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A... ..A. _.A O . _ .0 0 I .. 0 .I o O ‘ '00 10.0. . A . . 0 ‘. 00 LIBRARY Michigan State University This is to certify that the dissertation entitled INVESTIGATING A NOVEL FUNCTION OF HISTONE H3 IN MITOTIC CHECKPOINT CONTROL IN SACCHAROMYCES CEREVISIAE presented by Jianjun Luo has been accepted towards fulfillment of the requirements for the PhD. degree in Biochemistry and Molecular Biolggy Mwfle Major Professor's Signature ‘7 // r/z 0/ D Date MSU is an Affirmative Action/Equal Opportunity Employer H PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K‘IProj/AccS-ProlelRC/DateDueJndd INVESTIGATING A NOVEL FUNCTION OF HISTONE H3 IN MITOTIC CHECKPOINT CONTROL IN SACCHAROMYCES CEREVISIAE By Jianjun Luo A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Biochemistry and Molecular Biology 2010 ABSTRACT INVESTIGATING A NOVEL FUNCTION OF HISTONE H3 IN MITOTIC CHECKPOINT CONTROL IN SACCHAROMYCES CEREVISIAE By Jianjun Luo Erroneous segregation of eukaryotic genome causes aneuploidy that is the leading cause of first-trimester miscarriages and is also intimately linked to certain developmental defects and cancers. To prevent missegregation, dividing cells monitor both kinetochore-spindle attachment and the tension between sister chromatids. Mitotic tension is a result of bipolar attachment, that is, spindles emanating from opposite spindle pole bodies attach to and pull each of the two sister kinetochores. Although it has been firmly established that many interphase nuclear functions, including transcriptional regulation, are regulated by chromatin and histones, how mitotic progression and quality control might be influenced by histones is still less well characterized. Our studies indicated that histone H3 plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis. Lack of tension due to erroneous attachment activates the spindle assembly checkpoint, which corrects the mistakes and ensures segregation fidelity. A histone H3 mutation (Gly44 to Ser) impairs the ability of yeast cells to activate the checkpoint in a tensionless crisis, leading to missegregation and aneuploidy. The defects in tension sensing result directly from an attenuated H3-Sgo1p interaction essential for pericentric recruitment of 8901 p. Reinstating the pericentric enrichment of 8901 p alleviates the mitotic defects. Histone H3, and hence the chromatin, is thus a key factor transmitting the tension status to the spindle assembly checkpoint. Further studies on characterizing the interaction between H3 and Sgo1p led to the discovery that the tension sensing function depends critically on a unique “tension sensing motif", or TSM, of histone H3. Lys42, Gly44, and Thr45 of H3 form a core of a motif executing a mitotic function that is pivotal for cells to respond to the lack of tension between sister chromatids. This motif functions through physically recruiting or retaining Sgo1p at the pericentromeres. lntriguingly, the histone acetyltransferase activity of Gcn5p likely plays a negative role in H3 TSM-Sgo1p function. These results reveal that a TSM of histone H3 is a key player in faithful segregation of mitotic chromosomes, and that interaction with a chromatin modifying enzyme may be an important part of the mitotic quality control process. ACKNOWLEDGMENTS I gratefully acknowledge my advisor, Dr. Min-Hao Kuo, for his years of advice, supervision, and contribution to my research. This work would not have been possible without his encouragement and support. I would also like to acknowledge my committee members, Dr. Dean DellaPenna, Dr. R. William Henry, Dr. Laura R. McCabe, and Dr. John L. Wang, for their continuous guidance and support. Many thanks go in particular to Xinjing Xu, our previous lab manager/research scientist, for her selfless assistance in my research and life. I would like to thank many Kuo lab members, Dr. Yang Liu, Dr. Asha Acharya, David Almy, Xiaobo Li, for their valuable discussion and help in my research. I also want to thank Dr. David N. Arnosti, Dr. Zachary Burton, and Dr. Colleen Doherty for their comments and suggestions in my research. I would like to indicate my gratitude to Dr. Pamela J. Fraker, Dr. Louis King, and Dr. Joseph Leykam for their guidance and technical assistance. I also want to thank the Department of Biochemistry and Molecular Biology and the GEDD group for the financial support. Finally, I want to express my gratitude wholeheartedly to my wife Xiangyi Wang, my son Gerald Yanming Luo, my parents and parents-in-law for their support and love. TABLE OF CONTENTS Images in this thesis/dissertation are presented in color List of Tables .................................................................................... vii List of Figures ................................................................................... viii CHAPTER I: LITERATURE REVIEW ....................................................... 1 Part I: Nucleosome structure and roles of histones in cell cycle progression ....................................................................... 2 Part II: Histone post-translational modifications ................................. 6 Part III: Budding yeast mitotic spindle checkpoint and tension sensing mechanism ..................................................................... 16 Part IV: Research interests and significance .................................... 21 References .............................................................................. 23 CHAPTER II: Histone H3 Exerts a Key Function in Mitotic Checkpoint Control. 40 Abstract ................................................................................... 42 Introduction .............................................................................. 43 Materials and Methods ............................................................... 46 Results .................................................................................... 55 Discussion ................................................................................ 67 Acknowledgements .................................................................... 74 Tables ..................................................................................... 75 Figures .................................................................................... 77 References 112 CHAPTER III: Characterization of a Tension Sensing Motif of Histone H3 in Saccharomyces cerevisiae ................................................ 1 18 Abstract .................................................................................. 1 20 Introduction ............................................................................ 121 Materials and Methods ............................................................ 124 Results ................................................................................... 128 Discussion .............................................................................. 137 Tables ................................................................................. 140 Figures .................................................................................. 143 References ............................................................................. 162 APPENDICES ................................................................................... 166 APPENDIX I: Linking H3 phosphorylation to a Saccharomyces cerevisiae 14-3-3 protein Bmh1 p ......................................................... 167 Introduction ............................................................................ 167 Materials and Methods ............................................................ 169 Results ................................................................................... 1 71 Discussion .............................................................................. 174 Tables ................................................................................. 176 Figures .................................................................................. 1 77 References ............................................................................. 184 APPENDIX ll: Further investigation of the interactions between H3 and Sgo1p in mitotic tension sensung 187 Introduction ............................................................................ 187 Materials and Methods ............................................................ 188 Results ....................................................................... ' ............ 1 89 Discussion .............................................................................. 191 Tables ................................................................................. 1 93 Figures .................................................................................. 194 References ............................................................................. 199 vi LIST OF TABLES CHAPTER II: Histone H3 Exerts a Key Function in Mitotic Checkpoint Control Table 2-1. Yeast strains used in this study ...................................... 75 Table 2-2. Plasmid constructs used in this study ............................... 76 CHAPTER III: Characterization of a Tension Sensing Motif of Histone H3 in Saccharomyces cerevisiae Table 3-1. Yeast strains used in this study ..................................... 140 Table 3—2. Plasmid constructs used in this study .............................. 142 Appendix I: Linking H3 phosphorylation to a Saccharomyces cerevisiae 14-3-3 protein Bmh1p Table A-1. Yeast strains used in this study ..................................... 176 Table A-2. Plasmid constructs used in this study ............................. 176 Appendix II: Further investigation of the interactions between H3 and Sgo1p in mitotic tension sensing Table B-1. Yeast strains used in this study ..................................... 193 Table B-2. Plasmid constructs used in this study ............................. 193 vii LIST OF FIGURES CHAPTER II: Histone H3 Exerts a Key Function in Mitotic Checkpoint Control Figure 2-1 The G448 mutation confers pleiotropic phenotypes ............ 77 Figure 2-2 The G448 mutation causes chromosome instability ............ 80 Figure 2-3 G448 mutant cells activate the spindle checkpoint in response to benomyl toxicity ................................................................. 83 Figure 2-4 The G448 mutation impairs the tension sensing function ..... 85 Figure 2-5 G448 mutant mitotic phenotypes are suppressed by overexpressing 8901 p ................................................... 86 Figure 2-6 The G448 mutation selectively downregulates the pericentric recruitment of Sgo1p in vivo .................................................. 89 Figure 2-7 Physical interaction between H3 and Sgo1p is attenuated by the G448 mutation ............................................................... 92 Figure 2-8 Reestablishing pericentric Sgo1p recruitment by BD fusion...94 Figure 2-9 Bromodomain fusion partially rescues the tension sensing defects of G448 mutant cells ............................................ 96 Figure 81 G448 cells exhibited slower growth rate 99 Figure 82 Sgo1p interacts histone H3 in different contexts ............... 100 Figure 83 G448 mutation is semi-dominant ................................... 102 Figure 84 2pm 8601 does not rescue a histone H4 allele, hhf1-20, that causes temperature sensitivity and centromeric function defects ........................................................ 103 Figure 85 Mcd1 p recruitment to selective centromeres, pericentric regions, and chromosome arms is not affected by G448 mutation .................................................................. 104 Figure 86 No discernable transcriptional defects were observed in the G448 cells ................................................................ 105 viii Figure 87 2pm 8601 suppression is independent of the PP2A activity ................................................................... 109 Figure 88 Genetic interactions between the Aurora kinase, histone H3, and 8601 ......................................................... 110 CHAPTER III: Characterization of a Tension Sensing Motif of Histone H3 in Saccharomyces cerevisiae Figure 3—1 The Tension Sensing Motif (TSM) residues function through Sgo1p ....................................................................... 143 Figure 3-2 K42A and T45A mutant cells responded normally to benomyl treatment by activating the spindle assembly checkpoint 147 Figure 3-3 The K42A and T45A mutant cells exhibited chromosome Instability ................................................................... 148 Figure 3-4 Defects in responding to tensionless crisis in K42A and T45A Mutants ..................................................................... 1 51 Figure 3-5 The K42A and T45A mutation selectively downregulates the pericentric recruitment of 8901 p in vivo ............................. 154 Figure 3-6 The K42A and T45A mutation attenuates the affinity of H3 to Sgo1p in vitro ............................................................. 156 Figure 3-7 Over-expression of 8601 in wild-type cells expands the existing pericentric domain of Sgo1p ............................... 157 Figure 3-8 The HAT activity of Gcn5p is a negative regulator of the tension sensing function of H3 ........................................ 159 Figure 3-9 The negative regulation of Gcn5p on H3 tension sensing function depends on functional SGO1 .............................. 161 APPENDIX I: Linking H3 phosphorylation to a Saccharomyces cerevisiae 14-3-3 protein Bmh1p Figure A-1 Tethered catalysis approach to phosphorylate N-terminal tail of histone H3 ............................................................. 177 Figure A-2 Bmh1p specifically interacts with phosphorylated histone H3 ........................................................................... 178 Figure A-3 Residues important for the interactions between Bmh1p and phosphorylated H3 ...................................................... 180 Figure A-4 No defective phenotypes observed in mutants of histone H3 and/or H2B ................................................................. 182 Appendix II: Further investigation of the interactions between H3 and Sgo1p in mitotic tension sensing Figure B-1 Screen for intra-genic mutations of 8601 that rescue the benomyl hypersensitivity of H3 G448 ................................... 194 Figure B-2 Schematic list of mutations identified in the intra-genic suppressor screen ........................................................... 195 Figure B-3 Western Blot analysis against Sgo1p fused HA tag shows no major increase of 8901 p level among intra-genic suppressors when compared to wildtype 8901 p ............................................... 196 Figure B-4 Chromatin immunoprecipitation analysis of the association of intra-genic mutants of 8901 at centromere and pericentric region ..................................................................................... 197 CHAPTER I Literature Review Literature Review Part I: Nucleosome structure and roles of histones in cell cycle progression Nucleosome composition, positioning and centromeric nuclesome In eukaryotes, the enormous length of genomic DNA needs to be packaged into a higher order nucleoprotein complex termed chromatin, whose basic repeat unit is the nucleosome. The nucleosome core particle consists of 146 base pairs (bp) of DNA wrapped in 1.65 left-handed superhelical turns around a histone octamer, which is formed by four histone partners: an H3-H4 tetramer and two H2A-H28 dimers (99). Histones are small basic proteins consisting of a globular domain and a more flexible and charged NHz-tenninal tail that protrudes from the nucleosome. The distinct levels of chromatin organization are dependent on the dynamic higher order structuring of nucleosomes (65). Nucleosomes are connected to each other via a linker DNA of characteristic length. Linker histone H1 is bound to the nucleosome at the point where the DNA enters and exits the core, and to the linker DNA. The internucleosomal associations also can be mediated by direct interactions between histone H4 amino-terminal tail and the acid patch of the H2A/H2B (25, 141). Due to the organization into nucleosome and the highly compacted chromatin structure, genomic DNA sequences can be more or less accessible to transcription, DNA repair or DNA recombination machinery (9, 42, 70, 135). Recently, genomic technologies have been widely used to map nucleosome positioning across genomes, and results similarly point out that nucleosomes are highly phased near the 5’ end of genes, a nucleosome-depleted region close to the transcriptional start site is flanked on both sides by positioned nucleosomes. The depletion of nucleosome at the promoter region favors the assembly of transcriptional machinery and the initiation of transcription (67, 106, 168, 176). In the centromeres of the eukaryotic chromosome, the H3 variant CENP-A (Cse4p in budding yeast) is assembled into the centromere specific nucleosome (109, 143). Cse4p shares 64% identity to canonical H3 and exhibits similar DNA- binding characteristics as H3 (75, 147). Recently, Camahort and colleagues examined the composition of Cse4p-containing nucleosome and their results further supported the notion that Cse4p simply replaces H3 in an octameric nucleosome that contains Cse4p, H2A, H2B, and H4 (14). A kinetochore protein, 8cm3p, originally thought to form a unique centromere specific hexameric nucleosome with Cse4p and H4 (112), may just be required when Cse4p protein levels are limited. 8cm3p is not an essential component of Cse4p-containing centromeric chromatin (14). Known histone alleles and chromatin function So far, many histone mutations have been identified, and studies on these histone mutants have rapidly expanded our understanding of a variety of chromatin functions. The sin (switch-independent) alleles [H3 E105K (Glu105 to Lys mutation), H3 R116H, H3 T118l, H4 V43l, H4 R45H, H4 R45C], which sufficiently alter nucleosome mobility, nucleosome structure or higher order chromatin folding, partially bypass the need for the SWl/SNF chromatin remodeling complex in transcriptional regulation (33, 52, 88, 116, 133). The Irs (Loss of rDNA Silencing) alleles (H3 72-83, H4 78-81) cause a loss of repression of genes specifically embedded in transcriptionally silent regions of the genome (e.g., ribosomal DNA locus or telomeres) (122, 142). Although each of SIM and LR8 domain consists of histone H3 and H4 L1 and L2 loops that form a DNA- binding surface at either superhelical location (SHL) :l: 2.5 (LRS) or SHL :l: 0.5 (SIN), these two domains are functionally distinct. Indeed, mutations in the LRS domain do not cause the sin phenotypes, and vice versa (35). In addition to sin and Irs alleles which mainly affect the globular domain of histones, studies with deletions or single mutations on the flexible histone tail region have also provided evidence for chromatin functions in cell cycle progression. For instance, deleting the N-terminal domain of H3 or H4 delays progression through the G2/M periods (114). Four lysine residues in the N- terrninal domain of histone H4 (K5, K8, K12, K16), which are subjected to reversible acetylation, are required for the maintenance of genome integrity, and are important for DNA replication and nuclear division (107, 108). In an endeavor to explore essential functions of H3 and H4 amino terminus, Ma et al. (102) carried out a genetic screen for second-site mutations that are lethal when combined with a H3 amino-terminal deletion, and identified H8L1 and HSL7 (histone synthetic-lethal gene). Hsl1p and Hsl7p are negative regulators of 8we1 p (the S. Cerevisiae homolog of Wee1 p) kinase. The 8we1 p kinase inhibits mitotic Cd028p kinase activity and destroys the function of morphogenesis checkpoint required for delaying mitosis when bud formation is impaired. Therefore, these cell cycle regulators function in a pathway upstream of H3 to modulate histone functions in the cell cycle (102). Moreover, a histone H4 allele, hhf1-20 (H4 T82l A89V double mutant), compromises the interaction between H4 and the centromere specific H3 variant Cse4p, thus impeding the functions of centromere and mitotic progression at the restrictive temperature (144). Two alleles of histone H2A (H2A 820F, H2A G30D) cause cold-sensitive growth defects, a significant increase in ploidy, increased chromosome loss rate, and altered chromatin structure over centromeric DNA, suggesting a critical role for histone H2A in centromere function (127). The hyperploidy phenotype can be suppressed by mutations affecting a histone deacetylases, HDA1 (71). Together, these studies suggest a prominent role for histones in chromosome segregation; however, little is known about how histones proactively regulate the mitotic progression and cell cycle regulation. Part II: Histone post-translational modifications Each core histone contains an accessible N-terminal tail that is subjected to different post-translational modifications including methylation, phosphorylation, acetylation, ubiquitination, etc. (20, 64, 140, 148). Changes in chromatin structure and dynamics by histone post-translational modifications, chromatin remodeling, and DNA methylation, are correlatively associated with transcriptional regulation. The reversible acetylation of histones by histone acetyltransferases (HATs) and deacetylases (HDACs) regulates the accessibility of DNA and recruits select proteins for regulating transcription. Histone methylation, catalyzed by histone methyltransferases (HMTs) and cleared by a variety of demethylases (23, 82), acts primarily as docking sites for different proteins whose recruitment regulates the biochemical and structural characteristics of the underlying chromatin loci. DNA methylation exerts mighty effects, both locally and spanning an expansive chromatin locus, on gene activity (123). Histone methylation The majority of biological methylation reactions are catalyzed by methyltransferases that transfer the methyl group from S-adenosyl methionine (8AM, 8AMe, or AdoMet) to the acceptor molecules including DNA, RNA, and selective arginine or lysine residues in proteins. Methylation of DNA and histones has tremendous impacts on local and global gene activities, DNA repair, and epigenetic inheritance (17, 132, 151, 157). Synthesis of SAM involves the methionine adenosyltransferases (MATs), ATP, and methionine. Epigenetic regulation involving SAM includes methylation of DNA, histones, and several nonhistone transcriptional regulators, such as the tumor suppressor protein p53 and the general transcriptional factor TAF 10 (55). DNA methylation represses transcription, and is essential for the maintenance of chromatin structure and genomic stability. Alterations of the promoter methylation have been associated with changes of transcription profiles of cancers. Consistently, transgenic mice with one of the two methionine adenosyltransferase genes (MA T1A) deleted revealed stunning development of hepatocellular cancer (HCC) by 18 months of age (97). Deleting MA T1A leads to drastic decrease of the liver SAM level and increase of serum methionine concentration. However, it should be noted that, it is unclear whether the cancerous development was ascribed to misregulation of DNA or histone methylation, or both. In this regard, it is interesting to note that the budding yeast Saccharomyces cerevisiae does not have DNA methylation, and hence may be a good model to delineate the effect of deleting the methionine adenosyltransferase genes (SAM1 and 8AM2) on protein methylation and gene expression (100). In higher eukaryotes, H3 K4, K9, K27, K36, K79, and H4 K20 can be methylated (17). In general, methylation at H3 K4, K36, and K79 is associated with transcriptional activation, whereas methylation of H3 K9, K27, and H4 K20 are marks of repressive chromatin states (110, 130). In vitro studies showed that tri- methylation of H4 at lysine 20 increased the ability of nucleosomal arrays to fold and condense, suggesting that histone methylation not only creates binding sites for proteins, but also likely affects higher order chromatin structure directly (98). Studies carried out in HeLa cells indicated that trimethylation on H3 K9 and H4 K20 in late 62 is necessary for proper pericentric heterochromatin formation and chromosome segregation during mitosis (50). In fission yeast, H3 K9 methylation provides binding sites for heterochromatin protein (HP1) in RNAi-dependent heterochromatin assembly processes (44). In budding yeast, only H3 K4, K36, and K79 are known to be methylated by histone lysine methyltransferase enzymes, 8et1 p, Set2p, and Dot1 p, respectively (1 1 1). Histone phosphorylation Phosphorylation of histone H3 plays an important role in the regulation of gene expression and in chromosome condensation. The long standing mystery for H3 serine 10 (810) phosphorylation is that this modification appears to be involved in two structurally opposite processes: transcriptional activation (21, 61) which requires chromatin fiber decondensation, and chromosome compaction during cell division in which chromatin condensation is indispensible (48, 54, 160). H3 810 phosphorylation begins during prophase, with peak levels detected during metaphase, and a general decrease in the amount of phosphorylation during the progression through the telophase (46). A similar correlation can be observed during meiosis and a lack of H3 810 phosphorylation inhibits meiosis in Tetrahymena thennophila (166, 167). Surprisingly, in budding yeast, mutation of 810A does not result in a major defect in chromosome transition, suggesting phosphorylation of H3 at 810 is not indispensible for cell-cycle progression in yeast (54). Since the first discovery that phosphorylation of histone H3 810 was associated with chromosome condensation and segregation during mitosis and meiosis (46), mitosis-specific phosphorylation of H3 has also been observed at H3 828 (38) and at H3 threonine 11 (129). Although the H3 831 residue exists only in histone variant H3.3, phosphorylation of H3 831 occurs at centromeric regions of metaphase chromosomes (47). Interestingly, S. cerevisiae contains only one type of canonical H3, which is most similar to mammalian H33, and also contains serine 31 (10). Mitosis-specific phosphorylation of H3 810 starts in late G2 phase on pericentromeric chromatin only. As mitosis proceeds, phosphorylation of H3 spreads along the chromosome arms and completes its coating on the entire chromosome at prophase (39, 51). The dephosphorylation process starts at anaphase and finishes at telophase (39, 51). Members of the Aurora serine/threonine kinase family have been found to govern histone H3 phosphorylation at H3 810 during mitosis in several organisms (37, 54). In addition, Aurora B phosphorylates H3 828 in vitro (149) and in vivo in mammalian cells (39). In S. cerevisiae, the sole Aurora kinase encoded by the IPL1 gene (18) is required for high-fidelity chromosome segregation (34). Cells bearing only a temperature-sensitive allele (ipI1-2) of IPL1 show a reduction in the level of H3 810 phosphorylation when grown at restrictive temperature (54). 