t z l p has been shown to positively regulate gene expression by destabilizing nucleosomes at the promoters of genes (21). Loss of the nucleosome unpackages the promoter and allows it to interact with transcription binding factors, leading to gene expression. Whereas histone variants are substituting actual components of the nucleosome, ATP-dependent chromatin remodelers affect the position of the nucleosome with respect to D N A . There are a variety of remodeling complexes that are thought to function through slightly different mechanisms. The first of the ATP-dependent chromatin-remodelers to be discovered was from the SWI /SNF family, revealed through studies selecting for genes whose products counteract histone-mediated transcriptional repression (22). SWI /SNF complexes disrupt DNA-histone contacts, allowing uniform accessibility of D N A (23, 24). Another complex of the SWI /SNF family, R S C , is thought to function by transferring the entire histone octamer to another molecule of D N A (25). Yet another mode of chromatin-remodeling is observed in the IS WI family of complexes, which enable movement or sliding of the histone octamer to adjacent positions on D N A (26). The final mechanism of modulating chromatin structure is the use of post-translational covalent histone modifications, these include lysine and arginine methylation, lysine acetylation, serine phosphorylation, and ubiquitination (27). 1.1.3 The Histone Code Hypothesis Originally, post-translational histone modifications were generally thought to alter histone-DNA and histone-histone interactions, through the actions of charge neutralization and addition, thus altering nucleosomal structure (28). However, subsequent work has demonstrated a degree of complexity that cannot be explained solely by the direct effects of modifications 3 themselves. For example, histone acetylation, generally perceived as disrupting DNA-histone interactions through histone tail charge neutralization, is observed in transcriptionally silent regions of chromatin (28). This has led to the development of the histone code hypothesis, which states that post-translational histone modifications may modulate gene expression by encoding docking sites or recruitment signals recognized by other proteins. Evidence supporting the histone code hypothesis includes the identification of the bromodomain, a D N A binding motif that specifically recognizes acetylated lysines on the histone tail (27). Both SWI /SNF and S A G A (Spt, Ada, and Gcn5 Acetyltransferase) complexes contain the bromodomain, and preferentially associate with acetylated nucleosomes over unacetylated nucleosomes (28). A more recent example of the histone code is the discovery that Set2p methylation of histone H3 lysine 36 causes the recruitment of a histone deacetylase Rpd3S complex. This recruitment is dependent on a chromodomain of the Ea f i p subunit of the Rpd3S complex (29, 30). Although the implications of the histone code hypothesis are still only beginning to be understood, it is becoming increasingly clear that the modification of histones can cause widespread downstream effects including transcriptional activation and repression. 1.2 Histone Acetylation 1.2.1 Histone Acetylat ion The most well studied of all the post-translational histone modifications is acetylation. This modification is catalyzed by a group of enzymes called histone acetyltransferases (HATs) which transfer an acetyl group from acetyl co-enzyme A to lysine residues of histone proteins (31). The sites of modification reside primarily on the histone tails, though recent work has 4 shown the globular domains of histone proteins are targets of acetylation as well (32-34). The association between histone acetylation and transcription has existed for quite some time (35, 36), and the discovery that some transcription activators and co-activators, like Gcn5p (general control non-deprepressible 5), are actually H A T s only served to strengthen this view (37-40). However, more recent work has shown acetylation to be involved with both transcriptional activation and repression, indicating that the effect of histone acetylation cannot be entirely generalized (28, 41). Table 1.1 Sites of Histone Acetylation in Yeast (34, 42, 43) Histones H2A H2B H3 H4 Acetylation Sites K 5 K 5 K 5 K 8 K 9 K 9 K12 K12 K14 K15 K16 K18 K20 K23 K27 K56 Histone acetylation is generally believed to alter chromatin structure in two ways. The first is that acetylation of histone tails neutralizes the positively charged lysine residues, disrupting interactions with D N A causing altered nucleosomal folding and chromatin structure (44, 45). Although this model does fit with known histone-DNA interactions, the actual effect of acetylation on nucleosome structure directly appears rather modest (46). Additionally, the simplicity of this model fails to explain seemingly contradictory results concerning acetylation and gene expression. The second, mechanism of histone acetylation relates to its function within the histone code hypothesis, specifically that modified residues modulate the activity of 5 activators and repressors of gene activity (47). As has already been described, both SWI /SNF and S A G A complexes specifically recognize and bind acetylated histones. Histone acetyltransferase complexes are also subject to recruitment themselves. Yeast H A T s have been shown to associate with the transactivation domains of activators including V P 16 and Gal4 (48), and S A G A and N u A 4 are recruited to the nucleosome through a shared Tra lp subunit (49). It is thought that this sequence-specific targeting via DNA-binding proteins adds another level of control over transcription (50). 1.2.2 H A T a n d H D A C Complexes The H A T s responsible for post-translational acetylation of histones are categorized as type-A H A T s and are located in the nucleus. Type-B H A T s are found in the cytoplasm and acetylate histones prior to transport into the nucleus. In the yeast Saccharomyces cerevisiae there are at least eight different proteins shown to have H A T activity in in vitro or in vivo assays (34, 37, 51). Two of these H A T s relevant to this study are the H3 specific Gcn5p and Sas3p (something about silencing 3). Gcn5p is part of the G N A T (Gcn5p-related N-acetyltransferases) super family, which also includes Hat lp , Elp3p, and Hpa2p (37). Studies have shown that Gcn5p contains a bromodomain, Ada2p (adaptor deficiency or alteration) interaction domain, and a H A T domain essential for acetyltransferase activity (52, 53). In yeast, Gcn5p is closely associated with the adaptor proteins Ada2p and Ada3p (54). These three proteins form a trimeric module that forms the basis of all known Gcn5p H A T complexes, including S A G A , A D A , S L I K / S A L S A , and H A T - A 2 (55-59). A s shown in vitro, Gcn5p alone has the ability to acetylate free histone H3 at residue K14 (60). Once incorporated into H A T complexes, though, Gcn5p not only gains the ability to acetylate nucleosomal histones (59), but also expands the specificity of those residues 6 acetylated (61). Both S A G A and A D A have been shown to acetylate K 9 , K14 , K18 and K23 on histone H3. Table 1.2 Components of the Gcn5p Containing HAT Complexes SAGA, SLIK/SALSA, and ADA (42) and the NuA3 Complex (62, 63) Complex SAGA SLIK/SALSA ADA HAT-A2 NuA3 Subunits Gcn5 Gcn5 Gcn5 Gcn5 Sas3 Ada2 Ada2 Ada2 Ada3 Y n g l Ada3 Ada3 Ada3 Ada3 N t o l A d a l A d a l Tra l Tra l Ta f l4 Spt20 Spt20 Eaf6 Spt3 Spt3 Spt7 Spt7 Spt8 Taf5 Taf5 Taf6 Taf6 Taf9 Taf9 TaflO TaflO Ta f l2 Ta f l 2 S g f l l S g f l l Sgf29 Sgf29 Sgf29 Sgf73 Sgf73 Ubp8 Ubp8 Rtg2 Ahcl Originally identified in a screen for silencing defects in a sirlA background, Sas3p has subsequently been shown to function as both a positive and negative regulator of transcription depending on the gene locus (63). Sas3p belongs to the M Y S T (MOZ , Sas3p/Ybf2p, Sas2p, and Tip60p) family of histone acetyltransferases (37). As with Gcn5p, Sas3p possesses H A T activity, but can only acetylate nucleosomal histones when incorporated into N u A 3 , a 400 K D a complex made up of Sas3p, Y n g l p , N to lp , Taf l4p, and Eaf6p (Howe, L. unpublished data, (62, 63). NuA3 has been shown in vitro to preferentially acetylate histone H3 K14 and, slightly less effectively, K23 (64). 7 It is interesting to note that although histone acetylation is wel l conserved and seems to be involved in important cellular processes, only one H A T is essential, Esa lp of the H A T complexes N u A 4 and Piccolo N u A 4 (65, 66). It has also been shown that the combination of deleting GCN5 and SAS3 is synthetically lethal, so called because the lethal phenotype is being "synthesized" (64). This lethality is thought to be the result of a loss in some essential acetylation activity that requires at least one of Gcn5p or Sas3p. This lack of serious phenotypes in H A T mutants does suggest a certain degree of redundancy in their actions. Indeed, analysis of acetylated residues targeted by each H A T complex has revealed many similarities, for instance those between S A G A and A D A (61). Again, this serves as a reminder of the complexity and still developing understanding we have of histone acetylation. To counteract the effects of HATs , there exist histone deacetylase complexes (HDACs) . Just as histone acetylation has largely been associated with transcription, deacetylation has long been linked with silencing and repression (67). More recently, however, the Hos2p H D A C has been shown to deacetylate nucleosomes of coding regions during gene expression (68). In yeast, there are at least ten H D A C s in three phylogenetic classes, including Rpd3p, Hda lp , Sir2p, as well as the Hos and Hts proteins (69). One of the better characterized H D A C s is Rpd3p, which has recently been shown to be a part of two distinct complexes, Rpd3S and Rpd3L (29, 30). These two complexes have both been shown to deacetylate nucleosomes in coding regions, preventing spurious transcription. Rpd3S has been previously mentioned for its ability to be recruited by Set2p methylation in a transcriptionally repressive mechanism. 8 1.3 Sptl6p and the Yeast FACT Complex 1.3.1 The SPT Genes In yeast, the homologous recombination of Ty-retrotransposons with the genome can result in the insertion of 8-elements adjacent to or within genes (70). Two such genes include HIS4 and LYS2, resulting in the his4-9128 and lys2-1288 alleles (71). In normal cells, transcription of these genes is disrupted by the presence of promoter and termination sequences within the 8-element, for example, in the lys2-1288 allele, the 8-element inserts downstream of the start of the LYS2 gene. Transcription initiates and proceeds as normal but is prematurely terminated upon reaching the 8-element, causing the production of a non-functional LYS2 transcript, resulting in lysine auxotrophy (72). The mutation of SPT (suppressor of Ty) genes somehow alleviates the effects of 8-element insertion, rescuing production of a functional gene transcript (71). In terms of the lys2-128S allele, SPT gene mutants are thought to lose the ability to repress transcription from promoter elements in the 8-element. This cryptic initiation produces a truncated, though still functional LYS2 transcript, rescuing lysine auxotrophy. This loss of transcriptional repression is thought to be representative of a genome wide effect, as aberrant transcription has been observed at other loci besides lys2-1288 in mutants of SPT genes (73-75). SPT genes are categorized into two groups defined by the Ty elements they suppress (71). The first includes those genes that are related to TFIID function. The second, and more relevant to this study, are the histone related SPT genes. These include SPT11 and SPT12, later identified as the histone H 2 A and H2B encoding HTA1-HTB1 locus (76), SPT4, SPT5, SPT6, and SPT16 (77-79). These SPT gene mutants share similar characteristics, including counteracting the transcriptional defects of SWI /SNF mutants, and suppressing the loss of 9 upstream activator sequence (UAS) regions, needed for binding of transcriptional activators (71). Additionally, the histone related SPT gene mutants share many of the same effects as an imbalance in histone protein ratio and nucleosome disruption, suggesting that these SPT genes function to properly maintain nucleosome structure, and in so doing repress spurious gene expression (76). 1.3.2 The Yeast F A C T Complex - Sp t l6p and Pob3p The yeast F A C T complex (facilitates chromatin transactions - hereafter referred to as F A C T ) is comprised of two components, Sptl6p and Pob3p (polymerase one-binding 3) (80). Both subunits are essential in yeast, and show a high level of conservation throughout eukaryotes as seen in the human F A C T complex and the frog DUF1 complex (81, 82). The identification of SPT 16 actually occurred simultaneously through two different screens, one for suppressors of Ty, and the other for cell division cycle (CDC) mutants (78, 79). Surprisingly, both screens picked out the same G132D mutant of SPT16/CDC68. These initial screens demonstrated that SPT 16 could affect transcription of different genes both positively and negatively, providing the first clues to Sptl6p and F A C T function. Recent domain analysis of Sptl6p has shown it has three domains, a C-terminal (CTD), a mid, and an N-terminal domain (NTD) (83). The C T D was found to be essential for cell viability, suggesting it is required for F A C T ' s essential function, and the mid-domain is responsible for mediating interactions with the other F A C T subunit, Pob3p. A s for the N T D , it is not required for interactions within F A C T , and is suggested to interact with other proteins (83, 84). Pob3p is the smaller of the two F A C T subunits, and was identified through its ability to bind D N A polymerase a (85). One difference of the yeast Pob3p is that it lacks a high mobility group (HMG) box domain, a DNA-binding motif found in other Pob3p homologs SSRP and 10 DUF87 (in human F A C T and DUF1 respectively) (86). Instead, yeast F A C T utilizes the H M G 1 motifs of Nhp6p (non-histone chromosomal protein 6), a ubiquitous nucleosomal binding protein involved in altering nucleosome properties (86). However, strains with deletions of Nhp6p are viable (87), suggesting that the two essential subunits of F A C T proper do not require Nhp6p activity for function. 