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TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 p:/ClRC/DateDue.indd-p.1 INVESTIGATING FUNCTIONS OF GCN5 AND SNF1 IN HIS3 ACTIVATION By Yang Liu A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program 2006 ABSTRACT INVESTIGATING FUNCTIONS OF GCN5 AND SNF1 IN HIS3 ACTIVATION By Yang Liu GcnSp serves as the catalytic subunit of the SAGA histone acetyltransferase (HAT) complex and is critical for HIS3 activation in Saccharomyces cerevisiae. To identify molecular functions that act downstream of or in parallel with Gcn5 protein, an EMS and a minitransposon derived mutagensis libraries were screened for suppressors that rescue the transcriptional defects of HIS3 caused by a catalytically inactive mutant GcnSp, the E173H mutant. A truncated Reg] protein, Reg1(1-740), was found to be a dominant suppressor. The function of Reg1(1-740) protein requires an intact cis-acting element for the Gcn4 transcriptional activator, and doesn’t change the canonical HAT activity of Gcn5p. Regl protein functionally and physically interacts with a histone H3 kinase, Snfl p. Indeed, Snflp plays an important role for normal and Regl (1-740)-dependent HIS3 activation. However, substituting the phosphorylation residue in H3, i.e. SerlO, with alanine or glutamate neither attenuates nor augments the suppression phenotypes. These results argue against an essential role of H3 phosphorylation in HIS3 expression. Both Reg1(1-740) and overexpressed Snflp rescue the E173H allele of gcn5 selectively, suggesting physical interactions between GcnSp and these two proteins. In vivo co- purification experiments confirmed this notion. Moreover, GcnSp is a substrate of Snfl kinase in vitro and the phosphorylation ochnSp in vivo is dependent on the Snfl p. A quadruple mutation of GcnS (T203A/SZO4A/I‘21 lA/Y 212A, TSTY/4A) that diminished the in vitro phosphorylation also impairs HIS3 transcription in vivo. Interstingly, this mutant, as well as the snfl A mutation, is suppressed by deleting SPT3 or SPT8, suggesting that Snflp and TSTY residues of GcnSp function antagonized the inhibitory effects of Spt3p and Spt8p. Furthermore, Spt3 protein interacts with GcnS protein in vitro, which raises the possibility that Spt3p inhibits GcnSp function by direct interaction and such inhibition is relieved by the Snfl p-mediated phosphorylation of GcnSp or/and Spt3p. Our study might unravel an uncharacterized regulatory network intrinsic to the SAGA coactivator complex, of which the composition and molecular functions are well conserved in human cells. ACKNOWLEDGMENTS I want to thank my thesis advisor, Dr. Min-Hao Kuo for the unbelievable time and effort he spent on me during the last five years. Without his encouragement and support, all these work would not be accomplished. His guidance is a treasure for me for ever. I want to thank my thesis committee members, Dr. Triezenberg, Dr. Amosti and Dr. Henry, for their valuable suggestions and comments in my research. I want to thank J ing, our lab mom, for her selfless help in my research and life. And thank to my best friend in the lab, Dave Almy. I’ll never forget the pleasure and sadness we go through together. I also want to thank many lab members, Asha, Dawei, Jianjun, for their help in my research. I also want to thank peoples in Genetics program and Department of Biochemistry. And finally, I want to thank my family, my wife Weiwei, my son Max, my parents and parents-in-law. iv TABLE OF CONTENTS LIST OF TABLES ..................................................................... vii LIST OF FIGURES ..................................................................... viii CHAPTER 1: LITERATURE REVIEW ............................................. 1 Part I: Post-translational modification of proteins ................... 2 Part II: Histone modifications .......................................... 7 Part III: SAGA and histone acetyltransferase ........................ 13 Part IV: Snfl .............................................................. 16 Part V: Research interest and significance ............................ 19 References ................................................................ 21 CHAPTER II: HISTONE H3 SERl O PHOSPHORYLATION- INDEPENDENT FUNCTION OF SNF1 AND REG] PROTEINS RESCUES A GCN5' MUTANT IN HIS3 EXPRESSION ............................................................ 34 Abstract ..................................................................... 36 Introduction ............................................................... 37 Materials and Methods ................................................... 41 Results ...................................................................... 47 Discussion .................................................................. 58 Acknowledgement ....................................................... 64 References ................................................................. 65 Tables ...................................................................... 75 Figures ..................................................................... 78 CHAPTER III: SNFIP ACTIVATES HIS3 TRANSCRIPTION BY ANTAGONIZIN G THE INHIBITORY EFFECTS OF SPT3P AND SPT8P .......................................................... 93 Abstract ..................................................................... 95 Introduction ............................................................... 96 Materials and Methods ................................................... 100 Results ...................................................................... 105 Discussion .................................................................. 1 12 Future plan ................................................................ 115 Acknowledgement ....................................................... l 17 References ................................................................. 1 18 Tables ...................................................................... 123 Figures ..................................................................... 125 APPENDIX: IDENTIFICATION OF SUPPRESSORS THAT BYPASS THE GCN5 REQUIREMENT BY SCREENING AN EMS MUTAGENESIS LIBRARY CREATED FROM A GCN5' STRAIN ................................................................... 1 3 8 Introduction ............................................................... 1 39 Results ...................................................................... 143 References ................................................................. l 59 vi LIST OF TABLES CHAPTER II: HISTONE H3 SERIO PHOSPHORYLATION- INDEPENDENT FUNCTION OF SNFI AND REGI PROTEINS RESCUES A GCN5’ MUTANT IN HIS3 EXPRESSION Table 1 Yeast strain list ............................................................ 75 Table 2 Plasmid construct list ...................................................... 77 CHAPTER III: SNFIP ACTIVATES HIS3 TRANSCRIPTION BY ANTAGONIZIN G THE INHIBITORY EFFECTS OF SPT3P AND SPT8P Table 1 Yeast strain list ............................................................. 123 Table 2 Plasmid list .................................................................. 124 APPENDIX: IDENTIFICATION OF SUPPRESSORS THAT BYPASS THE GCN5 REQUIREMENT BY SCREENING AN EMS MUTAGENESIS LIBRARY CREATED FROM A GCN5' STRAIN Table 1 Summary of screening .................................................... 141 Table 2 Summary of spore analysis ............................................... 149 Table 3 Summary of dominance test .............................................. 153 vii LIST OF FIGURES CHAPTER II: HISTONE H3 SERIO PHOSPHORYLATION-INDEPENDENT FUNCTION OF SNF1 AND REGI PROTEINS RESCUES A GCN5' MUTANT IN HIS3 EXPRESSION Figure 1 Identification of a BGR suppressor rescuing the gcn5 E173H mutant... Figure 2 Characterization of the Reg1(1-740) BGR suppressor .................... Figure 3 The BGR suppressor is semidominant and selectively rescues the E173H defects of the GCN pathway ........................................ Figure 4 SNF1 is important for HIS3 expression and BGR phenotypes ............ Figure 5 H3 SerlO phosphorylation is not required for the BGR phenotypes Figure 6 Biochemical interactions of GcnS/Snfl and Gcn5/Reg1(1-740) proteins ........................................................................... Figure 81 Reg1(1-740) protein does not rescue san- 3-AT hypersensitivity Figure S2 Genetic interaction between GcnS and Snfl for HIS3 expression CHAPTER III: SNFIP ACTIVATES HIS3 TRANSCRIPTION BY ANTAGONIZIN G THE INHIBITORY EFFECTS OF SPT3P AND SPT8P Figure l Overproducing Snfl protein causes Gcn5p hyperphosphorylation in vivo .............................................................................. Figure 2 The TSTY/4A mutation of GcnS was suppressed by deleting SPT3.... Figure 3 Spt3p antagonizes Snflp function in HIS3 activation .................... Figure 4 Both gcn5 TS TY/4A and sanA are suppressed by deleting SPT8 ......... viii 78 8O 82 85 87 88 91 92 125 127 130 132 Figure 5 Snflp interaction with and phosphorylates Spt3p ......................... 133 Figure 6 Direct interaction between Gcn5p and Spt3p .............................. 135 APPENDIX: IDENTIFICATION OF SUPPRESSORS THAT BYPASS THE GCN5 REQUIREMENT BY SCREENING AN EMS MUTAGENESIS LIBRARY CREATED FROM A GCN5' STRAIN Figure l HIS3-IacZ reporter expression in selected BGR mutants ................. 142 Figure 2 Northern analyses of the bgr candidates .................................... 144 Figure 3A Strategy of dominance test ................................................. 152 Figure 3 Dominance test ................................................................. 154 Figure 4 Complementation test ......................................................... 156 ix CHAPTER I Literature Review Literature review Part I: Post-translational modification of proteins: The phenotypes and the behavior of an organism are not only decided by the genetic information it contains, but are also largely affected by the environment. The post- translational modification (PTM) of the gene products — proteins, is a strategy of the cell to adapt to the uncertain and frequently changing environment. There is a variety of protein PTMs in nature. Aside from protein degradation and restricted protease digestion, most easily observed modifications are adding a molecule covalently. These molecules can be very small, such as acetylation (lysine), phosphorylation (serine, threonine, tyrosine and histidine), methylation (lysine and arginine), nitrosylation (cysteine) and glycosylation (serine) etc., or relatively large, such as myristoylation (amino terminus), ubiquitylation (lysine) and sumoylation (lysine). These modifications might change the charge or the conformation of the protein, or in other cases, the attached group provides the better surface for the binding partners. In result, the activity or the cellular localization of the protein is altered. Post-translational modifications are involved in almost all cellular processes such as cell sorting, signal transduction, gene transcription, cell cycle regulation and DNA damage repair. The PTMs can be found almost everywhere in the cell, from the outer surface, to the very inside of the nucleus. Cell surface: Glycosylation and environmental sensing The first place to encounter extracellular environment change is the cell surface. These environmental factors may include small molecules like hormones and toxins or much larger particles like viruses, bacteria or other cells. Receptors located on the cell surface are sensors for those extracellular factors. However, the receptor per se is not sufficient for recognition. Carbohydrate groups, implanted by multiple glycosylation enzymes, are required for the function of receptors in most cases, and probably are the primary elements that interact with the outside signals (110). We can imagine that in the absence of or incorrect glycosylation will cause problems for cells in their toxin defense, cell-cell communication, and differentiation. Indeed, abnormal glycosylation patterns are known to be markers for, and in some cases the cause of, certain disease states including cancer (30). For exemple, in colonic cancer cells the mucin O-glycan chains, which are responsible for interactions between cancer cells and their microenvironment, are enriched in certain forms instead of a large range of structures (13). Moreover, different cancer cells display unique glycan epitopes, which could serve as targets for cancer diagnosis and treatment (19). Cytoplasm: phosphorylation and signal transduction Protein phosphorylation is probably the most common post-translational modification, as the protein kinases are the third largest protein family in the human genome and represent 2% of proteins (79). The roles of protein phosphorylation in signal transduction are evolutionarily conserved among eukaryotes and have been extensively studied over the years. Adding or removing the phosphate group can serve as an ON/OF F switch in responding to chemical or physical stress. Good examples are the MAP kinase pathways, in which the phosphorylation of the target protein causes increased kinase activity, or elimination of phosphatase activity to deliver the signal to downstream targets (27). Ser/Thr phosphorylation is thought to play intrarnolecular roles, such as promoting the conformational change or taking part in the catalysis. On the other hand, tyrosine phosphorylation is more likely involved in intermolecular interactions. Accurate phosphorylation regulation of the MAPK cascades is important for cells keep normal functions, including gene expression, cell proliferation, cell survival and death, and cell motility (20). The MAPK pathway is activated in virtually all melanomas (95). The oncogenic activation of tyrosine kinases is a common feature in cancer (93). Nucleus: The eukaryotic nucleus is the major place that DNA replication, gene transcription and DNA damage repair happens. More and more proteins involved in those cellular processes have been shown to require post-translational modifications for proper function. Some examples related to gene activation will illustrate the importance of PTMs. PTMs of activators: p53 The tumor suppressor protein p53 is the target of various post-translational modifications, which modulate p5 3 functions at different levels. There are at least 17 phosphorylation sites found in human p53 (12). Most of the known phosphorylation sites are located in the N-terminus of the protein (Ser6, 9, 15, 20, 33, 37 and 46, and Thr18, and 81), within or close to the transactivation domain (12). Multiple kinases show redundancy and each kinase may target several sites. Serine 15 phosphorylation of p53 can be achieved by the function of ATM/ATR (105, 130), DNAPK (6), ERK3 (114), and p38 kinase(71). Any of the above reactions will direct the cell to undergo apoptosis. Additionally, p38 kinase is able to mediate the phosphorylation of Ser33, Ser46, and Ser392. This phosphorylation causes either p53 stabilization or increased DNA-binding ability (12). As a multifunctional protein, phosphorylation of p53, although quite complicated, is insufficient to regulate the complexity of the processes it controls. Some other PTMs, such as acetylation (146), ribosylation (140), ubiquitylation (l4) and sumoylation (21) are also important features for accurate behavior of p53. Phosphorylation and acetylation generally result in activation of p53 (12). Sumoylation at lysine 386 was reported to repress gene transcription (83). These modifications are highly interactive. The ubiquitylation, which is the first step for p53 turnover, requires the deacetylation of p53 (59). Also, phosphorylation of several serines by CKl requires that Serl 5 is phosphorylated (106). The dysfunctional p53 proteins derived from incorrect modifications are always associated with different kinds of cancers. Besides p53, many other transcriptional activators/repressors and coactivator/corepressors are targets of post-translational modifications. In humans, p300- mediated acetylation of c-myc results in turn-over of c-myc (38). Phosphorylation of p300 by Akt kinase will increase its histone acetyltransferase activity and facilitates its gene activation function (56). In yeast, AMPK/Snfl will remove the negative effect of Migl repressor from gene promoters by phosphorylation under nutrient limiting conditions (96). Part II: Histone modifications Eukaryotic DNA is packed into chromatin by wrapping around the histone octamers. Such a highly compact structure forms a barrier for the DNA or chromosome related regulatory processes. Four approaches have been found to temporarily or permanently alter the organization of chromatin structure to overcome such obstacle. 1. Histone variants. Such as H33 and H2A.Z, which are incorporated in a DNA synthesis independent manner (2, 47, 61, 144). 2. ATP-dependent chromatin remodeling (121). 3. DNA methylation (111). 4. Covalent histone modifications (see below) The regulatory machineries ubiquitously utilize post-translational modifications to either alter the charge of the histones or to provide extra binding sites for regulators. It is hard to imagine how diverse and complicated the modification schemes are if a regulator needs to distinguish its target locus from millions of similar nucleosomes in the genome. Histone modification enzymes dynamically mark the nucleosomes with phosphorylation, methylation, acetylation, biotinylation and ubiquitylation etc., at different residues and in different combinations (29, 64, 99, 107). The histone modifications are also cross- regulated to modulate the chromatin functions more precisely. For example, histone H3 810 phosphorylation facilitates K9/K14 acetylation at the [NO] promoter (76). The K4 methylation and K9/K14 acetylation of histone H3 correlate very well through different developmental stages at chicken B-globin locus (75). Rad6-mediated ubiquitination of K123 of histone H2B is a prerequisite for H3 K4 and K79 methylations and is important for telomeric silencing in S. cerevisiae (91 , 127). A “histone code” hypothesis has been proposed, in which individual modifications or combination of modifications may extend the information potential of the genetic (DNA) code by recruiting different regulatory factors to the specifically modified chromatin loci (125) To realize a “histone code”, two classes of factors are required. The first class includes enzymes that generate or remove post-translational modifications. The second class includes proteins capable of interacting with histones bearing specific modifications. The extensively studies on histone post-translational modifications vote “YES” to the above question. Here I will summarize the two classes of factors identified using the examples of histone methylation and phosphorylation. Histone acetylation will be discussed in the next part. Histone methylation Histone methylation occurs on the side-chain nitrogen atoms of lysine or arginine residues. Neither lysine nor arginine methylation alters the charge status of the residues. Such chemical natures support the assumption that the modification is a “mark” for recruiting regulatory factors but not affects the nucleosomal structure. Lysine can exist in mono-, di- or trimethylated forms, while arginine is able to accept up to two methyl groups. In fact, different modification stages are reflected in different physiological functions (35, 80). Histone arginine methylation can be conducted by several protein arginine methyltransferase (PRMTs). Among them, PRMTl and CARMl catalyze asymmetric dimethylation of H4 R3 to facilite transcriptional activation (126, 138). PRMTS, a human SWI/SNF chromatin remodeler associated protein, functions as a repressor by symmetrically methylating R8 of histone H3 (94). On the other hand, methyl groups can be removed from arginine residues of histones by PADI4-mediated deimination reaction to create a citrulline residue (28, 139). So far, no methylated arginine specific binding motif has been found. However, PRMTI mediated H4 R3 methylation is essential and sufficient for histone acetylation at chicken