THE INVESTIGATION OF TRANSCRIPTIONRELATED PROTEIN-PROTEIN INTERACTIONS OF HISTONES IN SACCHAROMYCES CEREVISIAE ZHAO JIN NATIONAL UNIVERSITY OF SINGAPORE 2013 THE INVESTIGATION OF TRANSCRIPTION-RELATED PROTEIN-PROTEIN INTERACTIONS OF HISTONES IN SACCHAROMYCES CEREVISIAE ZHAO JIN (M Sc (Molecular Engineering of Biological and Chemical Systems), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013 ACKNOWLEDGEMENTS I would like to express my gratitude to all those who gave me the possibility to complete this thesis I want to thank the Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore for giving me the opportunity and scholarship to pursue my PhD study in the first instance I am deeply indebted to my supervisor Associate Professor Dr Norbert Lehming whose help, stimulating suggestions and encouragement helped me through this academic program Without him, this thesis quite simply would not have been possible It’s not that it might have come together in a different or lesser form – it’s that it would not have happened at all As far as I can tell, he is the most tolerant boss with a lot of patience and he’s also been a great friend I would also like to acknowledge and extend my heartfelt gratitude to my former colleagues from Dr Lehming’s Lab, who have supported me in my research work I want to thank them for all their help, support, interest and valuable hints Especially I am obliged to Dr He Hongpeng, Dr Chew Boon Shan, Dr Kevin Ang, Ms Lim Mei Kee, Ms Linda Lee and Madam Chew Lai Ming I have been truly blessed with caring parents and great friends whose patient love enabled me to complete this work Mr Oh Teck Fang is a great guy, who has accepted and cherished me as I am, bringing light and warmth to my darkest hours of life Mr Khin Maung Cho is a terrific adviser and a very supportive and generous person who have offered me so much advice and help over the years Madam Chew Lai Ming is like my mother in Singapore, always treating me as her own daughter Linda Lee is a very loyal friend, cheering for every progress I have made, no matter how little it is I want these people to know that their care has meant and means a lot to me Thanks to Maomi, the most excellent cat on earth, for being such a good company I TABLE OF CONTENTS ACKNOWLEDGEMENTS ································································· I  TABLE OF CONTENTS ···································································· II   LIST OF ABBREVIATIONS ······························································ IV   LIST OF TABLES ··········································································· IV   LIST OF FIGURES ·········································································· V   SUMMARY ··················································································· VI   CHAPTER INTRODUCTION ··························································· 1  1.1  Chromatin structure and regulation of transcription ······························ 1  1.2  Histone variants and gene control ······················································ 7  1.3  Yeast as a model eukaryote······························································ 13   1.4  Alanine-scanning mutagenesis ························································· 20   1.5  Phenotypic analysis ······································································· 21   1.1.1  Chromatin structure ····················································································· 1  1.1.2  Epigenetic control of gene expression ································································ 3  1.2.1  H2A.Z (Htz1) and the transcriptional regulation in Saccharomyces cerevisiae ··············· 12  1.3.1 Features of the yeast as a eukaryotic model organism ·············································· 13  1.3.2 Genetic nomenclature for Saccharomyces cerevisiae ··············································· 14  1.3.3 Yeast vectors and transformation······································································· 15  1.3.4 URA3 gene ································································································ 17  1.3.5 Plasmid shuffle ··························································································· 18  1.3.6 The yeast GAL genes and their expression control ·················································· 19  1.5.1 Antimycin A (AA) sensitivity – Indicator for defects in the transcription