1.3.3 F A C T Functions in Processes Involving D N A F A C T has been implicated in a variety of processes involving D N A . Initially, the complex was identified through its ability to facilitate R N A polymerase II transcription of nucleosomal D N A in vitro (88). Further in vitro research has shown that F A C T binds H 2 A - H 2 B dimers, destabilizing their interaction with (H3-H4)2 tetramers (82, 84, 89). In vivo, F A C T is distributed in parallel with R N A polymerase II, further supporting its role in facilitating elongation (73, 90). In addition, genetic studies have shown F A C T to be important in mediating nucleosome structure and transcription (86, 91). Currently, F A C T is believed to associate with the nucleosome, facilitating the displacement of H 2 A - H 2 B , disrupting nucleosomal structure and allowing R N A polymerase II progress (89, 92). F A C T also has a repressive effect on transcription, as demonstrated by the initial studies of SPT 16 as a repressor of aberrant transcription (78, 79). Further work has supported this role as spt 16 mutants allow the production of aberrant transcripts from other genes in addition to lys2-128S (74). It is suggested that F A C T mutants fail to properly maintain chromatin structure, thus allowing accessibility to normally non-permissive promoter regions. This may be a result o f improper re-packaging of the nucleosome following F A C T ' S role in transcriptional elongation (74, 92). This failure in preventing aberrant transcription may also be related to F A C T ' S putative role in moderating the fidelity of transcriptional initiation. It has been shown that, for certain 11 genes, F A C T is required for proper promoter association with the preinitiation complex, likely through nucleosomal alterations at promoter sequences (73). Mutants of SPT16, then, fail to prevent similar initiation from hidden promoters which are normally repressed. Other processes F A C T is associated with are D N A replication and D N A repair. In addition to the association of F A C T with subunits of the D N A polymerase a complex (80, 93), mutations of F A C T have been shown to interact genetically with genes important in D N A replication (94). Depletion of the F A C T complex in Xenopus oocytes has also been shown to compromise D N A synthesis in vitro (81). F A C T also interacts with the protein kinase C K 2 , which phosphorylates and activates p53 during times of replication stress (95). Whether these functions of F A C T are independent o f each other or representative of a more general function is currently not clear. Considering the above processes all involve D N A , and therefore must overcome the non-permissive state of chromatin, it is conceivable that F A C T is simply involved in maintaining nucleosome structure. Recent work has suggested that F A C T may be important in nucleosome reassembly, as mutations in SPT 16 and POB3 caused dependence on the Hir /Hpc nucleosome reassembly pathway (91). In any case, F A C T remains an integral facilitator of important cellular processes, although the mechanisms by which it acts are not completely elucidated. The research described in this study aimed to understand the possible relationship between histone acetylation and F A C T complex function, first suggested in recent studies showing a putative interaction between Sas3p (of NuA3) and Sptl6p (63, 96). In the first part of this thesis, we describe the confirmation of this interaction, and investigate F A C T ' S association with other H A T complexes and their possible role in F A C T function. The second half focuses on the novel sptl6A922 mutant, specifically examining the hypothesis that this partially truncated Sptl6p interferes with normal histone modifications. 12 Chapter 2 - Materials and Methods 2.1 Preparation of Yeast Strains and Plasmids The manipulation o f yeast strains in this study was carried out using standard protocols (97). These include all transformations, sporulation, screening, and plating. A l l yeast strains used in this study are listed in table 2.1, and all plasmids in table 2.2. The insertion of both T A P tags and H A tags downstream of HTB1, POB3, and AHC1 required additional P C R product. Consequently, 3x 100 uL reactions were concentrated to 40 uL for transformation into the cell. P C R purifications and gel purifications were done using the Wizard S V Gel and P C R Clean-up system (Promega Corporation, Madison, WI). The sptl6 null strain SNL314 and all SPT16 plasmids were gifts from our collaborators in the R.A. Singer Lab at Dalhousie University. The haploid sptl6::KANMX4 strain is maintained by a wi ld type SPT16 locus on the pSNLCDC68(3) plasmid. This plasmid consists of the 4.6 kb Bgl l /BamHI wi ld type SPT16 fragment from p 6 8 B S X l cloned into pSLncrABS. The pSNLCDC68(3) plasmid includes the URA3 gene and the ADE3 gene, allowing for selection against it using 5 ' - F O A plates (0.1% of 5'-fiuoroorotic acid) media and red/white colour-screening respectively. A l l pRS315 wi ld type and mutant SPT 16 plasmids were created by inserting a BamHI/SstI SPT16 fragment from a pRS316 plasmid into pRS315. The sptl6A922 truncation mutant was originally created using an Sstl/EagI SPT 16 fragment which was subsequently mutagenized and subject to in-frame deletion with C la l before KpnI/EagI digestion yielded the sptl6A922 gene fragment (98). The sptl6ANTD truncation mutant was originally created from a Nco l /B tg l digestion of full-length SPT16 on pJW212 (83). 13 The pSPT8HA plasmid contains the SPT8 ORF plus -1 ,000 bp of upstream sequences including the endogenous promoter, P C R amplified from genomic D N A . The fragment was then digested by Apa l /Sa l l and cloned into a CEN/ARS TRP1 vector pRS314 fused to a triple hemagglutinin ( H A ) - C Y C cassette. The wi ld type HHT2-HHF2 plasmid pHHT2 was created by Spel digestion of pDM18 (99), followed by ligation of the largest fragment into the Spel site of the vector pRS414 (100). A l l plasmids with HHT2 point mutations were created by site directed mutagenesis using Stragatene's QuickChange Site-Directed Mutagenesis K i t (101). 14 Table 2.1 Yeast Strains Used in This Study Strain Mating Type J \ j Genotype i SNL314 Mat a his4-912& leu2-3 Ieu2-112 Iys2-128b ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 j pSNLCDC68(3).URA „ „ „ ! YJS101 Mat a his4-9125 leu2-3 Ieu2-112 Iys2-1285 ura3-52 trplA 1 ade2::HISG ade3::HIS cdc68::KANMX4 \ HTB1TAP::TRP1 pCDC68NM2(4).LEU YJS102 Mat a his4-912S leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 j HTB1TAP::TRP1pCDC68NM3(4).LEU i YJS103 Mat a his4-9125 leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 | HTB1TAP::TRP1 pCDC68M3(12).LEU _ . J YJS104 Mat a his4-9128 leu2-3 Ieu2-112 Iys2-128b ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 | HTB1TAP::TRP1 pCDC68M3(19).LEU J YJS105 Mat a his4-912S \eu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 ! HTB1TAP::TRP1 pCDC68ANTD.LEU .. i YJS106 Mat a his4-912S leu2-3 Ieu2-112 lys2-128S ura3-52 trplA 1 ade2::HISG ade3::HIS cdc68::KANMX4 I PCDC68A4. LEU ' i YJS107 Mat a his4-912b leu2-3 Ieu2-112 Iys2-128b ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 | pCDC68NM2(4).LEU j YJS108 Mat a his4-912& leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 \ pCDC68NM3(4).LEU YJS109 Mat a his4-9128 leu2-3 Ieu2-112 Iys2-128& ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 pCDC68M3(12).LEU _ _ j YJS110 Mat a his4-912& leu2-3 Ieu2-112 Iys2-128b ura3-S2 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 \ pCDC68M3(19).LEU YJS111 Mat a his4-912S leu2-3 Ieu2-112 Iys2-128b ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 pCDC68ANTD.LEU j YJS112 Mat a his4-9125leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 \ PCDC68A922.LEU . _ J YJS113 Mat a his4-9128 leu2-3 Ieu2-112 Iys2-1288 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 \ POB3TAP::TRP1 pCDC68A4.LEU YJS114 Mat a his4-9125 leu2-3 Ieu2-112 Iys2-128& ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 | POB3TAP:TRP1 pCDC68ANTD.LEU I YJS115 Mat a his4-912Sleu2-3 Ieu2-112 lys2-1288ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 | POB3TAP..TRP1 pCDC68A922.LEU ) YJS120 Mat a his4-912b leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 POB3TAP..TRP1 pCDC68A4.LEUpAHCIHA.URA „ \ YJS121 Mat a his4-912& leu2-3 Ieu2-112 Iys2-1288 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 POB3TAP::TRP1 PCDC68ANTD.LEUpAHCIHA.URA , „. . j YJS122 Mat a his4-912Sleu2-3 Ieu2-112 lys2-128Sura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 POB3TAP::TRP1 pCDC68A922.LEU pAHCIHA.URA YJS127 Mat a his4-912S leu2-3 Ieu2-112 Iys2-1288 ura3-52 trp1A1 ade2::HIS<3 ade3::HIS cdc68::KANMX4 YJS128 Mat a his4-9125 \eu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 rpd3::TRP1 pCDC68ANTD.LEU YJS129 Mat a his4-9126leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 rpd3::TRP1 pCDC68A922.LEU _ YJS130 Mat d his4-912S leu2-3 Ieu2-112lys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 rpd3::TRP1 pCDC68NM2(4).LEU YJS131 Mat a his4-912& leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 rpd3::TRP1 pCDC68NM3(4).LEU YJS132 Mat a his4-912S leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 rpd3::TRP1 pCDC68M3(12).LEU YJS133 Mat a his4-912& leu2-3 Ieu2-112 Iys2-128& ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 rpd3::TRP1 pCDC68M3(19).LEU YJS134 Mat a his3A200 leu2Al lys2-1285ura3-52 trp1A63 cdc68::KANMX4 HTB1-3HA::HIS3MX6 POB3TAP::TRP1 pSNLCDC68(3).URA 15 YJS137 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 cdc68::KANMX4 HTB1- I 3HA::HIS3MX6POB3TAP::TRP1 pCDC68A4.LEU YJS138 Mat a his3A200 leu2A1 lys2-128Sura3-52 trp1A63 cdc68::KANMX4 HTB1-3HA::HIS3MX6, P0B3TAP..TRP1 pCDC68ANTD. LEU YJS139 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 cdc68::KANMX4 HTB1-3HA::HIS3MX6 P0B3TAP::TRP1 pCDC68A922.LEU ' \ YJS141 Mat a his3A200 leu2Al lys2-128Sura3-52 trp1A63 POB3-3HA::HIS3MX6 YJS145 Mat a his3A200 leu2Al lys2-128S ura3-52 trp1A63 HTB1-3HA::HIS3MX6 P0B3TAP::TRP1 ada2::KANMX4 ' ^ YJS146 Mat a his3A200 leu2Al lys2-128Sura3-52 trp1A63 HTB1-3HA::HIS3MX6 P0B3TAP::TRP1 sas3::KANMX4 „ _ _ YJS148 Mat a his3A200 leu2Al Iys2-1285ura3-52 trp1A63 (hht1-hhf1)::LEU2 (hht2-hhf2)::HIS3 Ty912S35::his4 rpd3::URA3 pHHT2(K14R). TRP YJS149 Mat a his3A200 leu2Al lys2-128S ura3-52 trp1A63 (hht1-hhf1)::LEU2 (hht2-hhf2)::HIS3 Ty912535::his4 rpd3::URA3 pHHT2(K23R):TRP „ „ YJS153 Mat a I his3A200 leu2Al Iys2-1285ura3-52 trp1A63 AHC1-3HA::HIS3MX6 YJS155 YJS156 Mat a Mat a his4-9126 leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 P0B3TAP::TRP1 pCDC68A4.LEU pADA2HA.URA his4-9125 leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 P0B3TAP::TRP1 pCDC68A4.LEU pSPT8HA. URA YJS157 Mat a his3A200 leu2A1 lys2-128Sura3-52 trp1A63 NT01-3HA::HIS3MX6 pFLAG-SPT16.TRP YJS158 Mat a his3A200 leu2A1 lvs2-128Sura3-52 trp1A63 YNG1HA::KANMX6pFLAG-SPTI6.TRP YJS160 Mat ct his4-9128 leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 pCDC68A4.LEUpAHC1HA.URA _._ YJS161 Mat a his4-912& leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 PCDC68ANTD.LEU pAHCIHA.URA YJS162 Mat a his4-912Sleu2-3 Ieu2-112 lys2-128Sura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 PCDC68A922.LEUpAHCIHA.URA , „ ... YJS163 Mat a his3A200 leu2A1 Iys2-1285 ura3-52 trp1A63 pAHCIHA. URA YJS164 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 pAHA2HA. URA YJS165 Mat a his3A200leu2A1lys2-1285ura3-52trp1A63pSPT8HA.URA YJS166 Mat a his3A200 leu2A1 Ivs2-128dura3-52 trp1A63 SAS3-3HA::KANpFLAG-SPT16.TRP . „ _ YJS167 Mat a his3A200 leu2A1 lys2-128Sura3-52 trp1A63 pFLAG-SPT16.TRP YJS168 Mat a ' his3A200 leu2A1 lys2-128S ura3-52 trp1A63 pFLAG-SPT16. TRP pAHCIHA.URA YJS169 Mat a his3A200 leu2A1 \ys2-1285 ura3-52Jrp1A63pFLAG-SPT16. TRP pADA2HA. URA _ YJS170 Mat a his3A200 leu2A1 Iys2-1285ura3-52 trp1A63pFLAG-SPT16.TRPpSPT8HA.URA YJS171 YJS172 Mat a Mat a his4-9126 leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP..TRP1 AHC1-3HA::HIS3MX6 pSNLCDC68(3).URA _ _ _ his4-9125 leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP..TRP1 AHC1-3HA::HIS3MX6 pSNLCDC68(3).URA YJS173 Mat a his4-912b leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP..TRP1 SAS3HA::HIS3MX6 pSNLCDC68(3).URA YJS174 Mat a his4-912b leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP::TRP1 SAS3HA::HIS3MX6pSNLCDC68(3).URA YJS176 Mat a his3A200 leu2Al lys2-128Sura3-52 trp1A63 AHC1TAP::TRP YJS177 Mat a his4-912h leu2-3 Ieu2-112 Iys2-1288 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 P0B3TAP::TRP1 NT01-3HA::HIS3MX6pSNLCDC68(3).URA YJS178 Mat a his4-912b leu2-3 Ieu2-112 Iys2-1286 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 P0B3TAP::TRP1 NT01-3HA::HIS3MX6 pSNLCDC68(3). URA YJS181 Mat a his4-9128 leu2-3 Ieu2-112 Iys2-128b ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 P0B3TAP::TRP1 pCDC68A4.LEUpYNG1HA.URA YJS182 Mat a his4-9125 leu2-3 Ieu2-112 Iys2-128h ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 P0B3TAP::TRP1 pSNLCDC68(3).URA pSAS3HA.LEU _ YJS183 Mat a his3A200 leu2A1 lys2-128Sura3-52 trp1A63 pYNGIHA.URA YJS184 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 pSAS3HA. URA YJS185 Mat a his4-912S leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP::TRP1 AHC1-3HA..HIS3MX6 pCDC68A4.LEU 16 I YJS186 Mat a his4-912S leu2-3 Ieu2-112 Iys2-1286 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP::TRP1 AHC1-3HA::HIS3MX6 pCDC68ANTD.LEU YJS187 Mat a his4-912& \eu2-3 Ieu2-112 Iys2-128& ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP::TRP1 AHC1-3HA::HIS3MX6 pCDC68A922.LEU YJS188 Mat a his4-9125 leu2-3 Ieu2-112 Iys2-128& ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP::TRP1 SAS3HA::HIS3MX6 pCDC68A4.LEU YJS189 Mat a his4-9125 \eu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP::TRP1 SAS3HA::HIS3MX6 pCDC68ANTD.LEU YJS190 Mat a his4-9125 leu2-3 Ieu2-112 Iys2-128& ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP::TRP1 SAS3HA::HIS3MX6 pCDC68A922.LEU YJS194 Mat a his3A200 leu2A1 lys2-128Sura3-52 trp1A63 POB3-3HA::HIS3MX6 pFLAG-SPT16.URA YJS195 Mat a his3A200 leu2A1 lys2-128Sura3-52 trp1A63 pYNGIHA.URA pFLAG-SPT16.URA I YJS196 Mat a his4-912B leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP::TRP1 pSPT8HA.URA pCDC68A4.LEU YJS197 Mat a his4-9128 leu2-3 Ieu2-112 Iys2-128& ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP::TRP1 pSPT8HA.URA pCDC68ANTD.LEU „ _ YJS198 Mat a his4-912& leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP::TRP1 PSPT8HA.URA pCDC68A922.LEU YJS199 Mat a his4-912& leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 pCDC68A4. LEU pSAS3. URA YJS200 Mat a his4-912& leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 pCDC68ANTD.LEU pSAS3HA.URA YJS201 Mat a his4-912Sleu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 pCDC68A922.LEUpSAS3HA.URA YJS202 Mat a his4-9126 leu2-3 Ieu2-112 Iys2-128& ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 P0B3TAP::TRP1 pCDC68A4.LEU pSAS3HA.URA YJS203 Mat a his4-912S leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 P0B3TAP::TRP1 pCDC68ANTD.LEU pSAS3HA.URA YJS204 Mat a his4-912Sleu2-3 Ieu2-112 