B-globin region (55), which suggests a modification specific recognition mechanism involved. Lysine methylations of histones are mainly detected in H3 and H4 (107). Several histone methyltransferases (HMTs) with the signature SET domain were identified in past 6 years. The first characterized HMT is Suv39h/Clr4 that conducts H3 K9 methylation (8, 67). Later, more H3 K9 HMTs were found, including G9a, ESET and EZH2 (31, 66, 129, 137, 143). Interestingly, G9a and ESET are di-methylases, whereas Suv39h is a tri-methylase. Consistent with their activity, Suv39h resides in heterochromatin, while G9a is largely localized to euchromatic regions and acts as either a transcriptional corepressor or coactivator (68, 92), implies the number of methyl-groups is important for the information read out. For instance, H3 K9 methylation is always linked to silenced chromatin. The specific recognition of lysine 9 methylated histone H3 ( mainly tri- methylated) by the chromodomain of HP] protein is a key step in heterochromatin formation (8, 67). Even mono- or di-methylated forms of K9 are usually associated with repressor complexes or inactivated chromatin (84, 98). Opposite to K9, lysine 4 methylation of H3 usually correlates with activated chromatin, especially the tri-methylated form (41, 108). The H3 K4 HMTs also contain the SET domains. The activity and substrate specificity of K4 HMTs are largely dependent on the protein complexes they are assembled into (107). Set] of the budding yeast creates permissive chromatin by adding the di-methyl mark (99). MLLI HMT is associated with coactivator complexes and catalyzes the tri-methylation of K4 to aid gene transcription (32, 141). Chromodomain proteins can also read the methylated K4 code, as Chdl from yeast and human is able to target K4 methylated H3 (43, 100, 120). WDRS, which contains WD40 repeats, is also shown to bind methylated K4 (141). However, these proteins can not distinguish tri-memethylation from (ii-methylated forms. Recently, several groups reported that the PHD finger of NURF and ING proteins specifically recognize trimethylated H3 lysine 4 (73, 97, 115, 142). Methylation of many other lysines in H3 and H4 N-terminal domains or core domains is also important for physiological fimctions. Set2 mediated H3 K36 methylation is correlated with transcriptional elongation (87, 136). H3 K79 methylation, catalyzed by non-SET HMT Dotl, is a mark to set up chromatin domains (39, 85, 90, 132, 145). Set8 10 catalyzed methylation of lysine 20 of H4 is involved in chromatin condensation in mitotic regulation (26, 102). In addition to methylation, lysine residues are the target for several other modifications, like acetylation, sumoylation, ubiquitylation and biotinylation, etc. (64, 103, 107). Indeed, these modifications may compete for the same residues, like lys9 of H3 is found in multiple modification forms (64, 103, 107). It raise the importance of reversal of the methylation. A few enzymes that remove methyl-group have been identified in last two years. LSD] is an F AD-dependent amine oxidase and capable of using methylated K4 as substrate (116, 117). The transcriptional repressor JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine 36 (26, 63). Histone phosphorylation Phosphorylation is another important feature of histones in chromatin. So far, only serines or threonines in histones are known to be phosphorylated. The phosphorylation sites are found in all of the 4 core histones (SI, T119, and $129 of H2A; 810, $14 and S33 of H2B; T3, 810, T11, 828 and S31 of H3; S1 of H4). Consistent with histone code hypothesis, phosphorylation of different residues results in different cellular regulations. For example, H4 81 and H3 810/828 phosphorylations play important roles in mitosis (9, 22, 54). S 14 of H2B is rapidly phosphorylated at sites of DNA double-strand breaks (40). Sterile 20 kinase mediated H2B SerlO phosphorylation is induced during apoptosis and meiosis in S. cerevisiae (3, 4). H3 810 phosphorylation is correlated with transcriptional 11 activation (1, 76). Many histone kinases have been identified to write the phosphorylation code. Apart from the Sterile 20 kinase for H2B 810 phosphorylation mentioned above, NHK-l phosphorylates T119 of H2A (5); Ipll/Aurora-B kinase is responsible for H3 S10/28 phosphorylation (54); 810 of H3 can be also phosphorylated by Snfl (76), Rsk2 ( 109), and MSKl (69). On the other hand, not many code erasers (phosphatases) have been reported, except the PPl/Glc7 that mediates dephosphorylation of S 1 O to counteract the Ipll kinase (54). Little is known about the proteins or motifs that specifically interact with phosphorylated histones. In vitro studies using phosphorylated peptide indicates the 14-3-3 module is capable of binding phosphorylated histone H3 (78). An independent study using tethered- catalysis yeast two-hybrid system (46) also detected the yeast 14-3 -3 proteins Bmhl and Bmh2 in the screening for phosphorylated H3 associating protein (Guo, et al, unpublished), which support the hypothesis that 14-3-3 proteins are phosphorylation code readers. 12 Part III: SAGA and histone acetyltransferase Histone acetylation is one of the best studied post-translational modifications. In the budding yeast Saccharomyces cerevisiae, the amino-termini of all 4 core histones possess multiple conserved lysine residues that are acetylated in vivo, like K9, K14, K18, and K23 in H3; K5, K8, K12, and K16 in H4; K4 and K7 in H2A; K11 and K16 in H2B (99, 103). The covalent linkage at an acetylgroup with the e-amino group of lysine residue not only alters the structure of the amino acid, but also neutralizes the positive charge. Based on sequence homology and architecture of the catalytic domain, histone acetyltransferases (HAT) can be divided into five superfamilies, GcnS-related acetyltransferase (GNAT), MOZ-Ybf2/Sas3-Sas2-Tip60 (MYST) related HATS, p300/CBP HATS, the general transcriptional HATs, and the nuclear hormone-related HATS (17). Most HATS existed in multisubunit protein complexes and that regulate gene transcription and other cellular processes. The MYST family member Esal is the HAT component of yeast NuA4 complex that plays roles in gene silencing, cell cycle progression and DNA repair of the double-strand break (7, 23, 24). The largest TFIID components, the Tafl protein, mediates histone acetylation in higher eukaryotic organisms to facilite transcriptional initiation (34, 52, 86). In contrast, the Elp3 acetyltransferase of the elongator complex modulates the transcriptional elongation process (50).. 13 The Gcn5 histone acetyltransferase of Saccharomyces cerevisiae is the catalytic subunit of multiple coactivator complexes, e.g. ADA, SAGA, and SALSA (44, 122). The ADA complex contains Ada2, Ada3/Ngg1, Gcn5 and Ahcl (36). This complex might regulate the basal expression of HIS3 gene (Kuo and Almy, unpublished). The SAGA and SALSA complexes are very similar except the Spt7 subunit is truncated and the Spt8 subunit is missing in SALSA complex. The yeast SAGA complex is a 1.8MDa protein complex that consists of at least 19 components. Based on the order of discovery, these components are divided into several groups. The first group is Suppressor of Ty (Spt) proteins, includes Spt3, Spt7, Spt8, and Spt20 (44). A subset of the TATA-binding protein (TBP) associating factors (TAFs, Taf5, Taf6, Taf9, Tafl O, and Tafl 2), initially identified as components of the TFIID general transcription factor, composes the second group of proteins in SAGA (44, 45). Transcriptional adapters are another group of proteins, including Adal/Hfi 1 , Ada2, Ada3/Ngg1, and Ada4/Gcn5 (l 1). Among them, Ada2, Ada3 and Gcn5 form a core module for HAT activity (123). Other than acting as a HAT that acetylates chromatin to stimulate gene transcription, SAGA also possesses other important activities. The Spt3 and Spt8 subunits interact with TBP and either inhibit or activate TBP function in gene transcription (10, 33, 37, 113). Chdl is a chromodomain-containing protein that recognizes lys4 methylated histone H3, which may aid the nucleosome specific recruitment (100). The pr8deubiquitylation activity was also found to copurify with SAGA complex (51, 58, 70). Moreover, the 14 newly found component of SAGA, ng73/Sca7, can be subject to polyglutamine- expansion, which is important for the complex to behave like an acetyltransferase (82). Several subunits play structural roles to maintain the integrity of the complex. Adal/Hfi l , Spt7 and Spt20 are such backbone proteins (123). The second group of architectural proteins consists of a few essential TAFs. Mutations in T AF 5 , TAFI 0, and TAF 12 also affect SAGA composition and integrity (45). Finally, the largest subunit, Tral , which is shared between SAGA and NuA4 complexes, is able to interact with the acidic transcription activators. Such activity is thought to be the mechanism for the coactivator recruitment to the gene promoter (7, 15, 42). About 10% of the Saccharomyces cerevisiae genes are controlled by SAGA (57). Most SAGA target genes are related to stress response and are regulated (57). Interestingly, mutations of the Spt3 module and the Gcn5 module affect different classes of genes, implying that the function of SAGA is more than that of a HAT (57). In most cases SAGA plays a positive role in gene transcription. However, SAGA is found to repress the ARC] gene when cells were grown in rich media (101). 15 Part IV: Snfl Sucrose non-fermenting l (Snfl) is the yeast homolog of the AMP-activated protein kinase (AMPK) family that play essential roles in regulation of glucose and lipid metabolism and cellular stress responses (16, 49, 62). The AMPK family is widely conserved through all eukaryotic organisms (49). By sensing the cellular AMPzATP concentration ratio, AMPK is activated to shut off energy consuming (anabolic) pathways and to stimulate energy generating (catabolic) pathways (16). The mammalian AMPK plays a central role in the regulation of energy balance at the whole body level by responding to hormonal and nutrient signals in the nervous system (62). Mutations in AMPK cause cardiac hypertrophy and arrhythmia (l 6). The prevalence of obesity and its associated diseases drive the AMPK energy gauge receiving rising attentions. Indeed, AMPK is becoming an important therapeutic target for type 2 diabetes (48). The functional AMPK complex exists in a heterotrimer that. The on subunit is the catalytic component of the complex. There is only one a subunit in Saccharomyces cerevisiae, which is encoded by SNF1 gene (49). Snf4 is the regulatory y subunit and is essential for activating the kinase activity (1 8, 72). Moreover, the complex requires one of the three B subunits, Ga183, Sipl or Sip2 to tether Snfl and Snf4 together (60). The three B subunits show great functional redundancy as the san' phenotypes can be only achieved with all three subunits deleted (112). Recent studies suggest that the selection of [3 subunit incorporation may affect the cellular localization of the Snfl complex (135). Upon glucose limitation, Gal83 directs the Snfl protein into the nucleus, and Snfl -Sipl 16 complex relocates around the vacuole, while the Sip2 associated kinase remains cytoplasmtic (135). Other than that, Ga183 has the ability to interact with the Sip4 transcriptional activator (133). Sip2 has been implicated to play a role in aging (74). Interestingly, the recent crystal structure of the Snfl kinase domain indicates that Snfl is able to form a homodimer (89, 104). Snfl is a 633 amino acid protein with a 330 residue kinase domain at the N-terminus. The C-terminal region contains a regulatory domain that interacts with other subunits of the complex. An auto-inhibition domain is located at residues 367-500. Once cells encounter glucose depletion or other stresses, the T210 residue in the activation loop of Snfl is phosphorylated by one of the three upstream kinases, Sakl , Elm] or Tos3, and subsequently a conformational change occurs to activate the kinase activity (53, 81, 88, 128). This activation process is inhibited by the Reg1/Glc7 protein phosphatase, as the T210 is constantly phosphorylated in the regIA strain (81). The activated Snfl will phosphorylate downstream targets with the consensus sequence (Hyd-X-Arg-X-X-Ser-X- X-X-Hyd ), in which the arginine at the P-3 position is crucial (49). Activation of the Snfl kinase is a key step in regulating genes related to utilization of alternative carbon sources. However, only a few examples have been established to illustrate what the downstream events are upon the phosphorylation by Snfl. In one case, the phosphorylation of Migl disrupts the Migl -Ssn6 repressor at the promoter and releases the repressor from DNA (131). In other cases, transcription might be stimulated by Snfl -mediated phosphorylation of activator. such as Cat8 and Sip4 (134). Snfl is also 17 a histone modification enzyme that regulates transcription at the chromatin level (76). The Snfl mediated phosphorylation of H3 serine 10 facilities the association of Gcn5 histone acetyltransferase. Such H3 810 phosphorylation, together with Gcn5 mediated K14 acetylation are important for yeast INOI expression (25, 76, 77). Genetic and biochemical studies indicated Snfl is able to active transcription by other mechanisms, such as interacting with mediator complex or TATA-binding protein directly (65, 118, 119). 18 Part V: Research interest and significance The yeast Gcn5 and its histone acetyltransferase activity in gene transcription have been extensively studied. However, the events following Gcn5-mediated histone acetylation are elusive. Moreover, little is know about how histone acetylation facilities transcriptional initiation. Furthermore, Gcn5 is likely to do more things than a HAT, since the catalytic inactive mutant and deletion strains show different expression profile in genomic studies (57, 71). To address the above questions, we sought to isolate extragenic suppressors that allow yeast cells to activate transcription in the absence of HAT activity of Gcn5. Characterizing these suppressors, which represent the factors functionally downstream or in parallel with Gcn5, will help us find out the molecular mechanism of how Gcn5 and its mediated histone acetylation modulates the transcription. Other than investigating the role of Gcn5 in regulating transcription, we are also curious about how Gcn5 is regulated. So far, the only known mechanism of regulating Gcn5 activity is that the recruitment of Gcn5 containing complex to target promoters by acidic activators. The HAT activity of mammalian histone acetyltransferase p300 is alleviated by Akt-mediated phosphorylation (56), which made us wondering whether Gcn5 is regulated in a similar way. Recent studies showed that Gcn5 is sumoylated in vivo, which raise the likelihood of cellular controlling of Gcn5 activity by post-translational modification, although the biological significance of Gcn5 sumoylation is not clear (124). 19 In our primary studies, we found that a suppressor, Reg1(1-740), may regulate Gcn5 by modulating Snfl -mediated phosphorylation of Gcn5. Moreover, our studies indicate that Snfl counteracts the function of Spt3, which is another SAGA component. Such observations suggest that Gcn5 is likely regulated in vivo via intra-complex mechanisms that mediated by Spt3, and by post-translational modifications, such as Snfl -mediated phosphorylation. As a conserved acetyltransferase across species, such regulations of Gcn5 may also exist in other higher eukaryotes. 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Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21(Wafl/Cip1). Mol Cell Biol 26:2782-90. 33 Chapter II Histone H3 Ser10 Phosphorylation-Independent Function of Snfl and Regl Proteins Rescues a gcn5' Mutant in HIS3 Expression Published in Mol Cell Biol. 2005 December; 25(23): 10566—10579. 34 Histone H3 Ser10 Phosphorylation-Independent Function of Snfl and Regl Proteins Rescues a gcn5' Mutant in HIS3 Expression Yang Liu,l Xinjing Xu,2 Soumya Singh-Rodriguez,2 Yan Zhao,2 and Min-Hao Kuol ,2* Program in Geneticsl and Department of Biochemistry and Molecular Biology, 2 Michigan State University, East Lansing, Michigan 48824 Received 22 August 2005/Accepted 14 September 2005 * Corresponding author. Mailing address: 401 BCH Building, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824. Phone: (517) 355-0163. Fax: (517) 353-9334. E-mail: kuom@msu.edu. 35 ABSTRACT: Gcn5 protein is a prototypical histone acetyltransferase that controls transcription of multiple yeast genes. To identify molecular functions that act downstream of or in parallel with Gcn5 protein, we screened for suppressors that rescue the transcriptional defects of HIS3 caused by a catalytically inactive mutant Gcn5, the E173H mutant. One bypass of Gcn5 requirement gene (BGR) suppressor was mapped to the REG] locus that encodes a semidominant mutant truncated after amino acid 740. Reg1(1-740) protein does not rescue the complete knockout of GCN5, nor does it suppress other gcn5‘ defects, including the inability to utilize nonglucose carbon sources. Reg1(1-740) enhances HIS3 transcription while HIS3 promoter remains hypoacetylated, indicating that a noncatalytic function of Gcn5 is targeted by this suppressor protein. Regl protein is a major regulator of Snfl kinase that phosphorylates SerlO of histone H3. However, whereas Snfl protein is important for HIS3 expression, replacing SerlO of H3 with alanine or glutamate neither attenuates nor augments the BGR phenotypes. Overproduction of Snfl protein also preferentially rescues the E173H allele. Biochemically, both Snfl and Reg1(1-740) proteins copurify with Gcn5 protein. Snfl can phosphorylated recombinant Gcn5 in vitro. Together, these data suggest that Regl and Snfl proteins function in an H3 phosphorylation-independent pathway that also involves a noncatalytic role played by Gcn5 protein. 36 INTRODUCTION Histone acetylation is a well-studied modification of chromatin (fl) and has been linked to transcriptional regulation, recombination, DNA replication, and damage repair (15). GNAT (Gcn5 protein-related N-acetyltransferases) and MYST (MOZ-Ybf2/Sas3-Sa52- Tip60) families of histone acetyltransferases (HATS) generate both targeted and global acetylation of the chromatin (28). Other HATs, such as TAF 1 (formerly TAFu250) and nuclear hormone receptor coactivators, though not belonging to either family, have also been shown to play critical chromatin-related functions via their HAT activities (78). The Saccharomyces cerevisiae Gcn5 protein is the catalytic subunit of several chromatographically distinct HAT complexes, including SAGA, ADA (3_2_), SALSA, and SLIK (ZQ, _7_1, 8_5_). SAGA is recruited to the promoter by certain transcriptional activators and causes promoter-specific nucleosomal hyperacetylation leading to transcriptional activation (4, 5, 48, 51, Q). The SAGA complex also performs HAT-independent functions, such as TATA binding protein (TBP) recruitment and histone deubiquitinylation (8, 2, 12, 24, 58, 44, 55, 25, 85). SAGA and SALSA/SLIK complexes share TBP-associated factors with TFIID (58). Low-resolution electron microscopic studies showed that the architectures of SAGA and TFIID complexes are highly similar (5, 14, 21, l_(_)_3). TFIID is critical for mostly housekeeping gene expression, and the SAGA-dominated genes (~10% of the nuclear genes) are largely stress-induced and are under the coordinated control of multiple chromatin and transcriptional regulators (43). 