14-3-3 proteins 14-3-3 proteins are a family of highly conserved, ubiquitously expressed, abundant acidic proteins in all eukaryotes studied (11). 14-3-3s were first recognized to interact with a discrete phosphoserine or phosphothreonine motif by forming homo- or heterodimers (174). While there are seven 14-3-3 isoforms in mammals, Bmh1p and Bmh2p are the only two 14-3-3 proteins in S. cerevisiae. Bmh1p and Bmh2p share 91% overall identity and >97% identity over their first 256 residues (36, 159). Simultaneous knockout of these two functionally redundant isoforms in S. cerevisiae is lethal (158). Bmh1p and Bmh2p are also required for a timely G1/S transition, DNA damage checkpoints and genomic stability (95, 96). Recently, Macdonald and colleagues reported that three human 14-3-3 isoforms (2, C, y) bind H3 (aa 1-20) peptides in a phosphorylation dependent fashion (103). Our data also showed that Bmh1p binds preferentially to phosphorylated H3 (Luo and Kuo, unpublished data). Histone acetylation 10 Acetyl coA (Ac-coA) is the acetate donor for acetylation reactions, in which the acetyl group is transferred from Ac-coA to the lysine s-amino group on the N- terminal tail of histones. In mammalian cells, Ac-coA can be synthesized via two routes. Ac-coA synthetase, like many fungal cells, condenses acetate and coenzyme A into Ac-coA. In the second pathway, the ATP-citrate Iyase (ACL) uses energy from ATP hydrolysis to convert citrate, a TCA cycle productfintermediate, and coenzyme A into Ac-coA and oxaloacetate. Boeke and colleagues used the budding yeast as the model to show that one of two Ac-coA synthetases in yeast, AcsZp, also regulates the global acetylation of histones, and that synthetic lethality results from deleting a key histone acetyltransferase (HAT), Gcn5p, in an acsZ-conditional mutant (153). Acetylation of core histones has been shown to enhance the affinity of transcription factors for nucleosomal DNA (69, 91, 163) and may cause a conformational change in nucleosome structure (3, 121). The acetylation of histones by HATs is thought to affect chromatin function through two different mechanisms. First, it neutralizes the positive charge of the lysine c-amino groups on the N-terminal tail of histones, which to some extent changes the structural characteristics of the histone or the binding to the DNA (3, 121). Second, this epigenetic mark serves as recognition sites for the binding of transcriptional factors to promote transcription (5, 16, 57). Histone acetylation is a reversible process, acetyl groups can be removed by histone deacetylases to reestablish the positive charge in the histones (84). 11 Histone acetylation has been linked to transcriptional activation. Studies showed that histone acetylation sites and HATs are indispensible for gene expression (29, 84, 86, 134). Genome-wide analyses on histone acetylation indicated that acetylation at a majority of lysine residues in histone H3 and H4 tails correlates positively with gene transcription (87, 94, 128). Since the identification of yeast transcriptional adaptor Gcn5p as catalytic subunit of type A histone acetyltransferase (13), Gcn5p has been shown the capability to acetylate H3 (at K9,14,18), H4 (K8, K16), and H2B (K11, 16) (85, 114, 150, 165, 178). The modulation of transcription by Gcn5p is independent of its known coactivator function (58). Recently, H3 K36 has also been identified as an acetylation site by Gcn5p, and ChlP-chip experiments demonstrated that H3K36 acetylation predominantly localized to the promoters of RNA polymerase II transcribed genes. This pattern is similar to that of other acetylation sites including H3 K9 and K14 but completely inversely related to that of H3 K36 methylation, suggesting the existence of an acetyl/methyl switch that governs chromatin function during transcription process (115). In addition, 28% of H3 in yeast contains acetylated K56 (172). H3 K56 acetylation by Rtt109 correlates with actively transcribed genes and associates with the elongating form of polymerase II in yeast (137). The temporal acetylation on N-tenninal tails of H3 and H4 is important for newly synthesized histones to be assembled into nucleosomes (145). For example, acetylation on H3 K56, which resides within the globular core domain of histone 12 H3, was demonstrated to be critical in nucleosome assembly following DNA replication and repair, implying the importance of H3 K56 acetylation on genomic stability (19, 27, 93, 177). Similarly, histone acetylation on H3 and H4 tails is also required for efficient origin activation during 8 phase, probably facilitating the loading of replication factor(s) at origins (155). Histone acetyltransferase Gcn5p The conserved histone acetyltransferase Gcn5p is present in multiple HAT complexes in yeast, including ADA and SAGA, each of which contains both unique and shared protein subunits (12, 30, 41, 138). SAGA (Spt-Ada—Gcn5- Acetyltransferase), a TAF (ID-containing multiprotein complex, is involved in transcription regulation in S. cerevisiae (41). The SAGA complex preferentially acetylates H3 K14, followed by H3 K18, and to lesser extents H3 K9 and H3 K23 within nucleosomes. The ADA complex is similar to SAGA, preferentially acetylating H3 K14 followed by H3 K18 (53). Studies indicated that Gcn5p is not essential for maintaining the integrity of SAGA complex, the absence of Gcn5p does not affect the association of any other proteins within SAGA, suggesting a peripheral location of Gcn5p in the complex (171). The mutation of gcn5 E1730 is not defective in Ac-coA binding and that substitution has no gross effect on Gcn5p protein structure, but dramatically affect the HAT activity of Gcn5p (90, 154). 13 Although GCN5 is not an essential gene in yeast, Gcn5p is required for mitotic gene expression (83). In the absence of GCN5, cells accumulate at 6le phase, indicating important roles of Gcn5p in cell cycle progression (178). Aside from Gcn5p, Sas3p is required for both the HAT activity and the integrity of the NuA3 complex, which preferentially acetylates H3 in nucleosomes (53, 68). The combined functional loss of GCN5 and 8AS3 results in an extensive, global loss of H3 acetylation and arrests cells at 6le phase (53), suggesting that histone acetylation is critical for cell cycle progression. Recently, Vernarecci and colleagues reported that Gcn5p is physically localized to the centromere and it might epigenetically regulate the functions of centromere/kinetochore in mitosis (162). Consistent with this observation, components of histone deacetylases complex (Hda1p, Hda2p, Hda3p) are also involved in the regulation of histone function at centromere and chromosome segregation (71 ). Cross-talk between histone modifications Different kinds of modifications on histones can cross—talk to act in concert to modulate gene expression. For example, monoubiquitination at K123 of histone H2B mediated by Rad6p is a prerequisite for trimethylation of both H3 K4 and H3 K79 by COMPASS and Dot1p methyltransferases, respectively, which then lead to the silencing of genes located near telomeres (26, 118, 120, 152). Asymmetric di-methylation of histone H3 R2 prevents the methylation of H3 K4 as well as binding to the H3 tail by MLL thereby inhibiting the transcriptional activation (45, 14 :r-—---—:. __ 56). Moreover, effects of combinatorial histone modifications on protein binding were also observed. For instance, the 14-3-3 proteins bind to phosphorylated H3 (810) with higher affinity when H3 K14 becomes acetylated (164, 170), whereas the double modifications of H3 (810 phosphorylation and K14 acetylation) are required to dissociate heterochromatin protein 1 from the methylated H3 tail (105). Modifications of histone tails considerably extend the information potential of the DNA code and gene regulation. Histone modifications can influence each other in synergistic or antagonistic ways, thus mediating gene regulation. 15 Part III: Budding yeast mitotic spindle checkpoint and tension sensing mechanism To maintain genomic stability during cell division, the spindle assembly checkpoint (SAC) ensures that all chromosomes make proper bipolar attachment to microtubules arising from opposite spindle pole bodies and come under tension before the transition from metaphase to anaphase (124, 146, 179). When cells encounter a problem of lack of tension between sister chromatids, or when the kinetochore is not attached to spindles, the mitotic checkpoint complex binds and inhibits cd020p, the substrate adapter for the anaphase-promoting complex (APC). The APC is a ubiquitin ligase complex required for the destruction of the securin (Pds1p in yeast). Pds1p inhibits the activity of the separase (Esp1p in yeast), which is the protease required for the cleavage of cohesin and the separation of sister chromatids (6). Thus, SAC inhibits the APC activity, and blocks cell cycle at Gle phase until the spindle/kinetochore defect is corrected. It has been controversial and difficult to determine whether signals of tension and microtubule attachment are separate or interdependent, and which one is the primary checkpoint signal (119, 124). Aurora B kinase, the enzymatically active component of the chromosomal passenger complex (CPC), appears to be able to distinguish these two checkpoint activators (7, 124). CPC and Aurora B kinasellpl1p 16 The budding yeast CPC consists of lpl1p (Aurora B), Sli15p (INCENP), Bir1p (Survivin), and Nbl1p (Borealin-like protein) (4, 117, 156). In metazoans, INCENP, Survivin and Borealin twin together and form a three-helix bundle before it recruits Aurora B. The localization of Aurora B thus depends on all the other CPC components (66). Upon entry into mitosis, the CPC starts to associate with the inner centromere, a region located between sister kinetochores. After the separation of sister chromatids in anaphase, CPC relocates to the spindle midzone (4, 15, 76, 136). During metaphase, the CPC plays vital roles in I recruiting kinetochore proteins and those that are responsible for kinetochore— microtubule interactions (1, 24, 31, 73, 89, 92). Thus, the CPC is thought to be one of the most upstream regulators of centromere/kinetochore function (76). Indeed, the analysis of a temperature-sensitive ipl1 mutant, showed that lpl1p is required to activate the spindle checkpoint in response to defects in the tension between sister chromatids (7, 8, 125). Sli15p provides the binding sites for lpl1p, and their association increases the kinase activity of lpl1 p. Consistent with that, sli15 mutant cells exhibit similar phenotypes as ipl1 cells (79). Although Bir1p and Nbl1p have little effect on the kinase activity of lpl1 p, they might contribute to its substrate specificity; accordingly, mutant cells of bir1 and ndI1 also have problems in achieving accurate segregation (117, 139). One possible mechanism by which lpl1p activates the spindle checkpoint in response to the absence of tension is that lpl1p promotes the bi-orientation by destabilizing improper kinetochore-microtubule interactions (125). 17 Shuogshinngo1 p Another protein required for checkpoint activation in a tensionless crisis is Shugoshin (60). The Shugoshin proteins were originally reported to be important for centromeric protection of cohesin in meiosis l (72, 80, 104, 131), although in vertebrate cells Shugoshin proteins also protect centromeric cohesin in the pre- anaphase stage of mitosis. The sole budding yeast Shugoshin protein, Sgo1p, is required for cohesin protection only in meiosis l but not in mitosis (60, 72, 104). The protection of the cohesin complex by Sgo1p requires the recruitment of a protein phosphatase 2A (PP2A) to centromeric region. PP2A functions by counteracting the phosphorylation process on cohesin subunit and prevents its untimely degradation (81, 126, 173). In meiosis II and mitosis, the tension between two sister chromatids might force the Shugoshin-PPZA complex to be relocated from inner centromeric region to the inner kinetochore, consequently permitting cohesion degradation and chromosome segregation (43). Mitotic roles of Sgo1p in budding yeast were revealed by studies on a particular sgo1 mutant, which was defective in activating spindle checkpoint because of the loss of tension between sister chromatids, and yet, mutant cells responded normally to microtubule depolymerization (60), implied that Sgo1p, like lpl1p, plays a critical role in tension specific checkpoint activation. 18 Fission yeast contains two Shugoshin proteins, Sgo1p and 8902p, Sgo1p is specifically expressed in meiosis and functions in the protection of cohesin, whereas 8902p is expressed in both mitotic and meiotic cell cycle (80, 81) and is required to ensure bipolar kinetochore-microtubule attachments by promoting the localization of Aurora B to kinetochores (73, 161). Whether budding yeast Sgo1p directs lpl1p to kinetochore for tension sensing is unclear, but studies on budding yeast indicated that in meiosis I, Sgo1p recruits lpl1p to centromeric region for the continued presence of PP2A and the protection of cohesin (113, 175). Additionally, the loss of sgo1 in budding yeast causes the inability of cells to reorient misattached chromatids, suggesting that 8901 p is required for the kinetochore biorientation (59, 77). The localization of 8901 p to the centromere relies on a spindle checkpoint protein Bub1p (32, 78, 80). In fission yeast, studies suggested that Bub3p, along with its binding partner Bub1 p, are required to promote the conversion from chromosome mono-orientation to bi-orientation (169). Recent studies in fission yeast indicated that Bub1p phosphorylates histone H2A at a conserved residue serine 121 and that this phosphorylation is important for the centromeric localization of Sgo1p. H2A 8121A mutation phenocopies Bub1 p kinase-inactive mutant bub1-KD in defects of kinetochore-microtubule attachment and centromeric protection. And the double mutant of H2A S121A and bub1-KD show no additive defects in chromosome segregation, suggesting that the 19 phosphorylation of H2A and Bub1p kinase activity function in the same pathway for the centromeric localization of 8901 p (32, 74). 20 Part IV: Research interests and significance Failures in the detection of bipolar attachment and the tension between two sister chromatids can lead to aneuploidy and genomic instability. H3 Ser10 phosphorylation occurs in all eukaryotes examined thus far. Defects in H3 phosphorylation are associated with chromosome missegregation (167). Such errors in chromosome segregation might result in aneuploidy (6), which is a hallmark of many cancers and might contribute to tumorigenesis (28, 63). Expression of the Aurora kinases is well coordinated with H3 phosphorylation during 6le transition (22). Aberrant regulation of Aurora kinases also has been associated with chromosome missegregation and tumor progression (49). So far, little is known about how chromatin proactively participates in mitotic progression and cell cycle regulation. Shugoshin protein family was recently identified as a cohesin protector in meiosis l (40, 81). It also contributes to the tension sensing function in mitosis by promoting the biorientation of kinetochore (73, 77). It has been observed that the downregulation of human Sgo1p leads to chromosome instability in colorectal cancer cells (62). The build-up of tension on all pairs of sister chromatids signals the successful establishment of bipolar attachment, which is the prerequisite for cells to initiate anaphase and maintain accurate chromosome segregation (2). 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Boeke3, Tony Hazbunz, and Min-Hao Kuo“ Department of Biochemistry and Molecular Biology, and 1Programs in Genetics and in Cell and Molecular Biology, Michigan State University, East Lansing, MI 48824 2Department of Medicinal Chemistry and Molecular Pharmacology, 575 Stadium Mall Drive, West Lafayette, IN 47907-2091 3High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205 Received 24 July 2009 lAccepted 3 November 2009 *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. 41 ABSTRACT It has been firmly established that many interphase nuclear functions, including transcriptional regulation, are regulated by chromatin and histones. How mitotic progression and quality control might be influenced by histones is less well characterized. We show that histone H3 plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis. Prior to anaphase, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. Lack of tension due to erroneous attachment activates the spindle assembly checkpoint, which corrects the mistakes and ensures segregation fidelity. A histone H3 mutation impairs the ability of yeast cells to activate the checkpoint in a tensionless crisis, leading to missegregation and aneuploidy. The defects in tension sensing result directly from an attenuated H3-Sgo1p interaction essential for pericentric recruitment of 8901 p. Reinstating the pericentric enrichment of Sgo1p alleviates the mitotic defects. Histone H3, and hence the chromatin, is thus a key factor transmitting the tension status to the spindle assembly checkpoint. 42 INTRODUCTION During mitosis, chromatin goes through significant compaction and condensation to form metaphase chromosomes for segregation. While there is a wealth of information on the crucial roles played by chromatin structures and histone modifications in controlling transcription, replication, repair, and recombination (30), much less is known about how individual histones contribute mechanistically to mitotic progression and regulation. Forward and reverse genetic studies have suggested that histones, rather than being merely a part of the cargo during mitotic segregation, may play key roles in cell cycle progression and regulation. A histone H4 allele, hhf1-20, compromises the interaction between H4 and the centromere-specific H3 variant Cse4p, thus impeding centromeric functions and mitosis at the restrictive temperature (50). Two alleles of histone H2A (44) cause cold-sensitive growth defects and a significant increase in ploidy. This hyperploidy phenotype can be suppressed by mutations affecting a histone deacetylase, HDA1 (19). Similarly, the Gcn5p histone acetyltransferase genetically interacts with several inner kinetochore components and is physically mapped to the centromeric regions (57). Deleting the flexible tail domain of H3 and H4 results in mitotic delay (36) via a mechanism that can be suppressed by inhibiting the spindle assembly checkpoint activity (J.L. and M.H.K., unpublished data). Together, these data 43 warrant a more thorough examination of how chromatin may proactively regulate the process of mitotic segregation. The center stage for mitotic segregation and control is the kinetochore, a large proteinaceous complex assembled on centromeres. The ultimate function of the kinetochore is to capture the spindle microtubules during mitosis. The kinetochore-spindle attachment drives the movement of chromatids to daughter cells, and error-free attachment is essential for even partitioning of the entire genetic complement. To prepare for segregation, S-phase cells first establish sister chromatid cohesion by loading the cohesin complex to centromeres, pericentromeres, and selective regions in the chromatin arms (8, 38). Cohesion prevents precocious segregation before alignment of chromosomes at the metaphase midplate. When cells enter the prophase, spindles are assembled in and emanate from the two spindle pole bodies and are captured by kinetochores. The opposing poleward pulling force then generates tension between sisters and causes the congression of bioriented chromosomes toward the midplane (11). Equatorial alignment of all chromosomes leads to activation of the anaphase- promoting complex/cyclosome, which catalyzes polyubiquitylation and degradation of the securin protein Pds1p and cyclins (23, 60), and activation of the separase Esp1p, which cleaves the Mcd1p/Scc1p subunit of the cohesin complex (37), and hence permits sister chromatid segregation. One of the most critical mitotic control mechanisms for segregation is the spindle assembly checkpoint, SAC (31), which monitors both the kinetochore-spindle attachment and the resultant tension between sisters (43, 61). The tension sensing function of the SAC is critical for cells to detect and correct the so-called syntelic attachment; that is, both sister kinetochores capture spindles emanating from the same spindle pole body. This condition meets the attachment requirement but does not generate tension. Aneuploidy will result if this type of error is not eradicated. One of the factors essential for cells to detect the tensionless crisis is the Shugoshin protein (890) (7, 15, 21). Downregulation of human Sgo1 expression is linked to about half of the colorectal cancer cases analyzed in one study (16). Yeast Sgo1p is localized to the centromeres and pericentromeres (22, 46). Centromeres and pericentromeres are the most likely loci in which tension is generated and monitored (3, 14). Here we show that histone H3 plays a key role in the pericentric recruitment of 8901 p for the tension sensing function in mitosis. 