lys2-128Sura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 P0B3TAP::TRP1 pCDC68A922.LEU pSAS3HA. URA YLH101 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 YLH102 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 YLH104 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 gcn5::HIS3 YLH106 Mat a his3A200 leu2Al lys2-128Sura3-52 trp1A63 sas3::HIS3MX6 YLH139 Mat a " his3A200 leu2A1 lys2-128S ura3-52 trp1A63 NT01-3HA::HIS3MX6 I YLH144 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 ada2::HIS3 YLH145 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 ada2::HIS3 sas3::HIS3MX6 YLH158 Mat a his3A200 leu2Al lys2-128Sura3-52 trp1A63 ahd::HIS3MX6 YLH160 Mat a his3A200 leu2Al lys2-128Sura3-52 ahd::HIS3MX6 sas3::HIS3MX6 YLH176 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 YNG1HA::KANMX6 YLH224 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 (hht1-hhf1)::LEU2 (hht2-hhf2)::HIS3 Ty912S35::his±__ YLH262 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 EAF6HA::HIS3MX6 YLH267 Mat a his4-912b leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1 TAP..TRP1 pSNLCDC68(3).URA „ . _ YLH268 Mat a his4-912b leu2-3 Ieu2-112 lys2-128S ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP..TRP1 pCDC68A4.LEU YLH269 Mat a his4-9125 leu2-3 Ieu2-112 Iys2-1285 ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 HTB1TAP..TRP1 pCDC68A922.LEU YLH286 Mat a his4-912& leu2-3 Ieu2-112 Iys2-128& ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 P0B3TAP::TRP1 pSNLCDC68(3).URA YLH307 Mat a his4-9128 leu2-3 Ieu2-112 Iys2-128& ura3-52 trp1A1 ade2::HISG ade3::HIS cdc68::KANMX4 rpd3::TRP1 pSNLCDC68(3J.URA ... YLH319 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 HTB1-3HA::HIS3MX6 YLH322 Mat a his3A200 leu2A1 lys2-128Sura3-52 trp1A63 HTB1-3HA::HIS3MX6 P0B3TAP::TRP1 YDM126 Mat a his3A200 leu2A1 lys2-1285ura3-52 trp1A63 SAS3-3HA::HIS3MX6 YDM147 Mat a his3A200 leu2A1 lys2-128Sura3-52 trp1A63 SAS3-3HA::KAN YDM175 Mat a his3A200 leu2Al lys2-128Sura3-52 trp1A63 (hht1-hhf1)::LEU2 (hht2-hhf2)::HIS3 Ty912S35::his4 pHHT2(K4R)HHF2. TRP 17 YDM176 Mat a his3A200 leu2A1 lys2-1285ura3-52 trp1A63 (hht1-hhf1)::LEU2 (hht2-hhf2)::HIS3 Ty912S35::his4 pHHT2(K14R)HHF2. TRP „ „. YDM177 Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 (hht1-hhf1)::LEU2 (hht2-hhf2)::HIS3 Ty912S35::his4 pHHT2(K23R)HHF2. TRP YDM203 Mat a his3A200 leu2A1 lys2-128Sura3-52 trp1A63 (hht1-hhf1)::LEU2 (hht2-hhf2)::HIS3 Ty912S35::his4 pHHT2(K36R)HHF2. TRP YDM204 I YDG010 Mat a Mat a his3A200 leu2A1 lys2-128S ura3-52 trp1A63 (hht1-hhf1)::LEU2 (hht2-hhf2)::HIS3 Ty912S35::his4 pHHT2(K4R, K36R)HHF2. TRP „ „ his3A200 leu2A1 lys2-128Sura3-52 trp1A63 (hht1-hhf1)::LEU2 (hht2-hhf2)::HIS3 Ty912S35::his4 pHHT2HHF2. TRP Table 2.2 Plasmids Used in This Study Plasmid Description Source or Reference pGSTSPT16 GST-Spt16 fusion protein for amino acids 1-464 on pGEX2T vector (63) pSNLCDC68(3) CDC68 insert cloned into pSLncrDBS vector (83) p£DC68A4 CDC68 WT fragment inserted into pRS315 vector R. Singer Lab pCDC68ANTD pCDC68A922 mutant cdc68 A2-484 inserted into pRS315 vector R. Singer Lab mutant cdc68 A6-306 inserted into pRS315 vector R. Singer Lab pCDC68NM2(4) mutant cdc68 E736G inserted into pRS315 vector R. Singer Lab pCDC68NM3(4) mutant cdc68 E857K inserted into pRS315 vector R. Singer Lab pCDC68M3(12) mutant cdc68 E763G. R784G, S819P inserted into pRS315 vector R. Singer Lab , .V.V.,„ V™™/™™**, * pCDC68M3(19) mutant cdc68 L804P, L946S, E1004G inserted into pRS315 vector R. Singer Lab pHHT2 HHT2-HHF2 locus on pRS414 vector _ .,„ J101) phht2K4R substitution mutation K4R of H3 on pHHT2 (101) phht2K14R substitution mutation K14R of H3 on pHHT2 (101) phht2K23R substitution mutation K23 of H3 on pHHT2 (101) , _ _ phht2K36R substitution mutation K36R of H3 on pHHT2 (101) phht2K4/36R pAHCIHA substitution mutations K4R and K36R of H3 on pHHT2 (101) _ AHCIORF on pRS314 fused to C-terminal triple HA cassette (58) PADA2HA ADA2 ORF on YCp88 fused to N-terminal triple HA cassette 1_Q02) pSAS3HA SAS3 ORF on pRS314 fused to C-terminal triple HA cassette M62) pSPT8HA SPT8 ORF on pRS314 fused to C-terminal triple HA cassette this study pSPT16FLAG full length SPT16 fused to C-terminal FLAG epitope (83) 18 2.2 PCR Techniques The tagging and deletion of genes was achieved using high efficiency transformation of P C R product. A l l P C R reactions were done on an Eppendorf Mastercycler Gradient P C R machine (Eppendorf North America, N Y ) using Taq polymerase enzyme. A 100 uL reaction was the normal volume for P C R product to be used for insertions or deletions, and typically contained 77 pi of s d H 2 0 , 10 ul of 10X Taq buffer, 8 ul of 2.5 m M dNTPs, 0.5 u M primers, 1 pi of template D N A , 1 pi of Taq enzyme. Screening for insertions or deletions was also done with PCR, using genomic D N A of transformed strains as a template. Standard elongation protocol was used for all reactions using Taq polymerase enzyme (92°C for 30 s. followed by 30 cycles of.92°C for 45 s., 55°C for 45 s., and 72°C for 1 min). 2.3 Plasmid Shuffle In this study, generation of spt 16 mutant strains was accomplished by transforming a pRS315 plasmid containing an spt 16 mutation into strains with the genomic copy of SPT 16 deleted and a wi ld type SPT16 gene carried on the pSNLCDC68(3) plasmid. The pSNLCDC68(3) plasmid contains both the URA3 and ADE3 genes, allowing for selection against it in two steps. First, transformants of the spt 16 mutant plasmid with LEU2 marker underwent red/white colour-screening. Selection of white colonies in an ade2::HISG, ade3::HIS strain indicated absence of ADE3 gene, and pSNLCDC68(3) plasmid. Growth on 5 ' - FOA plates (0.1% of 5"-flouroorotic acid) used to verify the absence of the URA3 gene on the pSNLCDC68(3) plasmid. 19 2.4 Preparation of Whole Cell Extracts for FLAG and TAP Pull-downs Strains were inoculated from freezer stocks into 5 m L of yeast extract-peptone dextrose (YPD) or, in the case of maintaining a plasmid, appropriate drop out media and allowed to grow overnight at 30°C with shaking. Fifty milliliter cultures were then inoculated using the 5 m L overnight culture to an OD600 of 0.5, and grown at 30°C with shaking to an OD600 of 2.0. Cells were harvested by centrifugation (4,000 rpm for 3 min) at 4°C, followed by l x wash with 25 m L distilled water. Harvested cells were resuspended in 500 uL of IPP 150 buffer (10 m M Tr is -C l pH 8.0, 150 m M N a C l , 0 .1% NP40 with 1 m M P M S F and 2 p.g/mL pepstatin A ) and an equivalent volume of glass beads was added. Samples were vortexed for 3 min using a Vortex Genie (Scientific Industries, Bohemia, N Y ) . Lysates were then clarified by centrifugation (14,000 rpm for 5 min) at 4°C. 2.5 Pull-downs of FLAG- and TAP-Tagged Proteins Whole cell extracts were normalized for bulk protein content by Bradford assay. IgG sepharose 6 Fast F low resin (Amersham Biosciences, Piscataway, NJ) or a F L A G M 2 resin (Sigma Chemical Co., St. Louis, M O ) was pre-washed 4x with cold IPP 150 buffer and aliquoted in 20 uL volumes for each pull-down. Four hundred microlitres of normalized whole cell extracts were added and allowed to rotate at 4°C for 2 h. Fol lowing binding, resin was washed 5x with 20 volumes o f cold IPP 150 buffer. Thirty microlitres o f 2.5x SDS loading buffer was then added to the resin and boiled for 5 min at 100 °C. Precipitated proteins were then analyzed using western blot analysis with anti -HA antibodies (Roche Diagnostics Canada, Laval, Qc). Normalization of T A P pull-downs was done by western blot analysis with peroxidase anti-peroxidase (PAP) antibodies (Sigma Chemical Co., St. Louis, MO) . 20 Normalization of F L A G pull-downs was done by coomassie staining of SDS polyacrylamide gel (PAGE) . 2.6 Preparation of Whole Cell Extracts for Calmodulin Pull-downs Strains used for calmodulin pull-downs were inoculated in 5 m L of Y P D and grown overnight at 30°C with shaking. One litres cultures o f Y P D were then prepared by inoculating the overnight culture and growing overnight at 30°C with shaking to an OD600 of 2.0. One litre cultures were harvested by centrifugation (10 min at 4,000. rpm) at 4°C and washed in 100 m L of distilled water. Cells from each 1 L culture was resuspended in 25 m L of calmodulin binding buffer (10 m M (5-mercaptoethanol, 10 m M Tr is -C l p H 8.0, 150 m M N a C l , 1 m M Mg-acetate, 1 m M imidazole, 2 m M C a C l 2 , and 0.1% NP40 with 1 m M P M S F and 2 p.g/mL pepstatin A) . Cells were broken by adding cell suspension to a 30 m L bead-beater chamber (BSP bead-beater, Biospec Products, Bartlesville, O K ) with - 1 0 m L of glass beads. Ten cycles of bead-beating (15s. bead-beating followed by 1 min rest) produced cellular lysates, which were then clarified by centrifugation (10,000 rpm for 15 min). Resultant supernatant made up whole cell extracts. 2.7 Calmodulin Pull-down Purifications of Protein Complexes For each calmodulin pull-down purification, 70 uL calmodulin affinity resin slurry (Stratagene, Cedar Creek, T X ) was pre-washed 4x with cold calmodulin binding buffer. Twenty-five mililitres o f whole cell extract was then added to the resin and allowed to rotate at 4°C for 2 h. Resin was washed 4x with 20 volumes of cold calmodulin binding buffer. Bound proteins were then eluted off the calmodulin resin by washing with 100 uL of calmodulin elution buffer (10 m M p-mercaptoethanol, 10 m M Tr is -C l pH8.0, 150 m M N a C l , 1 m M M g -21 acetate, 1 m M imidazole, 2 m M E G T A , and 0.1% NP40) except for A h c l T A P (purified A D A complex), which required 4 m M E G T A calmodulin elution buffer. Three elution washes were performed by rotating for 30 min at 4°C. Relative amount of purified protein in each elution was determined by western blot analysis with peroxidase anti-peroxidase (PAP) antibodies (Sigma Chemical Co., St. Louis, MO) . 2.8 Preparation of GST-Sptl6p Fusion Protein The pGSTSPT16 plasmid expressing GST fusion protein and was transformed into BL21 (DE3) cells and inoculated into 10 m L L B ampicill in media (50 u.g/mL ampicillin) for growth overnight at 37°C with shaking. Five hundred milliliters of L B ampicil l in were inoculated with 5 m L saturated overnight culture and grown at 37°C with shaking to an OD600 = 0.6. IPTG (isopropyl-beta-D-thiogalactopyranoside) was then added to the 500 m L culture to a final concentration of 0.1 m M causing induction of fusion protein. Cells were grown an additional 3 h. at 37°C with shaking. Cells were harvested by centrifugation (4000 rpm for 10 min) and washed with 25 m L l x phosphate buffered saline (PBS) solution. Cells were resuspended in 25 m L l x PBS with 1 m M P M S F and 2 u.g/mL pepstatin A and lysed by sonication using an Ultrasonic Processor X L (Mandel, Guelph, Ont) on setting 4 for 3x 30 s. with 1 min rests. Triton-X-100 was added to the lysates to a final concentration of 1% and allowed to rotate for 20 min at 4°C. Lysates were then clarified by centrifugation (10,000 rpm for 15 min at 4°C). GST-Spt l6p was purified using glutathione sepharose resin (Amersham Biosciences, Piscataway, NJ). Two hundred and fifty microlitres of glutathione resin was pre-washed 3x with l x PBS. Prepared lysates were then added and rotated for 2 h. at 4°C to allow binding. 22 Glutathione resin was washed 3x with cold l x PBS and transferred to a new 1.5 m L microfuge tube. 2.9 The Sptl6p Pull-down (in vitro analysis) In vitro pull-downs were performed using the prepared GST -Spt l6p fusion glutathione sepharose resin from above. Twenty microlitres each of purified N u A 3 , A D A , and S A G A (from calmodulin purification pull-down) were diluted into 180 uL of IPP 150 buffer (described above) and added to 10 uL of glutathione resin (containing immobilized GST-Spt l6p) . Tubes were rotated for 2 h. at 4°C to allow binding. Resin was washed 3x with 20 volumes of IPP 150 buffer. Thirty microlitres of 2.5x SDS loading buffer was then added to the resin and boiled for 5 min at 100 °C. Precipitation of A D A , N u A 3 , and S A G A with GST -Spt l6p was then detected by western blot analysis using peroxidase anti-peroxidase (PAP) antibodies (Sigma Chemical Co., St. Louis, MO) . 2.10 Histone H3 Acetylation Analysis Western blots for H3 acetylation were performed on isogenic wi ld type and spt 16 mutant strains. Strains were grown in 50 m L cultures and harvested as described previously. Whole cell extracts were prepared in 400 uL of IPP 150 buffer with P M S F and pepstatin. Bulk protein content of whole cell extracts was normalized using Bradford assay. Thirty microlitres of 5x SDS loading buffer was then added to 20 uL of normalized sample and boiled for 5 min at 100 °C. Western blots were performed using anti-acetyl-H3 antibodies (Upstate Biotech, Lake Placid, N Y ) . 23 Chapter 3 - Results 3.1 F A C T Interacts with the H A T Complexes N u A 3 and A D A The first clue to an interaction between the F A C T complex and a histone acetyltransferase came in a yeast two-hybrid screen (96). In it, gene fragments of SAS3, the catalytic subunit of N u A 3 , were pulled out using a LexA-Sptl6(i -464) fusion protein as bait. This was followed by evidence suggesting the entire NuA3 complex interacts with Sptl6p in vitro, using a GST-Sptl6(i_464) fusion protein and purified N u A 3 , and in vivo, using anti-Sptl6p polysera and HA-tagged Sas3p (63). Although the above mentioned studies strongly suggest a physical interaction between NuA3 and F A C T , no corresponding results have been published supporting the requirement of Sas3p for F A C T function. Mutants of the essential gene SPT 16 show a variety o f phenotypes, including sensitivities to hydroxyurea (HU) and 6-azauracil (6 -AU) , and an spt phenotype (63, 78, 79, 103). Yet, mutants of SAS3, or genes encoding other components of N u A 3 , show no such phenotypes. If these two complexes are in fact associating with each other for some process or mechanism, one would expect that disruption o f either complex would result in similar phenotypes. The fact that this is not the case suggested to us two possibilities. First, the Sas3p-Sptl6p interaction is an artifact and not actually indicative of a real N u A 3 - F A C T association. The second possibility is that F A C T is interacting with more than one H A T complex, and this other H A T acts in a role redundant to N u A 3 . However, before exploring this interesting hypothesis, we first aimed to eliminate any doubts regarding the interaction between NuA3 and F A C T . 