37 Although the promoter-specific histone acetylation function of Gcn5 has been firmly established (48, 5_l), which molecular activities are modulated by histone acetylation remains an open question. The best-known molecular event triggered directly by histone acetylation is the recruitment of bromodomain-containing proteins (29, 45, 55, _6_2). Besides this, however, little is known as to what other functions may be triggered or antagonized by histone acetylation. Identification of mutations that suppress defects associated with histone hypoacetylation may reveal factors downstream of histone acetylation. Thus far, the only reported screen for suppressors rescuing gcn5 null phenotypes was a multicopy suppressor hunt identifying ARG3 (_6_9_), which is likely involved in controlling the global chromatin structure via regulating the balance of nuclear polyamine. On the other hand, Gcn5 protein is important for only a portion of yeast genes (4_0, fl). Suppressors that display gene specificity, instead of global effects on chromatin structure, may shed light on the molecular basis for Gcn5-mediated transcriptional activation. In our first attempt to identify the bypass of Gcn5 requirement gene (BGR) suppressors, we isolated one such mutation mapped to the REG] gene. REG] (also called HEXZ and SRN 1) was identified in several genetic screens of glucose repression and RNA processing (58, 65. _6_6_, _9__6_). Regl protein associates physically and functionally with an essential and multifunctional protein phosphatase 1, Glc7 (5;, 51, 24), whose substrate specificity is apparently determined by association with different partners, including Regl protein. Mutations of REG] cause ectopic expression of several the Glc7 interaction domain of Regl protein derepress ADHZ and S UC 2 (28). A similar transcriptional repression defect caused by a glc7 mutation (T152K) can be suppressed by 38 overexpressing Regl protein (fl). These transcriptional derepression phenotypes are likely due to the inability of Glc7 to dephosphorylate the appropriate target protein(s) and consequently the ectopic increase of protein phosphorylation. Indeed, deletion of the Snfl protein kinase suppresses the derepression defects resulting from reg] or glc7 mutations (25, 58, 4_2_), indicating an antagonistic relationship between the Snfl kinase and the Regl-Glc7 phosphatase complex. Consistent with this notion, Regl protein interacts directly with Snfl protein in both yeast two-hybrid assays and affinity purification (6_1, 19). Furthermore, a hyperactive Snfl protein caused by regIA rescues the Spt- phenotypes of sp121 A cells (52). Curiously, the interaction between Regl protein and Snfl protein, at least within the yeast two-hybrid context, is enhanced in glucose starvation conditions (6_1 ), raising the possibility that Regl protein may have a positive role in Snfl protein action under certain conditions. Snfl protein acts as a cellular fuel gauge controlling responses to nutritional crises (3_7). The animal homologues of Snfl protein are activated by AMP and are referred to as AMP-activated protein kinases. In plants, Snfl protein-related kinases (SnRKs) fall into three large families, SnRKl , SnRK2, and SnRK3 (55). Snfl protein, AMP-activated protein kinases, and SnRKs are the catalytic (1 subunits of a trimeric complex composed of a scaffold [3 protein and a regulatory 7 subunit. In addition to bridging the a and 7 subunits, the [3 protein contributes to substrate selection as well. The 7 subunit of the yeast Snfl complex is encoded by SNF4 (14). At least three yeast genes encode the [3 subunits (25, 194). Snfl protein plays critical roles in controlling transcription of carbohydrate transporter and metabolism genes (89). Overexpression of Snfl protein also causes early aging. increased rRNA recombination. and loss of rRNA locus silencing (56), 39 a set of functions reportedly linked to histone H3 hyperphosphorylation. Indeed, several proteins can be phosphorylated by Snfl protein in response to glucose starvation, including Regl protein (22), Migl (22). and histone H3 (Q). The histone H3 phosphorylation activity of Snfl protein has been linked directly to transcriptional activation and TBP recruitment (58, 52). SerlO phosphorylation facilitates acetylation by increasing the affinity between Gcn5 protein and H3 (15, 18, 50). Both modifications are important for the expression of the [NO] gene in yeast (52, Q). In addition, genetic interactions between Snf 1 protein and Srb/mediator proteins (42, 84) and TBP (85) were reported. Whether these general transcriptional factors can be phosphorylated by Snfl protein is unclear. In this work, evidence that a gain-of-function BGR allele for Regl protein likely adopts a novel function in facilitating transcription of HIS3 is presented. This function appears to require a functional Snfl kinase. However, H3 phosphorylation does not play a critical role for the suppression, nor is it important for normal HIS3 activation. A unique allele specificity for a particular mutant Gcn5 protein is shared by the Regl suppressor and overproduction of Snfl protein. Indeed, both Snfl and Regl suppressor can be copurified with Gcn5 from yeast, linking these three proteins functionally and physically. 40 MATERIALS AND METHODS Yeast strains, plasmids, and genetic methods. Yeast strains used in this work are listed in Table 1. All genetic methods were according to reference 8_1. Yeast transformation was done with the lithium acetate method (22). Plasmids used in this work are listed in Table 2. To introduce gcn5 point mutations into the genome, the BamHI-Hindlll fragment from wild-type or mutant GCN5 was inserted into the same sites of YIplac21] (50) to generate pMK284. Constructs pMK284El 73H and pMK284F221A were linearized with NgoMIV and transformed into yeast. Integration results in two copies of GCN5 separated by the YIplac211 sequence containing a URA3 marker. 5-Fluoroorotic acid (5—FOA) selection and genomic PCR were used to obtain and verify the desired E173H and F221A mutations. The HIS3-lacZ reporter was introduced to yeast by transforming the Stul-linearized pMK334 that generates URA” integrants. pMK334 was constructed by inserting the EcoRI-Dral lacZ fragment of pLKC482 ($1) into the EcoRI-Hindlll sites of YIplac21 1, resulting in pMK333. An EcoRI-Bglll fragment containing the HIS3 promoter was isolated from pMK231 where a Bglll site was introduced at the 5' end of HIS3 open reading frame (ORF) and inserted into the EcoRI-BamHI sites of pMK333. A unique Stul site within the URA 3 gene was used for integrative transformation. All subsequent integrants were grown in the absence of uracil to maintain the integrated sequence. 41 To knock out the SNF1 gene, two disruptors were constructed. sanA-I ::LE U2 was generated by two-step subcloning. First, an ApaLI-HindIIl fragment upstream of the SNF1 ORF was inserted into the XbaI-Hindlll sites of pJJ252 (42) to create pMK452. The 3' flanking region of the SNF1 gene, an HpaI-Sacl fragment obtained by PCR, was inserted into the BamHI-Sacl sites of pMK452 to obtain pMK453. In the other disruptor (pYL45, snf] A-2::TRP1 ), the PstI—HindIlI fragment of SNF1 was first inserted into pBluescript KS+ (pMK449). The AflII-Bglll 200-bp fragment corresponding to amino acids 109 through 176 of SNF] in pMK449 was replaced with the EcoRl-Bglll fragment of pJJ248 containing the TRPI gene (42). To create 5an deletion strains, the HindIII- BamHI fragment of pMK453 or the EcoRI-BamHl fragment of pYL45 was obtained by restriction digestion before yeast transformation. To introduce the REG1(I -7-/0) allele, plasmid pYL31 was constructed by replacing the ClaI-Bglll fragment of pKD97 (25) with a ClaI-XhoI-digested PCR product that contains the open reading frame of REG] up to amino acid residue 740 followed immediately by a stop codon. The C laI-Kpnl fragment of pYL31 was cloned into HindIII-Kpnl sites of YIplac21 l to obtain pYL35. To replace the entire REG] ORF with REG1(]-740). pYL35 was linearized by SnaBI and integrated into the REG] locus by homologous recombination. The correct transformants were subjected to 5-FOA selection. Genomic PCR confirmed the correct genotype. The reg] A strains were generated by introducing a PCR fragment containing the KanMX6 cassette flanked by REG 1 sequences outside the ORF (1Q, 2). G41 8-resistant transforrnants were examined by genomic PCR to confirm the reglA genotype. 42 To create and test histone H3 mutations, strain J HY205 (2) was first made HIS3‘ by replacing the his3A] allele with the BamHI fragment of pJJ 217 (4'1) that contains the entire HIS3 gene, resulting in yDAl 0. Each histone H3 mutation was generated by the Quikchange method (Stratagene). using pJ H33 as the template. All mutations were confirmed by sequencing. The 2p SNF1 construct pYL4l was created by cloning the BamHI-Pstl fragment containing the entire transcription unit of SNF1 into EcoRI-Pstl sites of YEplacl 12 (_ZQ). Deletion of the general control-responsive element (GCRE) was as described previously (_5_1)- pMK547Gcn5 with an N-terminal hemagglutinin (HA) tag was created by cotransforming XbaI-linearized pMK547, derived from pAB8 with the G314 DNA binding domain deleted (54), and a PCR-amplified GCN5 open reading frame. The Gcn5- TAP fusion construct (pYL54) was generated by a strategy essentially equivalent to QuikChange mutagenesis protocol (Roche) except that the mutagenic primers were PCR- amplified TAP sequence (B) flanked with sequences around the stop codon of GCN5. pMK144 (52) was the template for mutagenesis and insertion of the TAP sequence. pYL67, a plasmid derived from pBSl479 (_74) by replacing the TAP sequence with eight Myc repeats, was severed as PCR template to amplify the MyczzTRP] cassette with flanking sequence correlated with residue 740 or the stop codon of REG] . Gel-purified PC R products were transformed into yeast cells to generate Regl-Myc fusions. HAT and kinase assays. Gcn5 protein amino acids 19 to 348 lacking the bromodomain were cloned into pET21a and expressed as a His-tagged protein (pMK515). 43 The desired point mutations were generated by the Quikchange method (Stratagene) and verified by sequencing. The recombinant protein was induced in the BL21 strain by adding 1 mM (final concentration) IPTG (isopropyl-B-n-thiogalactopyranoside) when cell culture reached an optical density at 600 nm (OD600) of 0.5/ml. Cell cultures were grown at 37°C for 3 h. Extraction and protein affinity purification were done according to reference 52. Kinase assays were done with the above Gcn5 protein incubated with glutathione S- transferase (GST)-Snf1 (wild-type or K84R) expressed and purified from yeast according to reference 55. The GST-SNFI constructs were kindly provided by D. Thiele (Duke University). Suppressor screening. The yeast genomic DNA library (#21) containing the an- lacZ/LE U2 intervening sequence was provided by M. Snyder (Yale University) (21). The DNA was prepared by cesium chloride gradient and digested by NotI before transforming into yMK995. Ten micrograms of the library DNA was digested and isolated by phenol- chloroform extraction and ethanol precipitation. Approximately 26,000 LE U transformants were replica plated to synthetic complete (SC)-His medium containing 20 mM 3-amino-l,2,4-triazole (3-AT) and incubated at 37°C for 3 to 5 days. 3-AT-resistant colonies were further transferred to nitrocellulose membranes, and the lac-Z level was tested according to reference 1. Colonies that showed blue color on the lacZ filter assays were grown in SC -Leu medium overnight and transferred to yeast extract-peptone- dextrose (YPD) (representing the repressed condition) or synthetic minimal medium (SD) containing 20 mM 3-AT for 4 h. Yeast cells (20 ml; ODW, of 0. l/ml) were then harvested 44 by centrifugation (10,000 X g for 5 min at 4°C ), washed, and suspended in extraction buffer (0.3 M sorbitol, 0.1 M NaCl. 5 mM MgC12, 10 mM Tris HCl, pH 7.4, 5 mM EDTA, Complete protease inhibitor cocktail [Roche]). Whole-cell extracts were prepared by vigorous agitation with glass beads using a bead beater (Biospec Products). 13- Galactosidase activity was quantified according to reference 1. One clone, renamed yMK1055 henceforth. repetitively showed elevated lac-Z expression in response to amino acid starvation and was further studied. yMK1055 was backcrossed to yMK1075 before 3-AT tests. To verify that a single an insertion event was responsible for the BGR phenotypes, yM K1055 was crossed to yMK1085. The diploid strain was subjected to sporulation and tetrad dissection; all trp_ segregants were tested for cosegregation of 3- AT resistance and leucine prototrophy. Recommended procedures were employed to map the integration site of the m'Tn-lacZ fragment (http:/ngacmed.yale.edu/mtn/insertion_librariesstm). Namely, yeast genomic DNA was isolated, digested by EcoRI, and subjected to intramolecular ligation prior to bacterial transformation. Plasmid DNA was isolated from Escherichia coli cells and sequenced across the junction between REG] and an-IacZ/LE U2 using a primer specific to lacZ. Northern analyses. Yeast cells were grown in appropriate selection media until the OD600 reached 0.5. Cells were then pelleted by centrifugation (5,000 X g, 5 min, 4°C) and transferred to either YPD (for basal expression) or SD supplemented with required nutrients and 40 mM 3-AT (for induced expression). Cell suspensions were further incubated at 37°C for 2 to 3 h before harvesting for RNA preparation. Although these relatively harsh conditions for induction were not essential, such treatment generally 45 generated more consistent results in HIS3 activation in our strain background. Procedures for RNA preparation and Northern blot hybridization were described previously (52). Interaction between Gcn5 and Snfl. To test the interaction between Gcn5 and Snfl proteins, a GST-Snfl expression construct (55) orjust GST (pYL44) was transformed to the strain carrying pMK547Gcn5. Purification of GST-Snfl was as described previously (55). Glutathione Sepharose 4B (30 pl; Amersham) was added to whole-cell extracts purified from 1.5 X 109 cells and incubated at 4°C for 3 h under constant rocking. Beads were pelleted and washed twice with HEMGT buffer (25 mM HEPES, pH 7.9, 12.5 mM MgC12, 150 mM NaCl. 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1>< Complete protease inhibitor cocktail [Roche]) followed by two more washes with HEMGT buffer containing 300 or 500 mM NaCl. The bound fractions were eluted by sodium dodecyl sulfate (SDS) loading buffer and resolved by 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The copurified HA-GcnS protein was detected by anti-HA antibodies (12C A5; Roche). For Gcn5-Reg1(l -740) copurification, whole-cell extracts from cells carrying pYL54 and C-terminally Myc-tagged Regl or Reg1(l-740) protein were prepared with the bead- beating method in FA lysis buffer (50 mM HEPES, pH 7.5. 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate, 1 mM phenylmethylsulfonyl fluoride, l>< Complete protease inhibitor cocktail [Roche]). Lysates from 3 X 109 cells were incubated with 30 ul ofimmunoglobulin G Sepharose 6 (Amersham) for 2 h at 4°C. After three washes with FA lysis buffer, the beads were boiled in SDS loading buffer and resolved by 8% SDS-PAGE. Western blots were conducted with an anti-c-Myc antibody (Roche). 46 RESULTS Screening and identification of a BGR suppressor. To screen for extragenic suppressors that rescue transcriptional defects caused by loss-of-function mutations of Gcn5 protein, we introduced a single mutation to the catalytic domain of Gcn5 protein. This mutation, E173H. is a Glu-to-His mutation at residue 173. Since Gcn5 protein participates in more than one complex. the use of this mutant likely maintains the integral architecture of these complexes (l_0_5). Previously, an E173Q mutation was shown by others to drastically reduce the in vitro and in vivo activity of Gcn5 protein (fl, Z5, 25). However, this E173Q mutant in our hands maintained significant activities in HIS3 expression after it was integrated back to the native GCN5 locus (data not shown). We thus designed a Glu-to-His mutation. With the slight positive charge of histidine under physiological pH, a more drastic reduction of the catalytic power of Gcn5 protein was expected (54, 82). The HAT activity of a bacterially expressed E173H mutant was tested using chicken histones as the substrates. As predicted, this mutation significantly reduced the in vitro HAT activity of Gcn5 protein (Fig. 1_A_). To test in vivo functions, the E173H allele was integrated to the native GCN5 locus to replace the wild-type allele. In parallel, another well-characterized F221A allele (52) was integrated in the same manner. Both alleles were controlled by the native GCN5 cis elements. Yeast strains bearing the wild- type, complete knockout. F221A. or E173H allele of GCN5 were then tested for responses to amino acid starvation. Each strain was patched to YPD medium and then replica plated to synthetic complete medium lacking histidine and supplemented with various concentrations of 3-AT, a competitive inhibitor of the His3 protein. Very minor growth defects were seen in gen)” strains when assayed at 30°C in medium 47 supplemented with 3-AT (Fig. 1_B_). However, when these cells were incubated at 37°C, 3-AT induced obvious growth defects of all three gcn5- strains. None of these cells were temperature sensitive (compare growth on YPD and SC -His without 3-AT). The clear growth defects of gen)" cells provide a platform for suppressor screening. We further modified the E173H mutant strain by introducing a HIS3-lacZ reporter to the ura3-52 locus. Insertion of HIS3-lacZ did not change the cellular sensitivity to 3-AT (Fig. 115, bottom two patches). This lacZ reporter, under the control of the HIS3 promoter, was also activated by amino acid starvation (Fig. 1C) and hence offered a convenient means to verify the 3-AT-resistant suppressor phenotypes. To identify suppressors, we used a minitransposon (an)-based mutagenesis approach (21). In this method, the an-lacZ/LEUZ sequence was integrated into a yeast genomic DNA library via transposition. Yeast DNA fragments along with the interrupting sequence were excised from the plasmid pool and transformed into yeast. Each mT n sequence integrated to the chromatin via homologous recombination between the flanking yeast sequence and the corresponding genomic locus. LEU transformants were replica plated to 20 mM 3-AT medium and grown at 37°C. All 3-AT-resistant clones were then screened for increased expression of B-galactosidase induced by amino acid starvation. From approximately 26.000 LE (E transformants, we identified 1 such colony (Fig. 12). Northern data clearly showed that the HIS3 expression was upregulated in this suppressor strain compared with the parental gcn5 E173H cells (Fig. 112). Similar complementation in transcription was seen in HIS 1, HIS6 (not shown), and HIS-l (Fig. 2_C) 48 as well. Genetic assays showed that a single mT n insertion event was responsible for the suppression phenotypes (data not shown and see Materials and Methods). To map the mutation. we rescued and cloned the m Tn insertion along with the flanking yeast sequences (see Materials and Methods). DNA sequencing across the junction revealed that the mutagenic fragment had inserted to the coding region of REG] (Fig. 2A), resulting in in-frame fusion of IacZ to residue 740 of Regl protein. While the expression of the Regl -an-lacZ fusion protein may have contributed to some [3- galactosidase activity shown in Fig. 11:. the HIS3 transcript quantification results (Fig. 1D) unequivocally demonstrated the rescue of gcn5' defects. Nonetheless, since the in- frame fusion of lacZ added a large mass to the truncated Regl protein, we were curious whether the B-galactosidase fusion was necessary for the suppression. By integrative transformation, we replaced the chromosomal copy of the native REG] with one that is truncated after residue 740 (without the lacZ fusion) and tested whether this “clean” REG] (1-740) allele was able to suppress the gen5 E173H mutation. Figure 2B shows that with a truncated Reg1(l-740) protein, the gcn5 E173H cells also exhibited significant resistance to 3-AT (Fig. 215. row 5) and restoration of HIS3 expression (Fig. 2B, lane 7), although the original an-lacZ insertion consistently showed better growth than Reg1(1- 740). The in-frame fusion of B-galactosidase enhanced but was not essential for suppression efficacy. We further deleted the REG] gene and found that the suppression phenotypes were lost (Fig. 215, left panel, row 4, and right panel, lane 4). Interestingly, in the presence of a functional Gcn5 protein, deleting the REG] gene does not seem to affect HIS3 expression 49 (Fig. 12, lanes 7 and 8). Together, these results showed that a Reg1(1-740) truncated protein is essential and sufficient for suppressing the gcn5 E173H mutation in HIS3 transcription. Gcn5, in the context of SAGA complex, is recruited to the HIS3 promoter by the transcriptional activator Gcn4 that binds the cognate cis element, GCRE (51). To test whether the Regl (1-740) suppressor exerted its function via Gcn4-GCRE or a novel Gcn4-independent mechanism. we replaced the GC RE 5' to the HIS3 gene with an irrelevant sequence (fl) and determined whether the suppression was affected. Comparison of mRNA transcribed from the GCRE-less HIS3 with the wild-type HIS-l control clearly showed that the GC RE was essential for Regl (l-740)-mediated suppression (Fig. _2£). indicating that Regl (1-740) protein modulates an activity downstream of the normal Gcn4 functions. One possible mechanism for the observed suppression is restoration of the HAT activity of the Gcn5 E173H mutant protein. To see if this was the case. we conducted chromatin immunoprecipitation using an antibody against histone H3 acetylated at Lys9 and/or 14 (g). Figure 22. lane 1, shows the expected hyperacetylation at the +1 nucleosome of HIS3 in the presence of a wild-type Gcn5 protein (51). The TATA element and the transcription initiation sites are within the +1 nucleosome. Promoter hyperacetylation was lost in the E173H background (Fig. 22, lane 5). When REG] was replaced with the REG1(]-740) suppressor allele. H3 remained hypoacetylated at the HIS3 promoter (Fig. 2_D, lane 7). suggesting that the canonical nucleosomal H3 acetylation by the Gcn5 acetyltransferase was not affected in the REG] (1-740) background. Reg1(l—740) preferentially rescues the E173H allele of gcn5 in a semidominant manner. To characterize the Reg1(1-740) suppressor further, we first tested whether this allele was dominant or recessive. Figure 5A shows that the REG] i/REGI(1-740) heterozygote retained a growth advantage over the parental gcn5 E173H REG] I homozygous strain at both 30 and 37°C. The strength of the suppression appeared to be somewhat weaker than the haploid strain, suggesting that the Regl (1-740) protein was a gain-of-function, semidominant suppressor. Besides E173H, the F221A mutation also causes quantitative defects of Gcn5 functions in vitro and in vivo (Fig. 115) (52 ). This mutation selectively impairs the ability of Gcn5 protein to bind acetyl coenzyme A (acetyl-CoA) (52, 25, 88), which is a prerequisite step for histone tail binding (88). It is thus likely that histone tails within the vicinity of the SAGA complex remain free from binding by the F 221A mutant protein. In contrast, based on the studies of the E173Q mutant (1Q) and our yeast two-hybrid tests comparing different disabled gcn5 mutants (M.