45 MATERIALS AND METHODS Yeast strains and plasmid constructs. The yeast strains and plasmids used in this work are listed in Tables 2-1 and 2-2. Most of the strains were derived from the W303 background (JHY200) (1 ). Initially, phenotypic characterization, including cellular responses to various stresses (eg. see Fig. 1A) and suppression by 2pm 8601, was conducted in parallel with both JHY200 and its 8288C counterpart JHY205 (1). Both strains exhibited literally identical phenotypes. Subsequent studies were thus focused on the W303 background strains. To delete SGO1, primers 0361 and 0362 were used to PCR amplify the Kluyveromyces Iactis TRP1 selective marker from plasmid pBS1479 (47), and the PCR product was transformed into yeast cells for tryptophan prototroph selection. To tag endogenous Pds1p with 13xMyc, 0323 and 0324 were used to PCR amplify pFA6a-13Myc-TRP1 for yeast integrative transformation. Integration was verified with PDS1- specific primers 0325 and 0326 and TRP1- specific primers 0375 and 0376. Mcd1p-13Myc was created in a scheme identical to that used for Pds1p-13Myc, except for the use of MCD1-specific primers 0JL21, 0JL22 (for integration), and 0JL23 (for verification). To introduce a carboxyl-terminal 46 six-hemagglutinin (6xHA) tag to Sgo1p, yeast strain A10652 (22) was used for a genomic PCR (primers 0363 and 0364) that amplified the 6xHA—TRP1 fragment flanked by 8601 sequences. The resultant PCR product was transformed into yMK1243 and yJL145 to knock in the HA tag and the TRP1 marker, creating yJL343 and yJL344, respectively. Contrary to Kitajima et al. (25), who analyzed Myc-tagged SGO1, we did not observe discernible phenotypes associated with carboxyl-terminal HA tagging. To place the MCD1 gene under the control of the GAL1,1O promoter (i.e., yJL171 and yJL172), the His3MX6-PGAL1 sequence (42) was amplified with primers 0388 and 0389. The PCR fragment was agarose gel purified and transformed into yMK1329 for histidine prototroph selection. Yeast genomic PCR was conducted using primers 0390 and 0391 (derived from the MCD1 locus) against 0392 or 0393 (derived from the His3MX6 sequence) for verification. The correct integration of the GAL promoter to the MCD1 5' untranslated region was further verified by the inability of cells to grow in the presence of glucose. To introduce the G448 mutation to a green fluorescent protein (GFP)-marked strain, SBY214, the (hht1-hhf1)A::KanMX allele from JHY200 was PCR amplified using primers 0396 and 0397 and transformed into SBY214 to knock out HHT1- HHF1 (creating yJL118). Correct integration was verified by genomic PCR using primers 0398, 0399 (from the HHT1/HHF1 locus), and mk133 (from KanMX). To replace the remaining copy of H3 and H4, i.e., HHF2-HHT2, with either wild-type 47 (WT) H3 or the G448 mutant with the URA3 selective marker, plasmid pMK622 (WT or G448 mutant) was cleaved with SnaB l and EcoR l for integrative transformation. Ura+ colonies were isolated for genomic PCR to verify the integration. Genomic PCR using primers 0404 and 0405 was done to amplify an HHT2 fragment for See I digestion (indicative of the G448 mutation) and sequencing to rule out the existence of any unwanted mutations. To create congenic strains that differ only at the HHT2 locus, both the WT and G448 mutant versions of pMK622 were digested for yJL118 transformation, resulting in yJL292 (HHT2-HHT2::URA3-KTR5) and yJL293 (th2 G448-HHF2::URA3- K TR5). The yeast chromosomal copy of HHT2 was mutated by integrating pMK622 bearing the desired mutation. To create pMK622, pMK621 was first generated by PCR to amplify from yeast genomic DNA part of HHF2 and K TR5 with primers 0400 and 0401. The PCR fragment was digested with EcoR I and Sph l and cloned into the same sites of pJJ244 (18). pMK622 was made by inserting the Spe l (blunted) and Aat ll fragment from pQQ18 bearing the G448 mutation into the Narl (blunted) and Aat ll sites of pMK621, resulting in pMK622 with two 140- bp direct repeats spanning the URA3 gene. Histone mutations were generated by two-step PCR site-directed mutagenesis. Briefly, the desired mutations were incorporated into two complementary oligonucleotides, and each was used for PCR against 017 (downstream of HHT2 48 open reading frame [ORF]) or 019 (downstream of the HHF2 ORF) that hybridized outside the HHT2/HHF2 genes in pQQ18. The two PCR fragments (amplified using pQQ18 as the template by Pfu Turbo DNA polymerase [Stratagene]) were agarose gel purified and subjected to a second round of PCR. The complementary sequence at the 3' ends of these two PCR fragments allowed annealing and extension. Primers 017 and 019 were included in the reaction mixture to exponentially amplify the entire HHT2/HHF2 gene with the mutation. The final PCR fragments were then digested with Sell and Spe l and used to replace the WT Sal l/Spe l sequence in pQQ18. The entire H3 and H4 genes were sequenced for verification. This second-step PCR usually did not work well with Pfu Turbo polymerase. Taq polymerase was used to circumvent this problem. However, Taq polymerase frequently introduced unwanted mutations that had to be revealed by sequencing and, if detected, set aside. The original G448 mutation was obtain fortuitously in this manner. pMK573, a 2pm URA3 SGO1 plasmid, was created by PCR amplifying the 8601 ORF from yeast genomic DNA with primers 0329 and 0330 that included 42 bp of homology to the vector pMK572 (see below) at the 5' ends. The PCR fragment was cotransformed with Hind III- and EcoR l-digested pMK572 into yeast strain yMK839 (32). Ura+ colonies were subjected to DNA isolation and bacterial transformation. Miniprep DNA was analyzed by restriction digestion and sequencing across the insertion junctions for confirmation of a correct insert. 49 To create pMK572 (a multicloning sequence flanked by the ADH1 promoter and terminator), ADH-Ras-ABamH l (4) was digested with Sma l and self-ligated to remove the Ras sequence. The resultant plasmid, pMK322, then was used as the template for PCR using primers 0327 and 0328 to amplify the ADH1 sequence and the multicloning sites. The PCR fragment was cotransformed with Hind III- and EcoR l-digested YEplac195 (a 2pm URA3 plasmid [10]), resulting in pMK572. The multicloning sequence contains unique Hind lIl, Sma I, Sal I, BssH ll, MIu l, Sac I, Not I, Eag I, Sfi l, Ball, and EcoR I restriction sites. To create a construct for glutathione S-transferase (GST)-HA-Sgo1p in Escherichia coli, the 8601 ORF was PCR amplified with primers 0JL25 and 0JL26. The PCR fragment obtained was digested with Not I and ligated to Not I- linearized pMK595, resulting in in-frame fusion of 3xHA and 8601 (pJL51). To further generate 3 GST fusion of HA-SGO1 for bacterial production, 3xHA-SG01 was isolated from pJL51 and inserted into pSP72 (Promega) at the Hind Ill and Xho I sites. The BamH I-Xho I fragment was then isolated and ligated to the BamH l and Xho I sites of pGEX4T-1, generating pJL55. Yeast methods. Yeast growth media, conditions, and transformation were based on standard procedures (49). When appropriate, a 5% concentration of Casamino Acids (CAA) was used to substitute for synthetic amino acid mixtures as a selective medium for a uracil, tryptophan, or adenine prototroph. Yeast transformation was done by the lithium acetate method (9). 50 Chromosome stability of the WT and G448 mutant strains was examined according to Spencer et al. (51), using plasmid pYCF1/CEN3.L cut with Bgl II and transformed into yMK1243 and yJL145. Ura+ transformants were grown in CAA- Ura medium overnight and then plated directly onto yeast extract-peptone- dextrose (YPD) plates to allow colony formation and scoring. A tension sensing test using the pGAL-MCD1 mutant strains was done precisely to reference 15, using strains yJL171 and yJL172. For Western analyses of yeast proteins, yeast extracts were prepared by directly boiling cell pellets in appropriate volumes of 2x sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE) loading dye for 5 min, followed by vortexing with 1 lysate volume of glass beads (0.45 pm in diameter; Sigma) at room temperature for 5 min. The mixtures were boiled again for 5 min and then centrifuged at 14,000 x g at room temperature for 1 min. The supernatant was transferred to another tube for SDS-PAGE. Suppressor screening. To screen for a multicopy suppressor of the G448 mutation, a YEplac195-based library was constructed by ligating 1 to 3 kb of Dpn l-digested yeast genomic DNA (enriched by sucrose gradient ultracentrifugation) to the BamH I site of YEplac195 (10). G448 mutant cells transformed with 0.3 pg of library DNA in each of 20 transformation reactions were plated onto CAA-Ura, and Ura+ colonies (estimated to be about 60,000 in total) were then replica plated onto YPD plates supplemented with 20 uglml benomyl and incubated at 30°C. 51 Initial benomyl-resistant colonies were regrown in YPD and streaked onto 5- fluoroorotic acid plates to select for clones that had lost the URA3 plasmid. Benomyl sensitivity tests were repeated to screen for clones displaying plasmid- dependent benomyl resistance. Candidates were subjected to DNA isolation for E. coli transformation. Plasmid DNAs were isolated for restriction digestion and retransformation into the G448 mutant strain to verify the suppression phenotypes, including hypersensitivity to benomyl, heat shock, and cold. The identity of the insert was revealed by DNA sequencing using universal M13 primers. Chromatin immunoprecipitation (ChlP). ChlP was conducted as previously described (28). To quantify the ChlP results, the semiquantitative PCR products were purified and resolved by 9% PAGE and stained with ethidium bromide. The captured gel images were then quantified by NIH Image. Intensities of the GEN/pericentric fragment were compared to that of a common internal control (the DED1 or, in some cases, the PGK1 locus). The ratio was further normalized to 0.1% input DNA (set at 1.0) for PCR amplifications carried out in parallel with all of the reactions. The ChlP data were obtained from at least three independent yeast cultures. Recombinant Sgo1p preparation. To express and purify GST-HA-Sgo1p from E. coli, 125 ml of BL21 codon+ cells (Stratagene) were subjected to induction (optical density at 600 nm of 0.5 to 0.6 in LB-ampicillin medium) with 1 mM 52 isopropyl-B-D-thiogalactopyranoside (IPTG) at 37°C for 4 hours. Cells were pelleted (5,000 x g for 5 min) at 4°C and sonicated in 5 ml of HEMGT buffer (25 mM HEPES, pH7.9, 12.5 mM MgCl2, 150 mM KCI, 0.1 mM EDTA, 0.1% Tween 20, 10% Glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) six times for 20 sec each time with 1-min chilling intervals. The soluble fraction was obtained by centrifugation at 10,000 x g for 15 min at 4°C. Sgo1p was purified by incubating the cytosol with 200 pl of reduced glutathione Sepharose 4B beads (Amersham) at 4°C for 1 hour. Bound Sgo1p was washed twice with 5 ml of binding buffer for 5 min each time, followed by another wash with 1.5 ml of binding buffer, and transferred to a microcentrifuge tube. The elution was done by gently rocking beads in 200 pl of 50 mM glutathione in the binding buffer for 30 minutes at 4°C. Eluate was collected, and the elution was repeated once under the same conditions. Two batches of eluate were separately dispensed and stored at -70°C. The yield and purity of GST-HA-Sgo1p were estimated by SDS-PAGE. Sgo1p interaction with H3, nucleosomal particles, and oligonucleosomes. Histones were prepared according to reference 6. Recombinant yeast histone H3 and reconstituted nucleosomal particles were gifts from K. Luger. For pulldown assays, approximately 5.5 pg of soluble recombinant Sgo1p was incubated with about 3.4 pg of yeast histones or oligonucleosome liberated by micrococcal nuclease digestion from a nuclear preparation with an A250 of 200 (WT and G448 mutant) in 150 pl of HEMGT buffer at 4°C for 1hour. Six microliters of glutathione 53 beads and 150 pl of the HEMGT buffer were added to the reaction mixtures, which were rocked gently at 4°C for another hour. Beads were washed with 500 pl of HEMGT buffer three times for 5 min each time, followed by boiling in 2x SDS-PAGE loading buffer for 5 min. Eluate was resolved by 15% SDS-PAGE and blotted for anti-H3 Western analyses. The anti-H3 antibodies were rabbit polyclonal serum made in house and raised against the peptide H2N- ClQLARRIRGERA-COOH. The Western assay used a 1:2,000 dilution of the primary antibody. For far-western assays, 0.3 pg of yeast histones was first resolved by 15% SDS-PAGE and then blotted onto a polyvinylidene difluoride (PVDF) membrane. Small strips of the membrane were blocked with 10% milk in TTBS (0.9% NaCl, 0.1% [vol/vol] Tween 20, 50 mM Tris, pH7.4) at room temperature for 30 min, followed by two 10-min TTBS washes. For the Sgo1p- histone interaction, the membrane was incubated with 0.3 pg of GST-HA-Sgo1 in 3 ml of TTBS buffer supplemented with 0.1% gelatin. The binding was conducted at 4°C with gentle rocking for 3 to 5 hours. The membrane was then subjected to regular Western blotting procedures using anti-HA monoclonal antibody 12CA5 (Roche) at a 1:750 dilution. 54 RESULTS An H3 mutation causes mitotic chromosome instability To identify histone H3 mutations that affect chromatin metabolism in the budding yeast Saccharomyces cerevisiae, we fortuitously encountered a Gly-to-Ser mutation of histone H3 at position 44 (referred to as G448 henceforth) that caused hypersensitivity to benomyl, cold temperature (14°C), heat shock (37°C), and hydroxyurea (HU; Fig. 1A). Mild growth defects were also caused by UV light; however, methyl methanesulfonate did not have an obvious effect. At 26°C, the doubling time of log-phase cultures of the WT and G448 mutant strains are 110.9 :I: 7.6 and 129.1 1: 8.8 min/generation, respectively (see Fig. S1 in the supplemental data). Fluorescence-activated cell sorter analyses of cultures grown at 14°C, 26°C, or 37°C did not reveal obvious defects in ploidy or cell cycle control (data not shown). These results demonstrated that the H3 G448 mutation conferred pleiotropic phenotypes and that cells were defective in dealing with certain stresses. It is worth noting that the some of the observed phenotypes, in particular, those related to mitosis (see below), were not specific to the original serine substitution, as an alanine mutation introduced at Gly44 resulted in literally identical phenotypes (data not shown). All of the analyses henceforth were conducted using the G448 allele. Residue G44 resides at the end of a unique region of H3 (K37-G44) that connects the flexible tail domain (residues 1 to 36) and the well-structured core 55 (residues 45 to about 130) (Fig. 1B) (34). The small region of K37-G44 inserts through the aligned minor grooves of two DNA gyres and then makes a sharp (3 turn into the nucleosome core. This conserved 8 turn structure brings the K42 carbonyl oxygen to a topographically favorable position to form a hydrogen (H) bond with the amide group of T45 (Fig. 1B, right) (58). In addition, the amide group of G44 may also H bond with the DNA phosphate backbone (34). By contrast, P43 does not appear to participate in H bond formation with any nearby atoms of DNA or histones, suggesting that G44 plays unique roles in determining the architecture of H3 as it transitions from the extended flexible tail domain into a rigidly structured nucleosomal core. Given the unique position of G44 and the cellular hypersensitivity of the mutant to such genotoxic insults as benomyl, HU, and UV light, we suspected that some of the observed phenotypes were results of aberrations of chromatin metabolism. This work focused on the mitotic functions of G44. To examine the role of H3 G44 in mitotic chromosome metabolism, we first measured the chromosome stability by introducing an artificial, non-essential chromosome into WI' and G448 mutant cells bearing the ade2-1 ochre mutant allele (51). adeZ yeast cells accumulate a red pigment and produce red colonies, whereas Ade” colonies are white. The artificial chromosome used in this study contained a mutant SUP11 tRNA gene that suppresses the ochre mutation. Recipient cells thus formed white colonies. The frequency of first-division chromosome loss of WT and G448 mutant cells was measured (Fig. 2A). 56 Compared with WT cells, G448 mutant cells lost the indicator chromosome at a much greater rate (71 losses versus <1 loss per 1,000 divisions). In addition, red sectoring of G448 mutant cells was commonly accompanied by indentation of the othenrvise round, smooth colonies (see closeups). This phenomenon may be due to slower cellular growth rates arising from aneuploidy of native chromosomes, and is consistent with the notion that the G448 mutation causes stochastic mitotic errors that retard growth (Fig. 81). We next investigated the molecular defects underlying the benomyl hypersensitivity of G448 mutant cells. Eukaryotes respond to benomyl-triggered microtubule depolymerization by activating the SAC, which stabilizes the securin protein (Pds1p in yeast) and arrests cells at Gle phase (31). When G448 mutant and WT cells were examined for the budding index and the Pds1p level after benomyl treatment, we observed a similar efficiency of Gle arrest (Fig. 3A) and upregulation of Pds1p for up to 180 minutes (Fig. SB), indicating that dissolving microtubules by benomyl was sufficient to trigger the normal spindle checkpoint responses. However, if yeast cells were plated on drug-free YPD medium to assess viability after 2, 4, or 6 hours of benomyl treatment, mutant cells lost viability more quickly (Fig. 2B). These results suggested that a lethal error, most likely missegregation (47), occurred when cells resumed mitosis after benomyl removal. To examine whether the segregation fidelity of G448 mutant cells was impaired after the brief benomyl shock, we used a GFP-marked haploid strain in which a lac operator array was integrated to the TRP1 locus 12 kb from 57 CEN4 (53). In this background, one of the two copies of H3 was deleted and the remaining one was replaced with the G448 allele. This new GFP-marked G448 mutant strain exhibited phenotypes comparable to those seen with the original, non-GFP-marked strain, including sensitivity to benomyl, cold, and heat (data not shown). When GFP-marked yeast cells were allowed to recover from the 2-hr benomyl shock, the WT strain exhibited a low rate of missegregation (1.1% t 0.2% of the G1-phase cells showed two GFP dots), whereas the G448 mutant cells showed 11.3% :t 0.7% co-segregation of both Chromosome IV sister chromatids (Fig. 2C). Without benomyl treatment, both strains had a low incidence of missegregation, although there seemed to be a mild elevation in the G448 background (1.5% :l: 0.6% versus 3.3% :l: 0.2%). These data suggested that the G448 mutant cells were unable to either detect or to correct the erroneous kinetochore-spindle attachment that frequently happens during recovery from benomyl toxicity (56). Together, the results in Fig. 2 strongly suggested that the G448 mutation caused chromosome instability by impairing a cellular control mechanism that ensured the bipolar microtubule-kinetochore attachment before committing to anaphase. G448 mutant cells are defective in tension sensing Prior to the anaphase onset, sister chromatids are held together by the cohesin complex (27, 39). Cohered chromatids resist the poleward pulling force from 58 bipolar spindles, hence generating tension. The SAC can be activated by attachment errors or by the lack of tension. Given that G448 mutant cells exhibited normal SAC activation upon microtubule depolymerization (Fig. 3), and that attachment errors likely eluded cellular scrutiny (Fig. 2C), we tested whether the SAC was activated in G448 mutant cells under a tensionless condition. To examine the tension-sensing function, we used a method (15) whereby the MCD1 gene encoding an essential cohesin component was placed under the repressible control of the GAL1 promoter (pGAL). Shifting cells from galactose to glucose medium represses MCD1 expression and hence perturbs the formation of sister cohesion, which consequently prevents tension establishment but does not influence the spindle-kinetochore attachment (2). This tensionless crisis activates the SAC. Using this approach, we compared the SAC activation of the WT and G448 mutant strains. Yeast cells bearing the pGAL-MCD1 gene were grown to log phase in YPgal medium and synchronized at G1 by o-factor. Cells were then released into either YPgal (Gal to Gal) or YPD (Gal to Glc) and harvested at different time points before the cells were again arrested at the next G1 stage by o-factor. Western blotting was conducted to compare the level of Pds1 p to see whether the tensionless crisis triggered the activation of SAC in G448 mutant cells (Fig. 4). Gal-to-Gal treatment allowed yeast cells to continue dividing, as evidenced by the degradation of Pds1 p when cells finished the first round of mitosis. In contrast, shifting WT cells from galactose to glucose (Gal to Glc) stabilized Pds1p, demonstrating the activation of the SAC. 0n the other 59 hand, G448 mutant cells degraded Pds1p about 60 minutes after release from the first G1 block under both Gal-to-Gal and Gal-to-Glc conditions, indicating that the G448 mutation impaired the ability of cells to detect or to respond correctly to the tensionless condition. Shugoshin is a high-copy-number suppressor of G448 tension-sensing defects The above results revealed that the G448 mutation of H3 caused a defect in the tension-sensing function in mitosis. To further delineate the molecular mechanism underlying this new function of histone H3, we conducted a high- copy-number suppressor screen for genes that could rescue the benomyl hypersensitivity phenotype. From about 60,000 transformants representing ninefold coverage of the S. cerevisiae genome, we obtained 20 independent clones. Sequencing results revealed five nonoverlapping genomic DNA inserts (data not shown). Histone H3 was one of the clones, thus validating the screening. Two multifunctional transcriptional activators, YAP1 and CAD1, were found multiple times in the screen. However, these two proteins have been shown to confer resistance to a variety of stresses, including benomyl (40, 59), and hence were considered unlikely to be relevant to the tension-sensing defects. Another candidate contained the intragenic region between the BIO1 and YGR287C genes without a discernible gene in this fragment. No further work was conducted on this clone. The last and most likely candidate contained the 8601 gene missing the first 34 amino acids and the 5' promoter element (data 60 not shown). Sgo1p plays a key role in mitotic tension sensing (15, 21). The identification of this gene, albeit slightly shorter, as a suppressor agreed well with the observed mitotic phenotypes of the G448 mutant. We posited that a cryptic promoter in the vector activated the expression of a slightly truncated but functional Sgo1p that was responsible for the observed suppression. To verify that 8901 p was a bona fide suppressor for G448, we cloned the SGO1 ORF, with or without the first 34 amino acids, downstream of the constitutive ADH1 promoter in a 2pm plasmid. G448 mutant cells transformed with different SGO1-overexpressing plasmids were tested for responses to benomyl, cold, HU, and UV light. Overexpressing Sgo1p alone rescued hypersensitivities to benomyl and cold but not to HU or UV light (Fig. 5A), indicating that the suppression was specific to mitotic defects caused by this mutation. Deleting the first 34 amino acids did not affect the suppression (data not shown), consistent with the notion that the original 34-amino acid truncation did not alter the normal function of Sgo1p. Critically, overproducing Sgo1p alleviated the phenotypes of benomyl- induced lethality, chromosome missegregation (Fig. 5B), and the inability to stabilize Pds1p under a tensionless condition (Fig. 5C). The expression of endogenous SGO1 was normal at both transcription and translation levels (Fig. 5D), arguing against the possibility that the G448 mutation downregulated the abundance of 8901 p. Together, these genetic data strongly suggested that Sgo1p acted downstream of H3 and that the G448 mutation undermined the function of Sgo1p in mitosis. 