2 4 3.1.1 NuA3 Interacts wi th F A C T To confirm an interaction between N u A 3 and F A C T , we first wanted to determine whether Sas3p was associating with the entire F A C T complex and not just Sptl6p. To this end, the C-terminus of Pob3p, a second subunit of F A C T , was TAP-tagged and tested for co-precipitation with Sas3p. The T A P tag consists of IgG-binding units o f protein A (ProtA) from Staphylococcus aureus and a Calmodulin Binding Peptide (CBP). Due to the interaction between ProtA and immunoglobins, proteins with a fused T A P tag w i l l bind IgG sepharose. Although not typical of a classical immunoprecipitation (IP), we w i l l be referring to the precipitation of TAP-tagged proteins with IgG resin as an IP. To detect Sas3p, an H A epitope tag was attached to the C-terminus of Sas3p expressed from its endogenous promoter on a centromeric plasmid (pSAS3HA), and was transformed into the Pob3TAP strain. Pull-downs were performed using strains expressing either Sas3HA3 or Pob3TAP by themselves (as negative controls), and a strain expressing both S a s 3 H A 3 and Pob3TAP. This was followed by anti -HA and peroxidase-anti-peroxidase (PAP) western blot analysis (Figure 3.1 A) . The anti-H A western blot clearly showed that Sas3HA3 was being co-precipitated with Pob3TAP. No Sas3HA3 signal was observed in the pull-down of the non-TAP tagged strain, revealing no visible non-specific interaction of Sas3HA with the resin. Although the ant i -HA antibody did recognize the T A P epitope attached to Pob3p in the IP lanes (presumably due to an interaction between the H A antibody and the protein A motif of the T A P tag), we chose to also use a P A P western to ensure equal precipitation of Pob3p between samples in this experiment. This in vivo interaction between Sas3p and Pob3p served to verify past data suggesting a Sas3p-FACT physical association. It also determined that the entire F A C T complex, and not just Sptl6p, was involved in this interaction. When taken together with the fact that GST-Pob3 fails 25 A. Input IP 4. + — + + — SAS3HA — + + — + + P0B3TAP Sas3HA Pob3TAP _ + + P0B3TAP Pob3TAP B. Input IP aHA NT01HA SAS3HA Spt16-FI_AG Sas3HA NtoIHA Spt16-FLAG Coomassie f Spt 16 FLAG Figure 3.1 NuA3 interacts w i th the F A C T Complex. (A) The interaction o f Pob3p with S a s 3 H A 3 was detected by ant i -HA western blot analysis following pull-down of TAP-tagged Pob3p (1% of input sample and 50% of IP). Precipitated samples were also probed by P A P western blot analysis to verify levels of Pob3TAP in samples (5% of IP). (B) S p t l 6 - F L A G was expressed on a plasmid and used in a F L A G immuno-precipitation experiments in strains expressing N t o l H A 3 and Sas3HA3. Interaction was detected by anti -HA western blot analysis (1% of input and 50% IP). The precipitation of S p t l 6 - F L A G was verified by coomassie blue staining o f IP samples (50% of IP). 26 to associate with purified N u A 3 in vitro (63) this data suggests that although Pob3TAP is pulling-down Sas3p, it is doing so through the interaction with Sptl6p. Although satisfied with the results of the Pob3TAP co-precipitation, we wished to determine whether it is the entire N u A 3 complex, not just Sas3p that is interacting with F A C T . To do this, we chose to demonstrate a N u A 3 - F A C T physical interaction using different subunits of each complex. In addition to Sas3p, the NuA3 H A T complex is known to contain at least four other subunits, Y n g l p , N to lp , TAF14p and Eaf6p (62, 63). Thus far, no other subunit of NuA3 has been shown to interact with either component of F A C T . Initially, we attempted to use a strain expressing HA-tagged Y n g l p in this set of immunoprecipitation experiments, however results lacked consistency. Furthermore, recent work has suggested that the H A epitope interferes with Y n g l p function (Martin et al., submitted). Consequently, we focused our attention on the N t o l p subunit of NuA3 in our attempts to further confirm a N u A 3 - F A C T association. In this experiment, a centromeric plasmid containing a C-terminal FLAG-tagged Sptl6p expressed from its endogenous promoter was transformed into strains expressing Sas3HA 3 (YDM147) and N t o l H A 3 (YLH139). Whole cell extracts were prepared from the resulting strains and S p t l 6 - F L A G was immunoprecipitated using a F L A G resin. A s seen in Figure 3.IB, we successfully showed another N u A 3 subunit, NtolHA3, interacts with Sptl6p in vivo. As a positive control, Sas3HA3 was also included in this immunoprecipitation experiment. Although significant non-specific binding of Sas3HA 3 was observed in the negative control, the signal was at least two-fold higher in the presence of S p t l 6 - F L A G . Coomassie staining for each IP sample was used to demonstrate equal levels of the S p t l 6 - F L A G fusion protein in the precipitates. After confirming the N u A 3 - F A C T interaction using tagged fusion proteins of both subunits of F A C T , and Sas3p and N t o l p of the N u A 3 complex, we were confident in discarding 27 the first possibility, that the Sptl6p-Sas3p interaction observed by two-hybrid is an artifact. Next, we moved onto exploring the second possibility that F A C T interacts with other H A T complexes. 3.1.2 Sp t l6p Interacts Direct ly wi th both N u A 3 and A D A If F A C T does have other interacting partners redundant with N u A 3 , it seemed logical that these partners share a similar function with NuA3 . Deletion of SAS3 results in only minor phenotypes, whereas deletion of SAS3 in combination with loss of GCN5 results in a synthetic lethality. Therefore, the possibility exists that NuA3 shares an overlapping function with a GC/V5-dependent H A T complex. Gcn5p is the catalytic subunit of at least four H A T complexes including S A G A , A D A , S L I K / S A L S A , and H A T - A 2 (55-59), although there is some debate as to whether the later two are bona fide complexes or subcomplexes of S A G A (55, 57, 104). Thus S A G A and A D A alone were selected as candidates for F A C T association. To investigate a possible interaction with F A C T , we chose to revisit the in vitro G S T pull-down system used in the original Sas3p interaction study (63). This would allow us to reproduce the initial data, while also testing the direct interaction between H A T complexes and Sptl6p in a highly purified system. Accordingly, GST (negative control) and GST-Sptl6(i_464) fusion proteins were expressed and. immobilized on glutathione sepharose beads. For purification of H A T s , strains were created containing TAP-tagged subunits of all complexes of interest: N t o l T A P (YLH134) for N u A 3 , A h c l T A P (YJS153) for A D A , and Spt8TAP (YLH339) for S A G A . H A T complexes were then purified using calmodulin affinity chromatography. The presence of purified H A T complexes bound the G S T beads was verified through P A P western blot analysis (Figure 3.2). Both N t o l T A P and A h c l T A P displayed some level of 28 <5^ i r P A P i r D_ Q_ O < CL g co Q. CO I Coomassie o p o 29 JC Q. < CO Figure 3.2 Sp t l6p interacts with puri f ied N u A 3 and A D A in vitro. GST and GST -Sp t l6 fusion proteins were bound to glutathione resin and used in co-precipitation experiments with purified NuA3 (Nto lTAP) , A D A ( A h c l T A P ) , and S A G A (Spt8TAP). Association of H A T complexes with GST and G S T - S p t l 6 was visualized through P A P western blot analysis (5% of input sample and 50% of pull-down) and the presence of GST and GST -Spt l6 fusion proteins was verified through coomassie blue staining (25% of pul l -down). 29 association with GST -Spt l6 , but not GST, suggesting that A D A , in addition to N u A 3 , interacts with Sptl6p. Interestingly, the interaction of GST -Sp t l6 appears to be stronger with A D A compared to N u A 3 , especially when levels of input signal are considered. This however could be due to differential effects of the T A P tags on each complex and on the binding of each H A T to Sptl6p. The results o f the test for S A G A - S p t l 6 interaction were less clear. Spt8TAP showed a significant interaction with the G S T beads alone and input samples also showed a considerably higher concentration of S A G A compared to both N u A 3 and A D A . This latter observation could either be a result of S A G A being more abundant in the cell , or better suited for the purification process. The significant level of non-specific binding observed for Spt8TAP with the GST alone may be a consequence of the higher concentration of S A G A in the input sample. Although the amount of Spt8TAP bound to GST -Spt l6 appeared to be slightly higher than the control, it was not definitive enough to argue for or against an interaction of S A G A with Sptl6p. The in vitro G S T - S p t l 6 co-precipitation experiment used a highly purified system to detect protein associations, thus allowing for analysis of the direct interaction these H A T complexes have with F A C T . In addition to again verifying the association of N u A 3 , results obtained were highly supportive of an interaction between A D A and Sptl6p, supporting the initial hypothesis that F A C T associates with more than one histone H3 specific H A T . Unfortunately, data regarding S A G A was not conclusive. To further investigate these results, we again turned to in vivo pull -down experiments. 30 3.1.3 The ADA HAT Complex Interacts with FACT in vivo Having already successfully used both TAP-tagged Pob3p and FLAG-tagged Sptl6p in verifying the interaction between NuA3 and F A C T , we decided to employ the same strategy in confirming the putative interaction with A D A , and the inconclusive standing of S A G A . For the Pob3TAP co-precipitation experiment, we transformed the same Pob3TAP expressing strain with centromeric plasmids carrying the ORFs of AHC1, ADA2, and SPT8 (expressed from their endogenous promoters) attached to a tr ip le -HA cassette (pAHCIHA, pADA2HA, and pSPT8HA respectively). Overnight cultures were grown in uracil drop-out media to ensure stability o f the plasmid, and subjected to pull-downs using the T A P tag. A s Figure 3.3A shows, both AI1CIHA3 and A d a 2 H A 3 exhibited a specific interaction with Pob3TAP, further suggesting an interaction between A D A and F A C T . Considering that A D A was observed to interact with the N-terminal fragment of SPT 16 in the GST -Spt l6 in vitro experiment, this result confirms that, like N u A 3 , A D A interacts with the entire F A C T complex via the Sptl6p subunit. Unfortunately, no such conclusions could be made regarding S A G A due to the fact that Spt8HA3 migrates similarly to Pob3TAP (-100 kD), which the ant i -HA antibody cross-reacts with (Figure 3.3A upper right panel). Careful scrutiny, however, does suggest Spt8HA 3 was not present at any significant level in the Pob3TAP pull-down lane. The P A P western blot shows relatively equal levels of Pob3TAP in all pull-down samples, as does the anti -HA signal associated with Pob3TAP in the upper panel. If observable amounts of Spt8HA3 were precipitating out with Pob3TAP, one would expect a slightly stronger ant i -HA signal in the Spt8HA 3 lane, which does not seem to be the case. 31 A. Input IP + — — + — — _ + _ _ + _ - - + - - + - - - + + + — AHC1HA — ADA2HA + SPT8HA _ + + + POB3TAP + - - + -— -§• "™~ Hh aHA Pob3TAP/Spt8HA A h d H A • Ada2HA PAP + + + P0B3TAP Pob3TAP B. Input IP 1 r + - + -- + - + - - + + - + -+ - + - + + aHA AHC1HA SPT8HA Spt16-FLAG _ Spt8HA ^ P - A n d HA Coomassie _ + + Spt16-FLAG Spt16-FLAG Figure 3.3 ADA interacts with the FACT complex. (A) Pob3TAP pull-downs from cells expressing AhclHA3, Ada2HA3, and Spt8HA3 probed by anti-HA western blot analysis (1% of input sample and 50% of IP). IP samples were also probed by P A P western blot analysis to verify precipitation of Pob3TAP in all samples (5% of IP). (B) S p t l 6 -FLAG was expressed from a plasmid and used in co-precipitation experiments using strains expressing AhclHA3 and Spt8HA.3 from plasmids. Interaction was detected using anti-H A western blot analysis (1% of input and 50% of IP). The precipitation of S p t l 6 -FLAG was verified by coomassie blue staining of IP samples (50% of IP). 32 As a final attempt to decipher whether S A G A interacts with F A C T , we utilized the S p t l 6 - F L A G pull -down once more. This time, pSPTl6-FLAG was transformed into strains containing plasmids expressing AhclHA3 and Spt8HA3. A s expected, AI1CIHA3 showed a clear interaction with Sptl 6 - F L A G . Spt8HA 3 ; on the other hand, failed to show any significant association with the FLAG-tagged F A C T subunit. Though there was a very faint signal in the IP sample, a similar level of association is seen in the negative control IP lacking Sptl 6 - F L A G (Figure 3.3B upper right panel), suggesting a certain level o f non-specific association of the H A -tagged Spt8p. Taken together with the previous data involving Spt8p, we concluded that, unlike NuA3 and A D A , S A G A does not interact with the F A C T complex. 3.1.4 Deleting the NTD of Sptl6p Causes a Loss of HAT Interaction Initial experiments started with a suggested interaction between N u A 3 and F A C T . After confirming this interaction, we demonstrated, through both in vitro and in vivo techniques that F A C T also interacts with the A D A complex, another histone H3 specific H A T . The next aim was to examine the possible function of the association between F A C T and the H A T s N u A 3 and A D A . The simplest way to ascertain the significance of F A C T ' S interaction with these H A T s would be to study an spt 16 mutant lacking this interaction. In order to do this, we wished to first map the H A T interaction domain of Sptl6p, thereby identifying an spt 16 mutant that might give us insight into which aspect of F A C T function N u A 3 and A D A are involved with. The N-terminal domain was a reasonable starting point in assessing which part of Sptl6p was required for H A T interaction for several reasons. First, the original yeast two-hybrid screen used the N-terminal 464 residues encoded by SPT16 as bait (96). Second, the GST -Spt l6 fusion protein used to identify an interaction with both N u A 3 (63) and A D A (this study) in vitro was made up of the same Sptl6p N-terminal residues. Third, domain analysis of Sptl6p has 33 suggested that the first 484 amino acids make up an independently folded N-terminal polypeptide that is non-essential for F A C T complex stability, and likely involved in other protein interactions (83). A strain expressing sp t l 6ANTD was received as a gift from our collaborators in the R.A. Singer Lab at Dalhousie University. Their recent work with the Sptl6p N-terminal domain revealed convenient restriction sites allowing for the generation of the sptl6ANTD mutant (83). This N-terminal truncation, lacking amino acids 2-484, was revealed to supply all essential functions of Spt l6p (83). A l l work using SPT16 and its mutants in this study used a "plasmid shuffle" system, where sptl6::KANMX4 strains were maintained by a wi ld type SPT 16 locus carried on a URA3 plasmid. This plasmid was then selected against during growth on 5 -FOA following transformation of spt 16 alleles carried on a LEU2 plasmid. In order to assess N u A 3 and A D A interactions with the F A C T complex in an sptl6ANTD mutant, a protein besides Sptl6p needed to be epitope tagged. Previous work has shown that the sptl6ANTD mutant does not affect F A C T stability, therefore we decided to employ the Pob3TAP co-precipitation experimental system used previously. To this end, Pob3 was TAP-tagged in a strain with the ANTD mutant spt 16 allele, and either carrying the plasmid expressing Sas3HA 3 (YJS203), or A h c l H A 3 (YJS121). A pull -down of TAP-tagged proteins was done to evaluate N u A 3 and A D A binding. A s seen in Figure 3.4A, all association between Pob3TAP and Sas3HA 3 was lost in the sptl6ANTD mutant strain, suggesting that the N T D of Sptl6p is required for interaction between F A C T and N u A 3 . Similarly, the sptl 6ANTD mutation resulted in loss of association between Pob3TAP and A h c l H A 3 o f A D A (Figure 3.4B). 