-H. Kuo, unpublished data), the E173H allele most likely prolongs its association with both substrates because the catalytic process stalls after the ternary complex is formed. Because of the possible differential effects on histone tail accessibility, we tested whether E173H, F221A. and a knockout allele of GCN5 responded differently to Reg1(1-740) suppressor. To test the allele specificity, the gcn5A and gcn5 F221A strains were engineered so that REG] was replaced with the REG] (1-740) allele, and the resultant strains were tested on 3-AT plates (Fig. 515). Neither the F221 A nor the complete knockout allele of gcn5 was rescued by Reg1(l-740) (Fig. 515, rows 6 and 8). The strong preference for the E173H 51 allele suggests that Regl protein may interact directly with Gcn5 protein or may control Gcn5 at a step(s) subsequent to the formation of the Gcn5-acetyl-CoA-histone ternary complex. We further tested whether Reg1(1-740) protein rescued other gcn5” phenotypes. Figure 59 shows that all three gcn5‘ mutants exhibited severe growth retardation in yeast extract-peptone-glycerol and yeast extract-peptone-ethanol media, as previously reported (58). Moreover, gcn5” cells were also sensitive to 100 mM hydroxyurea, an inhibitor of DNA replication. However, neither the m Tn allele nor the clean truncation of Reg1(1-740) protein was able to suppress any of these defects. The failure to suppress other phenotypes, such as caffeine sensitivity, and the inability to use galactose or sucrose were also observed (data not shown). In contrast, the sporulation defects (12) of a gcn5 E173H homozygous strain were partially rescued (not shown). We conclude that the Reg1(1-740) protein suppresses only a subset of Gcn5 target genes. Snfl protein plays a critical role for HIS3 expression. Regl protein is best known to inhibit the kinase activity of Snfl protein and consequently prevents the expression of many genes when glucose is abundant (see the introduction), a function termed glucose repression. Snfl protein derepresses the expression of these genes via several mechanisms, including histone H3 phosphorylation (52, 62). Phosphorylated H3 was shown to bind Gcn5 protein at a higher affinity (Q, 1_7_). It thus seems plausible that the hypoacetylation phenotype caused by gcn5 mutations may be compensated for by hyperphosphorylation of H3, which either helps anchor Gcn5 protein to the HIS3 promoter or by itself provides an environment suitable for stronger HIS3 expression. 52 To examine the link between Gcn5, Snf 1, and H3 phosphorylation, we first tested whether Snfl protein was involved in HIS3 expression. To this end, we created two deletion alleles of SNF] . The sanA-l ::LE U2 mutant had the entire ORF replaced with a LE U2 marker. However, this marker was incompatible with the an-lacZ-LE U2 insertion mutant; we thus created another allele, sanA-ZzzTRP] , that was truncated after amino acid 108. Figure 4 shows that both 5an mutations caused obvious growth defects on 3-AT plates (Fig. 4A, rows 2 to 4) as well as impaired HIS3 expression (Fig. 4B, lane 3. and data not shown), demonstrating that the Snfl protein was also a critical transcriptional regulator for HIS3. We next tested whether the Snfl protein was important for the suppression. Deleting SNF] from the original gcn5 E173H REG] ::an-IacZ suppressor strain significantly attenuated the suppression phenotypes (Fig. 4_A_, rows 6 and 7, and B, lane 5). Thus. Snfl protein is critical for normal and Reg1(1-740)-mediated HIS3 activation. To see how Gcn5 and Snfl proteins may genetically interact to activate HIS3, we examined whether overexpressing one of these two enzymes can rescue the HIS3 expression defects caused by a mutation of the other. While a 2pm multicopy GCN5 construct was unable to rescue the 3-AT hypersensitivity of the snflA-ZzzTRPI strain (Fig. 4_C_, left panel). overproduction of Snfl protein effectively rescued the E173H allele of gcn5 (Fig. 4_C, right panel, compare rows 3 and 4). On the other hand, overproduction of a catalytically inactive mutant of Snfl . K83 R, failed to rescue the gcn5_ phenotypes (data not shown), suggesting that the kinase activity was essential for the suppression. Intriguingly, neither deletion nor the F221A allele of gcn5 responded to the multicopy SNF] plasmid. Thus, the Snfl multicopy suppressor displays an allele specificity similar to that of Reg1(1-740). Furthermore. in the presence of a functional GCN5, overproduction of Snfl protein yielded higher resistance to 3-AT (Fig. E, right panel, row 2), very similar to the GCN5’ REG](]-740) strain (Fig. 515. row 2). Taking together the above results, as well as the reports that Regl and Snfl interact genetically and physically for transcription of several inducible genes (see the introduction), it seems likely that Snfl may be part of the mechanism by which Reg1(1- 740) protein suppresses the E173H mutant allele. H3 Ser10 phosphorylation is not responsible for bgr suppression. To test whether H3 Ser10 phosphorylation contributes to the BGR phenotypes. we used a yeast strain in which both copies of each of the four core histone genes had been deleted (2). Viability of the cells was supported by a low-copy-number plasmid bearing wild-type histone genes and a URA3 marker. The desired histone mutations can be introduced into an otherwise identical construct containing a LEU2 nutrient marker. After transforming the latter plasmid that delivered the specific histone mutation(s), the wild-type histone genes were shuffled out by 5-FOA selection, leaving the mutant allele as the sole copy for histone expression. Additionally. GCN5 and REG] were replaced with the E173H and REG1(1-740) alleles. respectively. 3-AT resistance was then compared among different LE U Ura- strains as shown in Fig. 5. In this genetic background, Reg1(1-740) also effectively rescued the E173H mutant. However. the SlOA mutation did not impose a discernible effect on cellular growth (Fig. 5. compare rows 3 and 4), ruling out a critical role played by phosphorylated Ser10 alone. Within the amino-terminal tail domain of histone H3, Ser28 and Ser31 share sequence similarity with SerlO (7ARKSTGG and 54 25ARKSAPSTGG). Although Snfl protein has not been shown to phosphorylate either serine residue, Ser28 can be phosphorylated by the Aurora family kinases for chromatin condensation during mitosis (15, 51, Q). We were curious about the possibility that the Snfl kinase activity might “spill over” to these two residues in the REG](]-740) strain. Thus, a triple Ser-to-Ala mutant, SlONS28A/S31A, was introduced to the gcn5 E173H REG1(]-740) background. These cells still exhibited robust growth in the presence of 3- AT (Fig. 5, row 6), further supporting the notion that H3 phosphorylation was unlikely to be the driving force for the observed BGR phenotypes. Consistent with this. neither single nor triple Ser-to-Ala mutations exacerbated the 3-AT hypersensitivity caused by the E173H mutation in a REG] 7 background (Fig. 5. compare rows 2, 8, and 10). We therefore conclude that Ser10 phosphorylation. though important for activation of several other genes, does not contribute appreciably to Gcn5 and Snfl protein-mediated HIS3 expression. Thus, Snfl protein most likely controls HIS3 expression by a novel, H3 phosphorylation-independent mechanism(s). While preventing H3 phosphorylation imposes no apparent effect on the Reg1(1-740) protein-generated suppression, we were nonetheless interested in knowing whether a constitutively phosphorylated H3 would be sufficient to bring about a chromatin environment that suppresses the gcn5 E173H transcriptional defects. Toward this end, Serl 0, Ser28, and Ser3l were replaced by aspartate or glutamate that mimicked the negatively charged phosphorylation state. Cellular growth in the presence of 3-AT was then assessed. While a single SlOE mutation yielded very few differences in REG] or REG] (1-740) background (Fig. 5. rows 5 and 9). the triple acidic mutation clearly brought about stronger resistance to 3-AT (Fig. 5. rows 7 and l 1). Since this phenotype 55 was independent of the REG] status, we conclude that constitutive negative charges at the amino terminus of H3 represent another bypass of Gcn5 requirement suppressor. Physical interactions of Gcn5, Snfl, and Reg1(l-740). The above data place both Regl and Snfl proteins to the regulatory circuitry of HIS3 and likely other amino acid starvation-inducible genes. The ability of Regl (1-740) protein and overproduced Snfl kinase to rescue preferentially the E173H mutant suggests an intriguing possibility that Gcn5 protein is a functional target for the Snfl kinase. To test this hypothesis, we purified a wild-type and a catalytically inactive (K84R) GST-Snfl protein from yeast (55) and incubated these two preps with recombinant Gcn5 protein expressed in E. coli. [y- 32P]ATP was included in the reactions to track the phosphorylation status of Gcn5. Figure 6_A shows that Gcn5 protein was indeed phosphorylated in the presence of the wild-type Snfl protein. The K84R mutation effectively diminished Gcn5 phosphorylation, indicating that Snfl protein was responsible for Gcn5 protein phosphorylation. Intrigued by the in vitro phosphorylation results. we further tested whether Gcn5 and Snfl proteins interacted in vivo. To this end, we epitope tagged Gcn5 with HA at its amino terminus. Two yeast strains expressing GST-Snfl or GST were transformed with the HA-GCN5 construct and subjected to one-step purification with a glutathione matrix. After extensive washing, the bound materials were resolved by SDS-PAGE and probed with an anti-HA antibody. Figure 515 shows apparent copurification of the HA-Gcn5 protein with GST-Snfl but not GST alone. Literally identical results were obtained in reciprocal experiments (i.e., immunoprecipitation with the anti-HA antibody. followed by 56 Western analyses to quantify Snfl protein in the precipitate) (not shown), confirming the in vivo association between Gcn5 and Snfl proteins. We then asked whether Regl protein also associated with Gcn5 protein. Figure 6_C shows that a Myc-tagged Reg1(1-740) protein was also present in the crude preparation of an epitope-tagged Gcn5 protein. Intriguingly, the full-length Regl -Myc protein was not detected under the same condition (Fig. 6C, first two lanes), consistent with the gain-of- function trait of the Reg1(1-740) suppressor protein. 57 DISCUSSION A putative noncatalytic function of Gcn5 protein. The histone acetyltransferase activity of Gcn5 protein is critical for the expression of multiple yeast genes. Point mutations that eliminate the HAT activity of Gcn5 protein cause defects in promoter acetylation and in transcriptional activation of such model genes as HIS3 and PHO5 (Z, 52, 73, 102). While these results provide solid evidence that Gcn5 protein uses its HAT activity to activate transcription, microarray studies also showed that a gcn5 knockout strain has transcriptional defects in more genes than does a strain expressing a catalytically inactive mutant (45), suggesting that Gcn5 protein may perform noncatalytic roles in gene expression. Indeed, Jacobson and Pillus showed that a catalytically inactive Gcn5 protein counteracts transcriptional silencing at subtelomeric loci (4_6_). Such noncatalytic functions of Gcn5 protein may be unveiled by characterizing point mutations that abrogate the catalytic power of Gcn5 protein but permit other functions to be exerted. This notion seems to be consistent with the data presented in this work. For example, HIS3 and H154 expression are effectively rescued by the Reg1(l-740) suppressor (Fig. 112 and 21:) in the E173H but not the knockout background. No restoration of histone H3 acetylation was detected, suggesting one possibility that the noncatalytic function of the E173H allele is selectively enhanced by Reg1(1-740) protein. This function is likely synergistic with its catalytic counterpart, as more pronounced resistance to 3-AT is exhibited by GC N5 7 REG] (1 -740) and GCN5; multicopy SNF] strains (Fig. 515 and 415). 58 It is also intriguing that the F 221A allele is refractory to Reg1(1-740) and higher doses of Snfl protein. Several other suppressors that are currently characterized by us do not show such unique allele specificity (Y. Liu, X. Xu, and M.-H. Kuo, unpublished data). Molecularly, E173H and F 221A mutations abrogate the HAT activity of Gcn5 via different mechanisms and may have different impacts on histone tails. F221A impairs acetyl-CoA binding (_7, __8, 25), whereas E173H blocks the nucleophilic attack on the bound acetyl-CoA (82). Association of acetyl-CoA is prerequisite to histone tail binding (88, 82). After the transfer of the acetyl group to histone within the ternary complex, the acetylated histone dissociates first and then follows the consumed coenzyme A. Thus, blocking the association between Gcn5 and acetyl-CoA by the F221A mutation likely prevents Gcn5 protein from binding to the substrate histone, rendering the latter susceptible to other unregulated or untimely chromatin binding and modulating activities. The E173H mutation, on the other hand, may lock Gcn5, acetyl-CoA, and the histone tail in a ternary complex, thus preventing possible usage or modifications of the histone tail by other activities. In addition, it remains a strong possibility that Gcn5 protein uses nonhistone protein substrates (58). If so, the retention of one of these proteins by the E173H mutant enzyme may exacerbate the histone hypoacetylation defects. Furthermore, only a subset of defects associated with gcn5_ mutants can be rescued by Reg1(1-740) (Fig. _3_C). Together, it is highly likely that Gcn5 uses multiple mechanisms to activate transcription in a target gene (or transcriptional activator)-dependent manner. Reg1(1-740) protein is a gain-of-function suppressor. Regl protein is a regulatory subunit for Glc7, an essential and multifunctional type 1 protein phosphatase (fl). Regl 59 protein also interacts with several other proteins. including Snfl (51, fl) and the yeast 14-3-3 homologues, Bmhl and Bmh2 proteins (22). The binding domains for these proteins are all within the first 500 amino acids that are conserved among Regl protein G homologues (22, 25, 51). These domain are preserved in our REG] (1 -740) suppressor allele, suggesting that the prototypical functions of Regl protein are not impaired by the C-terminal truncation. The Reg1(l-740) protein lacks about one third of the total length. The truncation occurs immediately before a stretch of acidic residues (15 of 19 residues are Asp or Glu), and the deleted portion is rich in serine, threonine, and acidic residues (16% Ser, 4.4% Thr, 8.8% Asp, and 7.3% Glu), Little is known about the molecular functions or potential partners of this part of the Regl protein. Preliminary sequence search reveals no clear homologues to this region across species (data not shown). Contrary to the gain-of- function BGR phenotypes, this C -terminal region is dispensable for glucose repression. For example, Dombek et al. showed that the C-terminal deletion of Regl protein (up to residue 693) does not cause appreciable derepression of ADH2 or S UC 2 (25). Shirra and Amdt reported that a Regl protein missing the last 80 amino acids is able to fully complement a recessive reg1-326 mutant (85 ). Indeed, we have no evidence of transcriptional derepression of those glucose-repressible genes in the REG] (1-740) background (Y. Liu and M.-H. Kuo, unpublished). It is possible that the carboxyl- terminal third of Regl protein interacts with a negative regulator(s), or another region of Reg] protein in cis, that restricts specifically the HIS3 expression-related functions of Regl protein. Perhaps this negative regulator selectively controls the residual non-HAT function of the E173H mutant of Gcn5 protein. Upon deleting this Ser-Thr-Asp-Glu-rich 60 domain, the negative effect of this regulator diminishes, hence unleashing the non-HAT function of Gcn5 protein for HIS3 activation. This view is consistent with the affinity purification data (Fig. 5) that the Reg1(1-740) but not the full-length Regl protein can be copurified with an epitope-tagged Gcn5 protein. It is important that the suppressing power of Reg1(l-740) protein is abrogated by deleting SNF] . While this result alone does not prove that Snfl protein acts downstream of the Reg1(l-740) suppressor. considering the well-established interaction between Regl and Snfl proteins. we suggest that at least part of the suppressor function of Reg1(1-740) protein is mediated through Snfl protein. However. we cannot rule out the existence of an intermediary step(s)/factor(s) for the suppression. One probable factor involved in the BGR phenotype is the type 1 protein phosphatase Glc7. Regl is one of several regulators of the essential Glc7 enzyme. Unfortunately, our attempts to link Glc7 protein to the BGR phenotypes failed to generate conclusive data. Using several known glc7 point mutations that cause phenotypes in glycogen metabolism and/or glucose repression, we indeed found a few able to confer strong resistance to 3-AT in the absence of a functional Gcn5 protein. However, such elevated 3-AT resistance was not accompanied by increased HIS3 transcription (Y. Liu and M.-H. Kuo, unpublished). This disparity probably arises from the fact that Glc7 protein controls multiple cytoplasmic and nuclear functions (e.g.. see references 87 and 101). Changes in the metabolism and flux of 3-AT may render yeast cells resistant to 3-AT with a low level of HIS3 transcription. The possible involvement of GLC7 in HIS3 regulation awaits further investigation when more mutant glc7 alleles are available. 