61 G448 mutation attenuates Sgo1p interaction and localization Based on the genetic interaction between SGO1 and H3 revealed by the high- copy suppressor screening, we suspected that the G448 mutation might affect a function of Sgo1p. Sgo1p is enriched at centromeres and pericentric regions, where tension is most likely monitored by the hitherto unspecified machinery (22). Mutations that eliminate the 8901 p recruitment to these loci also cause tension sensing defects (5). We thus used ChIP to examine whether the recruitment of Sgo1p was impaired by the G448 mutation. To this end, we tagged the chromosomal SGO1 with HA at the 3' end. The HA tag did not alter the benomyl (hyper)sensitivity of either WT or G448 cells (data not shown). Mitotic cells were harvested for ChIP, and immunoprecipitation efficiency compared by semiquantitative PCR. The ChlP results showed that Sgo1p was present at both centromeres and pericentromeres in WT cells (Fig. 6). However, pericentric Sgo1p was significantly reduced in G448 mutant cells, while the centromeric localization of 8901 p was normal. The pericentric Sgo1p domain was narrow, for PCR fragments 9.1 kb from CEN16 and 8.3 kb from CEN1 were so weak that it did not show a significant difference between the two strains (Fig. 63). At the SPT15 locus 313.3 kb from CEN5, there was essentially no Sgo1p detected in either background. The differential effects on centromeric and pericentric recruitment of 8901 p were consistent with the fact that the canonical H3 is replaced by Cse4p at centromeres (35). Mutating H3 thus had no effect on Sgo1p-centromere association. In conclusion, the ChlP data demonstrated that 62 the G448 mutation specifically diminished the pericentric localization of Sgo1p during mitosis. The genetic and ChIP data shown above predicted that histone H3 and Sgo1p interacted for mitotic tension surveillance, and that this association was attenuated by the G448 mutation. To test these predictions biochemically, we expressed GST and HA double-tagged Sgo1p in E. coli and subjected it to in vitro binding assays using core histones or oligonucleosomes purified from WT and G448 mutant yeast cells. In pulldown assays, histone H3 was incubated with 8901 p and trapped by glutathione beads. Anti-H3 Western blotting then was used to compare the relative abundance of WI’ and G448 mutant H3 trapped by recombinant Sgo1p. In a parallel far-Westem approach, the purified yeast histones were first resolved by SDS-PAGE and then blotted onto membrane. After incubating the blot with HA-tagged recombinant 8901 p, anti-HA antibodies were used to probe the trapped Sgo1p by each histones. Figure 7A (pulldown) shows that WT free and nucleosomal H3 bound Sgo1p efficiently, whereas G448 mutant H3 bound to Sgo1p with a diminished affinity. In the far-Western assay (Fig. 7B), HA-Sgo1p also bound more effectively to WT H3. lmportantly, the H3- Sgo1p interaction was independent of other histones, and 8901 p only interacted with H3 (Fig. 7B). Furthermore, chicken H3, recombinant yeast H3, and reconstituted nucleosomal core particles all bound Sgo1p (Fig. 82). It is noteworthy that the G448 mutation did not completely eliminate Sgo1p binding (compare the 0.6x and 1x histone doses in the left panel of Fig. 7A). The remnant 63 affinity for Sgo1p provided a molecular explanation for the dosage-dependent suppression seen in vivo. Together, these results demonstrated that the molecular basis for the G448 mitotic defects was an attenuated interaction between histone H3 and Sgo1p, resulting in the loss of 8901 p from pericentromeres. While the centromeric enrichment of 8901 p was not affected by the H3 mutation, the downregulation of pericentric Sgo1p enrichment was likely sufficient to cause the tension sensing phenotypes associated with the G448 mutation. Reinstating pericentric Sgo1p partially rescues G448 mitotic defects If the only purpose of the observed H3-Sgo1p interaction was to bring the latter to pericentromeres for tension surveillance, artificially tethering Sgo1p back to the pericentric loci should effectively eliminate the mitotic phenotypes of G448 mutant cells. On the other hand, if the interaction between H3 and Sgo1p was also important for mitotic control, forcing Sgo1p to pericentromeres by an approach other than the natural H3-Sgo1p association may, at best, only partially suppress the mitotic defects caused by the G448 mutation. The challenge of artificial tethering a protein to the pericentric regions in the budding yeast is that the pericentromeres do not form heterochromatin like other eukaryotic systems and that there is no known epigenetic mark specific to this region. Nonetheless, the ChlP data in Fig. 7A suggested that the establishment of an Sgo1p domain was likely nucleated from the H3-independent recruitment at the centromere, where the canonical histone H3 was absent. The physical association of H3 and Sgo1p then allowed the latter to spread outward to generate a pericentric domain (see Discussion for details). If this is so, fusing Sgo1p to a general chromatin- binding motif may reestablish the pericentric retention of Sgo1p and would allow us to assess the effect of pericentric recruitment of 8901 p in the G448 mutant background. To regenerate the pericentric enrichment of 8901 p in the G448 mutant background, we fused a bromodomain (BD) from Bdf1p to the carboxyl end of Sgo1p and a trimeric HA tag to the amino terminus. Bdf1p binds both histones H3 and H4 (41), and the bromodomain in different proteins has been shown to interact with acetyllysine (5, 17). Genome-wide ChlP analyses detected comparable histone acetylation between pericentromeres and the bulk of the yeast genome (29), suggesting that a general (acetylated) histone-binding module such as the bromodomain may complement the weakened affinity of Sgo1p for G448 mutant H3. As shown in Fig. 8A, this indeed was the case. When Sgo1p and Sgo1p fused to the bromodomain domain (Sgo1p-BD) were introduced to sgo1A mutant cells expressing the WT or G448 mutant allele of H3, Sgo1p—BD exhibited efficient pericentric enrichment (Fig. 8A and C), whereas Sgo1p without the BD remained underrepresented at pericentromeres. These two alleles of 8901 p were expressed at comparable levels (Fig. 88), indicating that the bromodomain fusion increased the affinity for a histone at the pericentromeres. lmportantly, the reinstatement of pericentric 8901 p was 65 accompanied by partial suppression of benomyl hypersensitivity, loss of viability following benomyl shock, and stabilization of Pds1p during MCD1 shutdown (Fig. 9A to C). Together, these results strongly suggested that dislodging Sgo1p from pericentromeres was the major underlying cause of the G448 mutant mitotic defects. In summary, histone H3 plays a critical role in Sgo1p recruitment to the mitotic pericentromeres, where the tension between sister chromatids is likely monitored. Genetic data strongly suggest an intimate association between H3 and Sgo1p, a notion supported by ChlP and biochemical assays. We thus conclude that the mitotic tension sensing function depends critically on the appropriate association between histone H3 and Sgo1p in the pericentric regions. 66 DISCUSSION Establishment of a pericentric Sgo1p domain This work uncovers a novel function of histone H3 in mitotic checkpoint control. By recruiting Sgo1p specifically to the pericentric region, histone H3 was shown for the first time to play a role key in segregation fidelity. As the G448 mutation selectively compromises the pericentric enrichment of 8901 p but apparently leaves centromeric 8901 p unaltered (Fig. 6), we suspect that 8901 p localization is nucleated by an H3-independent mechanism at the centromere/kinetochore, followed by spreading toward the pericentric regions via direct association with histone H3. This scenario is analogous to the mechanism of establishing the telomeric heterochromatin in yeast (12), in which Rap1 p nucleates the heterochromatin formation by binding to the telomeric C1-3A repeats. Silent Information regulator, or Sir, proteins are recruited by Rap1p. Via the direct association between Sir3p/Sir4p and the amino terminal tails of histones H3 and H4 (6), Sir proteins spread from the nucleation site to the neighboring region (45). Inserting the telomeric C1-3A repeats into an ectopic location is sufficient to create a new transcription silent domain, conclusively ruling out the need for a specific sis-acting element for Sir protein spread or retention (52). Similarly, relocating the centromere to an ectopic site establishes a new 8901 p domain (22). 67 The notion that the H3-Sgo1 p association is critical for mitotic regulation is also supported by the observation that the G448 allele caused mild benomyl sensitivity in the presence of the WT counterpart (Fig. 83). It is conceivable that the concomitant incorporation of wild type and G448 mutant H3 into nucleosomes in the pericentric region results in interrnedlate affinity for Sgo1p, thus resulting in visible but not as severe defects in coping with benomyl toxicity. The protein directly responsible for bringing Sgo1p to the centromere remains unidentified. Among the 30-plus kinetochore proteins and the SAC components, one of the likely Sgo1p recruiters is Bub1 p. Although deleting BUB1 causes loss of Sgo1p in centromeres and pericentromeres (7, 22), the physical evidence for Bub1p-Sgo1 p association is lacking to date. Moreover, Bub1 p is a kinase that is essential for SAC assembly at the kinetochore (33). It is thus also possible that Bub1p regulates the protein that physically brings Sgo1p to the centromere. Given the diverse posttranslational modifications of histones, it is tempting to speculate about an alternative model in which a novel epigenetic mark at the pericentromeres facilitates Sgo1p retention. For example, Gcn5p and Hda1p have been linked genetically to mitotic segregation (19, 57), and Ser31 phosphorylation of the human H3.3 variant is enriched at the pericentric heterochromatin (13). However, we do not believe this notion is very likely because Sgo1p interacts with bulk H3 from yeast and chicken, as well as recombinant H3 synthesized in E. coli (Fig. 82), arguing against an H3 68 modification essential for Sgo1p binding. Nonetheless, we do not rule out the possibility of an auxiliary function of an unidentified, pericentromere-specific modification. Nor do we disregard a notion that a chromosome arm-specific epigenetic mark prevents the H3-Sgo1p interaction outside the pericentric region. Due to the absence of functionally and structurally distinct pericentromeres in the budding yeast, the identification of this epigenetic mark presents a major technical challenge. Tension sensing-specific relationship between H3 and Sgo1p While the G448 mutation causes pleiotropic phenotypes (Fig. 1A), the genetic and physical interactions between H3 and Sgo1p are specific for tension sensing based on multiple lines of evidence. First, overexpressing Sgo1p does not have an effect on a histone H4 allele, hhf1-20, that impairs centromere/kinetochore functions (50) (Fig. S4). The hta1-300 allele of histone H2A, which causes defects in ploidy control, is also insensitive to SGO1 overexpression (I. Pinto, personal communication). Second, we have yet another H4 mutant allele, R358, that also causes benomyl hypersensitivity (J. Luo, X. Xu, and M.-H.Kuo, unpublished data). 2pm SGO1 constructs fail to suppress this mutant. In contrast, other candidates fished out of the original suppressor screens, such as CAD1 and YAP1, which have been linked to multidrug resistance, rescued the benomyl hypersensitivity associated with either H3 G448 or the H4 R358 mutations (data not shown). Third, although G448 mutant cells are sensitive to UV light and HU, neither defects is responsive to 8901 p overproduction (Fig. 5A), 69 indicating that, whereas the G448 mutation may also impair the control of DNA metabolism or damage repair, this is an SGO1-independent function. Indeed, deleting SGO1 does not render cells sensitive to UV light or HU (data not shown). Lastly, the G448 mutation does not affect the pericentric recruitment of the cohesin component Mcd1 p (Fig. 87), indicating that pericentric H3 is a specific interaction target for Sgo1p, whereas Mcd1 p (i.e., the cohesin complex) is recruited to the pericentric region via a different pathway (8). The last notion is consistent with the fact that cohesin also forms clusters in the chromosome arms whereas 8901 p is absent therein (19). One might argue that the G44 mutation causes a general transcriptional defect that indirectly impairs Sgo1p recruitment or acts in a different pathway that leads to the tension sensing defects observed in this work. We do not think this notion likely because when we examined the transcription of a variety of genes representing functions in mitosis, checkpoint control, nutrient responses, mating, and transcriptional silencing, we did not detect any discernible differences between WT and G448 mutant cells (Fig. 86). The only transcription defect that we found associated with G44 mutations is the deregulation of transcription driven by cryptic promoter elements within certain ORF (20) (EMH, and JB, submitted). However, this phenotype was not affected by SGO1 overexpression (data not shown), further strengthening the mitosis-specific relationship between H3 and Sgo1p. 70 While the current work focuses on the G44 allele of H3, which was obtained fortuitously in an attempt to create other mutants with mitotic phenotypes, our preliminary data indicated that residues surrounding Gly44 of H3 perform similar mitotic functions and that targeted mutations thereof lead to tension sensing defects similar to those of the G448 mutant (J. Luo and M.-H. Kuo, unpublished data). We suspect that G44 is part of a "tension-sensing motif" that makes physical contact with 8901p. Biochemical and molecular studies are in progress to test this hypothesis. Other factors related to Sgo1p and tension sensing Sgo1p belongs to the Shugoshin family proteins found in many eukaryotes. Several conserved proteins, including Bub1p, PP2A, and microtubules, are linked to Shugoshin functions. For example, Bub1 homologues are critical for centromeric and pericentric localization of Shugoshin in different organisms (7, 22, 24, 55). Human and yeast Shugoshin proteins collaborate with a specific form of protein phosphatase 2A (PP2A) to protect meiotic cohesin (26, 46, 54). The Sgo1p-PP2A cooperation appears to be independent of the Sgo1p-H3 interaction, for deleting CDC55 or RTS1, the key B and 3' regulatory subunits of PP2A complexes, does not affect the ability of the 2pm SGO1 to suppress the benomyl hypersensitivity phenotype of G448 mutant (Fig. S7). The human and Xenopus Shugoshin bind and stabilize mitotic kinetochore microtubules (48). It is unclear whether budding yeast 8901 p does so. However, this microtubule stabilization function is clearly a postbiorientation activity preceded by the tension 71 surveillance function. Therefore, we believe that histone H3 is the first protein shown to act directly upstream of Sgo1p in mitotic tension sensing. A key player in correcting tension defects is the Aurora B kinase encoded by IPL1 in budding yeast. lpl1p destabilizes spindle-kinetochore attachment before anaphase nucleation, hence permitting correction of attachment errors (2). We observed synthetic phenotypes when lpl1p or one of its partners, Sli15p, was overexpressed in an sgo1A or H3 G448 background (Fig. 88). These genetic interactions suggest that increasing the detachment activity of lpl1p/Sli15p brings about a molecular defect (i.e. spindle detachment) similar to that caused by benomyl treatment. Lacking of functional Sgo1p or intact H3 causes growth defects. It is critical that the BD-Sgo1p fusion protein can be recruited to the pericentromeres. However, close examination of the efficacy of suppression revealed that Sgo1p-BU only partially corrected the mitotic defects (Fig. 8). The enrichment of recombinant 8901 p is consistent with the "nucleation-and-spread model" depicted above for establishing the 8901 p domain. However, the inability of the Sgo1p-ED chimeric protein to completely restore benomyl tolerance suggests two possibilities. First, the bromodomain may somehow interfere with the tension sensing function of Sgo1p but not the ability to interact with the nucleating protein for recruitment. Alternatively, the physical interaction between 8901p and H3 at G44 might be critical for the tension sensing function. For 72 example, if Sgo1p docks directly to G44 and nearby residues, the bromodomain may place Sgo1p toward the far end of the amino terminal tail domain of H3 or even at a different spot on the nucleosomal particle where the H4 tail resides. If this is so, the activity of Sgo1p in tension sensing may be attenuated even though it has been directed back to the pericentromeres. Structural and biochemical experiments are required to examine these questions. 73 ACKNOWLEDGMENTS We are grateful for C. David Allis, Angelika Amon, Sue Biggins, Jennifer Gerton, Phil Hieter, Karolin Luger, Mitchell Smith, and Fred Winston for generous supply of materials; Sue Biggins for advice; and lnés Pinto for sharing unpublished results. We thank Sue Biggins, Sharon Dent, and John Wang for critical reading of the manuscript, Yang Liu for creating the original G448 allele, and David Almy, No-Ya Hung, and Andy Lin for technical assistance. This work was partly supported by a grant (CMB 0315542) from the National Science Foundation to M.-H.K. J.L. contributed to all of the experimental data, except the following. X.X. conducted suppressor screening and initial characterization of 2pm SGO1. Genetic interactions among IPL1, H3 and SGO1 were contributed by H.H. and TH. Work related to cryptic promoter regulation was contributed by E.M.D. and J.D.B. M.-H.K. coordinated the project. There is no conflict of interest for this work. 74 TABLE 2-1. Yeast strains used in this study Source . or Strain Relevant genotype r efer en c e A10652 W303 MA Ta SGO1-6HA (22) JHY200 MATa ade2-1 cam-100 his3-11,15Ieu2-3,112trp1-1 ura3-1 hht1- (1) hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2-htb2zzHPH pJH33 [ARS CEN URA3 HTA1-HTB1 HHT2-HHFfl SBY214 MATa ad92-1 bar1A can 1-100 his3-11::pCUP1-GFP12-Iacl12::HI83 (2) Ieu2,3-112 lysZA trp1-1::IacO::TRP1 ura3-1 yJL118 MA Ta ade2-1 bar1A can1-100 his3-11::pCUP1-GFP12-lacl12::HI83 This Ieu2,3-112 IysZA trp1-1::IacO::TRP1 ura3-1 hht1-hhf1::KAN study yJL145 MATa ade2-1 can1-100 hi53-11,15leu2-3,112 trp1-1 ura3-1 hht1- This hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2-htb2::HPH study pMK4396448 [ARS CEN LEU2 HTA1-HTB1 hht2-G448-HHF2] yJL171 MA Ta ade2-1 bar1A can 1-100 his3-11,15::pGAL-MCD1::HIS3 This Ieu2-3,112 trp1—1::PD81-Myc13::TRP1 ura3-1 hht1-hhf1::KAN study hht2-hhf2::KAN hta1-htb1::Nat hta2-htb221HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] yJL172 MA Ta adeZ-1 bar1A can 1—100 his3—11,15::pGAL-MCD1::H/83 This Ieu2-3,112trp1—1::PD81-Myc13::TRP1 ura3-1 hht1-hhf1::KAN study hht2-hhf2::KAN hta1-htb1zzNat htaZ-htb2::HPH pMK4396448 [ARS CEN LEU2 HTA1-HTB1 hht2-G448-HHF21 yJL292 MA Ta adeZ-1 bar1A can1-1 00 his3-11::pCUP1-GFP12-lacl12::H/S3 This Ieu2,3-112 IysZA trp1-1::IacO::TRP1 ura3-1::hht2-hhf2::HHT2- study HHF2::URA3 hht1-hhf1::KAN yJL293 MA Ta ad92-1 bar1A can1-100 his3-11::pCUP1-GFP12-lacl12::H/S3 This leu2,3-112 lysZA trp1-1::IacO::TRP1 ura3-1::hht2-hhf2::hht2-G448- study HHF2::URA3 hht1-hhf1::KAN yJL343 MA Ta ade2-1 can 1—100 his3—11, 15 leu2—3, 112 trp1—1::SGO1- This 6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat study hta2-htb2zzHPHpQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] yJL344 MATa ad92-1 can 1—100 his3—11,15Ieu2—3,112 trp 1-1::SGO1- This 6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat study hta2-htb2zzHPH pMK439G44S [ARS CEN LEU2 HTA1-HTB1 hht2- G448-HHF2] yMK1243 MATa ade2-1 can1—100 his3-11,15 leu2-3,112 trp1—1 ura3-1 hht1- This hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat htaZ-htb222HPH pQQ18 study iARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] yMK1329 MA Ta ad92-1 can 1-100 his3—11,15/eu2-3,112 trp1-1::PDS1- This Myc13::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2.-:KAN hta1-htb1::Nat study htaZ-htb222HPH pJH33 [ARS CEN URA3 HTA1-HTB1 HHT2-HHF2] yM K839 MA Ta leu2-3 trp1 ura3-52 (32) 75 TABLE 2-2. Plasmid constructs used in this study . . Source or Plasmid Main features reference JH33 pRS316-HTA1-HTB1 HHT2-HHF2 (1) pJL51 2micron 3xHA-SGO1 This stud pJL53 ARS1 CEN4 URA3 pADH1-3xHA-SGO1-tADH1 This study pJL55 pGEX-4T-1 3xHA-SGO1 This study ARS1 CEN4 URA3 pADH1-3xHA-SGO1-Bromodomain- pJL76 tADH1 This study pMK4396448 pRS315-HTA1-HTB1 hht2 G448-HHF2 This study pMK572 2micron URA3 vector with ADH1 promtoer and terminator This study MK573 2micron URA3 SGO1 with ADH1 promtoer and terminator This study pMK621 pJJ244 URA3-HHF2-KTR5 insert This study MK6226448 pJJ244 hht2 G448-HHF2-URA3-HHF2-KTR5 This study MK622WT pJJ244 HHT2-HHF2-URA3-HHF2-KTR5 This stud pQQ18 pRS315-HTA1-HTB1 HHT2-HHF2 (1) pYCF1/CEN3.L YRp14/T EL cassette (pYCF1) with a CEN3 insert (51) 76 FIG. 2-1. The G448 mutation confers pleiotropic phenotypes. (A) Yeast cells bearing the G448 allele as the sole copy of H3 were tested on YPD medium under the indicated conditions. Sixfold serially diluted log-phase cells were spotted for growth tests. All drug tests were conducted at 30°C. (B) Position of G44 of H3 within a nucleosomal core particle. Left panels: Two views of the crystal structure (Protein Data Bank entry: 1ID3) based on White et al. (58). Right panels: closeup view of the “Lys-Pro-Gly-Thr 8 turn (top) and the secondary structural domains (bottom) of histone H3. Numbers below the secondary structure are amino acid residues at the junctions of the indicated domains. The green dotted lines in the closeup represent possible hydrogen bonds between the carbonyl oxygen of K42 and the amide groups of G44 and Thr45. Hydrogen is not included. DNA is omitted for clarity. 77 Fig. 2-1 IHU, 75 mM -- HU 100 mM ‘-' uv 100 J/m2 --r ,;. uv 150 J/mz m_~ MMS, 0 01% MMS, 0.05% 78 Fig. 2-1 continued H2A m H28 O Gly44 of H3 (IN «1 L1 «2 L2 «3 1*135 45 56 64 78 86 114121 131 79 FIG. 2-2. The G448 mutation causes chromosome instability. (A) Mitotic chromosome stability tests. Representative pictures of colonies on YPD plates are shown at the top. The bar graph was prepared from three independent experiments with standard error of the mean. Colonies with at least 50% continuous red sector were counted as the first-division chromosome (Chr.) loss. Colonies that were totally red, as a result of chromosome loss prior to cell plating on YPD, were excluded. (B) G448 mutant cells lose viability faster after short benomyl exposure. Log-phase cells were treated with 60 pglml of benomyl for the indicated time, washed, counted, and spread onto benomyl-free YPD plates. Percent viability was calculated by dividing total number of colonies by the number of cells inoculated (counted microscopically) and was normalized to that of the T0’ samples. Results were from at least 3 independent experiments. (C). Higher missegregation is associated with the G448 mutation. WI' and G448 mutant cells with the TRP1 locus marked by GFP were treated with 30 pglml benomyl for two hours, collected, and regrown in YPD medium containing a factor for two hours before fixation for microscopy. At least 200 unbudded cells with GF P dots were scored in four independent cell cultures of each strain. Green and red bars represent WI' and G448 mutant cells, respectively. Error bars show standard deviations. Randomly selected images of two-dotted wild type and G448 cells (marked by white triangles) are shown on the right. w/o, without. 80 Fig. 2-2 100 F 35 3'32 80 oz 31: *3 60 52 40 20 l 81 G448 Fig. 2-2 continued B 100.0 fl T) 0 80.0 2 .Q (0 s 60.0 E 8 5 40.0 CL 20.0 0.0 0 2 4 6 Time in Benomyl (hr) C 14 IWT UG44S r: 12 T .9 E 75 10 8 E? '- a) (‘1’ g 8 .2 2 6 4 1' 2 _ ,, , L_ w/o benomyl 2-hr recovery from benomyl 82 FIG. 2-3. G448 mutant cells activate the spindle checkpoint in response to benomyl toxicity. (A) Comparable budding indices were obtained from WT and mutant cells. Benomyl (60 pglml) was added to log-phase cells. The percentage of large-budded cells (with daughter cells at least half the diameter of their mothers) was determined microscopically. (B) Pds1p was activated and stabilized in both normal and G448 mutant cells in the presence of benomyl. Comparable numbers of a-factor arrested G1 cells were released at T0’ into YPD medium containing 0 or 30 pglml of benomyl. The same volume of cell suspension was taken at the indicated times for boiling and whole-cell extract preparation, followed by anti-Myc Western blotting to quantify the abundance of Pds1p. 83 Fig. 2-3 A 120 £ 8 100 - ”WI .8 IG44S '0 80 ‘ 8 .9 60 — 8. ,3 40 ~ o\° 20 - ,_JZI__ 0 2 4 Time in Benomyl (hr) B Time, min 0 30 60 75 90 105 120 135 150 180 Benomyl ‘“~N.