activation of the GAL genes by Gal4p ·································································································· 21  1.5.2 6-Azauracil (6-AU) sensitivity – Indicator for defects in transcription activation by Ppr1p of the URA3 gene or defects in transcription elongation ······················································ 22  1.5.3 3-Amino-1, 2, 4-triazole (3-AT) sensitivity – Indicator for defects in transcription activation of the HIS3 gene by Gcn4p ······················································································· 23  1.6  Multicopy suppressor screening························································ 24   1.7  Split-ubiquitin system ···································································· 25   1.8  Chromatin immunoprecipitation assay ··············································· 27  1.9  Implications of histone modifications in human diseases ························· 31  1.10  Aim of the study ··········································································· 33   CHAPTER MATERIALS AND METHODS ·········································34  II 2.1 Materials························································································ 34   2.1.1 Yeast Strains ······························································································ 34  2.1.2 E coli strains ····························································································· 35  2.1.3 Plasmids ··································································································· 35  2.1.4 Primers ···································································································· 36  2.1.5 Media ······································································································ 36  2.2 Methods ························································································· 37   2.2.1 Construction of histone single-point mutants (for alanine scanning) ······························ 37  2.2.2 Phenotypic test (spot assay) ············································································ 38  2.2.3 Multicopy-suppressor screening ······································································· 38  2.2.4 Western blotting ·························································································· 39  2.2.5 RNA isolation ···························································································· 40  2.2.6 Quantitative reverse-transcription PCR ······························································· 40  2.2.7 Chromatin immunoprecipitation ······································································· 41  2.2.8 GST pull-down assay and immunoprecipitation ····················································· 41  2.2.9 Split-ubiquitin assay ····················································································· 42  CHAPTER ALANINE SCANNING AND PHENOTYPIC TESTS FOR HISTONE H2A AND H4 ····································································43   3.1 Abstract ························································································· 43   3.2 Results··························································································· 43   3.2.1 Alanine-scanning mutagenesis and phenotypic analysis of histone H2A ························· 43  3.2.2 Alanine-scanning mutagenesis and phenotypic analysis of histone H4 ··························· 50  3.2.3 The H2A mutations R30A and E57A weakened the interaction with Gal4p and caused the gal phenotype ········································································································ 57  3.3 Discussion ······················································································ 58   CHAPTER INVESTIGATION OF THE GLUCOSE REPRESSION DEFECTIVE PHENOTYPE OF HISTONE MUTANTS ····························63  4.1 Abstract ························································································· 63   4.2 Results··························································································· 63   4.2.1 Glucose repression defective histone mutants ························································ 63  4.2.2 Suppressor screening ···················································································· 65  4.2.4 H4 K91Q, but not H4 K91R, caused a glucose repression