61 Regl protein was recently shown to be purified in a complex containing two yeast 14-3-3 homologues, Bmhl and Bmh2 proteins, and heat shock proteins Ssdl and Ssd2 (22). Deleting BMH] or BMH2 did not appreciably alter the ability of Reg1(1-740) protein to rescue the gcn5 E173H mutant (X. Xu and M.-H. Kuo, data not shown), indicating that these two proteins are not part of the suppression mechanism. Altematively, functional redundancy between Bmhl and Bmh2 proteins (92% identical) (28) may account for the lack of phenotypes in bmhin and bthA strains. Interestingly, the gain-of-function nature of the Reg1(1-740) suppressor, as well as the phenotypic similarity between Reg1(1-740) and overexpressed Snfl protein, are at odds with the well-characterized antagonistic relationship with Snfl protein (see the Introduction). We suggest that the functional relationship between these two proteins may be gene dependent. One precedent for this type of functional variation was reported for Spt3/8 proteins on TBP recruitment. Spt3 protein genetically and physically interacts with TBP (25). While Spt3 protein is required for TBP binding to the TATA elements of GAL] and ADHZ (8, 2. 2_4, 55). it also plays a negative role in TBP-TATA interaction in other cases (Z, 105). Snfl protein activates HIS3 in an H3 phosphorylation-independent mechanism. Snfl protein is a member of the AMP-activated protein kinase family that serves as a metabolic sensor in eukaryotic cells (51). It thus seems reasonable that Snf 1 protein also contributes to the regulation of amino acid biosynthesis genes as shown in this work. Despite the functional interaction between Gcn5 and Snfl proteins for [NO] activation (15, 1], 58-_6__Q), the H3 phosphorylation function of Snfl protein is unlikely to be a major 62 determinant in HIS3 expression (Fig. 5). However, we cannot rule out the possibility of phosphorylation at other residues or histones by Snfl protein. In addition, genetic data showed that Srb/mediator complex and TBP are also potential substrates of Snfl kinase (.49.. 8.3)- It is interesting that Snfl protein can modify a recombinant Gcn5 protein and that these two proteins are copurified from yeast (Fig. _6_). We do not yet know the site(s) modified by Snfl protein in vitro, nor has it been tested whether Gcn5 protein is phosphorylated in vivo. The human Gcn5 protein was shown to be modified and inhibited by the DNA- dependent kinase (6). In our hands, the in vitro-phosphorylated Gcn5 also seems to exhibit a slightly lower activity on histones H3 and H4 (X. Xu and M.-H. Kuo, unpublished). However, it remains an open question as to whether a phosphorylated Gcn5 protein behaves differently within the context of native complexes. In conclusion, combining the data presented here and those reported by others, we propose a simple model that that Reg1(1-740) protein uses its newly adopted affinity for Gcn5, while maintaining the Snfl interaction domain (12), to mediate the interaction between Gcn5 and Snfl proteins. When Snfl protein is brought to the vicinity of Gcn5, phosphorylation of Gcn5 protein or another factor(s) within or near the SAGA complex may provide the noncatalytic function that rescues the E173H mutation for effective activation of a subset of Gcn5 target genes. ACKNOWLEDGMENTS We are grateful to the following people for generously supplying materials: D. Almy for DAlO; C. D. Allis, M. Smith, and J .-Y. Hsu for the histone knockout strain and plasmids; K. Dombek and E. Young for REG] constructs; D. Thiele for GST-SNF] constructs; M. Snyder for the an library; K. Tatchell for mutant strains of GLC 7; A. Acharya for chicken nuclei; and M. Carlson for SNF] constructs. 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Molecular architecture of the S. cerevisiae SAGA complex. Mol. Cell 15:199—208. 73 104. Yang, X., E. J. Hubbard, and M. Carlson. 1992. A protein kinase substrate identified by the two-hybrid system. Science 257 :680—682. 105. Yu, Y., P. Eriksson, L. T. Bhoite, and D. J. Stillman. 2003. Regulation of TATA- binding protein binding by the SAGA complex and the th6 highmobility group protein. Mol. Cell. Biol. 23:1910—1921. 74 TABLE 1. Strain yMK839 yMK842 yMK984 yMK986 yMK988 yMK995 yMK1055 yMK1085 YL232 YL328 YL338 YL351 YL352 YL3 75 YL376 YL558 YL559 YL585 YL603 YL610 JHY205 DAIO Yeast strain list Relevant genotype MA T atrp] leu2-3,1 12 ura3-52 MA Tatrp] leu2-3.112 ura3-52 gcn5A22hisG MA Tatrp] 1e142-3,1 12 ura3-52 gcn5 F221A MA Tatrp] leu2-3, 112 ura3-52 gcn5 E173H MA Tatrp] leuZ-3,112 URA3::H]S3-lacZ MA Tatrp] leuZ—3,1 12 URA3::HIS3-lacZ gcn5 E173H M4 T atrp] leu2-3,112 URA322111S3-1acZ gcn5 E173H reglzzan MA Tatrp] leu2-3.112 ura3-52 gcn5 E173H pMK125(CEN GCN5 TRP] ) MAT atrp] leu2-3,112 ura3-52 snf‘IA-I ::LE U2 MA Tatrp] lad-3,112 ura3-52 gcn5 E173H REGlzzan MA Tatrp] leuZ-3.] 12 ura3-52 gcn5 E173H regIAzzKanMX6 MA Tatrp] leu2-3,112 ura3-52 gcn5 E173H REG](1-74()) MA Tatrpl leu2-3,112 ura3-52 gcn5 F221A REG](1-740) MA Tatrp] leu2-3,112 ura3-52 REG1(]-740) MA Tatrp] leu2-3,]12 ura3-52 gcn5A::hiSG REG](1-740) MA Tatrp] leu2-3,112 ura3-52 snf]A-2::TRP1 MA Tatrp] leuZ—3,112 ura3-52 gcn5 E173H REGlzzan snf] A-2::TRP1 MA Tatrp] 1e112-3.112 ura3-52 GCN5 REG] (1-740) sanA- 2::TRP1 MATatrp] leuZ—3,112 ura3-52 gcn5 E173H REG/(L740)- myc::TRP1 MA Tatrp] 1e112-3. 112 ura3-52 gcn5 E173H REG]- myczzTRPl MA Tahis3A] leu2A0 met] 5A0 ura3 .510 hht] -hhf1 : :KA N hhf-2hh12: :NA T hta] -htb1 : :HPH hta2-htb2: :NA T pJH33[CEN URA3 HTA l-HTB] HHTZ—HHFZ] MA Taleu2A0 metl5A0 ura3A0 hhtl-hhflzzKAN hhf- 2hht2zzNA T hIaI-htblzzHPH hta2-htb2zzi’VA T pQQ18[CEN 75 Source or reference Q g This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study IN D. Almy, unpublished Strain YL3 72 YL381 YL407 YL408 YL409 YL410 YL457 YL458 YL459 YL460 Relevant genotype LE U2 H TA 1 -HT B] HHTZ-HHF 2] MA TaleuZAO met] 5.30 ura3A0 hht]-hhf]::KAN hhf- 2hht222NAThta1-htb1zzHPHhta2-htb2::NATgcn5 E173H pQQl 8[CEN LE U2 HTA] -1-1TB] HHT2-HHF2] MA T aleuZAO met] 5 A0 ura3AO hht]-1211f] : :KAN hhf- 2hh1222NA T hta] -htb1::HPH hta2-htb2: :NA T gcn5 E173H REG1(1-7-IO) pQQ18[CEN LEU2 HTA 1-HTB1 HHTZ- HHF 2] MA TaleuZ-AO met] 5A0 ura3A0 hht] -hhf1 ::KAN hhf- 2hh12: :NA T hta] -htb1 : :HPH htaZ-hth: :NA T gcn5 E173H REG] (1-740) pMK439S 1 0A[CEN LEU2 HTAI-HTBI hhtZ-S 1 0A -HHF 2] MA TaleuZ-AO met] 5A0 ura3A0 hht] -hhf1 ::KAN hhf- 2hht2: :NA T hta] -htb1 : :HPH hta2-htb2: :NA T gcn5 E173H REG]('1-740)pMK439S10E[CENLEU2 HTAI-HTBI hht2-S 1 OE-HHF 2] MA Taleu2AO met] 5 A0 ura3A0 hht] -hhf1::KAN hhf- 2hht2: :NA T hta] -htb1 : :HPH hta2-htb212NA T gcn5 E173H pMK439S 1 0A[CEN LE U2 H TA 1 -H TB] hht2-S10A- HHF 2] MA T aleuZAO met] 5 A0 ura3AO hht] -lzhf] : :KAN hhf- 2hht2: :NA T htaI-htb1zzHPH hta2-htb2: :NA T gcn5 E173H pMK439S 1 0E[CEN LEU2 HTA I-HTBI hht2-S10E— HHF 2 ] MA Taleu2A0 met] 5 A0 ura3A0 hht] -hhf]::KAN hhf- 2hh12: :NA T hta] -htb1 : :HPH hta2-htb2: :NA T gcn5 E173H pMK439S 10A/S28A/S31A[CEN LEU2 HTA I-HTBI hht2-S] 0A/S28A/S3 1 A -HHF 2] MA T aleuZAO met15A0 ura3A0 hht] -hhf] : :KA N hhf- 2hht2: :NA T hta] -htb1 : :HPH hta2-htb2::1\~’A T gcn5 E173H REG1(1-740) pMK439SIOA/S28A/S31A[CEN LEU2 H TA l-H TB 1 hht2-S10A/S28A/S31A -HHF2] MA TaleuZAO met] 5 A0 ura3A0 hht] -hhf1 ::KAN hhf- 2hht2: :NA T hta] -htb1 : :HPH hta2-htb2: :NA T gcn5 E173H pMK439SlOE/S28D/S31D[CEN LEU2 HTA 1-HTB] hht2- S 1 0E/SZ8D/S3 1 D-HHF 2] MA T aleuZAO met] 5 A0 ura3A0 hht] -hh/] : :KAN hhf- 2hh12: :NA T htal-htb1zzHPH hta2-htb221NA T gcn5 E173H REG1(1-740) pMK439SIOE/SZ8D/S31D[CEN LEU2 H TA l-H TB] hht2-S] ()E/SZ8D/S3 lD-HHF 2] 76 Source or reference This study This study This study This study This study This study This study This study This study This study TABLE 2. Plasmid construct list Plasmid pMK125 pMK284 pMK284E173H pMK284F221A pMK334 pMK449 pMK453 pKD97 pYL3 l pYL35 pYL4 1 pYL42 pYL44 pYL45 pYL54 pYL67 pQQ18 pJH33 pMK439SIOA pMK439S10E pMK439SlOA/828A/S31A pMK439SIOE/S28D/S31D pMK515 pMK547Gcn5 Description pRS4 1 4-GCN5 Integration construct for introducing point mutations to GC N5 Integration construct for introducing E173H to GCN5 Integration construct for introducing F 221A to GCN5 Integration construct for introducing HIS3-lacZ reporter to URA3 locus pBluescriptKS-SNF] snf] A: :LE U2 disruptor pRS3 1 6-HA -reg1;16 pRS3 16-HA-REG1(1-740) Integration construct for introducing REG1(1- 740) mutation YEplacll2-SNF] pYEX-4T-GST-Snfl vax—4r-Gsr snf] AxTRP] disruptor pYEX-4T-Gcn5-TAP 8 X MyczzTRP] for tagging proteins with 8 Myc repeats pRS315-HTA1-HTBI HHT2-HHF2 pRS3 l6-HTA1-HTB] HHTZ-HHFZ pQQ18 with an H3 S10A mutation pQQ18 with an H3 SlOE mutation pQQ18 with an H3 SlOA/S28A/S31A mutation pQQl8 with an H3 SlOE/SZ8D/S31D mutation pET21-6xHis-Gcn5 protein 3xHA-Gcn5 oxerexpression 77 Source or reference 52 This study This study This study This study This study This study 25 This study This study This study 55 A. Acharya, unpublished This study This study This study MIN This study This study This study This study This study This study FIG. 1. Identification ofa BGR suppressor rescuing the gcn5 E173H mutant. (A) In vitro HAT assays. Chicken core histones were acetylated by recombinant Gcn5 proteins, and the 3H-labeled acetylated histone was detected by fluorography (right panel). C BR. C oomassie blue R250. (B) Temperature-dependent hypersensitivity to 3-AT is generated by three gcnf alleles. The E173H and F 221A alleles were introduced to the native G(,']\"5 locus under the control of its own cis elements. Each strain was patched to YPD and grown at 30°C for 2 days. Cells were then replica plated to SC -His medium supplemented with various concentrations of 3-AT and incubated at 30 or 37°C for 3 to 4 days. Relevant genotypes are listed on the right. (C) The BGR suppressor rescues both 3-AT hypersensitivity and HIS3-lacZ expression. Both plates were from cultures grown at 37°C. The B-galactosidase activities (U/mg of total proteins/min) were measured from cultures grown in YPD or SD (minimal) medium to early log phase. Errors represent variation from two independent cultures of each strain. (D) Northern blot hybridization. Log-phase cells were harvested from rich or minimal medium supplemented with 20 mM 3-AT before RNA preparation and hybridization. 18S rRNA was used as the internal control. The ratio of HIS3 to 18S rRNA was measured and normalized to the basal expression of a GCNT strain. A, activated; R, repressed. Figures of gel staining, fluorography, and culture plates were scanned with a flatbed scanner and acquired by Photoshop 7.0. 78 Fig. 1. A c 6‘ 6‘ 4% e) «6 (152‘ 0Q? '3 ((5% 0‘00 ‘0 e- 45 ‘0 e H3 H2A ’ H4 ’ CBR staining Fluorography C SC-His 10 mM 3-AT "“3; GCN5 E173H . . E173H BGR YPD SD GCN5 7.9 a 0.3 13.4 :- 1.8 Et73H 0.5 :t 0.1 0.9 a. 0.2 Et73H BGR 7.6 a 0.6 13.0 a 1.3 79 SC-His + 3-AT (mM) 30° 37° 517314 . 4, WT URA3::HISS-lac2 517311 UFlA3::H/S3-IacZ BGR regtzt G CN5‘ E 1 73H E173H GCN5‘ A Fl A R A F] A R 1 2 3 4 5 6 7 8 HIS3 » , . . . , 188 as ea: as an aid sat as“ HIS3/18S: 9.7 1 2.9 0.8 5.81.3 9.0 1.3 FIG. 2. Characterization ofthe Reg1(l-740) BGR suppressor. (A) DNA and protein sequences across the an-lacZ integration site. Insertion of the an-lacZ fragment at nucleotide 2122 results in an in-frame fusion between the Regl protein and the Tn3 long terminal repeat (LTR; lowercase) and the lacZ gene. Ser740 of Regl protein is marked. The nucleotide and amino acid residue numbers are relative to the start codon of the REG] open reading frame. (B) Regl (1-740) protein is essential and sufficient for the BGR phenotypes. The left panel shows 3-AT tests (37°C) ofisogenic strains bearing different alleles of REG]. The right panel shows Northern hybridization results. All samples were obtained from induced conditions (see legend to Fig. m). The 111S3/l 8S rRNA ratio of each sample was calculated and then normalized to that of the gcn5 E173H strain (lane 2 or 6). (C) Reg1(l -740) protein-mediated suppression requires the Gcn4 activator binding site. GCRE. Shown are reverse transcription- PCR results. PGK] is an internal control. (D) Promoter H3 hypoacetylation is not affected by the Regl (1-740) suppressor. Shown are quantitative PC R results of chromatin immunoprecipitation using an antibody against H3 acetylated at Lys9/l4. ACT] open reading frame was used as the internal control 80 Fig. 2. A REG1§—-—-| Tn3LTR l—-—->Iacz -‘ :42 -_ ... -. . '1"“-_--1v." ‘_. B REG, GCN5: WT E173H WT £17311 " FtEGt: WT WT an .1 WT WT (1-740) WT 1 2 3 4 5 6 7 H183 I (H O u a an . H g I I A 185 lawman-nu] (1-740) HIS3/1833.0 1 1.8 0.7 2.1 1 1.4 c D El73H GCN5: WT REGt: WT WT an an GCRE(HISS): WT WT WT mut GCN5: WT E173H FiEG1: FL (1-7421 FL (1-7402 1 2 3 4 5 6 7 8 ”Raw.“ “5‘“ ACT1 Ac.H3 Unduut.1_ _. HIS3 +1 ACT1 H~~H~~4H~H Input ‘ HISS+1 ARARARAR 81 FIG. 3. The BGR suppressor is semidominant and selectively rescues the E173H defects of the GCN pathway. (A) Semidominant features of the REG] (1 -740) allele. Diploid strains bearing different combinations of GCN5 or REG] alleles were tested for their resistance to 3-AT at 30 or 37°C. (B) Allele specificity ofthe suppression. The REG ] (1-740) allele was integrated to the chromosome to replace the wild-type REG 1 gene in different gen)" strains. Resultant strains were then spotted to 3-AT medium and grown at 37°C. (C) Growth defects in different conditions caused by gcn5" mutations are not affected by the Reg1(l-740) protein. Indicated strains were grown to log phase in YPD. spotted to yeast extract— peptone-glycerol, yeast extract-peptone-ethanoI, or YPD containing 100 mM hydroxyurea (HU). and incubated at 30°C for 3 to 4 days IS 11:11 11619111 protein Fig. 3. mummth-s —His Trp 10 mM 3-AT 10 mM 3-AT GCN5 / GCN5 WT/E173H E173H/E173H E173H/E173H E173H/E173H E173H/E173H mkmmé 10 mM 3-AT M WT WT WT (1-740) E1 73H WT E173H (1-740) 21 WT .1 (1-740) F221A WT F221A (1-740) 83 REG] / REG] WT/ WT WT/ WT WT/ WT WT/an WT/an Fig. 3. 84 GCN5 lVT EfiGH EHGH EHBH EZHA EZNA W7 HEGt WT WT an (1-740) WT (1-740) A FIG. 4. SNF] is important for HIS3 expression and BGR phenotypes. (A) Deleting SNF1 causes hypersensitivity to 3-AT in REG] and REGlzzan strains. The snf]A-2::TRP] allele contains a TRP] insertion at amino acid 109. The snf/A- ] ::LE U2 allele lacks the entire ORF of SNF] . (B) Northern hybridization of some of the strains shown in panel A. Only induced transcription is shown. The ratios of HIS3/l 8S rRNA were normalized to the gcn5 E173H sample (lane 2) and are shown at the bottom. (C) Overexpression of Snfl protein also selectively rescues the E173H allele of GCN5. (Left panel) A multicopy GCN5 construct does not rescue sanA-I . (Right panel) gcn5 E173H was selectively rescued by a multicopy SNF1 construct. Growth at 37°C is shown. Fig. 4. A 5 mM 3-AT GCN5 Fr‘EGt SNF1 1 WT WT WT 2 WT WT .1—1::LEU2 3 WT WT A—2::TRP1 4 WT WT A—2::TRP1 5 E173H an WT 6 E173H an .1-2:.-T1=1P1 7 . E173H an A—2::TRP1 B GCN5: WT E/H WT E/H E/H REGt: WT WT WT an an SNF1: WT WT a—I WT .1—2 1 2 3 4 5 H183 ’ 8 188 ‘11 at u. it HIS3/18S: 6.5 1 0.6 2.1 0.3 C —His 10 mM 3-AT GCN5 snf1J—l + 1 3‘AT GCN5—211M vector 1 ' Vector 2p GCN5 2 SNF1 2p 8th 3 Vector 4 SNF1 5 Vector s SNF1 7 Vector 8 SNF1 86 Fig. 5. REG1 H3 1 WT WT 2 WT WT 3 (1-740) WT 4 (1-740) 810A 5 (1-740) S10E 6 (1-740) S10A/28A/31A 7 (1-740) 810E/280/31D 8 WT S10A 9 . WT S10E 1o . g o a s;- .- WT 810A/28A/31A 11 g o o 4‘ t O \E‘ WT S10E/280/31D FIG. 5. H3 Serl 0 phosphorylation is not required for the BGR phenotypes. Yeast strains expressing the desired H3 mutants were tested on 3-AT plates at 37°C. Strains derived from this background (2) were more sensitive to 3-AT. The choice Asp or Glu in site- directed mutagenesis was based on whether a restriction site could be generated. 87 FIG. 6. Biochemical interactions ochnS/Snf] and Gcn5/Reg1(l-740) proteins. (A) Recombinant Gcn5 is phosphorylated by the wild-type but not the K84R Snfl protein. GST-Snfl or GST alone was purified from yeast and incubated in the presence of [y-33P]ATP with recombinant Gcn5 protein. (B) Copurification of Gcn5 and Snfl proteins from yeast. HA-GcnS and GST—anl or GST alone were expressed in yeast, and the whole-cell extracts were subjected to glutathione affinity purification. The bound materials were washed with 0.3 or 0.5 mM NaCl prior to SDS-PAGE and Western analyses using an anti-HA antibody (top). (C) Copurification of Reg] (1 -740) and Gcn5 proteins from yeast. Gcn5 was C- terminally tagged with protein A and coexpressed in yeast strains with Regl-Myc or Regl(1-740)-Myc recombinant proteins. \ll-v’hoIe-cell lysates were bound to immunoglobulin G beads and analyzed by SDS-PAGE and anti-Myc Western analyses. 88 Fig. 6. 7.5% int-wt Bound. Bound. A SNF1: WT K84R WT K84R FGST-Sflf‘l 2...... ........,~ a—Hisxe-Gcns Autorad Coomassie B = = ‘ ‘ m w m m s a g g c a a 5 5 a 2 ‘2“ ‘2“ 2 2 -a%-~v a a o O a O” C). c c ' . E U u ._ ,2 E E a 5 5 I: 2 a a 2 ‘3 ‘3 i N Western 1.: .‘ ‘M—HAGcns ‘ f ' , Reg1(1-740)—Myc—’ ' ..._ -..- 1<——GST-Snf1 1 can é “ 1 1 - H. . , +—GST he 123456 89 Supplementary Data: Snfl and Reg1(1-740) likely function together for the bgr phenotypes That deleting GCN5 or SNF1 abolishes the bgr phenotypes demonstrates the importance of both Gcn5 and Snfl proteins. While the unique allele specificity for the E173H mutation suggests that Reg1(1-740) may act directly on Gcn5, it is unclear whether Snfl is a target for the Reg1(1 -740) suppressor protein. Given that Regl and Snfl can interact physically and functionally (see Introduction), and that the Snfl interaction domain of Reg] remains intact in the bgr allele (79), it is tempting to speculate that the Reg1(1-740) protein elicits the bgr phenotypes via the Snfl kinase. If so, deleting SNF1 from a GCN5 reg] (1-7-10) strain will eliminate the suppressing power of the Reg1(l-741) protein. To test the above hypothesis. we introduced the reg] (1-7-10) mutant into a 3-AT sensitive GCN5 sanA strain and tested the cellular growth on 3-AT plates. Unlike the strong bgr phenotypes for E173H, the Reg1(1-740) protein fails to rescue the 3-AT sensitivity of the snflA strain. Although at 10 mM 3-AT, a slightly better growth was observed in the GCN5 reg] (1 - 741)) snf]::TRP1 strain. this improvement in 3-AT resistance is minute compared with the SNF1+ cells (row 3, Figure SI). Similarly, knocking out SNF] from the 3-AT resistant GCN5 reg] (’1- 740) strain sensitizes drastically the cellular growth on 3-AT plates (data not shown). These results suggest that Snfl is likely a key co-factor for Reg1(1-740) to carry out the bgr functions. 90 Fig. S]. 10 mM 3-AT E173H REG] E]73H (1-740) SNF1 GCN5 REG] snft‘ GCN5 (1-740) snft— \tmmth-a Figure S1. Reg1(1-740) protein does not rescue snfl— 3-AT hypersensitivity. The reg] (1 - 740) allele was introduced to the GCN5 snf] :.'TRP1(snf1“) strain. After confirming the correct genotype by genomic PC R, three integrants (rows 5-7) and other control strains were tested on 3-AT plates at 37°C. 91 Fig. $2. 2.5 mM 3-AT '. . a a} “ WTsnrr- [YCp50-SNF1] ' WT snft‘ [YCp50] E173H snft- [YCp50-SNF 1] E173H snf1' [YCp50] F221A snft— [YCp50-SNF1] F221A snt1’[YCp50] gcnSA snt1- [YCpSO-SNF 1] 901154 snf1— [YCp50] CDNQU'IAODN-i 5 mM 3-AT ' " WT snr1- [YCp50-SNF1] WT snf1” [YCp50] E173H snf1“ [YCp50- SNF 1] £173H snft‘ [YCp50] F221A snft‘ [YCpSO-SNF 1] F221A sntt“ [YCp50] gcn5A snf1‘ [YCpSO-SNF 1] A gcnSA snftr [YCp50] (DVOUTACDI’O-d Figure S2. Genetic interaction between Gcn5 and Snfl for HIS3 expression. A. Gcn5 and Snfl are likely part of two pathways for HIS3 expression. SNF1 was deleted from the indicated ganT strains (all snf] strains used in this panel were snf1::TRP1), and the resultant strains were transfomied with YCp50, an ARS CEN plasmid, or YCpSO-SNF] that contained a wildtype SNF1 gene under the control of the native SNF] regulatory elements. 92 CHAPTER III Snflp activates HIS3 transcription by antagonizing the inhibitory effects of Spt3p and Spt8p (Manuscript for Mol. Cell. Biol.) Snflp activates HIS3 transcription by antagonizing the inhibitory effects of Spt3p and Spt8p Yang Liul, Xinjing Xuz, and Min-Hao Ku01‘2* . . 