~.~-~~—‘ 0 WT ~~ufi~~~-~-’ 30pg/ml Time, min 0 -30 60 75 90 105 120135150“ 180 Benomyl 0 G44S 30 pglml Pds1p-13xMyc 84 Fig. 2-4 YP-galactose arrest in Gal back at 60’ T . TSO' a factor G1 0/" \ a factor added /" YP-glucose Time.min 0 4O 50 60 70 80 90 0 40 5O 60 70 80 90 Gal-to-Gal Gal-to-Glc J Gal-to-Gal Gal-to-Glc 5 ] 13xMyc E: t. i In L T ’ a ‘wv- , . ' ' , . - " . . ' 5 .' -- ' .1 ~ . . ~ - o 7 ‘ '. 1 . . I .' ‘E ... . .’.. . .3... A. .A . . . v r o , . . 4 1 1 L i 1 ._. I: . G-6-PDH ado-pint.- -wv-o'fiv—ww“'“"o w . . ‘) -.- x." .... v: by" .1” ‘ nu - s . w]. u 1 ...; ...... . 9 5 a l- . . .- - V}. v- n. ...-u ~ WT G448 FIG. 2-4. The G448 mutation impairs the tension sensing function. MCD1 was placed under the control of the GAL1 promoter for glucose repression. The abundance of Myc-tagged Pds1p was analyzed by Western blotting in the presence (Gal-to-Gal) or absence (Gal-to—Glc) of Mcd1 p. Experimental schemes are shown above the Western blot assay results. G-6-PDH: glucose-6-phosphate dehydrogenase. 85 FIG. 2-5. G448 mutant mitotic phenotypes are suppressed by overexpressing Sgo1p. (A) 8901 p overproduction specifically rescues the mitotic phenotypes of G448 mutant cells. The SGO1 ORF was cloned into a 2pm plasmid bearing the promoter and terminator sequences of ADH1. WT and G448 mutant cells transformed with SGO1 or the corresponding vector were tested under the indicated conditions. (B) Left panel, cell viability test after benomyl treatment. This plot was generated from three independent experiments, and the error bars depict the standard deviations. Right panel, chromosome missegregation assay. Shown are percentages of two-GFP-dotted G1 phase cells. Data were collected from three or four independent cultures, and error bars represent standard deviations. See the legend to Fig. 23 and C for details. (C) Tension sensing defects conferred by the G448 mutation are alleviated by SGO1 overexpression. G448 mutant cells receiving a 2pm plasmid with or without the ADH1-driven SGO1 gene were tested for the molecular response following MCD1 shutdown. Experiments were done exactly as those shown in Fig. 4. (D) Neither SGO1 transcription nor protein abundance is affected by the G448 mutation. Left, reverse transcription (RT)-PCR shows normal expression of SGO1 in WT and G448 mutant strains. Right, Western blotting of a C'-6xHA-tagged Sgo1p demonstrates the equal abundance of Sgo1p in these two strains. The Loading control is a cross-reacting band from both yeast lysates. 86 Fig. 2-5 A YPD + 15 pglml YPD, 30° Benomyl YPD, 14° - _ . ,1 '- WT + 2p vector teem:- QQ- WT+2pSGO1 ' G44S + 2p vector 6448 + 2p SGO1 UV hypersensitivity Va” 150 2:5 200 J/mz 250 J/mz / + 2 vector '1'" .._ ' ;, ' WT+ 2p vector + 2“ SGOI G448 + 2p vector B 100 14 :02 —I—WT+2p VC C 8 —O -WT+2p SGO1 2.9.. 12 g g 80 “ —O—G44S+2p vc g ,g —o -G44S+2p $601 93 10 - > 8’ F .2 8 : 8 E 0'.) 40 - E 5 ‘ 8 (D 4 - CL 20 ~ T 2 a O I 0 " 0 2 4 6 WT+ WT+ G44S+G44S+ Time in Benomyl (hr) vect SGO1 vect SGO1 87 Fig. 2-5 continued Time, min 0 .619 ”513433; .7 MY": :'..' -' , 5? . ' .’ G448 + vector - - 80 90 (3443 + 2p SGO1 A ' Pds1p-13xMyc G448 G448 SGO1 \ 8PT15 -> RT-PCR 88 ,- j . . {“1“ . n7. ”. .- -_. ,- "1. . ‘. 1 ‘ .' '. - ; ‘ .' .I; ....“ ,' O 40 50 60 7O 80 90 Sgo1-6HA ’ 2 Loading control Western: o-HA FIG. 2-6. The G448 mutation selectively downregulates the pericentric recruitment of Sgo1p in vivo. (A) ChIP analysis of HA-tagged Sgo1p. Samples in all of the panels are arranged in the same order. The common internal control (marked by the star on the right) for multiplex PCRs is from within the ORF of the DED1 gene 386.2 kb to the right of CEN15. Targets of the PCR fragments and their distance to the cognate centromeres are listed at the top of each gel image. R: right; L: left. SPT15 is 313.3 kb to the right of CEN5. (B) Quantification of ChIP results. Ethidium bromide-stained DNA gel images were quantified by NIH Image. The intensities of each CEN or pericentric fragment was compared to that of the DED1 internal control (star). The ratio was then normalized to 0.1% input (INP) DNA (set at 1.0). Error bars represent standard deviations from at least three independent cell cultures for ChlP. Ab, antibody. 89 Fig. 2-6 ‘ . r - . > . . .1? . . i . . . '. t"- . .31}:- . y , o . j . 1‘;L:.'-;¢_;=;-2¢'.‘s... .:. . ‘l. . ‘ -:- - n: .1"-“'a yet-i W W21 - - ,i‘.-§-'. 'jigfif“ l: ,, ‘x ‘1: l'! .r‘x 1‘ A -" x ... 1.. ‘. 3’. 1.00 0.13 0.36 8.24 8.68 1.00 0.27 0.46 2.151.68 INP WT G/S WTG/S INP WT G/S WTG/S NO Ab a-HA NO Ab a-HA . O o 'n V CEN16L 0.3kb CEN16R 0.4kb CEN16L 1.7kb :."'-‘.- ..., 1.00 0.22 0.10 1.12 0.03 1.00 0.13 0.07 0.94 0.51 1.00 0.00 0.06 0.73 0.03 CEN1R 8.3kb CEN16R 9.1 o .' . . ‘ , .. f' w. I.‘ 7.32:“.- j .. .I_ ..... .' A31. .1 - t .R.’ ..5.3? “ii - a... : '.: ;_ sens (313kb) 1:53;; :‘- ~ 2': . n '. .. - . .. .:. “F ‘I - I III I O 1 ‘il‘- .1. I- .an Q <0 us. I e . n I hogs-v .... ‘11 ... . . . ,.._. ,. ..z . r: =.... .1. . . _. . ' .. . . " :2 :t; "'..'.: :F-O- ' -~ mi“ ,, < mug, , 5 1.00 0.33 0.27 0.35 0.52 1.00 0.22 0.10 0.26 0,211.00 0.11 0.11 0.35 0.31 90 Fig. 2-6 continued B 9 . I0.1% input as . Eth, no Ab E E36448. noAb \° 5L. 7 . th,ChlP g DG44S,ChIP E s. *5 a: 5 l 4 d 3 I 2 1 . 1 1 . _ II I ] 1 1!} || :. . ' .5 I I . ”I l o . EL: I IE I a“: I a I H- L! :61 I '5': II ..n. I CEN3 CEN16 CEN16L CEN16R CEN1R CEN16R CEN1R CEN16R SPT15 0.3kb 0.4kb 1.2kb 1.7kb 8.3kb 9.1kb 91 FIG. 2—7. Physical interaction between H3 and Sgo1p is attenuated by the G448 mutation. (A) Pulldown assays assessing the interactions between bacterially expressed GST-3xHA-Sgo1p and histones and oligonucleosomes purified from WT or G448 mutant yeast cells. Trapped H3 was quantified by anti-H3 antibodies. (B) Far-Western assays detecting direct binding between H3 and Sgolp. Yeast core histones were resolved and blotted onto a PVDF membrane, and incubated with GST-3xHA-Sgolp. Sgo1p, trapped via association with H3, was probed by anti-HA antibodies. A parallel gel was stained with Coomassie blue R250 (CBR) to reveal the relative mobility of yeast histones. 92 Fig. 2-7 A WT G448 WT G448 . 0.6x u aun- Anti-H3 Matrix: histones f . GST-HA-Sgo1 f fl W Anti-H4 " 1x - histones g I Anti-HA Matrix: 1x GST - histones Input C'- ‘l' Anti-H3 Input m ‘ 0.2x ______,———— histones .- o Anti-H4 Western: o-H3 Free core histones Oligonucleosomes B WT G448 WI" G448 H3 cl- - /H .. m ‘ :Hza H4 . .... “... H2A \H4 Western: a-HA CBR 93 FIG. 2-8. Reestablishing pericentric Sgo1p recruitment by BD fusion. (A) ChlP assays assessing the distribution of Sgo1p and Sgo1p-BU at selective loci. G448 sgo1A mutant cells were transformed with an ARS CEN plasmid containing 3xHA-Sgo1p or 3xHA-Sgo1p-BD genes and analyzed by anti-HA ChlP. The assay conditions were identical to those described in the legend to Fig. 6. A non- specific PCR fragment ("N") amplified along with the CEN16L 0.3 kb is marked with an arrow. (B) Comparable expression of 3xHA-Sgo1p and 3xHA-Sgolp-BD. G448 sgo1A mutant cells bearing the indicated recombinant SGO1 construct were examined by anti-HA Western blotting. Even loading was evidenced by Coomassie blue R250 staining (not shown) and by proteins that cross-reacted with the anti-HA antibodies. (C) Quantification of the ChlP results was done as detailed in the legend to Fig. 68. 94 N \ “Mtg. :35} _ CEN16L 0.3kb ’ CEN16R-1.7kb 1..000372.771..96157 1.000.110.7..60110761.00 0.370.790.380.89 A lnp - WTG/S G/S SGO1 SGO1- BD B m <-HA-SGO1-BD “HA-SGO1 Cross- reacting bands SGO1SGO1-BD CEN16R 4. 0kb 7 CEN16R 9.1kb 6.00 5.00 4.00 3.00 Ratio to 0.1% input 2.00 1.00 0.00 1000160780250431.000.190.32017049 IINP InoAb CEN16 CEN16LCEN16RCEN16RCEN16R 0.3kb 1.7kb 4.0kb 9.1kb 95 FIG. 2-9. Bromodomain fusion partially rescues the tension sensing defects of G448 mutant cells. (A) The indicated yeast strains expressing Sgo1p with or without the bromodomain were tested for resistance to benomyl (A), for viability after benomyl exposure (B), and for the Pds1p level after MCD1 shutdown. See the legends to Fig. 2 and 4 for details. In panels B and C, only data from G448 mutant cells are shown. 96 Fig. 2-9 Benomyl YPD, 30° 7.5 ug/ml 100 10 pglml H3 SGO1 Plasmid WT WT none WT A Vector WT A SGO1 WT A SGO1-BO G/S WT none G/S A Vector G/S A SGO1 G/S A SGO1-8D G/S A SGO1 6/8 A SGO1-8D 80~ O) 0 Percent Viable Cells A O N O l -—o - G448 sgo1A +SGO1 —I— G448 8901A +8601 -80 2 Time in Benomyl (hr) Time, min 0 40 50 60 70 80 90 3 0 4O 50 60 70 80 90 G448, I ‘ h u .... SGOl ' G448, _ . .. ___ SGO1-BO A ~5. ...—----- Pdslp-13xMyc 97 ADDITIONAL EXPERIMENTAL PROCEDURES EMSA. Interactions between Sgo1p and reconstituted nucleosomal core particles (NCP) were examined by electrophoretic mobility shift assays (EMSA). 1.4 pg of NCP was mixed with 0.24 pg of GST-HAx3-Sgo1p (see above) or 0.3 pg of GST in HEGT buffer without MgCl2. For supershift of NCP mobility, 2 or 4 pg of 12CA5 (Roche) anti-HA monoclonal antibodies were included. After mixing all reactants on ice, reactions were loaded to 4% polyacrylamide gel (0.5 X TBE). The gel was run at constant 25 V for 6 hours. Free DNA and NCP complexes were visualized by EtBr staining. 98 Supplemental Data Fig. S1 10.00 1.00 ODGOO 0.10 0.0 4.0 8.0 12.0 16.0 20.0 24.0 Time (hr) 0.01 FIG. 81. G44S cells exhibited slower growth rate. Optical density at 600 nm (ODsoo) was assessed at the indicated times after making appropriate dilution of starting log-phase WT and G448 mutant cultures. Three independent growth tests were conducted. This plot shows typical results with error bars representing standard deviations measured at each time point. 99 FIG. 82. Sgo1p interacts histone H3 in different contexts. (A) Bacterially expressed yeast H3 and acid-extracted chicken H3 interact with Sgo1p. Pull- down assays using GST-HA-Sgo1p (left panel) or GST alone (middle panel) were carried out by incubating acid extracted yeast (lanes 1 and 2) or chicken (lane 3) core histones, or recombinant H3 expressed in E. coli (lane 4). Histone bound to the matrix were resolved and probed by anti-H3 antibodies. 12% input of each histone samples is shown in the right panel. Note that lanes 1 and 2 are identical to Figure 7A (see main text). (B) 8901 p interacts with reconstituted nucleosomal core particles (NCP). Interactions between GST-HAx3-Sgo1p and NCP were examined by EMSA assays. Moderate retardation was observed in different batches of recombinant 8901 p with NCP (compare lane 4 with lane 2). Adding the anti-HA monoclonal antibodies to reactions containing the HA-tagged Sgo1p resulted in further NCP mobility retardation (lanes 6 and 7). Neither GST nor anti-HA antibodies (lanes 3 and 5, respectively) changed the mobility of NCP. 100 Fig. 82 A GST-HA-Sgo1 ligand GST ligand Input ' - - O. 1 2 3 4 1 2 3 4 1. WT yeast histones 2. G448 yeast histones 3. Chicken histones 4. Recombinant H3 8 free DNA . Nucleosomal core particle 1 2 3 4 5 6 7 8 GST - - + - - — — - GST-HAXS-SGO1 - - — + — + + .. Anti-HA Ab - - — — + + + _. (2119) (2119) (4:19) 101 Fig. S3 HHF2 HHT2 wt or 6448 fi r—-> Galactose Glucose 0 pg/ml 10 pg/ml 15 pglml benomyl benomyl benomyl FIG. 83. G448 mutation is semi-dominant. Yeast cells with the chromosomal copies of all core histone genes were deleted and were transformed with two plasmids each bearing a single copy of genes encoding all four core histones. In the "top" plasmid, H3 (HHT2) and H4 (HHF2) were expressed from the native, divergent promoter. The H3 was either wild type or the G448 allele. In the "bottom", second plasmid, the H3/H4 promoter was replaced with the GAL1, 10 promoter, rendering the expression of these two genes repressible by glucose. Log phase YPGal cultures were spotted to the indicated plates containing either glucose or galactose. Cellular growth was then compared two to three days later. 102 Fig. S4 YPD 30° YPD 34° YPD 37° Beno. 10pg/ml (DO-hODN-A 1. yMK1243WT 2. MSYP726 hhf1-20 3 & 4. MSYP726 hhf1-20 + 2p vc 5 & 6. MSYP726 hhf1-20 + 2p SGO1 FIG. 84. 2pm SGO1 does not rescue a histone H4 allele, hhf1-20, that causes temperature sensitivity and centromeric function defects (16). The hhf1-20 strain (MSYP726) was a kind gift from M. Smith. 103 Fig. 85 CEN3 CEN3R 0.3kb CEN16L 0.3kb if? M .... ‘13" 7 .- . . . . I. ' 31:31.2“ 2’33 .2 & “I‘SI' IO'QH... HILJLJ=N ..J‘WLJU * o I , . . ‘ '1') " t INP No Ab WT GIS INP No Ab WT G/S INP No Ab WI' G/S INP No Ab WI" G/S CEN1R 8. 3kb CEN16R 9.1kb SPT15 ACT1 H H it“ - - w 3&- U“ . ' - U U U a; 2 ‘ "'I. , "II-I ““4 U U ' -‘ ...... U ’~—- if ya u Huh-1...: u w u 1.4-ca Id U Vt INP No Ab WTG/S INP No Ab WTG/S INP No Ab WTG/S INP No Ab WTG/S fi' DED1 FIG. S5. Mcd1p recruitment to selective centromeres, pericentric regions, and chromosome arms is not affected by G448 mutation. ChlP assays were conducted in the wildtype and G448 strains bearing C'-Myc-tagged Mcd1p. Logarithmically growing cultures were used. All PCR reactions contained two pairs of primers with DED1 as the common internal control for PCR reactions. Specific PCR targets and their distance from the centromere are indicated on top of each gel image. SPT15 is 313.3 kb on the right of CEN5, and ACT1 is 93.8 kb on the left of CEN6. 104 FIG. 86. No discernable transcriptional defects were observed in the G448 cells. Transcripts of different classes of yeast genes were analyzed by quantitative RT- PCR. (A) Mitosis-related genes. Each panel is clustered based on the major functions of the assayed genes. Loading control, SPT15, is shown in panel (B). (C) Representative inducible genes were analyzed by RT-PCR. SUCZ and GAL1 are induced by shifting carbon source from glucose to sucrose and from raffinose to galactose, respectively, whereas ARG1, H181, HIS7, and TRP3 are amino acid biosynthesis genes that are induced by amino acid starvation. SPT15 was used as the common internal control for each quantitative PCR reaction. (D) Mating type-specific genes are expressed and repressed normally in the 6448 cells. Both the wildtype and the G448 cells were of the a mating type (MA Ta). Thus, a-specific genes (e.g. a1 and the a pheromone receptor STE2) are expressed in MA Ta cells. In contrast, o-specific genes, such as (11 and the a pheromone receptor STE3, are repressed in MATa cells. Genomic DNA was also used in separate PCR reactions to demonstrate that the lack of signal in 01 and STE3 RT—PCR was due to the lack of the corresponding transcripts but not to a systemic failure in PCR amplification. 105 Spindle checkpoint components GLC7 BUB 1 IPL1 MAD1 SLI15 MP8 1 Kinetochore and spindle Cohesin complex components NDC80 MCD1 TUB1 SMC1 1x 5x 1x 5x WT G448 .. 1x 2x 4x 8x 106 Fig. 86 continued SUCZ SPT15 a #4 \~—J U H . “WM-“'4 GAL1 Carbon SPT15 “we WI' G443 WT G448 WI" G443 WT G448 Glucose Sucrose Raffinose Galactose u— “ ARG1 hot—n HIS 1 U "'"‘ HIS7 hath-d l ‘ TRP3 l Haw-J '3. . ‘ .1 WT G448 WT G448 SPT15 . I ‘.4- ." Basal Induced 107 J Amino acid biosynthesis Fig. 86 continued D RT— PCR a-speciflc genes a-specific genes (mating) (transcriptional silencing) a1 01 i“ H W N... STE2 STE3 bu. h a“; a 1x 5x 1x 5x 1x 5x 1x 5x WT G448 WT G448 SPT15 1x 2x 4x 8x 1x 2x 4x 8x WT G448 Genomic DNA PCR 31 a1 STE2 STE3 SPT15 u u 1x 5x 1x 5x 1x 5x 1x 5x1x 5x 108 Fig. S7 15 pglml Benomyl (DCDVCDUT-wa—l AA ‘0 _s N 2p DNA vector SGO1 vector SGO1 vector SGO1 vector SGO1 vector SGO1 vector SGO1 RTS1 + DDDD + CDC55 + DDDD FIG. S7. 2pm SGO1 suppression is independent of the PP2A activity. Shown are benomyl resistance of WT and G448 cells in the presence or absence of two PP2A components, RTS1 and CDC55, after transforming with 2pm vector or SGO1. Deleting either PP2A subunit does not affect the suppression by SGO1 overexpression. 109 FIG. 88. Genetic interactions between the Aurora kinase, histone H3, and SGO1. (A) IPL1 and SLI15 are overexpressed in wildtype, 6448, or sgo1A cells. 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J Cell Sci 115:3547-3555. 117 CHAPTER III Characterization of a Tension Sensing Motif of Histone H3 in Saccharomyces cerevisiae (Manuscript in preparation) 118 Characterization of a Tension Sensing Motif of Histone H3 in Saccharomyces cerevisiae Jianjun Luo, Xinjing Xu, and Min-Hao Kuo“ Department of Biochemistry and Molecular Biology, and 1Programs in Genetics and in Cell and Molecular Biology, Michigan State University, East Lansing, MI 48824 Running title: chromatin role in mitosis Key words: histone H3, Chromatin, mitosis, Shugoshin, Saccharomyces cere visiae *Corresponding author. 401 BCH Building, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824. E-mail: &m@msu.edu; Phone: 517-355-0163; FAX: 517-353-9334 119 L1 ABSTRACT To ensure genomic stability during cell division, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies before segregation. The tension between sister chromatids generated by the poleward pulling force is an integral part of Chromosome biorientation. Our previous studies indicated that Gly44 of H3 is critical for recruiting/retaining 8901 p at the pericentromeres for monitoring the tension status of sister chromatids during mitosis. Studies carried out in this work showed that Lys42, Gly44, and Thr45 of H3 form a core executing a mitotic function that is pivotal for cell survival under benomyl stress. Consistent with the G448 mutant phenotypes, K42A and T45A mutant cells failed to respond to the lack of tension between sister chromatids. The benomyl hypersensitivities of these mutants can be suppressed by the overexpression of SGO1. Lys42, Gly44, and Thr45 thus form a tension sensing motif (TSM). This motif functions by physically recruiting or retaining 8901 p at the pericentromeres. lntriguingly, the histone acetyltransferase activity of Gcn5p likely plays a negative role in H3 TSM-Sgo1p function. These results reveal that a TSM of histone H3 is a key player in faithful segregation of mitotic Chromosome, and that interaction with a chromatin modifying enzyme may be an important part of the mitotic quality control process. 120 INTRO lllslor chron cycle roles recor chror cont: To e chec altar cohe whic Cell The allaa ShUg Derlc mom; INTRODUCTION Histones are basal structural and functional components of eukaryotic Chromosome. The flexible N-terminal tail of H3 and H4 is required for normal cell cycle progression (24, 25). Although emerging data have shown the important roles of chromatin structure and histone modification in controlling transcription, recombination, DNA replication, DNA repair and establishment of sister- chromatid cohesion (2, 21), it is less clear as to how individual histones contribute mechanistically to mitotic progression and regulation. To ensure faithful segregation of sister-chromatids, the spindle assembly checkpoint (SAC) ensures the establishment of bipolar kinetochore-spindle attachment, which results in tension between sister-chromatids that are held by cohesin complexes (11, 28). Lack of attachment or tension activates the SAC, which then arrests cell cycle by inhibiting the anaphase-promoting complex (28). Cells resume mitosis after the error is corrected. The tension sensing function ensures that the two sister kinetochores are attached to spindles emanating from opposite spindle pole bodies. The Shugoshin proteins in different organisms are critical for this function (15, 18, 33, 34). Sgo1p in yeast and mammals is enriched at the centromeres and pericentromeres (17, 29, 30), where tension is believed to be generated and monitored (4). The recruitment of S. pombe Sgo1p is achieved by the 121 phosphorylation of Ser121 of H2A by the Bub1 p kinase, and heterochromatin markers at the pericentromeres (16, 33). In S. cerevisiae, the Bub1p kinase and the H2A Ser121 phosphorylation also are essential for localizing Sgo1p to the centromeric region (9, 16, 17). We recently reported that histone H3 is an essential player in mitotic tension surveillance and that the pericentric, but not the centromeric, enrichment of 8901 p is critically dependent on H3. Yeast cells harboring a unique Gly44-to-Ser (G448) mutant allele of H3 exhibit phenotypes >1 typical of those resulting from tension sensing defects, including Chromosome instability, missegregation, and inability to activate the SAC when tension buildup is artificially prevented. Overexpressing 8901 p or artificially tethering 8901 p to the pericentromeres can restore the tension sensing function of H3 mutant cells (23). Histone acetyltransferases such as Gcn5p are best known for their roles in facilitating transcription (12, 20). Other chromatin functions of Gcn5p are beginning to emerge. For example, deleting GCN5 causes cell cycle defect and accumulation of 6le cells (13, 35). The notion that Gcn5p plays a mitotic function is supported by the observations that GCN5 genetically interacts with genes encoding several inner kinetochore components, and that Gcn5p is present at the centromeric regions (32). These results suggest that Gcn5p may regulate the kinetochore function or structure that is critical for mitotic control and progression. 122 In this work, we defined the Tension Sensing Motif of histone H3 that is strategically positioned at the junction between the flexible tail and the structured histone core domain. lmportantly, genetic data suggested that this novel tension sensing function is regulated by the histone acetyltransferase Gcn5p. 123 MATERIALS AND METHODS Yeast strains and plasmid constructs. The yeast strains and plasmids used in this work are listed in Tables 3-1 and 3-2. To assess the effects of histone H3 mutations from lysine 36 to lysine 56 (Fig. 1A) in our strain background (yMK1243 and equivalents), plasmids (TRP1*) harboring only H3 and H4 genes with histone mutations created by the Boeke group were co-transformed with pJL74 (a LEU2+ plasmid bearing only histones H2A and H2B) into yMK1141 (contains pMK440, a URA3” plasmid bearing all four core histone genes) (5). 5-FOA selection was conducted to select for yeast cells that had lost the plasmid pMK440. pJL74 was a self-ligated product of the linearized pMK439 cutted by Sall (blunted) and Pstl (blunted), which removed H3 and H4 genes from pMK439. All other studies were carried out with histone mutations generated in pMK439 by two-step PCR site—directed mutagenesis (23). Yeast methods. Yeast growth media, conditions, and transformation were based on standard procedures (31). When appropriate, 5% casamino acids (CAA) were used to substitute for synthetic amino acid mixtures as selective medium for uracil, tryptophan, or adenine prototroph. Yeast transformation was done with the lithium acetate method (10). Chromosome stability assays were conducted by measuring the mating behavior 124 of diploid strains bearing wild-type or selective tension sensing mutants. Diploid strains with histone mutations were constructed via plasmid shuffling, yMK1174 (MA Ta/a, contains pMK440 URA3”) were used for transformation to harbor histone mutations generated on pMK439 (LEU2+), 5-FOA selection was followed to select for yeast cells that had lost the plasmid pMK440. To assess the chromosome stability, cells were first patched onto YPD plates and incubated at 30° for 2 to 3 days till saturation. Cell patches were again replica plated to #- another fresh YPD plates pre-spreaded with a or a tester cells and were allowed to mate at 30° for 10 hours, followed by further replica plating to minimum medium plates to select for cells that had mated successfully. Mating between the tester and the subject strains resulted in complete complementation of nutrient requirement, that is, cells were able to survive in the minimal medium without any amino acid or nucleoside addition. Tension sensing tests using the PGAL1-MCD1 strains were according precisely to reference (15) using strains yJL171, yJL487 and yJL492. For Fluorescence Activated Cell Sorting (FACS) analyses, logarithmically growing cells (0.500500) were collected, washed once with 1ml water, and fixed overnight at 4°C with 1ml 70% ethanol. Cell pellets were washed with 1ml 10mM Tris (pH 7.4), 15mM NaCl, and resuspended into 300pl Tris/NaCl buffer containing 0.1mg/ml RNase A. After the incubation at 37°C for 2 hours, cell pellets were resuspended into PBS (pH7.4) at 1X108 cells/ml. For propidium 125 iodide staining, 10pl of cell suspension was mixed with 490pl of PBS containing 40pg/ml propidium iodide. 