defect ································· 70  4.2.5 Glucose repression defects of HDAC gene deletion strains ········································ 71  4.3 Discussion ······················································································ 74   CHAPTER INVESTIGATION OF THE INTERACTIONS BETWEEN HISTONES AND MIG PROTEINS ······················································75   5.1 Abstract ························································································· 75   5.2 Results··························································································· 75   5.2.1 Mig1p interacts with core histones ···································································· 75  5.2.2 Several histone H4 mutant proteins that are defective for glucose repression are also defective for the interaction with Mig1p ················································································ 76  5.3 Discussion ······················································································ 77   III CHAPTER HTZ1 INTERCONVERTS CHROMATIN STATES ················79  6.1 Abstract ························································································· 79   6.2 Results··························································································· 79   6.2.1 Transcriptional activation causes short-term memory ··············································· 79  6.2.2 A mixed population of repression-deficient and repression-competent HTZ1 cells ··········· 85  6.2.3 The transcription status of the episomal GAL1 promoter in HTZ1 cells is stable·············· 87  6.6.4 The transcription status of the chromosomal GAL1 promoter in HTZ1 cells is also stable··· 88  6.6.5 The growth on U plates and F plates reflects the GAL1 transcription status ····················· 91  6.6.6 Galactose induction evicts nucleosomes from the GAL1 locus ···································· 94  6.6.7 Nucleosome occupancy reflects the transcription status of the GAL1 locus······················ 96  6.3 Discussion ······················································································ 98   CHAPTER CONCLUSION ···························································· 104   REFERENCES·············································································· 107   LIST OF ABBREVIATIONS AA: antimycin A ChIP: chromatin immunoprecipitation HAT: histone acetyltransferase HDAC: histone deacetylase HPTM: histone post-translational modification NTP: nucleoside triphosphate ORF: open reading frame RNAII: RNA polymerase II TBP: TATA-binding protein UAS: upstream activation sequence USP: ubiquitin specific protease 3-AT: 3-amino-1, 2, 4-triazole 6-AU: 6-azauracil LIST OF TABLES Table 1.1 Names of the genes and their encoded proteins in this study ……………… 15 Table 4.1 Screen for suppressors of glucose repression defective histone mutants … 65 Table 4.2 Genes located on the multi-copy plasmids that suppressed the glucose repression defect caused by H4 N25A and H4 K91A ……………………… 69 IV LIST OF FIGURES Figure 1.1 Architecture of a chromosome 2  Figure 1.2 Crystal structure of the nucleosome core particle 3  Figure 1.3 GAL genes transcription upon galactose induction 20  Figure 1.4 Split-ubiquitin system 26  Figure 1.5 Summary of chromatin immunoprecipitation methodology 30  Figure 3.1 Growth defects and temperature sensitivity of cells expressing histone H2A alanine point mutant proteins in place of wild-type H2A 45  Figure 3.2 Antimycin A sensitivity of histone H2A alanine point mutant strains 46  Figure 3.3 3-Aminotriazole sensitivity of histone H2A alanine point mutant strains 47  Figure 3.4 6-Azauracil sensitivity of histone H2A alanine point mutant strains 49  Figure 3.5 Growth defects and temperature sensitivity of cells expressing histone H4 alanine mutant proteins in place of wild-type H4 53  Figure 3.6 Antimycin A sensitivity of histone H4 alanine point mutant strains 54  Figure 3.7 3-Aminotriazole sensitivity of histone H4 alanine point mutant strains 55  Figure 3.8 Summary of phenotypes displayed by histone H2A mutants 56  Figure 3.9 Summary of phenotypes displayed by histone H4 mutants 56  Figure 3.10 H2A R30A and H2A E57A mutant strains were defective for the proteinprotein interaction with Gal4p 58  Figure 4.1 Histone mutant strains defective for glucose repression 65  Figure 4.2 Multi-copy suppressors of the glucose repression defect caused by H4 N25A…………………………………………………………………………………… 66 Figure 4.3 Multi-copy suppressors of the glucose repression defect caused by H4 K91A….