1 . . . . Program In Genetics and Department of Biochemistry and Molecular Btologyz, Michigan State University, East Lansing, Michigan 48824 * Corresponding author. Mailing address: 401 BCH Building, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824. Phone: (517) 355-0163. Fax: (517) 353-9334. E-mail: kuom@.msu.edu. 94 ABSTRACT Transcriptional activation of the Saccharomyces cerevisiae HIS3 gene requires both Gcn5 histone acetyltransferase (HAT) and Snfl kinase. Our studies suggest Gcn5 protein is a likely target for Snfl in HIS3 regulation because the phosphorylation of Gcn5 in vivo is dependent on the Snfl dosage. Moreover, the Gcn5 residues (T203, S204, T211 and Y212; TSTY) that are important for Snfl mediated phosphorylation are critical for HIS3 activation as well. Interestingly, the HIS3 transcriptional defect caused by substitution of the above essential residues is suppressed by deleting the Spt3 component of the Gcn5 related coactivator complexes, which indicates that Spt3 protein plays negative roles in HIS3 transcription. The notion that Snfl and TSTY residues of Gcn5 protein are in the same regulatory pathway is further supported by the observation that deleting SPT3 also rescues snflA in HIS3 activation. As expected, knocking out Spt8, which is fiinctionally related to Spt3 protein, is also able to rescue the impairment caused by snflA and gcn5 T STY/4A (TSTY residues substituted to alanine) mutants. The in vitro pull-down assays demonstrate direct interaction between Gcn5 protein and Spt3 protein, suggesting that Spt3 protein inhibits Gcn5 function by physical association. Thus, one possible function of Snfl is to disrupt the interaction by phosphorylating Gcn5 protein or Spt3 protein or both. INTRODUCTION Eukaryotic DNA is packaged into a structure called chromatin. Chromatin modifying enzymes, e. g. ATP-dependent chromatin remodelers and covalent histone modification enzymes, dynamically alter the chromatin structure and/or chromatin surface to change the accessibility of genetic information that is carried by DNA. Histone acetylation is a well studied covalent modification involved in the regulation of many cellular processes (5). In particular, the promoter acetylation of histones is usually coupled with gene activation; and histone deacetylases (HDACs), the enzymes that mediate deacetylation reactions, are considered to be one of the most promising targets of anticancer drugs (31). In Saccharomyces cerevisiae, the Spt-ADA-GcnS-acetyltransferase (SAGA) complex is one of the histone acetyltransferase complexes that regulates transcription of about 10% of yeast genes (18). The 1.8 MDa SAGA complex is composed of more than 19 subunits and possesses multiple activities related to transcriptional regulation. The SAGA complex has two known histone modification activities. Gcn5, together with the adapter proteins Ada2 and Nggl/Ada3, mediates histone acetylation (12, 22, 23, 34). In addition, SAGA is able to remove ubiquitin from histone H2B through the activity of pr8 and ng11 subunits (16, 19, 24). Polyglutamine extension of Sca7/ng73 protein is critical for HAT activity of SAGA (30). The Tral protein, the largest SAGA component, interacts with acidic activators for promoter recruitment of the complex (3, 4). The chromodomain containing protein C hdl might be involved in substrate recognition by interacting with 96 methylated lysine 4 of histone H3 (33). Three backbone proteins, Hfil/Adal, Spt7 and Spt20, are essential for SAGA integrity (42). A group of TBP association factors (TAFs) that are shared between TFIID and SAGA (Taf5, Taf6, Taf‘), Taf10 and Taf12), are also thought to help maintain the architecture of the complex (13, 50). A module containing Spt3 and Spt8 proteins regulates gene transcription by modulating TBP activity (9, 42). Snfl is the yeast ortholog of the conserved AMPK kinase that is important for responses to metabolic stress (15). The kinase exists in several heterotrimers, each consisting of the catalytic or subunit, Snfl , the regulatory 7 subunit, Snf4, and one of the three scaffold 8 subunits, Ga183, Sipl or Sip2, which tether Snfl and Snf4 together (20). The three [3 subunits show functional redundancy as snf?" phenotypes are achieved when all three subunits are deleted (37). Recent studies suggest that the selection of 8 subunit incorporation may affect the cellular localization of the kinase complex (45). Moreover, Snfl protein is able to form a homodimer, based on the crystal structure study of the Snfl kinase domain (32, 35). Snfl is activated by upstream kinases, Sakl, Elml and Tos3, through phosphorylation of threonine 210 in the activation loop (17, 29). On the other hand, the Snfl activity is repressed by Regl protein, which helps recruit the protein phosphatase Glc7 that dephosphorylates and inactivates Snfl (8, 36). The roles of Snfl protein in regulating transcription of genes related to adaptation of alternative carbon sources have been well characterized. Snfl kinase may stimulate transcription by phosphorylating activators Cat8 and Sip4 (44). Snfl mediated phosphorylation of Migl protein disrupts the Mi g1 -Ssn6 repressor at the promoter, leading to derepression of the downstream genes (43). Genetic and biochemical studies indicated Snfl interacts with the 97 mediator complex or TATA-binding protein directly (2], 39, 40). Interestingly, Snfl also phosphorylates Ser 10 of histone H3 for INOl activation (27). H3 phosphorylated at Ser 10 exhibits a stronger affinity for Gcn5 protein. linking H3 phosphorylation to acetylation (7, 27,28). Our previous studies showed that Snfl protein positively regulates HIS3 transcription, as deletion of SNF] abolished the HIS3 activation under amino acid starvation conditions. In addition, we showed that SNF1 is a multi-copy suppressor of the E173H mutation of GCN5 (26). Instead of acting as a histone kinase, we found that Snfl protein more likely regulates the function of the SAGA coactivator complex, since replacing Ser10 of H3 with alanine or glutamate neither attenuates nor augments the suppression phenotypes. Moreover, Snfl protein interacts with Gcn5 protein in vivo and phophorylates Gcn5 protein in vitro (26). We suggested that Gcn5 phosphorylation is critical for HIS3 activation, We now present evidence for Snfl -dependent Gcn5 phosphorylation in vivo. The residues in Gcn5 protein that are important for Snfl -mediated in vitro phosphorylation are important for Gcn5 in viva functions as well. Interestingly. transcription defects resulting from the mutations of the putative phosphorylation sites are rescued by deletion of another SAGA component, SPT3. Deletion of SPT3 also suppresses the defect in HIS3 activation caused by 511/121. Similar to Gcn5, Spt3 protein copurified with Snfl in vivo. Moreover, in vitro pull-down assays revealed that Spt3 protein can bind Gcn5 protein directly. Together, we suggest that Spt3 protein performs a negative role in HIS3 98 activation by interacting directly with Gcn5 protein. and this inhibition is alleviated by Snfl -mediated phosphorylation of Gcn5 protein or Spt3 protein. 99 MATERIALS AND METHODS Yeast stains, plasmids and genetic methods. Yeast strains used in this work are listed in Table 1. All genetic methods were performed according to reference (3 8). Yeast transformation was done using the lithium acetate method (1 l). Plasmids used in this work are listed in Table 2. The spt3A strains were created by introducing a PC R fragment containing the KanMX6 cassette flanked by SPT3 sequences outside the open reading frame (46). G418-resistant transformants were examined by genomic PCR to confirm the 319132] genotype. The spt8A strains were generated with similar strategy except the SPT8 open reading frame is replaced by the TRP] gene. Gcn5-myc and Snfl -myc strains were generated by transforming the gel-purified PCR products using pYL67 (26) as template to amplify the 8xmycssTRP1 cassette with flanking sequences of GCN5 or SNF1. The integration was confirmed by genomic PCR and expression was confirmed by westem blot with or-c-myc antibody (9E10, Roche). pYL89 with an N-terminal hemagglutinin (HA) tag was created by co-transforming XbaI-linearized pMK547 (26), and a PCR-amplified SPT3 open reading frame. The HA- Spt3-TAP fusion construct (pYL99) was generated by co-transforrnation of the large 100 fragment of BamHI-NgoMIV digested pYL54 and a PCR amplified HA-SPT 3 fragment from pYL89 flanked with pYL54 sequences outside the GC N5 open reading frame. The PCR product containinig HA-SPT3 was digested with EcoRI-Xhol and inserted into the same sites of pET21a to create pYL90 that expresses HA-Spt3 in bacteria. All of the above constructs were verified by PCR and the expression was tested by Western blot with or-HA antibody (12CA5. Roche) or Rabbit antiserum (for TAP tagged construct). Plasmids with Gcn5 mutations were created by following the QuikChange mutagenesis protocol (Roche). RNA preparation and RT-PCR. Yeast cells were grown in appropriate selection media until the OD600 reached 0.5. Cells were then pelleted by centrifugation (5,000 X g. 5 min, 4°C) and transferred to SD supplemented with required nutrients and 40 mM 3-AT (for induced expression). Cell suspensions were further incubated at 37°C for 2 to 3 h before harvesting for RNA preparation. Procedures for RNA preparation was described previously (23). 10 pg of RNA was treated with lO—unit DNaseI (Roche) in lOOpL (50mM Tris-HCl, pH7.5, SmM MgClg), and incubated at 37°C for 1 hour. The cDNA was created following the instruction of ImpronII reverse transcriptase (Amersham) kit using of 30 ng of poly(dT) for priming. The resultant cDNAs were diluted 50 fold. PCR reactions contained 50 mM KC], 10 mM Tris-HCI (pH 9.0) at 25°C, 1% Triton X-100, 2 mM MgC13, 0.1 mM each dNTP, 0.5 pM each primer, and 1.25 U Taq DNA polymerase (Promega), and 101 appropriately diluted DNA templates. PCR parameters were (94°C, 4 min; 50°C, 4 min; 72°C, 30 sec) for 2 cycles; (94°C, 45 sec; 50°C, 45 sec; 72°C, 30 sec) for 24 cycles; and 72°C, 3 min. PCR products were resolved in polyacrylamide gels followed by Ethyldium Bromide staining. In vitro kinase assays. Kinase assays were performed with recombinant Gcn5 protein incubated with glutathione S—transferase (GST)-tagged Snfl (wild-type or K84R) expressed and purified from yeast according to reference (14). The GST-Snfl constructs were kindly provided by D. Thiele (Duke University). Affinity purification and Immunoblot. Snfl and Gcn5 interaction was tested as previous described (26) except the Gcn5 was myc-tagged at the c-tenninus within the chromosomal copy. To test the interaction between Spt3 and Snfl proteins, a GST-Snfl expression construct (14) or just GST (pYL44) was transformed to the strain carrying pYL89 (HA-Spt3). Purification of GST- Snfl was performed as described previously (26). Glutathione Sepharose 4B (10 pl; Amersham) was added to whole-cell extracts purified from 3 x 109 cells and incubated at 4°C for 1 hour with constant rocking. Beads were pelleted and washed three times with HEMGT buffer (25 mM HEPES, pH 7.9, 12.5 mM MgC12, 150 mM NaCl, 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, l>< Complete 102 protease inhibitor cocktail (Roche)). The bound fractions were eluted with SDS loading buffer and resolved by 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The copurified Gcn5-myc protein or HA-Spt3 protein was detected by or-c-myc (9E10, Roche) or or-HA antibodies (12CA5, Roche). In vitro interaction. The recombinant protein was induced in the E. coli BL21 (DE3) strain by adding 0.1 mM (final concentration) IPTG when cell culture reached an optical density at 600 nm (ODb-oo) of 0.5/m1. Cell cultures were grown at 16°C overnight. Extraction and protein affinity purification were done according to reference (23) except the E. coli cells containing the recombinant HA-Spt3 were first broken with buffer containing 2% SDS, and diluted 20 fold afterwards to solve the insolubility problem. The in vitro pull down assays were done by incubating the immobilized protein with bacterial whole cell lysates containing the protein of interest at 4°C for 1 hour. After washing 3 times with HEMGT buffer, the proteins were eluted with SDS loading buffer and subjected for western analyses with or-HA (12CA5, Roche) or a-His-tag (BD Scientific) antibodies. Phosphoprotein staining. To detect in vivo phosphorylation of proteins, the yeast cells were grown in appropriate media at 30 °C until late log phase. Then the cultures were harvested by centrifugation and washed with PBS buffer. Whole cell lysate (WCL) from 3 x 109 cells was prepared 103 by agitate beating with a mini-bead-beater (BioSpec Inc.) as described (26), except the 800 pL PiPT buffer (50 mM potassium phosphate, pH 7.5; 140 mM potassium chloride; 0.1% TritonX-100; 1 mM DTT; 1 mM EDTA; 1 mM sodium orthovanadate; 10 mM sodium fluoride; 1 tablet/20 mL Protease Inhibitor Cocktail (Roche); 1 mM PMSF) was used. Affinity purification was done by incubating the 500 pL of WCL with 20pL of IgG sepharose 6G (Amersham) at 4°C for 1 hour. The IgG beads were collected with gentle centrifugation and washed twice with PiPT buffer without protease inhibitors, followed by two more washes with PiPT buffer with higher concentration of potassium chloride (500 mM). The IgG sepharose affinity purified proteins were eluted from the beads with SDS loading buffer and resolved on an SDS-PAGE mini-gel. Then the gel was fixed with 100 mL of 50% methanol/ 10% acetic acid (v/v) for 1 hour to over night. The residual methanol and acetic acid was removed by washing with 50 mL deionized water for 10 minutes with gentle agitation. After repeating the washing for 3 times, the gel was incubated with 50 mL of Pro-Q Diamond Phosphoprotein Gel Staining solution (Molecular Probes) for 1 hour, with gentle agitation in dark. To reduce the background and non-specific staining, the gel was treated with destaining solution [50 mM NaOAc, pH 4.0; 20% acetonitrile (v/v)] for 30 minutes with 3 repeats. Finally, the gel was washed twice with deionized water at room temperature for 5 minutes per wash. The staining was detected by Molecular imagerFX-PRO Plus (BioRad). Commassie staining was performed after the gel was scanned to check the evenly loading. 104 RESULTS Overproducing Snflp causes Gcn5p hyperphosphorylation in viva. Previously we showed that Snf 1p phosphorylates Gcn5p in vitro and that Snflp and Gcn5p co-purified from yeast extracts (26). It is likely that Snflp phosphorylates Gcn5p in a physiological environment. To test this hypothesis, we partially purified TAP-tagged Gcn5 protein from yeast with IgG affinity matrix. Phospho-staining with Pro-Q Diamond (see Materials and Methods) was used to examine the Gcn5 phosphorylation status (Fig. 1A). Clear staining of Gcn5-TAP was observed (Lane 2, about 70 KDa) and the band shifted upon TEV protease digestion that removed the Protein A moiety of the TAP-tag (Lane 1, about 55 KDa). The phospho-staining is specific, as the fluorescence signal was diminished upon it protein phosphatase treatment (Lane 3). These results clearly demonstrate that Gcn5p can be phosphorylated in vivo. To test whether Snfl protein is responsible for Gcn5 phosphorylation in vivo, we compared the phospho-staining intensity of Gcn5-TAP proteins isolated from strains containing different doses of Snfl protein (Fig. 1B). While Gcn5-TAP protein isolated from a snflA strain showed positive, albeit weak, phospho-staining, a much stronger signal was seen in the Snfl overproducing background. We thus conclude that Gcn5p is a substrate for the Snfl kinase in vivo. The weak phospho-staining of Gcn5p in the .9an21 background also suggest a Snfl-independent phosphorylation of Gcn5p in vivo. Potential phosphorylation sites in Gcn5p are essential for HIS3 induction To understand the biological function of Gcn5 phosphorylation, we first mapped the phosphorylation sites in Snflp modified recombinant Gcn5 by mass spectrometry. The preliminary data suggested that T203, 8204, T2] 1 and Y212 might be phosphorylated (not shown). Because Snfl p is a Ser/Thr kinase, we tested the in vitro phosphorylation efficiency of Gcn5p bearing mutations at T203, S204 and /or T211 (Fig. 2A). While none of the single mutations caused significant reduction in Gcn5 phosphorylation, T203A/S204A and T203A/S204A/T21 1A mutations drastically diminished the in vitro labeling, indicating that all 3 residues are likely phosphorylated by Snfl p. We consistently observed elevated phosphate labeling of T203A and T21 1A mutants. If Gcn5 phosphorylation is physiologically relevant to HIS3 activation, we expect that the mutation of phosphorylation site(s) will affect HIS3 transcription. Indeed, a quadruple mutation (T203A/S204A/T211A/Y212A, TSTY/4A) impaired the cellular ability to survive under 3-AT stress and HIS3 induction as well (Fig. 2B and 2C). Without visible defects in rich (YPD) and histidine dropout media (SC -His), the TSTY/4A allele caused poor growth upon 3-AT treatment. Consistent with the 3-AT growth defect, the HIS3 expression was barely detectable by RT-PC R (Fig. 2B). These results indicate the T203/S204/T21 l/Y212 residues of Gcn5p, which are important for Snfl p-mediated phosphorylation in vitro, are essential for Gcn5p function in HIS3 transcription. 106 The gcn5 T S T Y/4A mutant is suppressed by spt3A Spt3p is a subunit of the SAGA complex, and it interacts with the TATA-binding protein, TBP (9). Under the non-inducing condition, HIS3 expression is repressed by Spt3 protein as the deletion of SPT3 derepresses HIS3 transcription in synthetic complete media (1). Gcn5p HAT activity is still required to active HIS3 transcription as gcn5/.1 spt3A and gcn5E173Hspt3A strains are sensitive to 3-AT (Fig. 2B). Different from the gcn5A and gcn5E173H mutations. the gcn5 T STY/4A mutation was suppressed by spt3A (Fig. 2B and 2C). The gcn5 T STY/4A 5171321 strains showed robust growth under a harsh amino acid starvation condition, as evidenced by resistance to 30 mM 3-AT. HIS3 mRNA level increased significantly in a double mutation strain as well. The negative effect of Spt3p is further confirmed by complementation of the gcn5 TS TY/4A spt3A strain with a multicopy plasmid carrying the wild type SPT 3 gene. By overexpression of SPT3, the suppression phenotype is lost (Fig. 2D, row 8). Notably, increasing the copy number of SPT 3 caused cellular sensitivity to 3-AT, even in a strain background containing wild type Gcn5p (Fig. 2D, row 4; and Fig. 3C, row 2). This result provides another piece of evidence that Spt3p functions as a repressor. In summary, the results above confirmed the negative roles of Spt3 protein in HIS3 expression, which are related to Gcn5 TSTY functions. Such functions related to TSTY residues of Gcn5p might be activated by Snfl mediated phosphorylation, as these residues are essential for in vitro phosphorylation by Snfl. The TSTY residues of Gcn5 might represent an activity different from the canonical HAT function, since the 107 gcn5E] 73H and gcn5A are not rescued by removing SPT3. Moreover, the allele specific suppression of spt3A on gcn5 TSTY/4A implies that Spt3 protein might physically interact with Gcn5p. The defect of HIS3 expression in snfl A strains is rescued by deleting SPT3 That the mutations of the likely Snflp targets (i.e. TSTY/4A) can be rescued by spt3A raises a possibility that spt3A might also rescue the snflA defects. Indeed, in a snf] A spt3A double deletion strain, the HIS3 mRNA level is much higher than that of the sanA single mutation (Fig. 3A). The 3-AT sensitivity test results conserved with the RNA quatitation data (Fig. 3B). Moreover, re-supplying SPT3 to the snflA spt3A cells attenuated resistance to 3-AT (Fig. 3C). which means the spt321 is a genuine suppressor. Importantly, the suppression by 5171321 requires the presence of Gcn5 HAT activity, since the introduction of E173H mutation eliminates the suppression thoroughly (Fig. 3B). Together, these data suggest that Snflp functions upstream of Gcn5p, and that the HAT activity of Gcn5p might be subjected to regulation by both Snfl p and Spt3p. The gcn5 T S T Y/4A mutation and snf] A are suppressed by spt8A. Spt8p is functionally similar to Spt3p in the regulation of TBP recruitment to the promoter, as null mutations in SPT8 are suppressed by some Spt3 mutations (10). Similar to Spt3p, Spt8p inhibits HIS3 and TRP3 basal expression (1). However, Spt8p and Spt3p 108 are not equivalent in all situations. For example, Spt8p is not required for TBP recruitment at GAL] promoter (2). Furthermore, Spt8p is absent in the SAGA like coactivator complex SLIK/SALSA (41). Accordingly, we were curious as to whether Spt8p behaved similarly to Spt3p in regulating Gcn5p and Snfl p. To address this question, SPT8 was deleted from sanA and gcn5 TS TIC/4A strains. The resultant strains were then analyzed for their 3-AT sensitivity. Fig. 4 shows that spt8A is also a suppressor for the sanA and gcn5TSTlC/4A mutations. These results implicate that Spt3p and Spt8p function together to achieve the inhibitory function. Snflp interacts with and phosphorylates Spt3p in vivo Previously, we showed that Snflp and Gcn5p interact in vivo (26). The in vitro kinase assay suggests that Snfl p is able to bind Gcn5p directly, although this association might be transient. Because both Snflp and Gcn5p are associated with multi-subunit complexes, we are unsure if the Snfl p complex binds to free Gcn5 protein or in the context of SAGA or SLIK/SALSA. By examining the interaction between Snflp and other components of the Gcn5p related complex, we believe Snflp is likely associate with the Gcn5p in complex. In Fig.5A, an HA-epitope-tagged Spt3 protein was co-expressed in yeast with GST-Snfl or GST. With the partial enrichment of the GST-Snfl protein by incubating the whole cell lysate with glutathione beads, the Spt3 protein was copurified with GST- Snfl (Lane 2) but not with GST alone (Lane 4). 