20,000 cells were subjected to FACS analysis on a BD Vantage SE in Flow Cytometry Facility (Biochemistry, MSU). Western analyses of yeast proteins were conducted as mentioned in reference (23). Home-made H3 antibody H3-182 was used at 1:20.000 dilution at 4°C for overnight. Chromatin immunoprecipitation (ChlP). ChlP was conducted as previously described (19, 23). To quantify the ChlP results, the semi-quantitative PCR products were purified, resolved in 9% polyacrylamide gel electrophoresis, and stained by ethidium bromide. The captured gel images were then quantified by NIH Image. Intensities of each CEN/pericentric fragment were compared to a common internal control (DED1 or PGK1). The ratio was further normalized to 0.1% input DNA (set at 1.0) for PCR amplification of all reactions carried out in parallel. The ChlP data were obtained from at least three independent yeast cultures. Sgo1p interaction with H3. Histones were prepared according to reference (7). Recombinant Sgo1p was prepared as previously described (23). For pull-down assays, approximately 5 pg of soluble recombinant 8901 p were incubated with about 3 pg of yeast histones in 150 pl of HEMGT buffer at 4°C for 1 hour. 6 pl of glutathione beads (1 :1 slurry) along with 150 pl of the HEMGT buffer were added 126 to the reactions and rocked gently at 4°C for another hour. Beads were washed with 500 pl of the HEMGT buffer for 3 times, 5 min each, followed by boiling in 2x SDS-PAGE loading buffer for 5 minutes. Eluate was resolved by 15% SDS- PAGE and blotted for anti-H3 Western analyses (23). To study the functions of GCN5 on the H3-Sgo1 p tension sensing function, H3 mutations (on pMK439 LEU2”) were introduced into yJL486 (gcn5A, pMK440 URA3+) via plasmid shuffling and 5-FOA selection, resulting strains yJL506 to yJL510 (see Table 3-1). yJL486 was constructed by transforming 4.6kb gcn5::URA3 fragment, which is released from pMK147 by Xho I and Xba I, into yMK1141 (pMK440 URA3“). Dominant negative HAT inactive mutants of gcn5, gcn5E173H or gcn5F221A were carried on pMK144 and were directly transformed into cells bearing H3 mutations (For SGO1 series, parent strains were yMK1243, yJL145 etc.; for sgo1A background, parent strains were yMK1361, yJL17O and others bearing indicated H3 mutation, see Table 3-1) 127 RESULTS Mutations of a cluster of amino acid residues of H3 cause mitotic chromosome instability Our initial findings that Gly44 of H3 is important for 8901 p interaction and tension sensing (23) suggest that this residue is part of a functional motif that acts as the loading dock for 8901 p recruitment. Indeed, Boeke and colleagues reported in their systematic phenotypic Characterization of histone mutations that K420 and R49A of H3 render cells hypersensitive to benomyl (5), one of the diagnostic phenotypes of our tension sensing defective G44S allele (23). To test whether Gly44 is part of a Tension Sensing Motif, amino acid substitutions of residues K36 (the end of the tail domain) through K56 (the end of the (IN helix of the histone core) were introduced to yeast cells, and the growth under varying concentrations of benomyl was assessed. Figure 1A shows that K42A, G44A and T45A caused severe hypersensitivity to benomyl whereas H39A, R40A, Y41A, and R49A were moderately sensitive to this spindle poison. While both K36 and K56 are known to be acetylated and are important for several nuclear functions (6, 26), alanine substitution of these residues did not affect benomyl tolerance. lntriguingly, although P43 is flanked by K42 and G44, alanine substitution at P43 clearly is functionally neutral. This observation is consistent with the crystal structure shown in Figure 18 (right) in that the imino acid ring structure of P43 points away from the side chains of K42 128 and T45, and sits flat on the beta turn. It is possible that G44 plays a more prominent role than does P43 in permitting the formation of this beta turn structure. Additionally, like 6443, the benomyl hypersensitivity of these new H3 mutants also can be suppressed by the overexpression of SGO1 (Fig. 1D and 1E). Together, these data suggest that K42, G44, and T45 form a core executing a mitotic function together with 8901 p that is pivotal for cell survival under the benomyl stress. I q That the K42A mutation phenocopies G448 (as well as G44A) suggests one possibility that acetylation at K42 controls G44-related mitotic functions. This notion appears to be supported by the observations that the K420 mutation that mimics a constitutively acetylated state also causes benomyl hypersensitivity (row 9, Fig. 10), whereas K42R that mimics a constitutively unacetylated state was apparently phenotypically neutral (rows 3, Fig. 10). Two inferences can be drawn here. First, the positive charge of this position has to be maintained by either a lysine or an arginine residue. Alternatively, K42 acetylation might be a natural negative regulator of TSM. If the latter hypothesis is correct, we predict that K42R would suppress the phenotype of G448 that undermines the Sgo1p interaction and recruitment. Contrary to this prediction, the G448 benomyl hypersensitivity phenotype could not be suppressed by K42R (compare row 3 with 2, 4, 5, Fig. 1C), arguing against that the uncorroborated acetylation of K42 was functionally relevant. 129 Benomyl depolymerizes microtubules, which in turn activates the spindle assembly checkpoint, SAC, that arrests cells at 6le phase. Hypersensitivity to benomyl may be a result of the failure of activating the SAC. However, both K42A and T45A mutants can stabilize the securin protein Pds1p when treated with benomyl (Fig. 2), ruling out the possibility that the SAC itself is defective in these mutant cells. Together, these phenotypic comparisons suggest that the short stretch of H3 from K42 to T45 is a functional or physical target of a mitotic regulator. Chromosome instability conferred by K42, G44, and T45 mutants Mitotic defects frequently trigger instability of chromosomes. To see if this is true for the new H3 alleles, we compared the mating behaviors of wild-type, K42A, G448, and T45A cells. As an additional control, we included another novel histone mutant allele, H4 R358, which is also hypersensitivity to benomyl (Luo and Kuo, unpublished data). In the budding yeast, diploid cells inherit the transcriptionally active MA Ta and MA Ta loci on chromosome III from the two haploid parents. Concomitant expression of these two loci represses genes essential for mating. Normal a/q diploid thus is considered a non-mater. If there is an increase in chromosome instability, random loss of one of the two chromosome III homologues will enable cells to mate, regardless of the ploidy for the rest of the genome. Gaining the ability to mate is thus an easy method to examine the chromosome stability. Figure 3A shows that a substantial portion of 130 diploid cells bearing each of these H3 mutant alleles were able to mate with either MA Ta or MA Ta haploid tester strains, while wide-type or H4 R358 mutant cells maintained their diploid non-mating character. Genomic PCR using primers differentiating MA Ta and MA Ta loci demonstrated that the ability to mate as haploid cells correlated well with the loss of either of the two copies of chromosome III (Fig. 3B). To rule out the possibility that these maters were a result of meiosis that would have generated mating-capable haploids, we did Fl fluorescence activated cell sorting (FACS) analysis to examine the overall DNA content of cells from the patches on minimal medium plates. Figure 30 shows that prior to mating with the tester, all starting diploid cells contained 2N or slightly higher than 2N DNA. Upon mating with the haploid mater, the DNA content further increased (marked by the arrows of the G1 peaks), thus manifesting the fusion of two sets of genome. Consistent with the conclusion that these H3 mutant diploid cells had the aneuploidy phenotype, we observed that K42A, G44A, G448, and T45A diploid cells either had very low sporulation efficiency, or, if tetrads were formed, low germination rate (data not shown). Finally, in contrast to the H3 mutants, the H4 R358 mutation behaved like wild-type cells, thus arguing against the possibility that all benomyl hypersensitive mutants suffer from chromosome instability. We thus surmise that K42, G44, and T45 are important for cells to maintain mitotic chromosome stability. 131 K42A and T45A mutations compromise tension sensing function and 8901 p recruitment The physical proximity of K42, G44, and T45, as well as the similar phenotypes in benomyl hypersensitivity and chromosome instability strongly suggest that the new K42A and T45A alleles also are defective in monitoring the tension between sister chromatids. To address this question, we analyzed the Pds1 p securin abundance after repressing the expression of an essential cohesin complex I4 component, Mcd1 p/Scc1 p. In the absence of Mcd1 p, the cohesion between L4 sister chromatids is lost, thus eliminating the tension without affecting the spindle-kinetochore attachment (3). Shifting cells from galactose to glucose medium represses the PGAU-controlled MCD1 expression and prevents cohesin complex formation (see panel A of Fig. 4), Figure 4B shows that wild-type cells stabilized the Pds1p, whereas K42A (Fig. 4C) and T45A (Fig. 4D) mutant cells failed to do so. The inability of cells to respond to the lack of tension is in excellent agreement of the documented tension sensing defects caused by the G448 mutation (23). We conclude that K42, G44, and T45 together form a tension sensing motif. Yeast and other eukaryotic cells monitor the tension status via the Shugoshin family proteins. Recruitment of Sgo1p, the budding yeast Shugoshin protein, to the pericentromeres is essential for the tension sensing function. Given that overexpressing SGO1 suppresses the benomyl hypersensitivity phenotype of both K42A and T45A mutants (Fig. 10), we suspected that these two alleles 132 damaged the ability of H3 to retain Sgo1p at the pericentric region, hence compromising the tension sensing activity. We first examined the steady-state 8901 p concentration by western blotting, and found that all strains expressed Sgo1p at a comparable level (Fig. 50). We then conducted ChlP to see if the localization of 8901 p is affected by the new TSM mutations. Figure 5 shows that Sgo1p was recruited successfully to CEN16 in all strains tested. However, the pericentric 8901 p was significantly reduced in K42A and T45A mutant cells. The abrupt drop of the SgoIp signal at as close as 0.3 kb to CEN16 in these two mutants strongly suggests that the recruitment of Sgo1p to the centromere, in which the canonical H3 is replaced by a centromere-specific H3 variant Cse4p, is realized by a mechanism different from that for pericentric Sgo1p enrichment. This notion is further confirmed by in vitro pull-down assays that examined the physical interaction between H3 and Sgo1p. Figure 6 shows that the wild-type H3 purified from yeast was able to interact with a GST-tagged Sgo1p. In stark contrast, K42A and T45A mutant H3 exhibited much weaker affinity for the recombinant 8901 p. Consistent with genetic test, P43A mutant H3 binds Sgo1p similarly as wild-type. We thus conclude that both K42A and T45A mutations compromise severely the ability of H3 to interact with 8901 p in vitro and in vivo, and that the tension sensing motif of H3 functions through physically recruiting or retaining 8901 p at the pericentromeres. Expanding the pericentric domain of Sgo1p 133 One of the possible mechanisms for Sgo1p enrichment at the pericentric region is that a centromeric factor nucleates the initial recruitment. Excessive Sgolp molecules then spill over sideway to interact with histone H3 in nucleosomes adjacent to the centromere. This model does not require an obligatory pericentromere-specific epigenetic mark for 8901 p binding. The fact that bulk H3 associates effectively with Sgo1p in vitro is consistent with this notion. If correct, this model predicts that overexpressing SGO1 would lead to expansion of existing Sgo1p domain. Indeed, yeast cells receiving a 2p plasmid harboring the SGO1 gene exhibited a wider Sgo1p domain around CEN16 when compared with those expressing SGO1 from an ARS CEN plasmid (Fig. 7). Moderate Sgo1p signals can be seen as far as 20 kb from CEN16 if Sgo1p was overexpressed (Fig. 7B). This increase in Sgo1p association was not seen at the SPT15 locus, more than 300 kb from CEN5. Because all ChlP PCR reactions were quantified with an internal DED1 control (see gel pictures in Fig. 7A), the effect we saw on the pericentric Sgo1 p domain Clearly was not due to an increase of non-discriminating genome-wide association of Sgo1p to chromatin. The HAT activity of Gcn5p likely regulates TSM function While it is highly likely that 8901 p was first recruited to the centromeres and then spreads to pericentromeres, it remains enigmatic how 3901 p is excluded from the chromosomal arms where H3 is nearly ubiquitously present. One intriguing possibility is that the H3-Sgo1 p interaction is subjected to direct or indirect regulation such that only the pericentric H3 is amenable to Sgo1p association. 134 Because bacterially expressed Sgo1p and H3 interact well [(23) and data not shown], it seems likely that the H3-Sgo1p interface is negatively regulated by a reversible modification. The Gcn5p histone acetyltransferase, though well-known for its roles in transcriptional activation, has also been shown to exert a centromere-related function during mitosis (32, 35). Suspecting that Gcn5p may be involved in the H3 mitotic function exerted via K42, G44, and T45, we examined the effect of deleting GCN5 from each of these H3 mutants. Figure 8A fl 1.....- - shows that deleting GCN5 effectively suppressed the benomyl hypersensitivity of G44S, G44A and K42A strains. Surprisingly, the T45A mutant was refractory to this suppression. The latter result is critical in that it rules out the possibility that deleting GCN5 resulted in a global change in benomyl flux or metabolism. To further delineate whether the suppression was related to the HAT activity of Gcn5p, we overexpressed two dominant negative alleles of GCN5, gcn5 F221A and gcn5 E173H (22), that were devoid of the catalytic activity. Figure 8B shows literally identical results as those in Figure 8A. That is, the hypersensitivity to benomyl of K42A and G44A (G448) mutant cells was suppressed by the catalytically inactive alleles of GCN5, whereas the T45A mutant resisted this suppression. In addition to benomyl hypersensitivity, we noticed that mutations at K42, G44, and T45 also caused cellular hypersensitivity to a nucleoside analog, hydroxyurea (HU) (Fig. 88). However, overexpression of the GCN5 HAT inactive mutant had no effect on the HU hypersensitivity of above mentioned H3 mutant, strongly suggesting that the genetic interaction between Gcn5p and H3 is specific to the regulation during mitosis. The notion that Gcn5p is a negative 135 regulator for the H3 mitotic function was further supported by the observation of enhanced benomyl sensitivity when the wild-type Gcn5p was overexpressed in the K42A, G44A, and G448 mutants (Fig. 8B). Moreover, this negative regulation of H3 function by Gcn5p HAT activity requires a functional Sgo1p. Upon removing SGO1, we see that the suppression by gcn5 F221A and gcn5 E173H was completely reverted (Fig. 9), suggesting that Gcn5p regulates H3 function in the same pathway as Sgo1p and that Gcn5p controls both H3 and Sgo1p in their , mitotic functions. 136 DISCUSSION This work uncovered a tension sensing motif in histone H3 that is important for mitotic checkpoint control. Three core residues of this motif, Lys42, Gly44, and Thr45 are all important for interactions with 3901 p both in vitro and in vivo. Alanine substitutions of these three residues share similar mitotic phenotypes, and are all susceptible to the overexpression of SGO1, suggesting that they function in the same pathway for monitoring tension between sister chromatids. Among the three residues, Lys42 and Thr45 are known to be modified under certain conditions. Methylation of Lys42 is important for transcriptional regulation in yeast (E. M. Hyland and J. D. Boeke, personal communication). However, transcriptional defects caused by K42A cannot be rescued by SGO1 overexpression (our unpublished observation), arguing against the notion that the mitotic function involving Lys42 is modulated by its methylation. Consistent with this notion, K42R, which prevents such modifications as acetylation and methylation, is functionally neutral for mitotic control (Fig. 1C). Thus, we do not believe that Lys42 modification is functionally linked to mitotic regulation. On the other hand, it is intriguing that deleting GCN5, or simply inactivating its acetyltransferase activity, is sufficient to suppress the benomyl hypersensitivity phenotype associated with K42A, G44A, and G448 mutations, but not the T45A mutation. It is tempting to speculate that Thr45 is a downstream target of Gcn5p. 137 Threonine acetylation, though not yet known by Gcn5p; has been documented in at least one report (27). Another possibility is that a yeast Thr45 kinase is regulated by the HAT activity of Gcn5p. When H3 Thr45 is phosphorylated, the H3-Sgo1 p interaction will be inhibited, thus compromising the H3 function for tension sensing. Thr45 phosphorylation has been documented recently by the Kouzarides group (14). They showed that phosphorylation of Thr45 is elevated in apoptotic neutrophils, and postulated that this modification somehow facilitates h the nick and fragmentation of the surrounding DNA during apoptosis (14). Recently, Grant and colleagues also reported the phosphorylation of H3 Thr45 in S. cerevisiae. H3 Thr45 phosphorylation peaks during DNA replication, and is mediated by the S phase kinase CdC7-Dbf4 complex (1). It will be interesting to see whether phosphorylation of Thr45 by Cdc7-Dbf4 complex depends on the functional Gcn5p, and whether that phosphorylation is linked to the H3 TSM- 8901 p mitotic function. Studies in fission yeast indicated that Bub1p phosphorylates histone H2A at a conserved residue Ser 121. H2A S121A mutant cells phenocopy Bub1p kinase- inactive mutant bub1-KD in defects of kinetochore-microtubule attachment and centromeric protection. The phosphorylation of H2A and Bub1p kinase activity function in the same pathway for the centromeric localization of Sgo1p (8, 16). It will thus be interesting to examine whether pericentic H2A also actively regulates 8901 p localization, and whether the phosphorylation level of H2A is affected by the HAT activity of Gcn5p. 138 All tension sensing mutants selectively eliminate the pericentric Sgo1p but leave the centromeric Sgo1p unaltered, suggesting that 8901 p is first recruited to the centromere/kinetochore, and then spreads to the neighboring pericentric H3 TSM for protein-protein interactions. Moreover, overexpressing Sgo1p widens the pericentric domain of Sgo1p. Therefore, we propose a Spillover model explaining the establishment of the 8901 p domain at pericentromeres and how chromatin modulators such as Gcn5p may affect the H3-Sgo1p interaction that is central to the tension sensing function of H3. In this model, we suggest that a centromere factor, likely the Bub1p kinase and the phosphorylated H2A (16) nucleates the recruitment of Sgo1p at centromere. Excessive 8901 p molecules then spill over to the pericentric region where they are retained by the TSM of H3, hence establishing the pericentric domain by which the tension status is monitored. The spreading or maintenance of Sgo1p at the pericentromeres is also hypothesized to be regulated by Gcn5p. Genetic data suggested that Gcn5p negatively regulates the H3 TSM-Sgo1p mitotic function. It is possible that the HAT activity of Gcn5p prevents pericentromeres from recruiting/retaining of Sgo1p. 139 TABLE 3-1. Yeast strains used in this study Strain Relevant genotype Source or reference yJL170 MA Ta ad92-1 can 1—100 his3—11,15 leu2-3,112 trp1-1.':sgo1::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb112NathtaZ- htb2::HPH pMK439G44S [ARS CEN LEU2 HTA1-HTB1 hht2-G44S- HHF2] This study yJL171 MA Ta ad92-1 bar1 A can 1—100 his3—1 1, 15.‘.'pGAL-MCD1.'.'HIS3 Ieu2- 3, 1 12 trp1-1::PDS1-Myc13:: TRP1 ura3-1 hht1-hhf1.‘.’KAN hhtZ— hhf2.'.'KAN hta1-htb1ZZNat htaZ-hthIIHPH pQQ18 [ARS CEN LEU2 H TA 1 -HTB1 HHT2-HHF2] (23) yJL339 MA Ta/a his3A1 Ieu2A0 met15A0 ura3A0 hht1-hhf1.':KAN hhf- 2hht2.‘.'NA T hta1-htb1.'.'HPH htaZ-htb2::NA T pQQ18 [CEN LEU2 H TA 1 -H TB 1 HHT2-HHF2] This study yJL340 MA Ta/a his3A1 Ieu2A0 met15A0 ura3A0 hht1-hhf1.‘.'KAN hhf- 2hht2.'.'NA T hta1-htb1::HPH hfaZ-htb2.'.‘NA T pMK43QG44S [CEN LEU2 H TA 1-HTB 1 hht2-G44S-HHF2] This study yJL343 MATa adeZ-1 can 1—100 his3—11,15leu2—3,112 trp1-1::SGO1- 6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2-htb212HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] (23) yJL369 MATa ad92-1 can 1-100 his3-11,15 leu2—3,112 trp1-1 ura3-1 hht1- hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nathta2-htb212HPH pMK439P43AjARS CEN LEU2 HTA1-HTB1 hht2-P43A-HHF2] This study yJL431 MA Ta ad92-1 can 1—100 his3—11,15 leu2-3,112 trp1—1::sgo1zzTRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK439644A [ARS CEN LEU2 HTA1-HTB1 hht2-G44A- HHF2] This study yJL467 MA Ta ad62-1 can 1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 hht1- hhf1::KAN hht2-hhf2.‘:KAN hta1-htb112Nat htaZ-htb222HPH pMK439K42A [ARS CEN LEU2 HTA1-HTB1 hht2-K42A-HHF2] This study yJ L468 MA Ta ade2-1 can 1-100 his3-11,15 Ieu2-3,112 trp1-1 ura3-1 hht1- hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2-htb2::HPH pMK439K4ZQ [ARS CEN LEU2 HTA1-HTB1 hht2-K4ZQ-HHF2] This study yJ L469 MA Ta ad92-1 can 1—100 his3-11,15 Ieu2-3, 112 trp1-1 ura3-1 hht1- hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat htaZ-htb2::HPH pMK439K42R [ARS CEN LEU2 HTA1-HTB1 hht2-K42R-HHF21 This study yJ L470 MA Ta ad92-1 can 1-100 his3-11,15 Ieu2-3,112 trp1-1 ura3-1 hht1- hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2-htb2zzHPH pMK439K42R/G44S [ARS CEN LEU2 HTA1-HTB1 hht2-K42R/G44S- HHF2] This study yJL471 MA Ta ad62-1 can 1-100 his3—11, 15 leu2—3, 112 trp1-1 ura3-1 hht1- hhf1.'.'KAN hht2-hhf2.'.'KAN hta1-htb1zzNat htaZ-hthIIHPH pMK439T45AiARS CEN LEU2 HTA1-HTB1 hht2-T45A-HHF2] This study yJL475 MA Ta/a his3A1 Ieu2A0 met15A0 ura3A0 hht1-hhf1.'.'KAN hhf- 2hht2.'.‘NA T hta1-htb1::HPH htaZ-htb2.'.'NA T pMK439K42A [CEN LE U2 H TA 1 -HTB1 hht2-K42A-HHF2] This study yJ L479 MA Ta/a his3A1 Ieu2A0 met15A0 ura3A0 hht1-hhf1.’:KAN hhf- 2hht2.‘.‘NA T hta1-htb1.'.'HPH htaZ-htb2xNA T pMK439T45A [CEN LE U2 H TA 1 -H T81 hht2- T45A-HHF2] This study 140 TABLE 3-1 Cont yJ L480 MA Ta adeZ-1 can 1—100 his3—11,15 leu2—3, 112 trp1-1::sgo1::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat htaZ- htb2::HPH pMK439K42A [ARS CEN LEU2 HTA1-HTB1 hht2-K42A- HHF21 This study yJ L481 MA Ta adeZ-1 can 1—100 his3—11, 15 leu2—3, 112 trp1-1::sgo122TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat htaZ- htb2::HPH pMK439T45A [ARS CEN LEU2 HTA1-HTB1 hht2-T45A- HHF2] This study yJL486 MA Ta ad92-1 can 1—100 his3—11,15Ieu2—3,112trp1—1 ura3-1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2-htb2::HPH pJH33 [ARS CEN URA3 HTA1-HTB1 HHT2-HHF2] This study yJL487 MA Ta ad92-1 bar1A can 1—100 his3—11,15::pGAL-MCD1::HIS3 leu2- 3,112 trp1-1::PDS1-Myc13::TRP1 ura3-1 hht1-hhf1::KAN hht2- hhf2::KAN hta1-htb1::Nat htaZ-htb2::HPH pMK439K42A [ARS CEN LEU2 HTA1-HTB1 hht2-K42A-HHF2] This study yJL492 MA Ta ad62-1 bar1A can 1—100 his3-11,15::pGAL-MCD1::HIS3 leu2- 3 112 trp1-1. :P-DS1 Myc13.. TRP1 ura3-1 hht1-hhf1. .KAN hht2- hhf2. :KAN hta1-htb1: .N..athta2-htb2 HPH pMK439T45A [ARS CEN LEU2 HTA1- HTB1 hht2-T45A- -HHF2] This study yJL506 MA Ta adeZ-f can 1-100 his3—11,15Ieu2—3,112 trp1-1 ura3-1..gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] This study yJL507 MA Ta adeZ-1 can 1-100 his3-11, 15 Ieu2-3,112 trp1-1 ura3-1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pMK43QG44S [ARS CEN LEU2 HTA1-HTB1 htf2-G44S-HHF2] This study yJL508 MA Ta ad92-1 can 1—100 his3-11, 15 Ieu2-3, 112 trp1—1 ura3-1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pMK43QG44A [ARS CEN LEU2 HTA1-HTB1 htt2-G44A-HHF2] This study yJL509 MA Ta ad92-1 can 1-100 his3—1 1, 15 Ieu2-3, 112 trp1-1 ura3-1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pMK439K42A [ARS CEN LEU2 HTA1-HTB1 htt2-K42A-HHF2] This study yJL510 MA Ta ad62-1 can 1-100 his3-1 1, 15 Ieu2—3, 1 12 trp1—1 ura3-1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat htaZ-htb2::HPH pMK439T45AjARS CEN LEU2 HTA1-HTB1 htt2-T45A-HHF2] This study yJL540 MATa ad92-1 can 1—100 his3—11,15Ieu2—3,112 trp1—1.78601- 6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pMK439K42A [ARS CEN LEU2 HTA1-HTB1 hht2- K42A-HHF2] This study yJL543 MA Ta adeZ-1 can 1—100 his3—11, 15 Ieu2-3,112 tlp1-1.