………………………………………………………………………………….67 Figure 4.4 The H4 K91A mutation reduced the protein interactions with the four core histones 70  Figure 4.5 The role of H4 K91 in transcriptional regulation 71  Figure 4.6 Glucose repression defects of HDAC gene deletion strains 73  Figure 5.1 Mig1p interacts with core histone proteins 76  Figure 5.2 Protein-protein interaction of histone H4 with Mig1p, Mig2p and Mig3p 77  Figure 6.1 The repression status of the GAL1 promoter in HTZ1 cells is stable 81 Figure 6.2 A Titration scheme: transcriptional activation causes short-term memory.… 82 Figure 6.2 B Titration scheme: transcriptional repression is stable.…………………… 83 Figure 6.2 C Titration scheme: transcriptional derepression is stable……………………84 Figure 6.3 The repression status of the chromosomal GAL1 promoter in HTZ1 cells is stable 90  Figure 6.4 The growth on U and F plates reflects the transcription status of the GAL1 promoter 93  Figure 6.5 Galactose induction evicts nucleosomes from the GAL1 locus 95  Figure 6.6 Nucleosome occupancy reflects the repression status of GAL1 97  Figure 6.7 Htz1 is required to establish glucose repression in all cells 103  V SUMMARY Alanine-scanning mutagenesis of the histones H2A and H4 was performed in the model eukaryote Saccharomyces cerevisiae Wild-type histones were replaced by the mutant proteins via plasmid shuffle, and the resulting mutant strains were screened for growth defects reflecting defects in transcriptional activation and repression of reporter genes Histone mutant proteins that conferred phenotypic defects were tested for defects in protein-protein interactions with the help of the split-ubiquitin system H2A E57A, which conferred the gal phenotype when expressed in place of wild-type H2A (indicative of defects in transcriptional activation of the GAL genes by Gal4p), was also defective for the protein-protein interaction of H2A with Gal4p One possible explanation of this result is that Gal4p has to interact with H2A when it binds to its sites in the enhancers of the GAL genes H4 Y51A, which caused mis-expression of a reporter fusion of the GAL1 promoter to the URA3 open reading frame under repressive conditions (indicative of defects in transcriptional repression of the GAL1 promoter by Mig1p), was also defective for the protein-protein interaction of H4 with Mig1p One possible explanation for this result is that Mig1p has to interact with H4 when it binds to its sites in the silencer of the GAL1 gene H4 K91A, which also caused a glucose repression defect of the GAL1 promoter, was defective for the protein-protein interactions with the other core histones One possible explanation of this result is that glucose repression requires stable nucleosomes The H2A variant H2A.Z was found to be required for both galactose induction and glucose repression of the GAL1 gene The GAL1 mRNA was rapidly degraded when cells were exposed to glucose, which explains why the role of H2A.Z in glucose repression had not been observed in previous studies VI CHAPTER INTRODUCTION 1.1 Chromatin structure and regulation of transcription 1.1.1 Chromatin structure If laid out end to end, the DNA in a single human individual would stretch to 5×1010 km, 100 times of the distance between the Earth and the Sun (Latchman, 2010) Clearly, therefore, the DNA must be compacted in some way to fit inside a tiny cell In the eukaryotic nucleus, genomic DNA is wrapped around the histone octamer to form the basic repeating unit of chromatin, the nucleosome Each nucleosome is separated by a linker region of DNA of 55-75 bp in length that is bound by histone H1 (Bednar et al., 1998) When isolated under conditions of low ionic strength, chromatin in its extended form (10 nm fiber) looks like beads (nucleosomes) on a string (DNA) in the electron microscope The 10 nm fiber is then wrapped into a 30 nm spiral called a solenoid, where histone H1 and regulation of histone modifications are involved to maintain the chromosome structure In heterochromatin and in the mitosis chromosome, the 30 nm fiber is further compacted by forming loops which are very closely packed, resulting in a 10,000-fold compaction of the genomic DNA (Hyde, 2009), (Figure 1.1) In the nucleosome, core histones function as spools for 147 base pairs of DNA to wrap around in ~1.7 left-handed superhelical turns (Kornberg and