34 Figure 3.4 The N-terminal domain of Sptl6p is required for NuA3 and ADA interaction. (A) Pob3TAP pull-downs of plasmid expressed Sas3FLA.3 in wi ld type and sptl6ANTD strains. Western blot analysis using ant i -HA antibodies was used to visualize S a s 3 H A 3 presence (1% of input sample and 50% of IP). Probing an identical blot with P A P was done to ensure precipitation of Pob3TAP in IP samples (5% of IP). (B) Pob3TAP pull-down of plasmid expressed A h c l H A 3 in wi ld type and sptl6ANTD strains. Western blot analysis using anti -HA antibodies was used to visualize AhclHA3 presence (1% of input and 50% of IP). A P A P western blot was used to verify the presence of Pob3TAP in IP samples (5% of IP). 35 These results suggested that the N-terminal domain described previously (83) is essential for the association of both A D A and N u A 3 with Sptl6p and the entire F A C T complex. Having now identified a mutant SPT 16 deficient in H A T binding, we wished to investigate possible phenotypes in order to gain insight into the function of F A C T ' s interaction with the NuA3 and A D A H A T complexes. 3.1.5 Phenotype Analysis Shows that Interaction with HATs is not Required for Known FACT Functions A s mentioned previously, mutants of SPT 16 have been observed to display sensitivities to H U , 6 - A U , and have an spt phenotype (63, 78, 79, 103). H U is a free-radical scavenger that inhibits ribonucleotide reductase, causing replication stress and sensitivity in strains deficient in processes related to replication. Sensitivity to 6 - A U , a uracil analog that causes an imbalance in ribonucleotide triphosphates, indicates defects in transcription elongation. The spt phenotype is observed in strains able to grow on lysine drop-out media through aberrant transcription initiation from a 8-element in the normally transcriptionally repressed lys2-1288 allele. Possessing an spt phenotype is often indicative of a failure to properly repress gene transcription in a chromatin regulated manner (76, 77). Therefore, these phenotypes have implicated Sptl6p and F A C T in a variety of D N A processes, including transcription initiation, elongation, and repression, and D N A replication. Our initial strategy was to test the spt!6ANTD mutant for these 36 Strain SPT16 spt16ANTD SPT16 spt16ANTD SPT16 spt16ANTD SPT16 sptWANTD •ft r Control 6-Azauracil Hydroxyurea -Lysine Figure 3.5 sptl 6J NTD shows no F A C T related mutant phenotypes. Wi ld type and sptl6ANTD strains were serially diluted ten-fold and spotted on Y P D (control), uracil drop-out media with 50 pg/mL 6-azauracil, Y P D with 100 m M hydroxyurea, and lysine drop-out media and incubated at 30°C for 3 days. 37 aforementioned phenotypes in the hopes they might suggest a function for the H A T interaction domain of Sptl6p. Accordingly, wi ld type and sptl6ANTD strains were subjected to tenfold serial dilution and spotted on appropriate media and Y P D as a control. A s Figure 3.5 shows, there was no observable difference in growth for any of the tested phenotypes. Previously, the sptl6ANTD mutant has been shown to be H U sensitive at 37°C (83), unfortunately we were unable to replicate this effect. The presumption that an Sptl6p mutant lacking its ability to interact with H A T s would show a mutant phenotype was based on the assumption that F A C T ' s interaction with A D A and NuA3 is essential for some process with measurable deficiencies. The fact that no phenotypes were observed may indicate that F A C T - H A T interaction is required for a function yet to be identified or the ability o f F A C T to interact with H A T s is redundant with the function of other factors in the cell. Just as A D A and N u A 3 may act superfluously in their association with F A C T , other aspects o f F A C T function may compensate for the loss o f function in the sptl6ANTD mutant. This would explain a lack of any genetic phenotypes upon loss of the N -terminal domain o f Sptl6p. However, the sptl6ANTD mutant could still be affecting interactions at the nucleosome at a level measurable biochemically. With this in mind, we chose to characterize F A C T ' s interaction with NuA3 and A D A using a biochemical approach. 3.1.6 NuA3 and ADA Mutants do not affect FACT Association with the Nucleosome The interactions between F A C T and H A T s raise another intriguing possibility that perhaps histone acetylation is required to mediate the interaction of F A C T with chromatin. This would be an example of the "histone code" in which histone post-translational modification serve as docking sites for factors that mediate chromatin remodeling. Although a direct interaction between F A C T and A D A or N u A 3 may facilitate this process, it may not be 38 absolutely critical, which may explain why loss of the Sptl6p N T D does not result in any known sptl 6 related phenotypes. To determine whether N u A 3 and A D A H A T activity are required to modulate the association of F A C T with the nucleosome, the null mutants sas3::HIS3MX6 and ada2::HIS3 were mated with the strain expressing H2BHA3 and Pob3TAP. The deletion of SAS3 causes a loss of both N u A 3 complex stability and H3 H A T activity (63), and deletion of ADA2 affects the H A T activity of the A D A and S A G A H A T complexes as wel l as S L I K and H A T - A 2 (61). A n attempt was made to construct an ada2Asas3A double mutant expressing both Pob3TAP and H 2 B H A 3 , however mating and random spore analysis failed to produce any strains carrying the necessary markers possibly indicating this strain was too sick. Pob3TAP pull-downs of H 2 B H A 3 were done followed by anti -HA western blot analysis. A s Figure 3.6 shows, no visible difference was seen in the levels o f association between Pob3TAP and H 2 B H A 3 in either o f the sas3A or ada2A mutants. This suggested that the H A T activity of N u A 3 , A D A , S A G A , SL IK , or H A T - A 2 is not involved in the modulation of F A C T ' s association with the nucleosome. That being said, failure to perform the co-precipitation experiment in an ada2Asas3A double mutant meant we could not disregard the possibility that activity o f these H A T s is compensatory to each other. Thus, although we were able to conclusively show that F A C T interacts with the NuA3 and A D A H A T complexes in vitro and in vivo, at this time we have no evidence that this interaction is required for F A C T function. 39 Figure 3.6 HAT Mutants do not affect the association between FACT and the nucleosome. ADA2 and SAS3 deletion strains expressing Pob3TAP and H2BHA3 were subjected to TAP-tag pull-down. Following co-precipitation, anti -HA western blot analysis was used to visualize levels of H 2 B H A 3 associating with Pob3TAP (1% of input sample and 50% of IP). P A P western blot analysis was used to verify precipitation of Pob3p of IP samples (5% of IP). 40 3.1.7 HAT Interactions with the Nucleosome are Unaffected in sptl6ANTD Mutants Our results thus far do not support the hypothesis that the function of F A C T is dependent on the ability of this complex to interact with H A T s . A n alternative hypothesis would be that F A C T is required to facilitate the function of the A D A and N u A 3 H A T complexes. Unfortunately the function of the A D A and NuA3 complexes in the cell is currently unknown and thus there are no suitable phenotypes available to analyze sptl6ANTD mutants for. We therefore decided to address this question using two independent biochemical approaches. One of the processes F A C T is implicated in is the binding and chaperoning of histone H 2 A - H 2 B dimers at the nucleosome during transcript elongation (89). Our results demonstrate that F A C T also interacts with N u A 3 and A D A , and thus the obvious question was whether F A C T is recruiting N u A 3 and A D A to the nucleosome for acetylation of histone targets. Research has already shown that the H A T s S A G A and N u A 4 interact with a number of acidic transcriptional activators and general transcription factors in a recruiting fashion to modulate their H A T activity (105, 106), it is possible that the F A C T complex has a similar function with respect to N u A 3 and A D A . In order to examine the association of N u A 3 and A D A with the nucleosome, we employed a chromatin pull-down assay that had been developed and used in our lab to show that Sas3p interaction with chromatin requires YNG1 (101). The histone protein H2B was C-terminally TAP-tagged in strains containing sptl6ANTD and SPT16. These were crossed with strains with integrated SAS3-3HA and AHC1-3HA tags to produce haploid strains expressing either Sas3HA 3 or A h c l H A 3 and H 2 B T A P with either wi ld type or mutant SPT16 alleles. Htblp-Sas3p co-precipitation experiments were done and ant i -HA and P A P western blot analysis was used to visualize results (Figure 3.7A and B). As Figure 3.7A shows, loss of the N-terminal H A T interaction domain of Sptl6p did not affect the level of Sas3HA 3 co-precipitated with H 2 B T A P . The same result was observed for 41 A. Input - + + + IP i r (xHA 1 + spt16ANTD + HTB1TAP Sas3HA PAP H2BTAP B. Input I 1 r aHA IP 1 — — + spt16ANTD — + + HTB1TAP A n d HA PAP H2BTAP Figure 3.7 Loss of the Sptl6p NTD does not affect NuA3 or ADA association with the nucleosome. (A) Chromatin pull-downs using H 2 B T A P of S a s 3 H A 3 in SPT16 and sptl6ANTD strains. Ant i -H A western blot analysis was used to visualize levels of S a s 3 H A 3 pulled-down (1% of input sample and 50% of IP). P A P western blot analysis of IP samples was used to verify precipitation of H 2 B T A P (5% o f IP). (B) Chromatin pull-down using H 2 B T A P of A h c l H A 3 in SPT 16 and sptl6ANTD strains. A n t i - H A western blot analysis was used to visualize levels of A h c l H A 3 pulled down (1% of input and 50% of IP). P A P western blot analysis of IP samples used to verify the precipitation of H 2 B T A P (5% of IP). 42 A h c l H A 3 (Figure 3.7B). In both pull-down experiments, input samples showed equivalent levels of the H A epitope tagged protein, and no non-specific binding was observed in the non-TAP-tagged control strain. Levels of A h c l H A 3 are slightly lower in the sptl6ANTD precipitate, although this minor difference was paralleled in the P A P western blot for H 2 B T A P levels (Figure 3.7B). These results suggest that the deletion of the N T D of Sptl6p has no effect on the association of N u A 3 and A D A with the nucleosome. 3.1.8 Global Histone Acetylation is Unchanged in the sptl6ANTD Mutant Although F A C T is not required for the binding of A D A or N u A 3 to chromatin, it may still be required for the H A T activity of these complexes. F A C T has been shown to remodel nucleosomes, and this remodeling may be required for N u A 3 or A D A to access the histone tails for acetylation. In order to directly determine whether F A C T is required for the H A T activity of NuA3 and A D A , an anti-acetylated H3 antibody was used to test levels of acetylated histone H3 in both SPT 16 and sptl6ANTD strains. Unfortunately, since the specific genes bound by A D A or N u A 3 have not been determined, we were limited to examining histone acetylation of bulk histones. The previous work of others have shown that both A D A and N u A 3 preferentially acetylate lysine 14 of histone H3 (61, 62), as well as other sites to a lesser extent, however an antibody that can detect this modification alone by western blot is not available. Therefore we chose to use an antibody raised against peptides corresponding to the histone H3 tail acetylated at lysines 9 and 14. Whole cell extracts from SPT 16 and sptl6ANTD strains were normalized for total protein levels and subjected to western blot analysis using these anti-acetylated H3 antibodies. Unfortunately, the results showed no significant change in the levels of acetylated-H3 in a strain lacking the Sptl6p N T D H A T interaction domain (Figure 3.8). However, as there 43 are other H A T s in the cell that target lysines 9 and 14, but do not interact with F A C T , it is still formally possibly that deletion of the N T D does result in minor losses of histone acetylation that are not detectable using this assay. Alternatively, F A C T may be required for acetylation of sites other than lysine 14 which have not been previously been detected due to absence of F A C T in the H A T assays used. In addition, there is evidence that certain H A T s also have non-histone targets (107-109), and this may be the case with N u A 3 and A D A . Thus at this time we have no evidence that F A C T is required for the function of A D A or N u A 3 in the cell , and although we have been able to conclusively show that F A C T interacts with the A D A and NuA3 H A T complexes in vitro and in vivo, we have been unable to reveal the functional relevance of this interaction. 44 Figure 3.8 Loss of the Sptl6p NTD does not affect global H3 histone acetylation. Whole cell extracts from strains with wi ld type SPT 16 and mutant sptl6ANTD alleles were normalized for bulk protein content using Bradford assay (10 mg/mL), and levels of H3 acetylation were determined by anti-acetylated H3 western blot analysis. Sample normalization was verified by P A P western blot analysis for H 2 B T A P . 