‘.’SGO1- 6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pMK439T45A [ARS CEN LEU2 HTA1-HTB1 hht2- rzsxerflszy This study yMK1174 MA Ta/a his3A1 Ieu2A0 met15A0 ura3A0 hht1-hhf1.‘:KAN hhf- 2hht2.'.'NA T hta1-htb1.'.'HPH htaZ-htb2::NA T pJH33 [ARS CEN URA3 H TA 1 -HTB1 HHT2-HHF2] This study yMK1243 MATa ad92-1 can1-100 his3—11,15Ieu2—3,112trp1—1 ura3-1 hht1- hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2-htb222HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2L (23) yMK1361 MA Ta ad92-1 can 1-100 his3-11, 15 Ieu2—3, 112 trp1—1::sgo1::TRP1 ura3-1 hht1-hhf1.‘.‘KAN hht2-hhf2.':KAN hta1-htb1zzNat htaZ- htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] This study 141 TABLE 3-2. Plasmid constructs used in this study Plasmid Main features Source or reference pJH33/pMK440 pRS316-HTA1-HTB1 HHT2-HHF2 (23) pJL51 2pm URA3 pADH1-3xHA-SGO1-tADH1 This study pJL52 ARS1 CEN4 URA3 pADH1-3xHA-tADH1 This study pJL53 ARS1 CEN4 URA3 pADH1-3xHA-SGO1-tADH1 This study pJL55 pGEX-4T-1 3xHA-SGO1 This study pJL74 pRS315—HTA1-HTB1 This study pMK572 2pm URA3 vector with ADH1 promtoer and (23) terminator pMK573 2pm URA3 SGO1 (23) pQQ18/Pmk439 pRS315—HTA1-HTB1 HHT2-HHF2 (23) 142 FIG. 3-1. The Tension Sensing Motif (TSM) residues function through Sgo1p. Yeast cells bearing the specific allele (wide-type or mutated as noted) as the sole copy of H3 were tested on YPD medium under indicated conditions. Six-fold serially diluted log-phase cells were spotted for growth test at 30°C for two days. (A) K42, G44, T45 are key residues of a TSM of H3. Single mutations of residues from K36 through K56 were compared for growth on benomyl plates. Arbitrary scores for benomyl hypersensitivity were assigned. -1 and -2 represent mild and severe hypersensitivity, respectively. (B) Structural view of the region that contains TSM. Left panel: Close-up view of the TSM (marked red from K42 to T45), showing that the B-turn and a preceding region (K37 to Y41, marked yellow) connects the flexible H3 N-terminal tail (Pink) and the rigid a N domain (Blue). Right panel: close-up view of the 42Lys-Pro-Gly-Thr B-turn. (C) Detailed benomyl hypersensitivity analysis of the TSM residues with focus on mutations on K42, G44, and T45, single or in combination. (D, E) Over-expression of SGO1 suppresses the benomyl hypersensitivity of tension sensing mutants. "—" sign indicates unaltered residues or no plasmids introduced. Yeast cells were transformed with 2pm SGO1 (pMK573) or the corresponding empty vector (pMK575), followed by spot assays to compare the benomyl hypersensitivity. VC, vector control. 143 Fig. 3-1 YPD, Benomyl Benomyl 30° 5pg/ml 10pg/ml Score . ... __ ,l. . coco-K cos 0 I51A, R52A, F54A, Q55A: too sick to score 144 Fig. 3-1 continued C YPD, Benomyl Benomyl K42 G44 T45 R R R A A Q C(DCDNO) (thON—8 A Vector 2p SGO1 K42 G44 T45 A ___ __ I Q .. ..- R l YPD,30° R s .. ..- A l A --- ... t g : : l Benomyl R S ___ 7.5 pglml ... --- A J 145 Fig. 3-1 continued YPD, Benomyl Benomyl 30° 7.5 pg/mI1O pglml YPD, Benomyl Benomyl 30° 7.5 pglml 10 pglml 146 H3 2p plasmid WT H39A H39A R40A R40A WT H39A H39A R40A R40A H3 VC VC SGO1 SGO1 SGO1 SGO1 SGO1 2p plasmid G44A Y41A Y41A R49A R49A G44A Y41A Y41A R49A R49A SGO1 SGO1 SGO1 SGO1 SGO1 Fig. 3-2 Time, min 0 45 60 75 90 105 120 135 150 165 Benomyl ---_____.......— 0 WT -------.- .. 30pg/ml &‘-~a~~m—a—.—I 0 K42A «3---flan--- 30pg/ml T45A ------‘-- 30pg/ml Pds1p-13xMyc FIG. 3-2. K42A and T45A mutant cells responded normally to benomyl treatment by activating the spindle assembly checkpoint. Pds1 p was activated and stabilized in wild-type, K42A and T45A mutant cells in the presence of benomyl. G1 arrested cells were released at T0’ into rich medium containing 0 or 30 pg/ml of benomyl. The same number of cells was taken at the indicated times for boiling and whole-cell extract preparation, followed by anti-Myc western blotting analysis to quantify the abundance of Pds1p. 147 FIG. 3-3. The K42A and T45A mutant cells exhibited Chromosome instability. (A) Diploid mating assays. Yeast diploid cells (yMK1174 derivative, defective in expression of tip, ura, his) bearing histone (H3 or H4) mutation as listed in the left to the image were first patched into YPD (rich medium) plates, and then replica plated into SD (minimum medium) plates containing haploid mating tester cells, three control strains (in an order as, MA Ta, MA Ta, and MA Ta/a) were included for comparison in the last row. Mating with the tester cells complemented the nutrient requirement for strains bearing histone mutations, thus selectively allowed the mated cells to grow on the SD minimal medium. (B) Diploid mating ability correlated well with the loss of one of two copies of chromosome III. Shown were genomic PCR results with primers flanking the mating loci to differentiate the existence of MA Ta or MA Ta. Except haploid control cells MA Ta and MA Ta, all others used mated cells to extract genomic DNA for PCR. Listed below gel image were images from their corresponding mating assays. G/S, G44S; WT, wild-type; KIA, K42A; T/A, T45A. (C) Representative images from Fluorescence Activated Cell Sorting (FACS) analyses. Logarithmically growing cells were stained with propidium iodide and subjected to FACS analysis. Dash lines were aligned to G1 peak of wild-type cells, and fine dot lines to G1 peaks of their parent strains (original diploid cells before mating), arrows used to highlight the shifts of G1 peaks. 148 Fig. 3-3 SD w/ a tester SD M a tester H4 R358 H3 6448 H3 WT H3 K42A H3 T45A Control , : I. II.“ l""|l| ...“. a a i'l'il-II W "In." "NIHIII-I . . . , j . ' . j. "" MA Ta 149 Fig. 3-3 continued Iv. .. . ..It i\ .H .. Aim-Hr t kkkkkk H 1 I4. . L W... ....“ w. . ..m m I -1---... ... N W In -. .1-.--- u I i M . I... p - y 8ll‘hfl.8. 8.18.88v88‘888li88i‘81ML 88888888888 Lm' 88888888888 +88 .-..-.» . . :1... . J .34 150 .90.: : \ . D l1 - m .1 .1818 . u . 8 1111‘. ~( g 81- . . ‘0 .44". m w J.- i ildni i i. .i i i...ii198553343:Ekfinificsfiidfiififie9fiaesswf . .0. rl Ill.” 1.4 I" -. - . i .- 11.2.1111 . 5‘ 1.. ... v. v, '4 8 I o I. , S I." 8’ .f FIG. 3-4. Defects in responding to tensionless crisis in K42A and T45A mutants. (A) Schematic drawing to elucidate the experimental design. The MCD1 gene, which encodes an essential cohesin component, was placed under the GAL1 ... . promoter control. Shifting G1-arrested cells from galactose (Gal) to glucose (Glc) represses MCD1 expression that is needed for cohesion in S phase. Tension will 1 not be built up without sister cohesion, even though the spindle-kinetochore attachment is unaffected. Normal cells (B) stabilize Pds1p to sustain the spindle checkpoint activity, whereas tension sensing defective mutant cells (K42A in panel C, T45A in panel D) fail to do so, giving rise to Pds1p oscillation in Glc media. At indicated times same number of cells was taken for boiling and whole- cell extract preparation. The abundance of Pds1p was quantified by normalizing with the loading control (western against G-6-PDH antibody). X axis, time in Cal or Glc; Y axis, relative Pds1p band intensity. Close diamonds, Gal-to-Gal; open circles: Gal-to-Glc. Note that the T45A cells grew more slowly, wider intervals was needed to detect Pds1 p fluctuation. 151 Fig. 3-4 A ('5 Gal: Glc: Attachment I+ 0.) Attachment (+) TENSION (1’) Tension (-) PGAL1-MCD1 K; 9 B Time, min 0 45 60 75 90 105 120135 150 165 Gal-to-Gal - - - a— -- ...- n- - a. Pds1p- ’ ‘ ‘ "' ”- 13xMyc Gal-to-Glc - - - - - - - - - Gal-to-Gal : '2; r: .°-; I . 7 ' G-G-PDH Gal-to-Glc 3: ,, Ratio -0— Gal-to-Gal -O- Gal-to-Glc WT 152 Fig. 3-4 continued C Time, min Gal-to-Gal Gal-to-GIC 0 45 60 75 90 105 120 135150 165 ‘ u 0 “ ' - r u‘ "a. —- ..-..zua-IC-‘O-I- Gal-to-Gal _; Gal-to-Glc Time, min Gal-to-Gal Gal-to-Glc Gal-to-Gal Gal-to-Glc "‘"’ Gal-to-Gal "'0" Gal-to-Glc K42A 0 65 80 95 110130150170190 210 ’0' Gal-to—Glc -."" Gal-to-Gal T4 5A 153 Pds1p- 13xMyc Ratio Pds1 p- 13xMyc G-6-PDH Ratio FIG. 3-5. The K42A and T45A mutation selectively downregulates the pericentric recruitment of Sgo1p in vivo. (A) ChlP analysis of HA-tagged Sgo1p. Samples in all of the panels are arranged in the same order. The common internal control (marked by the star on the right) for multiplex PCRs is from within the ORF of the PGK1 gene 24.3 kb to the right of CEN3. Targets of the PCR fragments and their distance to the cognate centromeres are listed at the top of each gel image. R, right; L, left. SPT15 is 313.3 kb to the right of CEN5. (B) Quantification of the ChIP results. Ethidium bromide-stained DNA gel images were quantified by NIH Image J. The intensity of each CEN or pericentric fragment was compared to that of the PGK1 internal control (star). The ratio was then normalized to 0.1% input (INP) DNA (set at 1.0 and showed with a dash line). Error bars represent standard deviations from at least three independent cell cultures for ChlP. Ab, antibody. (C) Sgo1p protein abundance is not affected by the K42A and T45A mutation. Western blotting of C’-6xHA-tagged Sgo1p demonstrates the equal abundance of Sgo1p in these three strains. The loading control is cross-reacting bands from yeast lysates. 154 Fig. 3-5 A Grill/16849.31, V . CEN16 I. 4 ‘* «I a. Q... urn-.'- L ' D ‘s M; L—u‘ w w w CEN15L.°:3kb . -.C.E..’.V1..6.E. 1:739- fin: "~'°’: . . . _ . . 7:3.3 .'.‘uuv‘h -- - . f ‘g Y? WTK/A T/A \ e9 a-HA Ratio to 0.1% Input 1....- 01 9.5!.‘(7555x5k . i. 5.3: , . M this. h“ *3..qu ' W . y .' . 1. - , 3.57.15. UWT, no Ab IWT, ChlP UK42A, ChlP IT45A, ChlP _ __________ ___________________ il in ..| Eu 1:. in CEN16 CEN16L CEN16LCEN16RCEN16RCEN16R SPT15 0.3kb 1.7kb .-. . -. 1 I“ W '- F..«“ fl 2345 H3: WT T45A K42A 4.0kb 6.5kb 9.1kb 4— Sgo1p-HA Cross-reacting Bands 155 Fig. 3-6 Matrix , GST L H I r- ~ * Bound GST-Sgo1p F. - . J‘ - J I ~- ...- - ‘ “I Input (20%) WT K42A T45A P43A WT K42A FIG. 36. The K42A and T45A mutation attenuates the affinity of H3 to Sgo1p in vitro. GST pull-down assays were conducted with bacterially expressed GST alone or GST-Sgo1p fusion proteins, as well as core histones purified from yeast cells expressing the wild-type, K42A, P43A, or T45A alleles of H3. Bound or input materials were resolved and blotted by anti-H3 antibodies. 156 FIG. 3-7. Over-expression of SGO1 in wild-type cells expands the existing pericentric domain of Sgo1p. (A) Yeast cells transformed with either a high-copy 2pm plasmid (pJL51) or an ARS CEN low-copy plasmid (pJL53) expressing HA- Sgo1p were analyzed by anti-HA ChlP. The assay conditions were identical to those described in the legend to Fig. 5. except the common internal control (marked by the star on the right) for multiplex PCRs is from within the open reading frame of the DED1 gene 386.2 kb to the right of CEN15. The CEN or pericentric PCR fragments were marked by the arrow on the right. (B) Quantification of the ChlP results was done as detailed in the legend to Fig. 5B. (C) Comparable expression of 2pm 3xHA-Sgo1p (pJL51) and ARS CEN 3xHA- Sgo1p (pJL53). H3 wide-type cells bearing the indicated recombinant SGO1 constructs were examined by anti-HA Western blotting. Equal loading was evidenced by Coomassie blue R250 staining (not shown) and by proteins that cross-reacted with the anti-HA antibodies. 157 Fig. 3-7 A CEN16 CEN16R 4.0kb CEN16L 8.5kb CEN16R 9.1kb . “ ““2. -. “"1 f‘ . I ...“... ‘3... heuuu.‘ L“ '. jEWI~-~W<:"" -. " ‘ 1:41, I ' . “be” a“ M.,. y-a “bu“ * Antibody: INP - + + HA-SGOII 2“ 2“ 31333; CEN16R12kb CEN16R 20kb SPT15 i... . ...-..4 i...- éwt... 4 Q 4... .... 3 B 3.0 - .. INO Ab 3 g 2.5 - I2p SGO1 $8 2.0 _ UARS SGO1 c5 9 1.5 - .9 1'6 CE 1.0 ~~ - 0.5 4 0.0 1 -: CEN16 CEN16R CEN16L CEN16R CEN16R CEN16R SPT15 4.0kb 8.5kb 9.1kb 12kb 20kb C -- .. ...... 4—HA-Sgo1p ’ Cross-reacting ::ch Bands 1 2 3 4 SGO1: 2p ARS/CEN4 158 FIG. 3-8. The HAT activity of Gcn5p is a negative regulator of the tension sensing function of H3. (A) GCN5 was deleted in the indicated H3 strains, which were then tested for benomyl tolerance. Note that the T45A is refractory to the gcn5A suppression. (B)'WiId-type and two catalytically inactive alleles of GCN5, gcn5F221A and gcn5E173H, were over-expressed in the indicated (H3 wild-type or mutants, shown at the top of images) strains, and the cellular resistance to benomyl and hydroxyurea (HU) were examined. 159 Fig. 3-8 YPD, 30° Benomyl 7.5 pg/ml Benomyl 10 pg/ml _H_3 GCN5 gcn5A GCN5 gcn5A 0005A Vector GCN5 gcn5 F221A gcn5 E173H Vector GCN5 gcn5 F221A gcn5 E173H Vector GCN5 gcn5 F221A gcn5 E173H Vector GCN5 gcn5 F221A gcn5 E173H Vector GCN5 gcn5 F221A gcn5 E173H G44S G44A 160 K42A GCN5 T4 5A YPD 30° Benomyl 7.5 pglml Benomyl 1O pglml HU 75mM HU100 mM Fig. 3-9 sgo1A G44A K42A T45A Vector ‘ GCN5 ~ 0 gcn5 F221 A ="?~ ‘3'”: ‘ . YPD 30 gcn5 E173H ' ‘ ' Vector GCN5 Benomyl gcn5 F221A - 5 99"” gcn5 E173H Vector GCN5 Benomyl gcn5 F221A . 0 337'- 75 pg/ml Gcn5 E173H O :23. ' , FIG. 3-9. 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Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase. Embo J 17:3155-67. 165 APPENDICES 166 Appendix I: Linking H3 phosphorylation to a Saccharomyces cerevisiae14-3-3 protein Bmh1p INTRODUCTION Phosphorylation of histone H3 at serine 10 (S10) has stimulated considerable interests since the first discovery that this modification is associated with chromosome condensation and segregation during mitosis and meiosis (3, 12, 26, 27). At interphase, H3 S10 phosphorylation is linked to transcriptional activation of a subset of inducible genes (22). Mitotic phosphorylation of H3 also has been found to occur at serine 28 ($28) (9). A newly discovered mitotic phosphorylation was mapped to serine 31 (831) of the H3 variant H33 (13). Interestingly, Saccharomyces cerevisiae contains only one type of H3, which is most similar to mammalian H33 and also contains S31 (1). However, the phosphorylation of H3 at $28 and S31 in S. cerevisiae has not been addressed yet. Mitosis-specific phosphorylation of H3 starts in late 62 phase on pericentric chromatin. As mitosis proceeds, phosphorylation of H3 spreads along the chromosome arms and completes its coating the entire chromosome at prophase. The dephosphorylation process starts at anaphase and finishes at telophase (10, 14). Recently, members of the Aurora serine/threonine kinase family have been shown to be important for mitotic phosphorylation of H3 at S10 (15) and $28 (10). 167 In S. cerevisiae, the sole Aurora kinase encoded by the IPL1 gene (4), is required for the high-fidelity chromosome segregation (6). Later on, studies indicated that lpl1p is required to activate the spindle checkpoint in response to defects in the tension between two sister chromatids (21 ). How the phosphorylation of H3 contributes to the mitotic progression and the proteins that interact directly with the phosphorylated H3 remain elusive. 14-3-3 proteins are a family of highly conserved, ubiquitously expressed, and abundant acidic proteins in all eukaryotes studied (2). 14-3-33 were first recognized to interact with a discrete phosphoserine or phosphothreonine motif by forming homo- or heterodimers (28). While there are seven 14-3-3 isoforms in mammals, Bmh1p and Bmh2p are the only two 14-3-3 proteins in S. cerevisiae. Bmh1p (267aa, Mw: 30kDa) and Bmh2p (273aa, Mw: 31 kDa) share 91% overall identity and >97% identity over their first 256 residues (7, 25). Simultaneous knockouts of these two functionally redundant isoforms in S. cerevisiae is lethal, while single knockout cells are viable (24). Bmh1p and Bmh2p are also required for a timely G1/S transition, DNA damage checkpoint and genome stability (16, 17). Utilizing the Tethered catalysis/Yeast two-hybrid (TC/Y2H) system that we developed for the identification of protein-protein interaction induced by a post- translational modification (11), we found that Bmh1 p interacts with phosphorylated H3 (Kuo, unpublished data). Recently, Macdonald and colleagues reported that three human 14-3-3 isoforms (5, C, y) bind H3 (aa 1-20) peptides in a phosphorylation-dependent fashion (19). In their studies, 14-3-33 168 are found to be recruited to the nucleosomes at inducible genes upon transcriptional activation. However, how this association contributes to the function of H3 phosphorylation in mitotic progression is still elusive. We set out to examine whether yeast 14-3-3 protein Bmh1p is involved in this regulation through interaction with the phosphorylated H3. Investigation of their interactions and other functionally related factors will further our understanding of the roles of H3 phosphorylation in mitotic progression and chromosome segregation. MATERIALS AND METHODS Yeast strains and plasmid constructs. The yeast strains and plasmids used in this work are listed in Tables A-1 and A-2. Mutations introduced in histone or BMH1 were generated by two-step PCR site- directed mutagenesis (18). Yeast two hybrid screen plasmids were constructed by N. Y. Hung and D. W. Guo as mentioned in reference (11); Biotinylated Bmh1 plasmid (pJL31) was constructed by PCR amplification of the entire BMH1 ORF region followed by subcloning that fragment into pAN6 plasmid at restrictions sites of BamH l and Kpn I in the C-terminus of Biotin Avitag peptide sequence (Avidity), the constructs were then transformed into AVB101 (Avidity) for biotinylated Bmh1 p expression; His-Bmh1 p plasmids (pJL33) was constructed by subcloning from pJL1 by Kpn I (blunted) and BamH I digestion into pET-28a plasmid (Novagen) on Hind III (blunted) and BamHI restriction sites and was 169 transformed into BL21 (DE3) (Sigma). Yeast methods. Yeast growth media, conditions, and transformation were based on standard procedures (23). When appropriate, 5% casamino acids (CAA) were used to substitute for synthetic amino acid mixtures as selective medium for uracil, tryptophan, or adenine prototroph. Yeast transforrnation was done with the lithium acetate method (8). Histone preparation. Histories were prepared from mid-log phase cultures in the presence of protease and phosphatase inhibitors as described (5). To obtain hypophosphorylated histones, the purified histones were treated at 30°C for 1h by A phosphatase (NEB) in the presence or absence of high concentration sodium phosphate (150mM, pH 7.4), which inhibits the activity of A phosphatase. Protein pull down assay. Biotinylated Bmh1 p and His-Bmh1p were purified from E. coli. Recombinant Bmh1 p and purified yeast histories were incubated in binding buffer (10mM HEPES, pH7.5; 150mM NaCI; 3.4mM EDTA; 0.05% NP- 40) at 4°C for 1hr, then Soft-link avidin resin (Promega) or Ni-NTA beads were added to trap tagged Bmh1p and associated histones at 4°C for an additional hour. Resins were washed with binding buffer for three times, and bound materials were eluted by 2 x sodium dodecyl sulfate (SDS) loading buffer and resolved by 15% SDS-polyacrylamide gel electrophoresis (PAGE). Western analyses were conducted as mentioned in reference (18). 170 RESULTS Bmh1 p specifically interacts with phosphorylated histone H3. To understand whether phosphorylated H3 (phos.H3) performs its biological function by recruiting specific associating proteins, we used the TC/Y2H approach (11) to screen for yeast proteins that interact specifically with phos.H3. Figure 1 shows that when H3 (aa 1-59) was fused to a wildtype IPL1 within the context of the yeast two hybrid system, H3 S10 was constitutively phosphorylated. An H3 fusion with lpl1p kinase inactive mutant was hypophosphorylated at S10. Due to the lack of appropriate antibody, we do not yet know whether H3 828 can be phosphorylated by the tethered Ipl1 p. Using the phos.H3-lpl1p bait, we collaborated with S. Fields and T. Hazbun (HHMI, University of Washington, Seattle) to screen for yeast proteins that specifically interact with phos.H3. Figure 2A shows that yeast 14-3-3 protein Bmh1p was identified as a potential phos.H3 interacting protein, and that mutations at either S10 and/or 828 diminished significantly the interactions between phos.H3 and Bmh1p. To characterize the physical interaction between Bmh1 p and phos.H3 biochemically, pulldown assays using purified histones were performed. Yeast histories were prepared and further treated with A phosphatase. Western analyses confirmed that A phosphatase effectively dephosphorylated H3 (at least at S10), whereas 150mM sodium phosphate (pH 7.4) inhibited the A phosphatase 171 activity and partially maintained the phosphorylation status of H3 (Fig. 2B). Phosphorylated and hypophosphorylated histories were then incubated with recombinant biotinylated Bmh1p expressed and purified in E. coli. Soft-link avidin resin was then used to purify the biotinylated Bmh1p and the associated proteins. Western analyses with anti-H3 antibody demonstrated that Bmh1 p interacts preferentially with phos.H3 (Fig. 20). Taken together, these data support the hypothesis that phosphorylation of H3 stimulates the Bmh1p binding to H3. Residues important for Bmh1p-phos.H3 interaction. To further investigate the molecular mechanism underlying Bmh1p-phos.H3 interaction, we constructed single, double, and triple alanine substitutions at $10, $28, and 831 of H3 and examined the specific involvement of these potential H3 phosphorylation sites. The Bmh1p binding capacity was compared by incubating comparable amounts of H3 wildtype or mutant histones with purified Hexahistidine tagged Bmh1 p. Proteins were recovered by Ni-NTA beads and eluates were subjected to SDS-PAGE and western analyses (Fig. 3A). Compared with the wildtype, H3 S28A and S10A/828A both failed to interact with Bmh1p, whereas S1OA and S10A/SZ8AIS31A showed much diminished association with Bmh1p. These results suggested that S28 phosphorylation is a major determinant for Bmh1p association. Interestingly, S10A/S31A, $28A/S31A, and S10A/828A/S31A all bound Bmh1 p better than their counterparts with wildtype $31. This surprising result suggests that the S31A mutation may suppress the defect induced by S10A and $28A. 172 Structural studies on phosphoserine binding pocket of the human 14-3-3C isofonn indicated that residues Lys49, Arg56, Arg60, Arg127, and Tyr128 are critical for 14-3-3s binding to the phosphate moiety (20, 28). Sequence alignment showed that these residues are completely conserved and are equivalent to Lys51, Ar958, Arg62, Argl 32, and Tyr133 of Bmh1p (2). Comparable binding assays were performed to examine the binding affinity among His-Bmh1 p wildtype and R58A/R62A, R132A/Y133A, R58A/R62A/R132A/Y133A mutants (Lys51 not included here). Figure 3B shows that R132A/Y133A completely and R58A/R62A/R132A/Y133A nearly completely caused the loss of their affinity for phos.H3, whereas the R58A/R62A mutation had no effect on Bmh1p binding to phos.H3. These results further supported the notion that Bmh1p-H3 association is stimulated by phosphorylation. Functional studies of the mitotic phosphorylation on H3. To understand how H3 S10 and 828 may contribute to the mitotic progression, I examined cellular growth in benomyl-containing media after mutations had been introduced into the sole copy of H3. Benomyl depolymerizes microtubules and triggers the spindle assembly checkpoint (SAC) which halts the cell cycle at G2/M. Sensitivity to benomyl can be used to evaluate the spindle or kinetochore function. Mutants defective for spindle or kinetochore function frequently show benomyl hypersensitivity. Figure 4 shows that all H3 mutants (S10A, $28A, S10A/828A, and S10A/828A/831A) behave similarly to wildtype. The lack of benomyl hypersensitivity of H3 S10A is consistent with the findings of others (15), 173 suggesting that phosphorylation at other sites or other histories substitutes for the important role of histone phosphorylation during mitosis in yeast. DISCUSSION TCIY2H results revealed that Bmh1p is a likely phos.H3 binding protein. Such observation was biochemically verified in this study as recombinant Bmh1p rather weakly binds purified hypophosphorylated H3 (Fig. 20). Slot blot assays using synthetic H3 (aa 1-20) peptides also substantiated that Bmh1p preferentially binds phos.H3 (data not shown). Given the 91% overall sequence identity between Bmh1p and Bmh2p, Bmh2p might perform similar functions as Bmh1p. It is widely accepted that phosphorylation of H3 at $10 (S10-P) is critical for mitotic chromosome segregation in Tetrahymena (27), however, in S. cerevisiae, H3 S10A by itself imposes no discernible mitotic phenotype (Fig. 4) (15), suggesting that phosphorylation at other sites of H3 can compensate for the loss of S10-P, or that other histories may substitute for this important role during mitosis in yeast. The binding assays shown in Figure 3 indicated that H3 828 phosphorylation is more important for Bmh1p association. The observation that binding between Bmh1p and H3 S1OA and/or $28A mutants was partially restored when alanine was introduced at 831 (Fig. 3A) imposes another layer of 174 complexity to their interactions. One possibility is that S31A induces compensatory phosphorylation at other sites. The observation that Bmh1 R132AIY133A and R58A/R62A/R132AIY133A mutants lost their affinity for phos.H3 is consistent with structural analyses from human 14-3-3C isoforrn (20, 28). The Bmh1 R58A/R62A mutant shows no defects in Bmh1p-phos.H3 interaction. However, it must be noted that Lys51 of Bmh1 might be critical and its mutation to alanine should be considered. However, mutations in phosphorylation sites of H3 (810, 28, 31) and H2B (S10) confer no defective phenotype tested thus far (Fig. 4), suggesting that phosphorylation at other sites of H3 (or H2B) or that other histones may substitute for this important role of H3 phosphorylation during mitosis in yeast. To summarize, Bmh1p was identified as a phosphorylated H3 binding protein. In vitro binding assays demonstrated that $10, $28 of H3 and R132, Y133 of Bmh1 are critical for the Bmh1p-phos.H3 interaction. 