Lorch, 1999) There are four core histones in all eukaryotes: H2A, H2B, H3 and H4 They are small, basic proteins (rich in lysine and arginine), with a net positive charge that facilitates their binding to the negatively charged DNA and neutralizes the net negative charge on the DNA molecule to allow further folding to occur Figure 1.1 Architecture of a chromosome (Courtesy: Darryl Leja, National Human Genome Research Institute) The DNA in each chromosome is wrapped around spools consisting of histone proteins, and the spooled DNA folds up still more Collectively, the DNA complexed to proteins is known as chromatin Amino acid sequences of histones are remarkably well conserved among eukaryotes, for example, the peptide sequence of histone H4 is 92% identical between yeast and human (Matsubara et al., 2007), indicating the key role of the histones in nucleosome structure Each of the four core histones has a similar structure, with an N-terminal tail and a helical region which consists of three -helices separated by two loops The helical region forms a specific structure, known as the histone fold, which allows individual histones to associate with one another The flexible N-terminal tails of histones, which extend beyond the surface of the nucleosome particle, are where most posttranslational modifications take place Based on the crystal structure of the nucleosome, the histone octamer is generally depicted as a kernel of a H32-H42 tetramer associated with two H2A-H2B dimers (Arents et al., 1991; Luger et al., 1997), (Figure 1.2) The significance of nucleosome structure in terms of gene regulation is that the positioning of DNA within the nucleosome dictates the accessibility of a gene to the Eickbush, T.H., J.E Godfrey, M.C Elia, and E.N Moudrianakis 1988 H2a-specific proteolysis as a unique probe in the analysis of the histone octamer J Biol Chem 263:18972-8 Elsaesser, S.J., A.D Goldberg, and C.D Allis 2010 New functions for an old variant: no substitute for histone H3.3 Curr Opin Genet Dev 20:110-7 Exinger, F., and F Lacroute 1992 6-Azauracil inhibition of GTP biosynthesis in Saccharomyces cerevisiae Curr Genet 22:9-11 Feng, Q., H Wang, H.H Ng, H Erdjument-Bromage, P Tempst, K Struhl, and Y Zhang 2002 Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain Curr Biol 12:1052-8 Fields, S., and O Song 1989 A novel genetic system to detect protein-protein interactions Nature 340:245-6 Fink, G.R 1964 Gene-Enzyme Relations in Histidine Biosynthesis in Yeast Science 146:525-7 Fraga, M.F., E Ballestar, A Villar-Garea, M Boix-Chornet, J Espada, G Schotta, T Bonaldi, C Haydon, S Ropero, K Petrie, N.G Iyer, A Perez-Rosado, E Calvo, J.A Lopez, A Cano, M.J Calasanz, D Colomer, M.A Piris, N Ahn, A Imhof, C Caldas, T Jenuwein, and M Esteller 2005 Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer Nat Genet 37:391-400 Ganesan, A., L Nolan, S.J Crabb, and G Packham 2009 Epigenetic therapy: histone acetylation, DNA methylation and anti-cancer drug discovery Curr Cancer Drug Targets 9:963-81 Gautier, T., D.W Abbott, A Molla, A Verdel, J Ausio, and S Dimitrov 2004 Histone variant H2ABbd confers lower stability to the nucleosome EMBO Rep 5:715-20 112 Gibbs, C.S., and M.J Zoller 1991 Identification of electrostatic interactions that determine the phosphorylation site specificity of the cAMP-dependent protein kinase Biochemistry 30:5329-34 Gligoris, T., G Thireos, and D Tzamarias 2007 The Tup1 corepressor directs Htz1 deposition at a specific promoter nucleosome marking the GAL1 gene for rapid activation Mol Cell Biol 27:4198-205 Goffeau, A., B.G Barrell, H Bussey, R.W Davis, B Dujon, H Feldmann, F Galibert, J.D Hoheisel, C Jacq, M Johnston, E.J Louis, H.W Mewes, Y Murakami, P Philippsen, H Tettelin, and S.G Oliver 1996 Life with 6000 genes Science 274:546, 563-7 Gromoller, A., and N Lehming 2000 Srb7p is a physical and physiological target of Tup1p EMBO J 19:6845-52 Guillemette, B., A.R Bataille, N Gevry, M Adam, M Blanchette, F Robert, and L Gaudreau 2005 Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning PLoS Biol 3:e384 Gunjan, A., and R.K Singh 2011 Epigenetic therapy: targeting histones and their modifications in human disease Future Med Chem 2:543-548 Guthrie, C., and G.R Fink 2004 Guide to yeast genetics and molecular and cell biology Part A Elsevier Academic Press, Amsterdam Hake, S.B., and C.D Allis 2006 Histone H3 variants and their potential role in indexing mammalian genomes: the "H3 barcode