45 3.2 The sptl 6A922 Mutant Causes Interference of Histone Acetylation It has been proposed that F A C T not only has the ability to destabilize nucleosomes and allow passage o f R N A pol II, but also to reassemble chromatin fol lowing transcription, which is required for proper repression of certain genes (89, 91, 103). This later hypothesis is based on the observations that in some SPT 16 mutants transcription can initiate from "cryptic" intragenic sites within multiple genes, such as lys2-128S. The fact that the sptl6ANTD mutant does not exhibit an spt phenotype is interesting when one considers that another well characterized mutant, sptl6A922, which has a smaller truncation to sptl6ANTD, does show this phenotype (83, 98). These results may suggest that while the sptl6ANTD mutant lacks the entire N -terminal domain of Sptl6p, the sptl6A922 mutant still expresses a remnant of this domain which becomes misfolded, and interferes with some aspect of normal chromatin structure regulation (83). In other words, the spt phenotype seen in an sptl6A922 mutant may not be due to a defect in FACT ' S ability to repackage chromatin, but rather to interference in the function of an unrelated chromatin modifying complex by aberrant Sptl6p structure (83). Due to the role of histone acetylation in transcription regulation, we postulated that it may be possible that the product of the sptl6A922 may be interfering with normal histone acetylation. The goal of our research with the sptl6A92'2 mutant was to investigate this interference model. 3.2.1 FACT-Nucleosome Association is not Affected in the spt!6A922 Mutant Our hypothesis was that the sptl6A922 mutant interferes with proper histone acetylation thus causing mutant phenotypes. Before testing this, however, we needed to ensure that the partial N T D mutant was not interfering with FACT-nucleosome interactions directly. To this end, we created wi ld type and sptl 6A922 strains which expressed HA-tagged H2B and T A P -46 tagged Pob3p. T A P pull-downs followed by H A western blot analysis were used to examine the interaction of F A C T with chromatin with the wi ld type and sptl6A922 strains. A s Figure 3.9 shows, no difference in the association of F A C T with the nucleosome was observed in the sptl6A922 mutant. This argued that the partial N-terminal truncation of this Sptl6p mutant was not interfering with normal FACT-nucleosome interactions. Although it is still formally possible that F A C T is able to bind but not modify chromatin structure in the sptl 6A922 strain, we were intrigued by the possibility that the mutant Sptl6p in the sptl6A922 strain was interfering with a F A C T independent function. 3.2.2 The sptl6A922 Mutant Shows a Loss in H3 Acetylation Structural predictions of the sptl6A922 mutant suggest that the partial N T D in the Sptl6p polypeptide is misfolded, possibly to an extent that interferes with other nearby functions (83). We proposed that this N T D fragment is interfering with the acetylation of nucleosomal histones, thus affecting normal chromatin maintenance and causing mutant phenotypes such as the spt phenotype. There are several lines of evidence that suggested histone acetylation might be affected by mutation of Sptl6. First, the F A C T complex is present at the nucleosome, as seen in its ability to bind nucleosomal H 2 A / H 2 B dimers (89) and would likely be in close proximity to any histone tail modifications, including acetylation. Second, it has been shown that certain spt 16 mutants acquire serious synthetic growth defects when combined with sas3A or gcn5A (91), and thus interference of histone acetylation by mutant Sptl6p may explain the sas3Agcn5A synthetic lethality. Finally the phenotypes of certain sptl6 mutants can be rescued by the deletion o f the gene encoding the histone deacetylase Rpd3p (86). These data 4 7 Figure 3.9 FACT association with the nucleosome is not altered in the sptl6A922 mutant. Strains expressing Pob3TAP with SPT16 and sptl6A922 alleles were subjected to TAP-tag pul l -down. Following co-precipitation, ant i -HA western blot analysis was used to visualize levels of H 2 B H A 3 associating with Pob3TAP. P A P western blot analysis was used to ensure equal precipitation of Pob3TAP. 48 demonstrate a possible connection between histone acetylation and F A C T function. To determine whether histone acetylation was affected in the spt!6A922 mutant, whole cell extracts were prepared from SPT 16 and sptl6A922 strains, normalized for protein concentration using a Bradford assay, and subjected to anti-acetylated H3 western blot analysis. As Figure 3.1 OA shows, the sptl6A922 strain showed a marked decrease in H3 acetylation, confirming the possibility that the partial N T D truncation of Spt l6p interferes with histone tail acetylation. Although the P A P western blot of H 2 B T A P suggested that each lane contained equal loadings of histones, the samples are expected to contain newly synthesized H2B, which is non-nucleosomal, and thus the H 2 B signal seen may not accurately represent the level of histone H3 present in the cell. Thus it is conceivable that mutating Sptl6p caused an alteration in the levels of nucleosomal histone H3, and not actual levels of histone acetylation. To resolve this problem, we chose to combine the H3 acetylation assay with an H2BTAP-tagged protein purification. This allowed us to purify significant quantities of nucleosomal histone H3 which could be normalized by coomassie staining. To this end, large scale calmodulin affinity purifications of nucleosomal histones from both SPT 16 and sptl6A922 strains were done using a TAP-tagged histone H2B. The histones were resolved by SDS P A G E and stained with coomassie blue (Figure 3.1 OB lower panel) which demonstrated equal levels of histone H3 in the lanes corresponding to SPT16 and sptl6A922. A s shown before, levels of H3 acetylation are noticeably lower in the truncated N T D mutant, indicated by anti-acetylated H3 western blot analysis. A P A P western blot showed proper normalization of H 2 B T A P , demonstrating that although H3 acetylation is decreased in the sptl6A922 mutant, presence of H 2 B T A P is equal in both strains (Figure 3.10). These results confirmed that sptl6A922 mutants exhibited decreased levels of histone acetylation. 49 oAcH3 P A P Figure 3.10 sptl6A922 mutant shows a loss of H3 acetylation. (A) Whole cell extracts from sptl6A strains carrying plasmids for SPT16 or sptl6A922 were normalized for bulk protein content using Bradford assay (10 mg/mL), and levels of H3 acetylation were probed by anti-acetylated H3 western blot analysis. Sample normalization was verified by P A P detection of H 2 B T A P . (B) Histones from wild type SPT16 and sptl6A922 were purified by calmodulin affinity chromatography of H 2 B T A P and subjected to anti-acetylated H3 western blot analysis. P A P western blot analysis was used to verify normalization of H 2 B T A P present. 50 We had previously shown that in an sptl6ANTD mutant, lacking the entire N-terminal domain, there was no change in H3 acetylation (3.2.2). However, in the sptl6A922 mutant, which has 182 more residues than sptl6ANTD, there was a loss in H3 acetylation. Although the idea of having more of the wi ld type Sptl6p present, and less than wi ld type H3 acetylation at first seems unusual, it does agree with the model of sptl6A922 interference. It suggests that the partial N-terminal truncation is actually gaining a function, albeit one that interferes with normal H3 acetylation, through the extra 182 residues. It also suggests that the F A C T complex is in a position at the nucleosome to affect histone modifications, supporting our hypothesis. Finally, this data may indicate that the spt phenotype seen in the sptl6A922 strain is a result of loss of histone acetylation as opposed to disruption of F A C T function. . 3.2.3 Histone H3 Acetylation is Altered in Multiple spt!6 Mutants To further examine the relationship between histone H3 acetylation and Sptl6p function, we chose to investigate whether other spt 16 mutants caused the same loss of histone acetylation as sptl6A922. If the sptl6A922 mutant was expressing a misfolded polypeptide that interferes with histone acetylation, we would expect there to be other spt 16 mutants with a similar effect, possibly due to mutation of residues important in folding. For that reason, the level of histone H3 acetylation was examined in four other spt 16 mutants, and compared to the wi ld type, spt!6ANTD, and spt!6A922 strains. Figure 3.11 shows a western blot analysis for levels of histone H3 acetylation in these strains. A l l four previously untested sptl 6 mutants, sptl6NM2(4), sptl6NM3(4), sptl6M3(12), and sptl6M3(19) are point mutations in various regions of the gene (see Table 2.2 for detailed description). A s seen before (in Figures 3.8 and 3.10 respectively), spt!6ANTD showed wi ld type levels of H3 acetylation, and sptl6A922 showed a loss in H3 acetylation. In the four point mutation sptl6 mutants, sptl6M3(19) showed 51 C(AcH3 PAP A s Ofo # # # 4 # # # £ # # AcH3 H2BTAP Figure 3.11 sptl6 mutants display altered levels of histone H3 acetylation. Whole cell extracts from sptl6A strains carrying plasmids for mutant sptl6ANTD, sptl6A 922, sptl6NM2(4), sptl6NM3(4), sptl6M3(12), and sptl6M3(19) were normalized for bulk protein content using Bradford assay (10 mg/mL), and levels of H3 acetylation were probed by anti-acetylated H3 western blot analysis. Sample normalization was verified by P A P western blot analysis of H 2 B T A P . 52 a significant loss in acetylation, and sptl6NM2(4) showed a slight loss. Both sptl6NM2(3) and sptl6M3(12) appeared to show near wi ld type levels of H3 acetylation. Although the underlying reasons for the loss of H3 acetylation in each of the spt 16 mutants is not known, the fact that altered acetylation was observed lended support to the theory of an sptl 6A 92'2 mutant interfering with normal histone acetylation. It was now clear that F A C T is in a position to affect histone modifications, as mutants of spt 16 were shown to alter histone H3 acetylation in Figure 3.11. In order to understand the importance of the sptl6A922 mutant's interference of H3 acetylation, our goal now shifted towards relating this biochemical data to an observable genetic phenotype. In this way, we aimed to not only understand spt!6A922, but also further elucidate the role of histone modifications in chromatin structure and function. 3.2.4 The spt Phenotype of sptl6 Mutants Correlates with the Loss in H3 Acetylation The first published data of the sptl6A922 mutant shows that the deletion of the N -terminal 300 residues decreases the need for Swi /Snf chromatin remodeling in transcription, and confers an spt phenotype (98). Both of these results suggest that the sptl 6A92'2 mutation is in some way compromising chromatin structure, which is well known in itself to be an important negative regulator of gene expression (110, 111). Our data showed that the sptl6A922 mutation also resulted in a significant loss in histone H3 acetylation (Figure 3.1 OA and B). We therefore wished to examine a possible connection between this altered acetylation and chromatin mediated gene repression. To do this, all six spt!6 mutant strains, containing the fys2-1283 allele, were screened for an spt phenotype through ability to transcribe the mutant lys2 transcript on lysine drop-out media. The results this far (Figure 3.12) confirmed the spt phenotype of sptl6A922 and also showed that sptl6ANTD did not confer such a phenotype. This paralleled the H3 acetylation 53 Strain Control -Lysine SPT16 sptWANTD spt16A922 spt16NM2(4) spt16NM3(4) spt16M3(12) spt16M3(19) Figure 3.12 sptl6 mutants confer an SPT phenotype. Iys2-128d strains containing the wi ld type SPT 16, six spt 16 mutants and the were serially diluted ten-fold and spotted on Y P D (control) and lysine drop out media to screen for the spt phenotype. Plates were incubated at 30°C for 3 days. 54 data of these two strains, and again argued that the extra residues on the sptl6A922 expressed polypeptide were interfering with some aspect of chromatin structure regulation. Amongst the four other spt 16 mutants, sptl6NM3(4) conferred the greatest spt phenotype, slightly stronger than that of sptl6M3(19). O f the two remaining mutants, sptl6M3(12) showed a noticeable level of growth indicative of an spt phenotype, though lesser than that of sptl6A922, especially when the amount of cells on the control plate are taken into account. The sptl6NM2(4) mutant displayed growth on lysine drop-out media marginally better than the wi ld type and sptl6ANTD strains, though not enough to indicate an spt phenotype. When the phenotype data of the various spt 16 mutants was compared with the results of the histone H3 acetylation westerns seen in Figure 3.11, a certain degree of correlation was observed between those mutants that showed loss of histone H3 acetylation, and those conferring an spt phenotype. Compared to the wi ld type, the mutants sptl6A922, sptl6NM3(4), and sptl6M3(19) all showed decreased histone H3 acetylation (Figure 3.11). These same mutant strains also demonstrated the greatest spt phenotypes. The fact that the spt phenotype is indicative of a defect in chromatin-mediated repression suggested to us that histone acetylation may have a novel role in proper transcription repression. Although this is contrary to the general view of acetylation and transcription, it is not entirely without precedent (41, 68). The correlation that we see between histone acetylation and spt phenotype, though, is not exact. For example, sptl6M3(19) showed the lowest level of H3 acetylation, whereas sptl6NM3(4) had the most prevalent spt phenotype. It can also be argued that the slight spt phenotype in the sptl6M3(12) mutant was not accompanied by any noticeable loss in H3 acetylation. These discrepancies are not surprising considering F A C T is a multi-domain and apparently multi -functional complex. It is conceivable that the spt phenotype is due to loss of histone acetylation in addition to disruption of another unrelated F A C T function. However, it is also possible that the spt phenotype and loss in histone H3 acetylation seen in some of the spt 16 mutants is a result 55 of disruption o f two independent F A C T functions. Therefore, we chose to further investigate a possible role for histone acetylation in the spt phenotype. We rationalized that i f the sptl6A922 mutant was interfering with the interaction of H A T complexes with histones, then loss of H A T -nucleosome interactions in this mutant may be observed. In addition, strains harbouring mutations in the genes encoding H A T s should also have an spt phenotype since blocking H A T activity and not having any H A T activity would be expected cause the same effect. 