175 TABLE A-1. Yeast strains used in this study Strain Relevant genotype Source or reference yJL111 MA Ta ade2-1 can 1—100 his3—11,15 Ieu2—3, 112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat htaZ-htb2::HPH pMK439 H3810/31A [ARS CEN LEU2 HTA1-HTB1 hht2- S10A/S31A-HHF2] This study yJL153 MA Ta ad92-1 can 1—100 his3—11, 15 Ieu2-3,112 trp1—1 ura3-1 hht1-hhf1.':KAN hhtZ-hhf2.‘.'KAN hta1-htb1IINat htaZ-hthIZHPH pMK439 H3S28A [ARS CEN LEU2 HTA1-HTB1 hhtZ-828A- HHF2] This study yJL166 MA Ta ade2-1 can 1-100 his3—11,15 leu2-3,112 trp1—1 ura3-1 hht1-hhf1.'.'KAN hht2-hhf2.‘.‘KAN hta 1 -htb 1: :Nat htaZ—hth: : HPH pMK439 H3S10/28A [ARS CEN LEU2 HTA1-HTB1 hht2- S10A/328A-HHF2] This study yMK1243 MA Ta ad62-1 can 1-100 his3—11,15 Ieu2-3,112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb222HPH pMK439 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] This study yMK1244 MA Ta ad92-1 can 1—100 his3-11, 15 Ieu2-3, 112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::NathtaZ-htb212HPH pMK439 H3810A [ARS CEN LEU2 HTA1-HT81 hht2-S10A- HHF21 This study yMK1246 MA Ta ade2-1 can 1-100 his3-11,15 Ieu2-3,112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2-htb222HPH pMK439 H3831A [ARS CEN LEU2 HTA1-HTB1 hht2-S31A- HHF21 This study yMK1249 MA Ta ad62-1 can 1—100 his3—11,15 Ieu2-3,112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2-htb2zzHPH pMK439 H3828/31A [ARS CEN LEU2 HTA1-HTB1 hht2- SZBA/SS1A-HHF2] This study yMK1250 MA Ta ade2-1 can 1—100 his3-11, 15 Ieu2-3, 112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2-htb2: :HPH pMK439 H3810/28/31A [ARS CEN LEU2 HTA1-HTB1 hht2- S10A/828A/S31A-HHF2] This study TABLE A-2. Plasmid constructs used in this study Plasmid Main features Source or reference p AN6 :3:ng expresses N-terminal AvnTag-protein Avidity pJL31 pAN6-BMH1 This study pJL32 pAN6-BMH2 This study pJL33 pET28a-BMH1 This study pJL34 pET283-BMH2 This study 176 Fig. A-1 Bait Constructs I " — ~ [X s \ \ 1 l $1 _ it GDBD H3(1-59) lpI1WI' HA GDBD H3(1-59) lp|1 HA (E1520. V153L) B IP: anti- HA anti- HA Janti- Phos. H3 (Ser10) ...th I <— lpl1: mut mut FIG. A-1. Tethered catalysis approach to phosphorylate N-terminal tail of histone H3. (A) Schematic drawing of the tethered catalysis histone H3 baits. GDBD: Gal4 DNA-binding domain; wt: wildtype. A trimeric hemagglutinin (HA) epitope tag was fused at the C-terminus of the chimeras. Point mutation of lpl1 at E152 and V153 results in a catalytically inactive form of |p|1 and is used as a control in the screen. (B) Auto-phosphorylation of the H3 N-terminal domain shown by western analyses. Yeast whole cell extracts from strains expressing wt and mut (mutant) Ipl1 fusion were used in western analyses using anti-HA and anti- phosphorylated H3 antibodies. Unpublished data of D. W. Guo and M. H. Kuo. 177 FIG. A-2. Bmh1p specifically interacts with phosphorylated histone H3. (A) TC/Y2H analyses indicated that Bmh1 p preferentially interacts with phosphorylated H3. Bait constructs that contain H3-lpl1 fusion or with mutations introduced at H3 serine 10, serine 28 or the Ipl1 catalytic domain were used for two-hybrid test. Prey construct contains Bmh1 fused to an activation domain. 8106 and/or 828A depleted the potential phosphorylation site(s) of H3. Yeast cells transformed with the indicated bait and prey constructs were grew to log phase and streaked into SC-Ade plate to detect the gene expression of ADE2 reporter. Unpublished data of X.J.Xu and M.H.Kuo. (B, C) Association of Bmh1 p with phosphorylated histone H3. (B) Purified wildtype histone was treated by A phosphatase in the presence or absence of 150mM sodium phosphate (pH 7.4). Histones were separated in parallel lanes and analyzed by western blot using anti-H3 or anti-phosphorylated H3 (serine 10 specific) antibody. (C) Western analyses of the in vitro pull down assays. The similar amount of biotinylated Bmh1 p was incubated with untreated wildtype histone, histones treated by A phosphatase in the absence or presence of sodium phosphate (150mM, pH 7.4) separately. Top: streptavidin-HRP western analyses showing evenly immobilized Bmh1p in the assay; middle: histones bound by immobilized Bmh1 p were detected by anti-H3 antibody; bottom: 10% of input histories were separated in parallel lanes and probed by anti-H3 antibody. 178 Fig. A-2 A Bafis _ ' Mme-m. I Prey: Bmh1 H3 (323A)_lpl1 H3 (S1OG/SZBA)-|p|1 B anti-H3 . ~ anti'Phos.H3 _ (serine 1o) 7* PPase _ + + NaPi _ _ + (150mM pH 7.4) C Immobilized Bmh1p Streptavidin-HRP Bound H3 anti-H3 10% input histone anti-H3 A PPase - + + NaPi - - + 179 FIG. A-3. Residues important for the interactions between Bmh1p and phosphorylated H3. (A) Potential phosphorylation sites at H3 critical for association with Bmh1p. Histones of wildtype and mutants (single or multiple serine to alanine substitution at H3 $10, $28, and 831) were prepared from yeast mid-log phase cells in the presence of phosphatase inhibitors and incubated with purified recombinant N-terminal His tagged Bmh1 p and were pulldown with Ni-NTA beads, the bound materials were eluted and subjected to SDS-PAGE and western analyses. Top: Ponceau S staining of immobilized Bmh1p; bound histones (middle) and 30% of the histone input (bottom, separated in parallel lanes) were probed by anti-H3 antibody. S represents Serine, A represents Alanine. (B) Residues in Bmh1p essential for its binding to phosphorylated H3. The same amount of phosphorylated histone was used for each binding assay. Top: Ponceau S staining of immobilized wildtype or mutant His-Bmh1p; bottom: bound histones were probed by anti-H3 antibody. Lane 1: Bmh1 wildtype; Lane 2: Bmh1 R58A/R62A mutant; lane 3: Bmh1 R132AIY 133A mutant; lane 4: Bmh1 R58A/R62A/R1 32A/Y 133A quadruple mutant. 180 Fig. A-3 30% input Histone H3 Ser10 Ser28 Ser31 Bmh1: R58 R62 R132 Y133 Immobilized Bmh1p Bound H3 181 >>>> Ponceau S anti-H3 Ponceau S anti-H3 anti-H3 FIG. A-4. No defective phenotypes observed in mutants of histone H3 and/or H28. Yeast cells bearing the sole copy of H3 or HZB (wild type or mutated as noted) were tested on YPD medium with indicated growth condition or additional chemical reagent. Sixfold serially diluted log-phase cells were spotted for growth for two days at 30°, 37° or seven days at 14°. (A) Alanine substitutions at H3 serine 10 and/or serine 28 cause no additional growth defects in response to stress of high temperature (37°), low temperature (14°), or benomyl. Cells tested on top panel are derived from yDA81 ($288C) genetic background, whereas bottom panel are derived from yMK1141 (W303) background. (B) Additional test similar as panel A to compare wildtype and triple mutant, H3 S10A1828A/S31A. (C) Combined mutations of H3 and H28 cause no defective phenotypes. Genetic features of strains used are listed below the image, as a control, a known mitotic defective H3 mutant, G44S, was included. WT: wildtype. 182 FKLIL4 H3 YPD 30° 37° 14° Benomyl15ug/ml N."_ Hg"- “" , .f, Ihr a yDA81 denvafives 828A S1OA/828A S1OA/828A “.0 >09 0% “0%” '.® :. 0'3 :.§ W_@ Pd "4, I? '. : I-N S10A S10A $28A $28A S10A/828A S10A/828A yMK1141 denvafives Benomyl H3 YPD 30° 37° 10ug/ml 15pg/ml wo' QEJE1331 S1OA/328A/S31A . i333 I.” YPD 30° 37° /][\ #5960653? neooam .2?- 6’6 0 ‘33 «'74.: cease? GGOOG@ H23 H3 H28 H3 WT WT WT G44S S10A WT S10E WT S1OA S10A S10E S1OD S10A $28A S1OE $280 S10A S1OA/828A S1OE 3100/8280 183 REFERENCES 10. 11. Brandt, W. F., and C. van Holt. 1982. The primary structure of yeast histone H3. Eur J Biochem 121:501-10. Bridges, D., and G. B. Moorhead. 2005. 14-3-3 proteins: a number of functions for a numbered protein. Sci STKE 2005:re10. Cerutti, H., and J. A. Casas-Mollano. 2009. Histone H3 phosphorylation: universal code or lineage specific dialects? Epigenetics 4:71-5. Chan, C. S., and D. Botstein. 1993. Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast. Genetics 135:677-91. Edmondson, D. G., M. M. Smith, and S. Y. Roth. 1996. Repression domain of the yeast global repressor Tup1 interacts directly with histones H3 and H4. Genes Dev 10:1247-59. Francisco, L., W. Wang, and C. S. Chan. 1994. Type 1 protein phosphatase acts in opposition to lpL1 protein kinase in regulating yeast chromosome segregation. 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Cell 91 :961-71. 186 Appendix ll: Further investigation of the interactions between H3 and Sgo1p in mitotic tension sensing INTRODUCTION Histones are the basic structural and functional components of eukaryotic chromosome. During mitosis, chromatin is decondensed for DNA replication, with the incorporation of newly replicated DNA, it condenses to form sister chromatids, which then will be segregated into daughter cells by pulling forces from spindles emanating from opposite spindle pore bodies. To monitor faithful segregation, spindle assembly checkpoint components function by delaying the metaphase to anaphase transition if either one of the following requirements is missing, bipolar attachment of kinetochores by spindles and tension that is generated between two sister-chromatids held by cohesin complex to remit the pulling forces of the spindles emanating from opposite spindle pole bodies. My previous studies showed that histone H3 proactively participated in the Sgo1p-mediated tension sensing checkpoint activation, H3 binds directly with 8901 p and that interaction was diminished largely by mutations in H3 tension sensing motif (3). It is still worthy to continue the investigation of the underlying molecular mechanisms of the interactions between H3 and Sgo1p, particularly in the side of 8901 p. This study was thus aimed to screen for intra-genic mutations in SGO1 that would strengthen or bypass the interactions with the mutated H3. 187 Genetic and biochemical analyses will be followed to characterize the obtained intra-genic suppressor. MATERIAL AND METHODS Yeast strains and plasmid constructs. The yeast strains and plasmids used in this work are listed in Tables B-1 and B-2. Yeast methods. Yeast growth media, conditions, and transformation were based on standard procedures (4). When appropriate, 5% casamino acids (CAA) were used to substitute for synthetic amino acid mixtures as selective medium for uracil, tryptophan, or adenine prototroph. Yeast transformation was done with the lithium acetate method (1). ChIP experiments were carried out as described (2), using anti-HA (120A5, Sigma) antibody. Intra-genic suppressor screen. To screen for $901 mutations that are able to rescue H3 G44S or G44A benomyl hypersensitivity, the error-prone PCR amplification approach was utilized to introduce random mutations into SGO1 ORF. To that end, primers MHK98 and MHK99 were used to amplify SGO1 ORF from pJL53 in a mixture of 1X PCR reaction buffer, 7mM MgClz, 1mM dNTP mixture, 0.4uM primers, 0.4mM MnClz, and 0.05unit DNA polymerase per 188 microliter reaction. PCR program is 30 cycles of 1min 94°, 1min 45°, and 2min 72°. The PCR product was gel purified and co-transformed with a Not I linearized basal expression plasmid (pJL52) into H3 6448 or G44A mutant cells with sgo1A background (strains yJL17O and yJL431). Transformants that were able to suppress the benomyl hypersensitivity caused not only by 390113, but also by H3 G44S or G44A mutation were processed for further analyses. Those transformants were patched into YPD and YPD plus 20pg/ml benomyl to screen for potential suppressors. For positive suppressors, 5-FOA selection was applied to pop out the plasmid bearing mutant $901 to verify the plasmid dependency. After the verification of the plasmid dependent suppression, plasmid DNA was extracted, sequenced with primer MHK98 or MHK99, and re-transformed into sgo1A H3 G448 or G44A mutant cells (strains yJL17O and yJL431), spot assays and chromatin immunoprecipitation approaches were followed to characterize these intra-genic suppressors. RESULTS Screen for $901 intra—genic mutations that can rescue H3 G448 tension sensing defect. My previous studies indicated that H3 plays important roles in sister chromatid tension sensing by either recruiting or retaining 8901 p at the pericentric region. The mutation of H3. G448 impairs the H3-Sgo1p association both in vitro and in vivo (3). Considering the fact that G44A phenocopies the G448 defects and that 189 Gly44 is part of a [3 turn in H3 N-terminal tail, we suspect that mutating Gly44 to serine or alanine causes a conformational change of H3 [3 turn, which then diminishes the H3-Sgo1p association. To better understand the interface of H3- 8901 p association, I carried out an intra-genic screen for mutants/suppressors that can rescue H3 G448 benomyl hypersensitivity, looking for some allele to allele specific mutations in $901 that can complement the low affinity of Sgo1p to H3 G44S. As shown in Figure 1, suppressors $68 and S71 can rescue H3 G448 benomyl hypersensitivity while 870 and S75 only slightly rescue that defect, although all suppressors $64, $65, $67, $68, 869, S70, S71 and S75 rescue the defective growth of H3 G448 at 14°. Suppressors 368, S71 were chosen for further studies. Similarly, other suppressors 853, A1, A5 (A1, A5 are from H3 G44A suppression screen) were identified, all characterized intra—genic mutations are mapped and listed in Figure 2. Overproduction of Sgo1p can rescue the H3 6448 benomyl hypersensitivity. To rule out the possibility that the observed suppression of benomyl hypersensitivity resulted from a higher protein level of Sgo1p, Western blot analysis was performed (Fig. 3). The variable Sgo1p protein levels in $901 intra-genic suppressors A5, SS3, 868, 871 did not exceed the expression level of wildtype Sgo1p (lane 3), Interestingly, suppressor S71 expresses a truncated Sgo1p and sequencing result also indicates that is the case (Fig. 2). 190 To examine whether these suppressors can restore 8901 p back into the pericentric region in H3 G448 cells, chromatin immunoprecipitation was performed. Figure 4A indicated that suppressor S53 is unable to restore the pericentric association of Sgo1p in H3 G448 cells. Similar patterns were also observed in Figure 48, where although A5, 871 (lane 6, 8 respectively) show comparable centromeric 8901 p association when compared to wildtype Sgo1p (lane 4, 5), these suppressors did not distinguish themselves from wildtype 8901 p in the pericentric (CEN16R 1.7kb) region. Suggesting the observed genetic suppression by these suppressors was not due to the restoration of Sgo1p to pericentric region in H3 G448 cells. DISCUSSION Throughout the studies of H3 6448 and its interaction with Sgo1p, we proposed a model in which the structural features of Gly44 and nearby residues allow that region to form a B turn, and a tension dependent conformational change of which regulates the binding of Sgo1p to H3. Histone H3 thus plays a structural role in tension sensing. I wished to examine this interaction in a great detail by characterizing sgo1 mutants that can restore the tension sensing function of the G448 and G44A mutants. With little structural information of Sgo1p in hand, it is hard to even predict how or which region of 8901 p is important for H3-Sgo1 p interaction. lntra-genic 191 suppressor screen of 8901 for H3 G448 (or G44A) benomyl hypersensitivity generated five good candidates. One important finding is that the removal of C- terrninal 270aa of Sgo1p (total 590aa) actually rescues H3 G448 benomyl hypersensitivity (suppressor S71 Y317X mutation). Additionally, mutations in the C-terminal region of 8901 p were also found in other four suppressors. Together, C-terminal region of Sgo1p is inhibitory for the interaction of Sgo1p and H3. It is likely that C-terminal region need to be processed for 8901 p to be mature or be activated for tension sensing function. The failure to observe the pericentric restoration of mutant 8901 p in ChlP analyses may also be explained in that these suppressors actually bypass the requirement to be pericentric localized. In that case, 8901 p is retained or recruited to the pericentric region by H3 at Gly44 and is further processed by currently unknown mechanism, the processed Sgo1p signals the generation of tension between two sister-chromatids, and leads to cell’s metaphase to anaphase transition. 192 TABLE B-1. Yeast strains used in this study Strain Relevant genotype Source or reference yMK1361 MA Ta ad92-1 can 1—100 his3-11, 15 Ieu2-3,112 trp1—1::sgo1::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1erat hta2- htb2::HPH pQQ18 [ARS CEN LEU2 HTA 1-HTB1 HHT2-HHF2] This study yJL170 MA Ta ad92-1 can 1—100 his3—11, 15 leu2—3, 112 trp1-1.':sgo1::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1zzNat hta2- htb2::HPH pMK439G44S [ARS CEN LEU2 HTA1-HTB1 hht2-G44S- HHF2] This study yJL258 MA Ta ade2-1 can 1—100 his3-11,15 Ieu2—3, 112 trp1-1 ura3-1 hht1- hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] pJL51 [2pm URA3 HA- SGO1] This study yJL431 MA Ta adeZ-1 can 1—100 his3-11, 15 Ieu2—3, 112 trp1-1::sgo1::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK43QG44A [ARS CEN LEU2 HTA1-HTB1 hhtZ-G44A- HHF2] This study yJL299 MA Ta ad92-1 can 1—100 his3—11, 15 Ieu2—3, 112 trp1-1::sgo1zzTRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1erat hta2- htb222HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] pJL53 [ARS URA3 SGO1] This study yJ L303 MA Ta ad92-1 can 1-100 his3-11, 15 leu2—3, 112 trp1-1::sgo1::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat htaZ- htb2::HPH pMK439644$ [ARS CEN LEU2 HTA1-HTB1 hht2-G44S- HHF2] This study TABLE B-2. Plasmid constructs used in this study Plasmid Main features Source or reference pJL51 2pm URA3 pADH1-3xHA-SGO1-tADH1 This study pJL52 ARS1 CEN4 URA3 pADH1-3xHA-tADH1 This study pJL53 ARS1 CEN4 URA3 pADH1-3xHA-SGO1-tADH1 This study 193 Fig. B-1 Benomyl Benomyl YPD, 7.5ug/ml 10ug/ml Genotype H3 WT $90113 + SGO1 H3 G44S SGO1 H3 6448 590113 + 5901-864 H3 G44S 590113 + sgo1-865 ’ H3 G44S 590113 + sgo1-SG7 H3 G448 sgo1A + 5901-868 H3 6448 sgo1A + Vector H3 G44S sgo1A + SGO1 H3 6448 sgo1A + 5901-869 H3 6448 sgo1A + sgo1-S7O H3 G44S sgo1A + sgo1-S71 H3 G44S 5901A + 5901-875 .1.- _ . .1" .r ' Q '- . 1% .3; 9 (,3 . 63 if (:1) 31?. :3 6 ice- FIG. B-1. Screen for intra-genic mutations of SGO1 that rescue the benomyl hypersensitivity of H3 6448. Spot assay were performed to verify the suppression of H3 G44S benomyl hypersensitivity by intra-genic mutants of $901. ARS CEN4 driven plasmids with either wildtype SGO1 or bearing random error- prone PCR amplified sgo1 ORF were transformed into sgo1A cells, 6 fold serial dilutions of cells were spotted into plates containing rich medium YPD with the absence or presence of benomyl as shown in the top. 194 Puree: 0 50 100 150 200 250 300 350 400 450 500 550 59083 30015’ if ’? ""3 ‘ TM”:*’ ,,z,: 7:‘ :‘“ . t‘ ] '109T ' L24OS ' 835512 A : A I A . . . ‘ . ‘ ‘ 5 ‘* * ., at w N31D ’ , j i _ ' 4 g -' ; N293S K345E . P483T A1 ;* ' * ** t: ' ‘ DZ48E . : P353L v ' ; N539K $53 * * * M215L DZ48N T324A H404R - L490F r ....................................................... *****1 ~ ’ 4 to Y317X ' ' 05190 FIG. B-2. Schematic list of mutations identified in the intra-genic suppressor screen . 195 Fig. B-3 C m» a. a... d <— Sgo1p Truncated Sgo1p 4 : H3 G448 590113 + sgo1-A5 : H3 WT 590113 + SGO1 : H3 G448 sgo1A + SGO1 : H3 G448 sgolA + sgo1-353 : H3 G44S sgo1A + sgo1-S64 : H3 G448 sgo1A + sgo1-868 ' H3 G448 sgo1A + sgo1-S71 : Zp Sgo1p positive control mfimmAwN-s FIG. B-3. Western Blot analysis against Sgo1p fused HA tag shows no major increase of Sgo1p level among intra-genic suppressors when compared to wildtype Sgo1p (lane 2, 3). Arrow indicates the size of Sgo1p, in suppressor S71 (lane 7) a truncated 8901 p was detected. Cell were grow to log phase, whole cell extracts were prepared by boiling cell pellets in 2X SDS loading dye and vortex for 5 minutes at room temperature, extracts equal to 0.2 00600 cells were loaded. Strains used lane 2: yJL299; lane 3: yJL303; lanes 4 to 7 were from suppressors Sis-$53, Sis-$64, Sis-$68, and Sis-S71 respectively. 196 FIG. B-4. Chromatin immunoprecipitation analysis of the association of intra- genic mutants of $901 at centromere and pericentric region. (A) ChlP analysis of H3 6448 $901 intra-genic suppressor $53 at CEN3, CEN16 and pericentric region 0.3kb left or 1.7kb right from CEN16. Star marks internal common PCR control at DEDl locus. Top bands are PCR signals from positions shown in top of each image. Strain used in lane 2 is yJL299, lane 3 is yJL303, and lanes 4, 5 are Sis-$53. (B) ChlP analysis of H3 G448 sgo1 intra-genic suppressors A5, 868, $71 at centromere of chromosome 16 and pericentric region 1.7kb right from CEN16. Strain used in lane 3 is yJL299, lanes 4, 5 are yJL303, and lanes 6, 7, 8 are suppressors Sis-A5, Sis-$68, Sis-S71 respectively. 197 Fig. 3-4 A CEN3 CEN16 mar-“"15: '5 . re“; '5'; ... 1' .‘ ' .: a '9;- . '..‘ ' . . ‘3 N Hun-a u H u. 5.-. LT; 5.5-J: 1 2 3 4 5 1 2 3 4 5 CEN16L 0.3kh__ CEN16R 1 7kb“ :5. ~ . 5...: .. Iv .. I. 't- -.. 3 1 2 3 4 5 1 2 3 4 5 1: H3 G443 sgo1A + sgo1-353 INP 2: H3 WT sgo1A + SGO1 3: H3 G443 $90115 + SGO1 4: H3 G443 sgo1A + 3901-353 5: H3 G443 sgo1A + sgo1-353 B CEN16 '7vfi _ ‘9. -- 1: H3 Wl' sgo1A + SGO1 lNP \- w ..‘J ...... ..-: 5. .5 5...: .1... 55a * ‘ 2: H3 WT sgo1A + SGO1 no Ab control '. ' ‘ ‘ ' . 3: H3Wngo1A + SGO1 ' ‘ ' ‘ ' ’ 4: H3 G443 sgo1A + SGO1 1 2 3 4 5 6 7 8 910 5:H3G443sgo1A+SGO1 6: H3 G443 sgo1A + sgo1-A5 CEN16R 17“" 7: H3 G443 $90115 + sgo1-368 ' 8: H3 G443 sgo1A + 3901-371 9: H3 G443 sgo1A + sgo1- -371 no Ab control .U ...; 3 ~ « . ' 'W 10: H3 G443 sgo1A + sgo1-S71 INP H5 .:.-54‘ 5,. w wig-.ad* 12345678910 198 REFERENCES 1. Gietz, D., A. St Jean, R. A. Woods, and R. H. Schiestl. 1992. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425. 2. Kuo, M. H., and C. D. Allis. 1999. In vivo cross-linking and immunoprecipitation for studying dynamic ProteinzDNA associations in a chromatin environment. 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