109 The LysS4-to-arginine substitution of Snflp is a dominant-negative mutation that eliminates the ATP binding capability (6). Presumably the K84R mutant will bind to its protein substrates more stably. Interestingly, we found a stronger interaction between Snfl K84R protein and Spt3p (Fig.5A, lane 3), but a weaker interaction between the K84R allele and Gcn5p (F ig.5B, lane 3). It suggests that Spt3p is another substrate of Snfl p in SAGA complex. Indeed. the partially purified HA-Spt3-TAP fusions were labeled by the Pro-Q Phosphoprotein staining solution (Fig. 5C). The construct bearing HA-Spt3-TAP cassette was transformed into strains in the absence of, with the chromosomal copy of or with overexpression of SNF] gene. By comparing the relative phospho-staining of Spt3p isolated from these strains, we observed a Snflp dosage dependent phosphorylation of Spt3p. These results indicate that Snflp is able to phosphorylates Spt3p and probably associates Spt3p in a direct manner in viva. Spt3p interacts with Gcn5p in vitro The allele specific suppression of gcn5TSSIV-IA by spt321 raises a possibility of direct interaction between Spt3p and Gcn5p. The Snfl p mediated phosphorylation may disrupt the association between Spt3p and Gcn5p to remove the inhibition from Spt3p. To test the direct interaction between Gcn5p and Spt3p, we performed in vitro pull-down assays, using bacterially expressed Gcn5p and Spt3 p. Such preparation decreases the chance of protein phosphorylation that might affect the interaction between Gcn5p and Spt3p. Spt3p was tagged with 3xHA repeats at the amino terminus, and Gcn5p was tagged with 6xHis-tag. The fusion HA-Spt3 protein was immuno-purified and bound to Protein G agarose beads. Then the immobilized Spt3p was incubated with bacteria lysate with 6xHis-tagged Gcn5p. The bound fractions were analyzed by immunoblotting against His-tag. As shown in F ig.6A, Gcn5p was effectively pulled down by HA-Spt3 fusion (Lane 3). As a control, the protein G beads in absence of Spt3p association did not pulldown Gcn5 protein (Lane 1). The a-His-tag signal is specific because the Spt3-beads can not enrich any signal at Gcn5 position by incubation a lysate without Gcn5-6xHis protein expressed. Consistently, in the reciprocal experiment the HA-Spt3 protein is preferentially pulled down by the Ni2+-Talon beads with the Gcn5-6xHis protein bounded, as shown in F ig.6B. These results demonstrated that Spt3p and Gcn5p are capable of interacting with each other directly. 1]] DISCUSSION Snfl protein, as a kinase regulating cellular stress responses, has been shown to play important roles in transcriptional control. It is not surprising that Snflp is also involved in gene activation upon amino acid starvation. In this study, we showed that Snfl p is able to interact with and phosphorylate the Gcn5 p related coactivator complex in vivo. The phosphorylation mediated by Snflp may antagonize the inhibitory function of Spt3p by a mechanism of altering the architecture of the SAGA complex. Snflp interacts with and phosphorylates SAGA or SALSA coactivator complexes Snfl kinase plays positive roles in transcription. Snfl -mediated phosphorylation of the repressor protein Migl p leads to the dissociation of the corepressor complex from the promoter (43). Snflp also phosphorylates activators, such as Cat8 and Sip4 (44). As we reported in this study, the coactivator proteins, another category of protein in transcriptional regulation, can be targeted by Snfl p. At least two components of the SAGA or SALSA/SLIK complexes, Spt3p and Gcn5p are co-purified with Snflp and are phosphorylated in vivo in a Snflp dosage dependent manner. SAGA and SALSA/SLIK are closely related coactivator complexes (41 ). The differences between these complexes include a SALSA/SLIK-specific truncated Spt7p subunit, and SAGA specific Spt8p subunit (41). We do not know as yet which complex(es) is targeted by Snfl p. One proposed experiment, the co-purification of full-length and truncated Spt7p with Snflp 112 might be helpful for addressing the question. However. our genetics results implicate Snflp is functionally associated with SAGA complex, since the deletion of SAGA specific component, SPT8, suppresses the sanA phenotypes. Spt3p and Gcn5p represent opposite functions in HIS3 activation. Spt3p and Gcn5p are two nonessential components of SAGA complex (42). In electron microscopy structure of SAGA, Spt3p and Gcn5p are located in two physically separate modules (50). Genomic studies showed that distinct groups of genes are regulated by Gcn5p and Spt3p (25). The different requirement of Spt3p and Gcn5p was further demonstrated by the observation that Spt3p but not Gcn5p is essential for GAL] expression (2) and the requirement is opposite toward HIS3 activation, e.g. the SPT3 deletion or point mutation spt3-401 derepressed the HIS3 gene at the non-induced condition by interacting TATA-Binding protein (1 ). Moreover, under certain circumstances, the two proteins have opposite functions. For example, the defect of H0 expression by gcn5A is suppressed by 319132] (51). Our results provide further supporting for the inhibitory role of Spt3p in HIS3 activation. By overproduction of Spt3p, we observed growth defects in the media containing 3-AT (Fig. 2D; Fig. 3C). On the other hand, deleting SPT3 has no effect on HIS3 induction (not shown and Belotserkovskaya et al, 1), which implies the Spt3 protein, although existed in the coactivator complex, possesses a negative effect on HIS3 expression. Interestingly. a specific gcn5 mutant allele TSTY/4A, rescues the defect of spt321 in alternative carbon sources usage (not shown), which indicates that Gcn5p has a possible negative effect on Spt3p. too. Such 113 inhibition is removed by an uncharacterized mechanism to exhibit the Spt3 function in these gene transcriptions. Spt3 may inhibit Gcn5 function by direct association The position of Gcn5p and that of Spt3 p are spatially separated upon electron microscopy data suggested architecture of SAGA complex (50). However, our genetics and biochemical data imply that the two proteins interact closely with each other. The in vitro pull-down studies clearly showed the capability of direct interaction of these two proteins. Thus, we hypothesize that Spt3 p binds to Gcn5p and blocks its activity in HIS3 induction. Given that the recombinant proteins are free of phosphorylation, we further hypothesize that Snflp mediated phosphorylation of Gcn5p or Spt3p or both may disrupt the Gcn5p- Spt3p interaction and consequently abolish the inhibition. Since Gcn5p is robustly copurified with Snfl p (26), we suspect that Snflp is present in the SAGA complex. Snfl is automatically activated during regular protein preparation procedure (47, 49), thus the Gcn5p or/and Spt3p in SAGA is likely in its phosphorylated form that leads to the disassociation of Spt3p and Gcn5p, which results in the separation of the two modules in the electron microscopy analyses (50). Moreover, the affinity purified SAGA complex in the Wu et al study is likely an active form and Spt3p is in a flexible domain (50), which reinforce the likelihood of Spt3p dynamically regulating Gcn5p function in an intra- complex manner. 114 Future study From the results and discussion above, we can clearly see the negative roles of Spt3p in HIS3 transcription. This inhibitory function is likely downstream of Snflp as HIS3 transcription defect of snf/A is suppressed by spt3A. Such suppression is gene specific since the transcription of another Snfl p target, GAL], is not rescued. The allele-specific suppression of the gcn5 TSTY/4A mutation by spt3A suggests that the inhibition by Spt3p is mediated through Gcn5p. The interaction between Gcn5p and Spt3p in vitro raises the possibility that Gcn5p is inhibited by Spt3p via direct association, and that Snfl kinase action alleviates this association. Several experiments are proposed to test the hypothesis in the near future. First, we want to test whether phosphorylation of Gcn5p prevents its binding to Spt3p. Towards this goal, we will use purified Snflp with Glutathione beads to phosphorylate recombinant Gcn5-Hisx6 (either purified or using bacteria lysate) in vitro and then test the ability to pull down Gcn5p by immobilized HA-Spt3 protein. We can not phosphorylate the Gcn5-Hisx6 bound to Nizf- beads because Snflp contains a l3xHistidine stretch that will associate with any Ni2+ matrix as well. Such that we can not distinguish the Spt3p bound to Gcn5p or bound to Snfl p. The phosphorylation status of Gcn5p can be monitored by isotope labeling or Pro-Q phosphoprotein staining. The importance of phosphate group(s) for Gcn5p-Spt3 p association could be characterized by phosphatase treatment after the kinase reaction. If the phosphorylation is critical for 115 blocking the Gcn5p-Spt3p association, the Gcn5p treated with the Snfl kinase will be sequestrated from the Spt3p and the precipitated matrix. and the interaction will be restored by incubation with phosphatase. Altematively. we will test the impact of Spt3p phosphorylation on Spt3p-Gcn5p interaction. Second, we are very curious about the biological function(s) that Spt3p has in HIS3 regulation. The most obvious hypothesis is that Spt3p blocks the HAT activity of Gcn5p. The reason is that Spt3p directly binds to Gcn5p, which suggests Spt3p may regulate Gcn5p function. In addition, the suppression of spt321 on gcn5 mutations in HAT domain (i.e. TSTY/4A, Fig. 2) implies HAT domain of Gcn5p maybe the target ofinhibition. To investigate whether Spt3 p association will affect Gcn5 HAT activity, we will perform in vitro HAT assays with recombinant Gcn5p in the presence or the absence of Spt3p. The recombinant Gcn5p, either full length or just the catalytic domain will be enriched by Nizi-talon beads followed by elution with imidazole. The recombinant HA-Spt3 protein can be affinity purified by a-HA antibody. Because the HA-Spt3 protein has poor solubility in E.coli cells. a newly created construct (pYL99) that expresses HA-Spt3-TAP proteins in yeast cells will be used as the backup source of Spt3p. In this case, the Spt3p could be obtained by IgG affinity purification and elution with TEV protease digestion. The Spt3 sample will be analyzed by or-Ada2 western before adding to the HAT reaction to ensure the complete removal of other HAT components that might regulate enzymic activity of recombinant Gcn5p. 116 ACKNOWLEDGMENTS We are grateful to the following people for generously supplying materials: D. Thiele for GST-SNF1 constructs; F. Winston for 2p SPT3 construct; and M. Carlson for SNF1 constructs. We also thank S. Triezenberg, D. Almy. and J. 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Yeast strain list. strains relevant genotype Source yMK839 MA Ta trpl leu2-3, 112 zrra3-52 (23) yMK842 MA Ta trpl leu2-3,112 ura3-52 gcn5A::hisG (23) yMK986 MA Ta trpl leu2-3,] 12 ura3-52 gcn5E] 73H (26) yYL232 MA Ta trpl leu2-3,112 ura3-52 snf]A::LE U2 (26) yYL515 MA Ta trpl leu2-3,112 ura3-52 spt3A::KanA-LY6 This study yYL516 MA Ta trpl leu2-3,112 ura3-52 gcn5E l 73 H Spt3A::KanMX6 This study yYL590 MA Ta trpl leu2-3. 112 ura3-52 GCN5-8xmyc::TRP1 This study yYL59l MA Ta trpl leu2-3, 112 ura3-52 SNF1-8xm_vc::TRP1 This study yYL600 MA Ta trpl lezr2-3. 112 zrra3-52 gcn5E] 73H SNF]-8xmyc::TRPl This study yYL607 MA Ta trpl lend-3,112 ura3-52 gcn5El 73H-8xmyc::TRP1 This study yYL622 MA Ta trpl leu2-3,112 ura3-52 SPT7-I3xmyc::TRP1 This study yYL682 MA Ta trpl leu2-3,112 ura3-52 gcn5/J spt3A::KanMX6 This study yYL683 MA Ta trpl lez.12-3,112 ura3—52 .snle::LE U2 Spt3A::KanMX 6 This study yYL782 MA Ta trpl leu2-3. 112 ura3-52 gcn5 TS TY/4A -8xmyc:: TRP] This study yYL783 MA Ta trpl 1e112-3, 1 12 ura3-52 gcn5 719TW4A-8xmycssTRPl spt3A::KanMX6 This study yYL786 MA Ta trpl 1e112-3,112 ura3-52 spt8A::TRP1 This study yYL787 MA Ta trpl leu2-3, 112 ura3-52 spt8A::TRP1 This study yYL788 MA Ta trpl lezt2-3, 112 ura3-52 sptrS’AxTRPl This study Table 2. Plasmid list. Plasmid Description Source pMKlOO pRSET-Gcn5-6xHis (23) pFW32 YEp-SNFI (48) pYL4] YEplacl lZ-SA’F] (26) pY L42 pY EX-4T-G ST-Sn fl (l4) pYL43 pYEX-4T-GST-Snf] K84R (14) pYL44 pYEX-4T-GST (26) pYL54 pYEX-4T-Gc115-TAP (26) pYL55 pYEX-4T-Gcn5El 73H-TAP This study pYL67 8xmyc::TRPl for tagging proteins with 8 myc repeats (26) pYL72 pMK547Gcn5. 3xHA-Gcn5 (26) pYL89 pMK547Spt3, 3xHA-Spt3 This study pYL90 pET2Ia-3xHA-Spt3 This study pYL93 pYEX-4T-Gcn5TSTY/4A-TAP pYL98 pRSET-GcnSTSTY/4A-6xHis This study pMK284TSTY/4A Integration construct for introducing This study T203A/S204A/T2l lA/Y212A to GCN5 pMK515 pET2l-6xHis-Gcn5 protein (26) FIG. 1. Overproducing Snfl protein causes Gcn5p hyperphosphorylation in vivo. (A) Gcn5 is a phosphoprotein. GcnS was fused to the Tandem Affinity Purification (TAP) tag consisting ofthe calmodulin binding protein (CBP), TEV protease cleavage site, and Protein A. Gcn5-TAP was expressed and purified from yeast by IgG beads. A fraction of the bound materials was treated with the TEV protease that specifically liberates Gcn5-CBP from the IgG beads (Lane 1). Proteins were resolved by SDS-PAGE prior to staining with the Pro-Q Diamond Phosphoprotein Gel Staining solution as described in Materials and Methods. The X-protein phosphatase with (Lane 3) or without phosphatase inhibitors (Lane 4) was used to treat an aliquot of sample to test the specificity of the assay. The gel was stained with Coomassie Blue R250 (C BR) after scanning with fluorometer to compare the amount of proteins loaded. (B) The level of in vivo Gcn5 phosphorylation is correlated with the copy number of SIV'FI . The Gcn5-TAP proteins were purified from strains without (Lane 6) or with overexpressed Snfl kinase (Lane 7) and subjected to phosphoprotein staining. Fig. 1 A h h .2 :2 .2 .e .C .C 1H C u C c -- c ._ 4’ + 0 + g o o E o o (B CD en *6 or m g 3 3 o S 3 u.1 o 9.- 4 Lu 0 9.- q. I'- C ¢< ¢< I— C ¢< << Gcn5-TAP . 1 ' , ‘ ‘ * —’ “"2”” ' q ‘ ' ' I W I“ J Gcn5-CBPj -3; . l’ Heavy Chain u _ . l 2 3 4 1 2 3 4 Pro-Q Diamond Coomassie Phosphoprotein staining B F F CL 1.:. o. u. ‘1: <1 2 ‘11 <1 2 I- F a) I- F U) 0 ”E :1. O "E :1. Z to N 2 to N Gcn5-TAP —" .x _‘ _ m g. u 1 5 6 7 5 6 7 Pro-Q Diamond Coomassie Phosphoprotein staining 126 FIG. 2. The TSTY/4A mutation of Gcn5 was suppressed by deleting SPT 3. (A) The T203, 8204, and T211 residues are important for Gcn5 phosphorylation in vitro. The indicated mutations were introduced into the Gcn5 HAT domain and were expressed in bacteria. Purified recombinant proteins were subjected to in vitro kinase assays in the presence of y-[32P] ATP and GST-Snfl purified from yeast. The SDS-PAGE gel was stained by CBR prior to autoradiography. (B, C) The substitution of T203, 8204, T211, and Y212 residues of Gcn5 with alanines (Gcn5 TSTY/4A) impairs HIS3 expression. The defect caused by Gcn5 TSTY/4A mutation was rescued by spt3A (B) RT-PCR. RNA was isolated from indicated strains that grew under induced condition and subjected to RT-PCR analysis to determine the HIS3 expression. PGK] was used as loading control. (C) 3-AT sensitivity test. Strains indicated were grown in YPD until late log phase. After adjusting to the same cell density, the different cultures were serially diluted and spotted onto plates in the presence or the absence of 3-AT. (D) 3-AT resistant phenotypes of gcn5 T STY/4A spt3A are suppressed by overexpression of SPT 3. The Spt3 overproduction construct YEp-SPT3/pF W32 was transformed into spt3A, gcn5 T STY/4A, and double mutation strains. The resultant transformants were subjected for 3-AT sensitivity test as in (C). (E) gcn5EI 73H and gcn5A are not suppressed by spt3A. SPT3 was deleted from strains containing gcn5E] 73H mutation or gcn5A and then examined for 3-AT sensitivity. Pictures were taken after the plates incubated at 30°C or 37°C for 2-5 days. 127 Fig. 2 Autorad f, ’1'] ’ CBR Autorad/CBR: 1.4 l 1 0.9 1.7 1.1 0.5 0.35 HIS3 PGKI HIS3/PGK1: 4.1 1 1.2 0.9 2.6 30mM3-AT '- o - WT gcn5EH gcn5 TS TY/4A gcn5 TS TY/4A spt3A 128 Fig. 2 10mM 3-AT YEpIacl95 YEp-SPT 3 YE plac195 YEp-SPT3 SC-His, Ura ' spt3A 3) gc cn5TSTY/4A arm-““NH YEpIacl95} gcn5TSTY/4A spt3A YEp-SPT 3 - 3-AT + 3"” gcn5EI 73H gcn5EI 73H spt3 A gcn5A Spt3 A FIG. 3. Spt3p antagonizes Snflp function in HIS3 activation. (A) RT-PCR. Yeast strains indicated were grown in YPD until late log phase and then were transfered to SD media with 40 mM 3-AT to inducing HIS3 transcription. RNA isolation and RT-PCR were perfomied as in Fig. 2B. (B) 3-AT sensitivity test. SPT3 gene was deleted in snf] A strains. The snf] A strain showed the decreased HIS3 transcription and 3-AT sensitivity as previously reported (26). The deletion of SPT3 rescues the snflA phenotypes as restored HIS3 mRN A level and wild type-like growth on 3-AT plate were observed in a double deletion strain. The Gcn5 HAT activity is required since the E173H mutation of Gcn5 in snf] A spt3A strain abolished the suppression phenotypes. (C) 3-AT resistant phenotypes of snf] A spt3A were suppressed by overexpression of SPT3. The SPT3 overproduction construct (YEp-SPT3) and epitop-tagged Spt3p (HA-Spt3-TAP) were transformed into snf] A spt3 A strain to complement the Spt3A phenotypes. 130 :elec'ai 41111 The Fig. 3 HIS3 PGKI gcn5EH WT sanA snflA spt3A snfl A spt3A gcnSEH SC—His, Ura 10mM 3-AT 1 O O D t WT+YEpIac195 2 O C ‘3 ‘1 WT+YEp-SPT3 3 ~39 .31:- ‘3: , snflA spt3A+YEplacl95 4 o 18 4:. snflA spt3A + YEp-SPT3 5 . . . sanA spt3A + 2p HA-SPT3-TAP 131 Fig. 4 WT snf] A ’ ? snflA spt3A i c115 TS T Y/A4-myc spt8A gcn5 TS T Y/4A-myc spt8A snf] A spt8A FIG. 4. Both gcn5 TS TY/4A and snf] A are suppressed by deleting SPT8. SPT8 was deleted from gcn5 TS TIC/4A and snflA strains. 3-AT sensitivity test was performed. While gcn5TSTY/4A and sanA cells grow poorly in the media in the presence of 3-AT, the further deletion of SPT 8 partially suppressed the growth defect. The spt8A, per se, has no obvious phenotype observed. FIG. 5. Snflp interaction with and phosphorylates Spt3p. (A) Snflp linteracts with Spt3p. HA-tagged Spt3 protein was co-expressed with GST-Snfl (WT, Lane 2 or K84R. Lane 3) in yeast cells. Glutathione-purified fractions were detected by anti-HA Westems to examine the abundance ofthe co-purified HA-Spt3 protein (top). HA-tagged Gcn5 protein (Lane I) and GST alone (Lane 4) were processed in parallel as the positive and negative controls. The C BR staining of the duplicate gel is shown at the bottom. (B) K84R mutation of Snfl p attenuates the interaction between Gcn5p and Snfl p. The chromosomal copy of GCN5 was tagged with 8xmyc tandem repeats. GST-Snfl was overexpressed in such background. Gcn5 proteins were analyzed by anti-c-myc Western after partially purified by Glutathione Sepharose (top). The blot was stained with Indian Ink after the Western analyses (bottom). (_C) Spt3p is phosphorylated in vivo in a Snflp dependent manner. The construct expressing HA-Spt3-TAP fusion was transformed into strain backgrounds without (Snfl A), with single copy (chromosomal copy. Chr.) or with multiple copies of S.