3.2.5 The Association of HATs with the Nucleosomes is not Affected in sptl6A922 To investigate the effect o f the sptl6A922 mutation on the association o f H A T complexes with nucleosomes we generated wi ld type and spt!6A922 strains which expressed Sas3HA3, AI1CIHA3, or Spt8HA 3 along with H 2 B T A P . These strains were subjected to T A P pull-downs, and western blot analysis was used to visualize the association o f Sas3HA3 (NuA3), A h c l H A 3 (ADA) , and Spt8HA3 ( S A G A ) with nucleosomes. These H A T s were tested as they all show specificity toward histone H3 in vitro. Regrettably, none of the H A T s tested showed any observable difference in nucleosomal associations between the wi ld type SPT 16 and mutant spt!6A922 strains (Figure 3.13), suggesting that the N-terminal fragment of the sptl6A922 mutant was not interfering with the interaction o f these H A T s with chromatin. However, it is still possible that although mutant Sptl6p does not interfere with the interaction of these H A T s with chromatin, it still blocks their ability to acetylate histones. We therefore decided to test strains lacking these H A T s for an spt phenotype. 56 C. Input IP _ + — — + spt16A922 4. 4. — + + HTB1TAP Figure 3.13 Loss of NTD does not affect NuA3 and ADA association with the nucleosome. Chromatin pull-down (using H 2 B T A P ) of Sas3HA 3 (A), A h c l H A 3 (B), and Spt8HA 3 (C) in SPT16 and sptl6A922 mutant strains. A n t i - H A western blot analysis was used to visualize levels of Sas3HA 3 pulled-down. P A P western blot analysis o f IP samples was done to verify equal levels of H 2 B T A P in precipitates. 57 3.2.6 Deletion of the Histone H3 Specific HATs Does not Confer an spt Phenotype To test whether H A T mutants have an spt phenotype, the genes encoding several subunits of S A G A , A D A , and N u A 3 were deleted from a strain containing the lys2-1285 allele and screened for an spt phenotype. These genes included GCN5 which encodes the catalytic subunit of S A G A , S A L S A , and A D A , as well as SAS3, which encodes the catalytic subunit o f NuA3 . The loss of either gene results in a loss of H A T activity for the respective complexes (59, 63). In addition, ADA2, important for Gcn5p H A T activity (61), and AHC1, essential for A D A complex stability (58), were deleted and screened. The results (Figure 3.14) revealed no spt phenotype in any of the null mutant strains. We also tested mutant combinations of both sas3Aada2A and sas3AahclA to rule out the possibility of functional redundancies between these HATs . Unfortunately, this screen was limited in its capacity to thoroughly test all combinations of H A T mutants. For example, a sas3Agcn5A mutant would have been ideal to test the effect of loss of the combined H A T activity of N u A 3 , A D A , and S A G A , however, such a mutant is synthetically lethal (64). We therefore concluded that, although the screen of H3 H A T mutants could not confirm the link between loss of histone acetylation and the generation of an spt phenotype, it did not eliminate the possibility. Currently, there is still much to be understood in regards to H A T complexes, their specific acetylation targets, and their overall downstream functions. It is thus inherently challenging to derive conclusions from our experiments involving the H A T complexes themselves. Therefore, we decided to adapt our approach in examining the sptl6A922 mutant interference hypothesis. Instead of focusing on the H A T s themselves, a genetic strategy would 58 Strain Control -Lysine spt16A922 gcn5A spt16A922 sas3A ada2A sas3Aada2A ahdA sas3Aahc1A $ # # £ # # # % Figure 3.14 H A T Mutants do not confer an spt Phenotype. Iys2-1286 strains containing H A T mutants gcn5A, sas3A), ada2A, sas3Aada2A, ahclA, and sas3AahclA were serially diluted ten-fold and spotted on Y P D (control) and lysine drop out media to screen for the spt phenotype. Plates were incubated at 30°C for 3 days. 59 be used to investigate the modulators of histone acetylation, and the histone targets of acetylation directly. 3.2.7 Genetic Interactions Suggest that the spt Phenotype of sptl6A922 is Due to Loss of Histone Acetylation The sptl6A922 mutant encodes an N-terminal domain partial truncation that is hypothesized to interfere with the access of histone modifying complexes with chromatin (83). In this study, we have so far shown that the sptl6A922 mutant did indeed show a loss of histone H3 acetylation, and that this altered histone modification showed a general correlation with the spt phenotype in other spt 16 mutants. To this point, attempts to confirm any functional relevance between these two observations from the perspective of H A T complexes failed to generate conclusive results. Here, we shifted our work from the modifier being interfered with, to the modification in question, histone acetylation. Histone acetylation of chromatin is counteracted by the activity of histone deacetylase complexes (HDACs) (112). If indeed the spt phenotype was a result of loss of histone acetylation then we rationalized that removal of deacetylase activity in the cell might alleviate it. The RPD3 gene encodes the catalytic subunit of the Rpd3 histone deacetylase complexes (113, 114), and has been implicated in the suppression of other F A C T mutant phenotypes (103). We created rpd3A strains containing the SPT 16, sptl 6A NTD, and sptl6A922 alleles and screened them for an spt phenotype alongside RPD3 strains of the same sptl6 mutants (Figure 3.15). Results show the spt phenotype of sptl 6A922 was significantly alleviated by the loss of the Rpd3 H D A C s . A s seen before, no spt phenotype was conferred in the sptl6ANTD mutant. These 60 rpd3A Strain Control SPT16 sptWANTD spt16A922 SPT16 sptWANTD . spt16A922 Figure 3.15 Deletion of RPD3 suppresses the spt phenotype of sptl 6J 922. RPD3 deletion strains carrying the SPT 16, sptl6ANTD, and sptl 6A922 alleles were serially diluted ten-fold and spotted on Y P D (control) and lysine drop out media to screen for the spt phenotype. The same sptl6 mutants containing the wi ld type RPD3 were plated alongside for comparison. Plates were incubated at 30°C for 3 days. 61 results suggest that the loss of acetylation observed in the sptl6A922 mutant, caused by the hypothesized interference of histone interactions, was in fact responsible for the development of an spt phenotype. The suppression of this phenotype through deletion of one of the H D A C s , though, argued that this interference was not complete, and perhaps acetylation is merely being slowed down. If acetylation was completely lost, deletion of RPD3 would have no effect. The most well understood targets of histone acetylation are on the N-terminal tails of histones. If, as suggested, histone H3 acetylation is important for chromatin mediated repression and preventing the spt phenotype, one would expect the mutation of acetylated residues to have a similar effect. Previous work in our lab has produced a number of histone H3 tail mutants (101). To generate these strains, a parent strain in which the genomic copies of both histoneH3 and H4 encoding genes, HHT1-HHF1 and HHT2-HHF2, are deleted was created, and maintained by an HHT2-HHF2 locus on a plasmid (pHHT2). Plasmids carrying mutant HHT2 were then introduced into this strain using a "plasmid shuffle". To further examine the sptl6A922 interference theory, we tested a variety of histone H3 mutants for an spt phenotype. We found that the mutation of lysine 14 and 23 to arginine conferred an spt phenotype (Figure 3.16). Several H A T s are thought to acetylate residues K14 and K23 , including N u A 3 , A D A , and S A G A (61, 64, 115). This result further supports the idea that histone acetylation is important for maintaining proper chromatin structure, and preventing an spt phenotype. Interestingly, the wi ld type plasmid also seemed to confer a slight spt phenotype in a certain population of cells, while no spt phenotype was observed in mutants of lysine 4 and 36. The spt phenotype observed in the wi ld type histone H3 strain is most likely due to altered stoichiometry of histones resulting from expression of histone H3 and H4 from a plasmid. However, why the K 4 R and K 3 6 R mutants do not show a similar phenotype is unknown. 62 Vector Control -Lysine pHHT2 phht2K4R phht2K14R phht2K23R phht2K36R phht2K4/36R Figure 3.16 Histone H 3 mutants confer an SPT phenotype. Strains with both the HHT1-HHF1 and HHT2-HHF2 loci deleted and transformed with the indicated hht2-hhf2 mutant locus carried on a plasmid were serially diluted ten-fold and spotted on Y P D and lysine drop-out media to test transcription from the lys2-128d allele. Plates were incubated at 30°C for 3 days. 63 Recent work has shown that K36 methylation acts in a pathway with Rpd3p in histone deacetylation (29, 30), and mutation of lysine 36 to arginine results in increased histone acetylation at specific loci . Thus the fact that mutation of K 3 6 can suppress an spt phenotype is consistent with the fact that the spt phenotype is due to loss of histone acetylation. In conclusion, our results support the hypothesis that loss of histone acetylation may be partially responsible for the spt phenotype of the sptl 6A922 mutant strain. The fact that the spt phenotype is indicative of loss of chromatin mediated repression suggests that in addition to facilitating transcription activation, histone acetylation may also be linked to the proper repression of specific loci . 64 Chapter 4 - Discussion 4.1 The Interaction of FACT with NuA3 and ADA This study began with an investigation into the interaction of the F A C T complex with histone acetyltransferases. Given that both F A C T and histone acetylation target the nucleosome (35, 88) and affect chromatin structure (1, 92), such a relationship is not at all surprising. Initial evidence of an interaction between Sptl6p of F A C T and Sas3p of the N u A 3 H A T complex came from a yeast two-hybrid screen (96) and co-precipitation experiments using GST-Spt l6p and anti-Sptl6p polysera (63). Interestingly, while F A C T mutants display a plethora of phenotypes, N u A 3 mutants share none of these, suggesting the possibility that other H A T s are interacting with F A C T in a redundant role. In the first part o f this study, we demonstrated that F A C T interacts with both the N u A 3 and A D A H A T complexes through the previously described N-terminal domain of Sptl6p (83). We then attempted to investigate the relevance of this interaction by characterizing an sptl 6 mutant lacking N u A 3 and A D A interactions. 4.1.1 The Importance of the HAT Interaction Domain of Sptl6p In our original search to identify a H A T interacting with F A C T and serving a redundant role as N u A 3 , we focused on Gcn5p dependent H A T s in part because o f a sas3A gcnSA synthetic lethality. It was reasoned that the shared essential function of Sas3p and Gcn5p may be related to the interaction between a Gcn5p dependent H A T , N u A 3 , and F A C T . However, it has since been shown that this assumption was flawed, as the synthetic lethality of Sas3p and Gcn5p is caused by non -HAT related activities (D. Grimes and L .J . Howe unpublished data). In 65 addition, the observation that the H A T s are interacting with a non-essential region of Sptl6p rules out the possibility that interaction with H A T s is required for F A C T ' s essential function. This would again argue that the function of N u A 3 and A D A with F A C T is either non-essential or redundant to some other process. To determine the functional relevance of the F A C T - H A T interactions, it was predicted that a loss of the H A T interaction domain, and consequent loss of N u A 3 and A D A interaction, would produce a mutant phenotype to study. Unfortunately, screening of the sptl6ANTD mutant produced no such phenotypes. Initially, it was somewhat surprising that an entire domain of the essential Sptl6p could be removed without serious effect, however this is not without explanation. It is possible that, in the context of FACT-mediated H A T complex recruitment, the N T D of Spt l6p is redundant with other mechanisms for the recruitment of H A T activity to chromatin. For example, the histone H3 specific H A T S A G A has been shown to interact with a variety of transcriptional activators (105), and could be compensating for a loss in N u A 3 and A D A recruitment in an sptl6ANTD mutant. Consistent with this, deletion of GCN5, which encodes the catalytic subunit of multiple histone H3 specific H A T s , including S A G A , results in severe growth defects in specific spt 16 mutants. However whether the spt!6ANTD mutant shows a similar genetic interaction has not been determined. Despite the lack of any sptl 6A NTD associated phenotype, it was still possible to investigate the effect a loss in F A C T - H A T interaction would have on N u A 3 and A D A association with the nucleosome. Our results using a chromatin pull -down assay, however, failed to show a loss in the association of N u A 3 or A D A with nucleosomes in the sptl6ANTD mutant. Again, this result, although negative, is not without explanation. First, the ability to see a loss in N u A 3 and A D A association is based on the assumption that the N T D of Sptl6p has a role in i f not al l , then a significant portion of N u A 3 and A D A interactions with the nucleosome. As mentioned above, it is possible that there are alternative pathways in the recruitment of these 66 HATs . The interaction of the N u A 3 complex with chromatin has been recently shown to be dependent on methylation of lysines 4 and 36 within histone H3. It is therefore possible that loss of Spt l6p-NuA3 interaction does not result in a net loss of N u A 3 from chromatin due. to a redundant interaction of N u A 3 with these methyl marks. Using multiple mechanisms to mediate the interaction of H A T complexes with various regions of the genome may be one way for a cell to fine tune histone acetylation levels to regulate transcription. Although the interaction between A D A and N u A 3 was not required for these complexes to interact with histones, it was still formally possible that these complexes require F A C T to access their nucleosomal substrates. To test this, the levels of histone H3 acetylation in the sptl6ANTD mutant was analyzed using anti-acetylated H3 antibodies raised against peptides acetylated at lysines 9 and 14. Although results did not show any loss in histone acetylation, this did not discount the possibility of a requirement for F A C T in mediating N u A 3 and A D A activity for many of the same reasons mentioned already. The assessment of this result is also made difficult by the fact that the actual lysines acetylated by N u A 3 and A D A in vivo are still not known. N u A 3 has been shown to acetylate K14, and to a lesser extent K23 in vitro (64), while the A D A complex acetylates lysines 14 and 18 in vitro (115). However, recent data has shown that deletion of GCN5 does not result in a loss of histone H3 lysine 14 acetylation, suggesting that A D A is not acetylating this site in vivo. In addition, it has recently been suggested that the anti-acetylated H3 antibody used in this study preferentially recognizes acetylated K 9 (D. Martin and L .J . Howe unpublished data). This could either be a result o f the antibody showing stronger affinity toward acetylated K 9 , or a significantly higher proportion of acetylated K 9 than K14 in the cell. In either case, it is possible that we failed to detect a loss of NuA3 or A D A acetylation in the sptl6ANTD mutant because the antibody used was not be suited for the assay. 