\='FI gene (O/E). The WCLs from the resultant strains were partially purified by IgG beads and resolved by SDS-PAGE. Then the gel was stained by Pro-Q Diamond Phosphoprotein staining solution as described in Materials and Methods followed by CBR staining. The signals were quantified by Image J and the relative ratio of Pro-Q and CBR signals were listed at the bottom. Fig. 5 A GST- Snfl Snfl K84R — HA- Gcn5 Spt3 Spt3 Spt3 Western: a-HA 1:22:42 ‘ 1 Bound Input (3%) CBR 1_200KDa _116 GST-Snfl --> - —-~ —96 1—66 1 1 4s GST_’_ __ 2_31 1 2 3 4 B Gcn5-myc Snf1 WT - K84R 01-ch - .4 ‘— Gcn5-myc lndialnk ‘ <— GST-Snfl , will "i ’ '- 1.. <— GST 134 Fig. 5. C HA-Spt3-TAP SNF1 A Chr. O/E ProQ CBR 135 FIG. 6. Direct interaction between Gcn5p and Spt3p. (A) Gcn5p is pulled- down by HA-Spt3 protein in vitro. Recombinant HA-Spt3 protein was obtained by expressing pYL90 in BL21 (DE3) cells. The HA-Spt3 fusion was enriched by IP with 01-HA antibody (3F10) followed immobilized on Protein G Sepharose 4G (Amersham). Then the beads were incubated with bacteria lysate with His-tagged full length of Gcn5 protein [pMKlOO in BL21 (DE3)]. The bound fractions (Lane 1-4) and the unbound fractions (Lane 5-8) were examined by Western blot with a-His-tag antibody (top) followed by a second western blot with a-HA antibody (bottom). (B) Spt3p is pulled-down by Gcn5 p in vitro. Ni2+-Talon beads bound with His-tagged Gcn5 was incubated with bacteria lysate containing HA-Spt3 protein. The bound fractions (Lane 1-4) and the unbound fractions (Lane 5-8) were examined by Western blot with or-HA antibody. Fig. 6 A Pull-down: or-HA/Spt3 Detection: or-His-tag/GncS Bound Unbound HA-Spt3: - - + - - + + Gcn5-Hisx6: + - + - + - a-His-tag . w : i «in. g ‘ , I. +Gcn5—6xHis l 2 3 4 5 6 7 8 B Pull-down: Ni2+-Talon/Gcn5 Detection: a-HA/Spt3 Bound Unbound Gcn5-Hisx6: - + - + - + - + HA-Spt3: + + - - + + - or-HA ‘ "" ' . . . <— HA-Spt3 137 APPENDIX Identification of suppressors that bypass the Gcn5 requirement by screening an EMS mutagenesis library created from a gcn5 strain 138 Identification of suppressors that bypass the Gcn5 requirement by screening an EMS mutagenesis library created from a gcn5 strain Introduction Gcn5-mediated histone H3 acetylation at lysine14 or lysine 18 is a key step in transcriptional activation of such genes as HIS3, [NO] and HXT 4 (6, 9, 14). Similarly, other histone modifications play important roles in different cellular process, such as H3 lysine 9 methylation is a mark of chromatin silencing (11). Serine phosphorylation is critical for cell cycle regulation. Such modifications may alter the charge of the histone tails to modulate the conformation of local chromatin. In other possibilities, these extra chemical groups may provide a different binding surface for other regulators association. The ‘histone code’ hypothesis was suggested based on the latter assumption, which is different modifications or combination of modifications may extend the information potential of the genetic (DNA) code by recruiting different regulatory factors to the specifically modified chromatin loci (13). Supporting the “histone code” hypothesis, many histone mark-specific binding proteins and binding motifs were found in the past few years, like bromodomain is a docking site for acetylated histories (2, 5). The chromodomain specifically interacts with lysine 9 methylated H3 (1, 4, 10). The PHD finger domain specifically recognizes methylated H3 139 lys4 (7, 12, 15). These facts explain the outcome of interaction between modified histones and regulatory factors perfectly. However, these findings cover only a small part of histone modifications. The proteins recognizing the phosphorylated histones and ubiquitylated histones, which are known important regulatory events, are still not unraveled. Moreover, although the “mark reader” was identified, like bromodomain may stick to acetylated H3, the significance of such association and the regulatory mechanism activated are elusive. To understand the regulatory mechanism associated with histone acetylation during the HIS3 activation, we initiated a suppressor screening in a strain background with a catalytic inactive mutation of Gcn5 (gcn5E1 73H, reference (8)). The extra mutations were introduced by two separate strategies. One is EMS mutagenesis (summarized in Table 1), in which the alkaline mutagen will cause the transition to introduce the point mutation into the yeast genome randomly. The second approach employed a minitransposon derived library. The minitransposon insertion-derived mutagenesis and screening are discussed in Chapter 2. The screening is based on the cell resistance to a chemical named 3-amino-triazol (3-AT), which is a competitive inhibitor of HIS3 gene product that requires the cells to express more HIS3 to achieve the histidine biosynthesis. Thus, when we remove histidine from the media to force the cells to activate histidine production, 3-AT will kill cells that fail to induce HIS3 gene (like gcn5’ cells). Using the random mutagensis approaches outlined above, we are seeking extragenic mutations that survived under 3-AT treatment even in the absence of Gcn5 histone acetyltransferase activity. Given the name Bypass of Gcn5 Requirement (BGR), these mutations may 140 represent the regulators working downstream or in parallel with Gcn5-mediated histone acetylation. By identifying these factors functionally linked with Gcn5, we expect to draw a much clearer picture of how histone acetylation facilitates HIS3 activation. In a more broad view, we are trying to find some clues to investigate the principles of how “histone codes” control the cellular progressing. As with other genetic screening, a lot of false positive candidates may be picked up because of the limitation of experimental set up. The most likely pseudo-suppressors are the factors that may affect the 3-AT transport in or/and out of the cell, e. g. alter the expression or activation of the 3-AT carrier. Other mutations may enhance the production of the substrate of the HIS3 protein, which makes 3-AT less competitive. To address this concern, B-galactosidase assay against the HIS3-lacZ reporter was performed after the 3- AT selection (Fig.1). Table 1. Summary of Screening: 65% cell death 108 colonies screened 144 candidates resistant to 3-AT 34 candidates verified by HIS3-lacZ reporter analyses The gcn5E173H mutation was created by Soumya Singh-Rodriguez. The EMS mutagenesis was performed by Dr. Kuo. The initial screening and verification with B-Galactosidase 141 assays were done by Xin-J in Xu and Xuqin Wang. I started the characterization of the mutations with the distinguished phenotypes. These assays include Northern analyses, spore analyses and isolation of mutations, dominance test, complementation test, and gcn5 deletion study. This chapter presents the results for these assays. B-Galactosidase Assay . 120 1100-: , ~ «-~ - - H - — — ; 80' ~~ ~ - WWW-w » ' 40 '- tLllililiiillilii llillllllli [Fill llfflllllihl Fig. 1. H1S3-lacZ reporter expression in selected BGR mutants. lacZ gene was translational fused to the 3’ of HIS3 promoter and then engineered into URA3 locus by homologous recombination. The strains were grown in 20 mL Yeast extract-Peptone- Dextrose (YPD) media to log phase and harvested by centrifugation. Then the cell pellet was resuspended and split into 10 mL YPD (for Basal expression) or Synthetic delete (SD, for inducing HIS3 expression) media and growing for 6 hours. The collected cells were broken by vortex with glass beads. 142 RESULTS: Northern analyses The HIS3-lacZ reporter assay provides us a simple and large-scale approach for analyzing HIS3 transcription. However, the ectopic copy of HIS3 promoter may not fully represent the endogenous gene transcription level since the local chromatin structure maybe different. Moreover, the HIS3-IacZ insertion at URA3 locus results in the reporter flanked by two repeated sequences. The high frequency of recombination in budding yeast will cause the looping out of the HIS3-1acZ fragment. Thus the culture is a mixture of cells with or without reporter, which may under evaluate the HIS3 induction. In such concerns, we decided to test HIS3 expression directly by measuring the mRNA level. Fortunately, we had narrowed the number of candidates with strong phenotypes to thirty four, which is the amount feasible for Northern analyses. Collaborating with Dave Almy in the lab, I grew different mutant strains along with the controls in YPD to log phase and then transferred the cells to SD with the supplement of 20mM 3-AT. After induction at 37°C for 6 hours, cells were harvested for RNA preparation. For each sample, 30pg of total RNA was used for Northern hybridization. The results are listed below. 143 Fig. 2. Northern analyses of the bgr candidates. The selected 34 candidates were divided into 3 sets. For each northern analysis, the strains indicated were grown to log phase and then transfered to SD media with 20mM 3-AT to induce HIS3 transcription. The RNA isolation and northern hybridization were followed the procedure described before (7). Results from two independent inductions for each set of samples are presented. The results were quantified by Phospholmager and the summarized results are shown as bar graph at bottom of each figure. 144 Setl #1 #2 #15 #18 #29 #32 #35 #37 #38 #68 #74 #79 WT gcnSEH .’. lg HIS3 :11. ’ + as 18$ up: 0.2 0.8 0.6 0.6 0.8 1.3 1.5 1.3 1.3 1.6 2.4 1.3 1.7 l #1 #2 #15 #18 #29 #32 #35 #37 #38 #68 #74 #79 WT gcnSEH 4 mss ”nfiufififlflfifififlfifi 18$ 1. fi 3 Tafl 93 fl 0.6 0.9 0.7 1.6 1.3 1.2 1.2 1.1 1.4 1.1 2.0 1.6 1.4 l EMS Sen ililllllllllll W4441>4>1ewera4§a HIS3/18S 2 l 145 Set2 #3 #4 #31 #34 #52 #58 #61 #93 #98 #99 #102 #103WTgcn5EH ”’33 ‘5‘ M at . fit an a III a u 18S .. 3e ...... g a. 9: 1.4 1.0 0.6 0.7 1.4 0.3 0.6 0.5 1.1 1.6 1.8 2.0 2.3 1 B #3 #4 #31 #34 #52 #58 #61 #93 #98 #99 #102 #103 WT gcnsEH 18S Hafiaééxeflflmuflfiflll 2.6 2.0 1.9 1.7 3.5 0.8 1.6 1.4 4.0 5.3 4.7 1.8 2.9 1 EMS Set2 HIS3/18$ ”P 4" #9 4? #4” #9 #9 #9 19‘" #9 $9” #6” $4539“ 146 Set3 #105 #111 #112 #113 #115 #122 #132 #133 #134 #136 #137 gcn5EH WT M my . W ‘ HIS3 .,,9 fl _*-'* " 7" . 18$ ”wwuww «(an wfifififi'flm.q¢y;# 0.03 0.2 0.7 0.6 1.1 0.9 0.3 0.2 0.2 0.2 1.0 1.0 2.3 #105 #111 #112 #113 #115 #122 #132 #133 #134 #136 #137 WT gcnSEH HIS3 h ’3. n ma: " N I 18${ .““‘* “W m at. W}! 9.x,“ l o 2.0 0.9 2.9 1.1 3.1 2.3 1.0 0.9 0.5 0.4 3.1 2.6 1.0 4.0 3.5 7* ,,, 3.0 2.5 4 2.0 — 1.5 1.0 0.5 6: x n 0 #9 %\v $3 $5 41¢” <9, 5: $4,»- 6 ’\ an 5’ ~10 4“ 5" 147 RNA analysis results indicate there are some false correlations between HIS3 expression and HIS3-lacZ expression. However, 18 candidates repeatedly show the enhanced HIS3 expression in both assays. Sporulation and isolation of the mutations EMS mutagenesis randomly introduces point mutations into yeast genome. Although the condition of EMS treatment is optimized to make most cells have only one hit with the mutagen, multi-mutations does happen inevitably. To distinguish the single mutation, spore analysis was performed for each candidate. The mutation strains were originally derived from a MA Ta gcn5A cell with a replacement of LE U2 gene by the gcn5EH gene, which provide an easy way to identify the gcn5 mutant by the Leu' phenotype. After crossing with a wild type MA Ta strain, the diploid strains carry the heterozygous of bgr alleles were subjected for sporulation. The spore analyses were performed by either random spore enrichment or tetrad- dissection. The isolated spores were patched on YPD plate to let cells growing for overnight then replica-plate to following plates: SC-Leu GCN5 genotype indicator SC-Ura HIS3-lacZ reporter indicator 3-AT bgr allele indicator YPD plates pre-spread with 227a or 70a. for matting type test. 148 Phenotypes on each plate were recorded and listed in Table 2. Table 2 Summary of Spore Analyses Mutant Number Total number of Leu' 3AT R:S* P value for 2:2 spores analyzed segregation #1 40 18:22 > 0.5 #2 53 25:28 > 0.7 #3 33 15:18 > 0.7 #4 25 11:14 > 0.5 #15 30 16:14 > 0.7 # 18 25 13:12 > 0.7 #29 29 20:9 _<_Q._Q§ #31 33 17:16 > 0.7 #32 236 119:117 > 0.9 #34 24 10:14 > 0.3 #35 161 81 :80 > 0.95 #37 27 18:9 > 0.05 #38 195 94: 101 > 0.5 #52 210 115295 >01 #61 N/D" #68 221 110:111 >0.95 #74 114 52:62 > 0.3 "#79 243 109:124 > 0.3 #93 23 17:6 M5 #98 204 104:100 > 0.7 #99 256 125:131 > 0.7 #102 242 123:119 > 0.7 #103 26 18:8 5__0._05 149 Mutant Number Total number of Leu' 3AT R:S* P value for 2:2 spores analyzed segregation #105 175 90:85 > 0.7 #111 185 100285 > 0.2 #112 209 792130 39M #113 240 1192121 >O.9 #115 236 1252111 >0.3 #122 246 1312115 >O.3 #132 33 23:10 <_002 #133 34 2529 <_0._01 #134 38 3028 < 0.001 #136 31 15216 >0.7 #137 235 1232112 > 0.3 * R: resistant; S: sensitive; A ND: not determined; italic and underlined: rejected by Chi- Square test By the statistical analyses of the above data, we noticed most of the mutations (27 out of 34) fulfill the hypothesis of 2:2 segregation for the 3-AT resistance phenotypes, which implies there is only one extra mutation besides gcn5E173H in these strains. For some of the candidates, like #133 and #134, which exhibit apparent differences for supporting the one mutation hypothesis, we think it’s still early to make conclusion from them because we haven‘t obtained enough spores yet. The only candidate that is significant different is #1 12, which gives 79 3-AT resistant and 130 sensitive spores. The apparent deviation can not be explained the linkage between 1e21222gcn5EH and the bgr allele, because that will make the spores with Leu' /3-AT'r dominate the population. Nor it can be simply explained by phenotypes require the existence of two mutations, since that will show an 123 segregation pattern in stead of 122. Before further evidence coming up, we hypothesis that, there are two mutations in candidate #112, the lethality of one mutation is suppressed by the other to accomplish the bgr phenotypes. Dominance Test Dominant/recessive is an important feature of a mutation. Knowing the dominancy of the mutations will be helpful for understanding the nature of the mutation, as the dominant mutations are always gain-of-function mutations. Also, the dominant mutations and recessive mutations will be treated with different strategies for mutation identifications. Based on the above thinking, I did the dominance tests for the 18 candidates that repeatedly showed the suppression of HIS3 expression (Figure 2). The mutants were backcrossed to the parental gcn5 mutant strain. The resultant diploid strains contain two copies of gcn5 E1 73H allele and heterozygous at the bgr loci. The diploid strain will show bgr phenotypes (3-AT resistant) if the mutation is dominant. If the diploid cell shows gcn5 phenotype (3-AT sensitive), it indicates that the mutation is recessive. 151 Fig. 3A Strategy of Dominance Test MA Ta >< MA Ta bgr gcnSE I 73H URA3 BGR gcn5EI 73H TRPI Select Ura+ Trp+ colonies MA T a/a BGR/bgr gcnSE I 73H/gcn5EH TRPI URA3 3-AT sensitivity test 3-AT resistant 3-AT sensitive bgr allele is dominant bgr allele is recessive 152 Table 3 Summary of Dominance Test Dominant Recessive #3 #52 #32 #74 #35 #98 #38 #103 #68 #111 #79 #115 #99 #105 #112 #113 #122 #137 153 20mM 3-AT * MK1075: MATa gcn5EH Fig. 3. Dominance test. BGR candidates were backcrossed and the resultant diploid strains were grown in YPD media to log phase. The harvested cultures were 5-fold serial diluted and spotted on the SC-His plates with or without 20mM 3-AT. The pictures were taken after 3 days of incubation at 37°C. The results were repeated 3 times. 154 Complementation test Complementation tests were done as described by F asser and Winston,1988 (3). The isolated recessive bgr' mutants with MA Ta matting type were transformed with YCplac33, which contains URA3 gene as selective marker. Similarly, the bgr‘ alleles with MATa matting type were transformed with YCplac22 to introduce T RP] marker. Then the strains with the same matting type were grown as set of stripes on plates dropped out uracil or tryptophene respectively. Then the two sets of stripes with different matting types were replica-plated perpendicularly onto one YPD plate. After the incubation at 30°C for 6 hours, the grid of MATa and MA Ta mutants were replica-plated onto the plate in the absence of both uracil and tryptophene to select the diploid strains. The complementation groups were defined by 3-AT sensitivity test. The diploids with the crossing between the different complementation groups, or in another word, the mutation(s) at the different genes, will sensitive to 3-AT since the heterozygosity of the bgr genes exhibits the dominant wild type phenotypes. MA Ta >< MA Ta gcn5El 73” bng- BGR? URA3 gcn5£1 73H BGRP bgr? TRPI Select l'ra+ Trp+ colonies MA Ta/a BGRl/bgrl‘ BGRl/bgrl- gcn5El 73H/3306131 73H TRPI URA3 3-.-\T sensitivity test 3-AT resistant: Fail to complement 3-AT sensitive: complement Same complementation group different complementation group MAT a x [MATa V e1}. 6:? 51.3: «‘22-... V _ 98a x 98a ”the 472:3?“ '* llla x 1110: 98a x 111a 111a x 9801 a cells x gcn5 EH (1 cells x WT Fig. 4. Complementation test. The recessive mutants with isolated MATa haploid and AM Ta haploid were matted to each other in pairwise. The resultant diploid cells were patched onto an YPD plate and grown for overnight. Then the patches were replica-plated to 3-AT plate and incubated at 37°C. The crossing with wild type strains were used as positive control. The diploid strains from backcrossing were also tested to ensure the recessive feature of the mutations. Self-crossing diploids are as expected to be resistant to 3-AT. However, neither 98a x 111a nor 111a x 98a grew well in the presence of 3-AT. Here “98a” represent the MATa isolation of EMS mutant #98. The same experiments as shown in Fig. 4 were done or will be done for all the 6 recessive bgr mutants. So far, we identified at least two complementation groups, #98 and # 111. And the MATa form of #1 15 complement both #98 and #111, which indicates #115 mutation may represents another group of suppressors. Also the #52 and the #74 mutation are not in the same complementation group. Whether the #52 and #74 mutations overlap with #98 group or #111 group are still under investigating. 156 GCN5 deletion study One group of expected suppressors is the mutations restore the HAT activity of Gcn5p. These mutations could be the reversal mutation of gcn5E1 73H, i.e. histidine 173 — to - glutamic acid, or an extra mutation within or at other loci of the gcn5 gene that enhances the HAT activity. To identify such kind of mutations, we performed the GCN5 deletion studies. By removing the entire open reading frame of gcn5, any factors altering the Gcn5 activity are eliminated. For instance, we got gcn5 deletion from 7 bgr candidates, which are #3, #35, #52, #68, #99, #102, and #103. The 3-AT test indicate all of them still possess the suppression power, which suggests these mutations indeed bypassed the requirement of Gcn5. Future plan: mapping the bgr mutations Other than finishing the above experiments, we are more interested in where do those mutations happen. For dominant and recessive mutation, we decide to apply different approaches to mapping the mutations. To those dominant mutants, we will make yeast DNA libraries from those strains. The clones in the libraries contain the DNA fragments that contribute the BHR phenotype will rescue the 3-AT sensitivity phenotype after being transformed to the parent strain carrying the gcn5 mutation. 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