67 We conclude that although F A C T is interacting with the histone H3 H A T s N u A 3 and A D A , the hypothesis that F A C T is recruiting these H A T s to the nucleosome cannot be confirmed or denied based on our evidence. With no observable sptl6ANTD phenotype, and no available technique to test for N u A 3 and A D A function, we can only speculate as to what the relevance of this interaction may be. 4.1.2 The Relevance of NuA3 and ADA Interaction with FACT - Alternative Models Since we were unable to provide support for the hypothesis of F A C T recruiting NuA3 and A D A to the nucleosome, we discuss here alternative models. One such model is that the H A T s NuA3 and A D A recruit F A C T to the nucleosome either directly or through their H A T activity. Precedence for such a model exists as nucleosome binding of the SWI /SNF and S A G A complexes has been shown to be dependent on histone acetylation (28, 116). A lso , recent work in our lab has demonstrated a role for histone modifications, specifically histone methylation, in the recruitment of N u A 3 to the nucleosome (101). Results in this study, though, fail to support this model as no change was observed in the association of F A C T with the nucleosome in either sptl6ANTD, or sas3A and gcn5A null mutants. This does not discount the possibility of the NuA3 and A D A dependent recruitment of F A C T acting in parallel with some other pathway. The work on sptl6ANTD and F A C T ' s role with N u A 3 and A D A in this study focused on functions relating to the nucleosome. This was the obvious choice since the only targets of acetylation yet to be identified for the N u A 3 and A D A H A T complexes are histone residues. However, there is a growing field of research indicating H A T s can acetylate non-histone targets (117). In regards to the sas3A gcn5A synthetic lethality, recent work has suggested that this phenotype is due to loss of acetylation of a non-histone protein, yet to be identified (D. Grimes and L .J . Howe unpublished data). It is possible that F A C T ' S association with N u A 3 and A D A 68 may be required for acetylation of this substrate. A s for non-histone targets of acetylation, the F A C T associated D N A binding protein, Nhp6p, is an interesting candidate. Nhp6p shares a D N A binding motif with the H M G B - 1 and 2 proteins (86), which are known to preferentially bend and bind distorted D N A (118). Furthermore, in vertebrates, acetylation of lysine residues in H M G B - 1 by C B P enhances its binding affinity to bent D N A (119). The association of N u A 3 and A D A with F A C T , then, may serve to modulate Nhp6p function. It should be noted, however, that although Nhp6p is not essential, loss off the protein does cause a number of severe phenotypes (87, 120). No such phenotypes were observed in an sptl6ANTD mutant, suggesting that i f N u A 3 and A D A are acetylating Nhp6p, this activity is not required for Nhp6p function. It should also be noted that, to this date, no evidence of N u A 3 or A D A acetylating non-histone residues has ever been published. The function of the N-terminal domain of Sptl6p remains largely unknown. Besides an interaction with H A T s , the only other previously described interaction is with the catalytic subunit of the protein kinase C K 2 , shown in vitro with human Sptl6p (95). C K 2 acts to phosphorylate p53 upon replication stress (121), however the significance of F A C T involvement in this process is currently unclear. In the research described here, we characterized the interaction between F A C T and two H3 H A T s , N u A 3 and A D A . Attempts to examine the relevance of this interaction, though, provided no conclusive results. Unt i l such time as the downstream functions of N u A 3 and A D A are elucidated, it w i l l remain difficult to learn more about the importance of their interaction with the F A C T complex. 69 4.2 The sptl6A922 Mutant and the Interference of Histone Acetylation The second part of this study focused on another Sptl6p N-terminal domain truncation mutant, sptl6A922. The mutant Sptl6p expressed by this strain has 182 more residues than sptl6A NTD, yet causes a number of mutant phenotypes, suggesting that the extra residues are interfering with normal functions (83). Because of F A C T ' s role in mediating chromatin structure, and the importance of histone modifications in a number of processes, our hypothesis was that the sptl6A922 mutant was interfering with normal histone modifications. Results showed that histone acetylation was indeed lost in the sptl 6A922, and not sptl6ANTD, supporting our hypothesis. In addition, deletion of the RPD3 histone deacetylase suppressed the spt phenotype of the sptl 6A922 mutant and mutation of modified residues within the H3 tail conferred a similar spt phenotype further supporting a model of histone acetylation interference. Although the above evidence supported the hypothesis that aberrant Sptl6p in the spt!6A922 strain was interfering with histone acetylation, biochemical co-precipitation experiments failed to demonstrate this interference with respect to the association of the H3 HATs , N u A 3 , A D A , and S A G A , with the nucleosome. However, in retrospect, the chromatin pull-down assay was not an ideal system to test this. First of al l , the sptl6A922 mutant may be interfering with H A T activity, and not H A T association, something we were not able to test directly. Secondly, it is possible that a variety of H A T s are subject to a small amount of interference undetectable when considering the complexes individually. The loss in histone H3 acetylation observed would then be a result o f the net loss in H A T activity o f multiple complexes. A final consideration is that not all H3 H A T s were tested in this study. Previous evidence showing spt 16 mutants having synthetic defects with sas3A and gcn5A (91) directed our focus on the Sas3p and Gcn5p dependent HATs . Additionally, the anti-acetylated H3 antibody used was designed to recognize acetylated lysines 9 and 14. Both of these points 70 prompted the testing of N u A 3 , A D A , and S A G A . However, evidence suggesting the anti-acetylated H3 antibody primarily recognizes lysine 9 suggests interference of N u A 3 and A D A are not as involved in the observed loss in H3 acetylation as both preferentially target K14 in vitro (61, 64). In the end, results did support the hypothesis that the sptl6A922 mutant was interfering with normal histone modifications, notably acetylation. The functional relevance of the loss in histone acetylation w i l l be discussed in the following section. 4.2.1 Histone Acetylation and Cryptic Initiation Suppression of the lys2-128 allele is due to a failure to properly repress cryptic initiation, presumably because of a defect in establishing repressive chromatin structure following transcription (75, 76). The current model of F A C T function involves facilitating the passage of R N A polymerase II during elongation by disrupting nucleosomal structure (82, 86, 89). The failure to properly repress transcription in F A C T mutants, then, may be the result of improper nucleosome reassembly or maintenance following elongation (92). Our data demonstrates a correlation between loss of histone acetylation in sptl 6 mutants and an spt phenotype, suggesting that histone acetylation may be involved in reestablishing repressive chromatin following transcription. However, the question becomes whether or not there is a direct relationship between these two observations or whether these may simply be two effects with the same root cause, interference of normal histone related processes. In argument for the latter, the correlation between histone acetylation and the spt phenotype that we saw was not exact. In addition, null mutants of several histone acetyltransferases failed to confer an spt phenotype, although this negative result can be explained by the inability to screen all possible combinations of H A T mutants. However, the spt phenotype of the spt!6A922 71 mutant was suppressed by deletion of RPD3 suggesting that this phenotype was a result of histone acetylation loss. Moreover, although histone acetylation is generally associated with increased levels of transcription (31, 122), there is precedence for acetylation causing a repression of transcription. Hos2p, another histone deacetylase in the same class as Rpd3p, is required for the expression of certain genes (68). It is proposed that following elongation, the permissive state of chromatin may be disrupted by histone acetylation, preventing further rounds of transcription. It is possible that histone acetylation, in this case, may serve as a marker for other factors important in repression to bind chromatin. Fol lowing this model, lysines 14 and 23 may be important in conjunction with histone acetylation for the association of these repressive elements. 4.2.2 The Possibilities of an Interference Model Besides acetylation, histones are post-translationally modified by methylation, ubiquitination, and phosphorylation (28). Recent studies have implicated methylation of lysine 36 on histone H3 in directing histone deacetylation by an Rpd3p complex known as RPD3S (29, 30). Mutation of two RPD3S complex subunits resulted in the appearance of aberrant transcripts, corresponding to an increase in acetylation in the FL08 and STE11 open reading frames (29). This pathway would seem to be an obvious candidate to be disrupted in the sptl6A922 mutant, especially with its link to the prevention of aberrant transcription. Results from this study, however, do not seem to agree. In our experiments, the deletion of RPD3 alleviated the spt phenotype, whereas the Set2p-RPD3S model describes Rpd3p as serving a repressive function. Additionally, the lysine 36 mutant failed to show an spt phenotype, however it is possible that the spt phenotype represents a more severe case of cryptic initiation, requiring the disturbance of several repressive mechanisms. Indeed, recent work with histone 72 modifications suggests complexity in the number and combination of histone residues important in the expression and repression of genes (41). In any case, the presence of pathways functioning to repress aberrant transcription does demonstrate that there are possible pathways to be interfered with. Another possible model of the sptl6A 922 mutant is that the misfolded protein is interfering with other general factors important in maintaining transcriptional repression. These factors may be chromatin remodelers themselves, or possibly binding factors that inhibit further transcription after R N A polymerase II passage. L ikely candidates would include other SPT genes that show similar mutant phenotypes as SPT16, such as SPT4, SPT5, and SPT6, all of which are in the same category of SPT genes. The proteins Spt4p and Spt5p form the D R B sensitivity-inducing factor (DSIF), and are involved in both transcriptional repression and elongation (123, 124). This is thought to occur in a mechanism involving negative elongation factor (NELF) and interactions with R N A polymerase II, regulated in part by methylation of Spt5p (125). It is possible that sptl6A922 may interfere with any stage of transcriptional regulation by DSIF and/or N E L F , or affect the modification of Spt5p and its elongation activation role. The possibility of Spt6p interference is more intriguing. Past research has shown that Spt6p functions to repress transcription from cryptic sites (74), while more recent work has paralleled initial data regarding the function of Spt l6p as a negative regulator o f gene expression (126). The absence of Spt6p alleviates the need for transcriptional activators at certain genes, much like initial studies showed spt 16 mutants alleviated the need for binding sequences for such activators (127). In addition, the genes identified as requiring Spt6p for repression include PH05, ADH2, and SUC2 (126), the latter of which has also been shown to be repressed by Sptl6p (78), suggesting the two SPT genes may act similarly for the same set of genes. Finally, Spt6p mediates these repressive effects by nucleosome reassembly at the 73 promoter following activation. These similarities to Sptl6p and F A C T may indicate a functional association between the two SPT genes. Spt6p could be recruited to the nucleosome following F A C T mediated elongation to counteract nucleosomal disruption and prevent aberrant transcription. F A C T mutants that cause cryptic initiation, such as sptl 6A 922, may not be directly affecting F A C T function, but preventing the activities of Spt6p. This may be through a loss of association between F A C T and Spt6p, or perhaps prevention of Spt6p-nuclesome association. Spt6p may also require association with specific modified histone residues, for example, lysines 14 and 23 may act as nucleosome docking site for Spt6p. The failure to repress aberrant transcription in spt 16 mutants has often been interpreted as a loss in F A C T function (75, 78, 98). Our data supports the hypothesis that the spt!6A922 mutant may be interfering with normal histone modifications, specifically, histone acetylation, and that this interference is in part causing an spt phenotype. F A C T , then, may not be directly responsible for preventing aberrant gene expression. Instead, F A C T mutants may be interfering with the normal processes and pathways required for proper nucleosome restructuring following transcription elongation. 4.3 Conc l ud i ng Statement The first part of this study focused on the interaction between F A C T and two H3 specific H A T complexes, N u A 3 and A D A . After confirming the putative interaction between Sas3p of NuA3 and Sptl6p, we went on to demonstrate both in vitro and in vivo that F A C T interacts with the A D A complex as well . The H A T interaction domain was then mapped to the N-terminal 482 residues of Sptl6p. 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Thesis/Dissertation
eng
Biochemistry and Molecular Biology
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Graduate
Investigating the role of histone acetylation in yeast FACT complex function
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