hypothesis" Proc Natl Acad Sci U S A 103:6428-35 Hampsey, M 1997 A review of phenotypes in Saccharomyces cerevisiae Yeast 13:1099-133 He, H 2008 The Effects of the histones and histone-interacting partners on transcriptional regulation In Microbiology Vol Ph.D National University of Singapore 113 Hedbacker, K., and M Carlson 2008 SNF1/AMPK pathways in yeast Front Biosci 13:2408-20 Henikoff, S., and K Ahmad 2005 Assembly of variant histones into chromatin Annu Rev Cell Dev Biol 21:133-53 Henikoff, S., T Furuyama, and K Ahmad 2004 Histone variants, nucleosome assembly and epigenetic inheritance Trends Genet 20:320-6 Hope, I.A., and K Struhl 1985 GCN4 protein, synthesized in vitro, binds HIS3 regulatory sequences: implications for general control of amino acid biosynthetic genes in yeast Cell 43:177-88 Hubert, J.C., A Guyonvarch, B Kammerer, F Exinger, P Liljelund, and F Lacroute 1983 Complete sequence of a eukaryotic regulatory gene EMBO J 2:2071-3 Hutchins, A.S., A.C Mullen, H.W Lee, K.J Sykes, F.A High, B.D Hendrich, A.P Bird, and S.L Reiner 2002 Gene silencing quantitatively controls the function of a developmental trans-activator Mol Cell 10:81-91 Hyde, D 2009 Introduction to genetic principles McGraw-Hill, New York, N.Y 870 p pp Iouzalen, N., J Moreau, and M Mechali 1996 H2A.ZI, a new variant histone expressed during Xenopus early development exhibits several distinct features from the core histone H2A Nucleic Acids Res 24:3947-52 Jackson, J.D., V.T Falciano, and M.A Gorovsky 1996 A likely histone H2A.F/Z variant in Saccharomyces cerevisiae Trends Biochem Sci 21:466-7 Jackson, J.D., and M.A Gorovsky 2000 Histone H2A.Z has a conserved function that is distinct from that of the major H2A sequence variants Nucleic Acids Res 28:3811-6 114 Johnsson, N., and A Varshavsky 1994 Split ubiquitin as a sensor of protein interactions in vivo Proc Natl Acad Sci U S A 91:10340-4 Johnston, M 1987 A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae Microbiol Rev 51:458-76 Johnston, M., J.S Flick, and T Pexton 1994 Multiple mechanisms provide rapid and stringent glucose repression of GAL gene expression in Saccharomyces cerevisiae Mol Cell Biol 14:3834-41 Kamakaka, R.T., and S Biggins 2005 Histone variants: deviants? Genes Dev 19:295-310 Keith, K.C., R.E Baker, Y Chen, K Harris, S Stoler, and M Fitzgerald-Hayes 1999 Analysis of primary structural determinants that distinguish the centromere-specific function of histone variant Cse4p from histone H3 Mol Cell Biol 19:6130-9 Kerkmann, K., and N Lehming 2001 Genome-wide expression analysis of a Saccharomyces cerevisiae strain deleted for the Tup1p-interacting protein Cdc73p Curr Genet 39:284-90 Kishore, G.M., and D.M Shah 1988 Amino acid biosynthesis inhibitors as herbicides Annu Rev Biochem 57:627-63 Klose, R.J., and A.P Bird 2006 Genomic DNA methylation: the mark and its mediators Trends Biochem Sci 31:89-97 Kornberg, R.D., and Y Lorch 1999 Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome Cell 98:285-94 Kouzarides, T 2007 Chromatin modifications and their function Cell 128:693-705 Kumar, A., and N Garg 2006 Genetic engineering Nova Science Publishers, New York 115 Kundu, S., P.J Horn, and C.L Peterson 2007 SWI/SNF is required for transcriptional memory at the yeast GAL gene cluster Genes Dev 21:997-1004 Lai, L.C., A.L Kosorukoff, P.V Burke, and K.E Kwast 2006 Metabolic-state-dependent remodeling of the transcriptome in response to anoxia and subsequent reoxygenation in Saccharomyces cerevisiae Eukaryot Cell 5:1468-89 Laser, H., C Bongards, J Schuller, S Heck, N Johnsson, and N Lehming 2000 A new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter Proc Natl Acad Sci U S A 97:13732-7 Latchman, D.S 2010 Gene control Garland Science, New York xvi, 430 p pp Latham, J.A., and S.Y Dent 2007 Cross-regulation of histone modifications Nat Struct Mol Biol 14:1017-24 Law, J.A., and S.E Jacobsen 2010 Establishing, maintaining and modifying DNA methylation patterns in plants and animals Nat Rev Genet 11:204-20 Leach, T.J., M Mazzeo, H.L Chotkowski, J.P Madigan, M.G Wotring, and R.L Glaser 2000 Histone H2A.Z is widely but nonrandomly distributed in chromosomes of Drosophila melanogaster J Biol Chem 275:23267-72 Lee, D.Y., C Teyssier, B.D Strahl, and M.R Stallcup 2005 Role of protein methylation in regulation of transcription Endocr Rev 26:147-70 Lehming, N 2002 Analysis of protein-protein proximities using the split-ubiquitin system Brief Funct Genomic Proteomic 1:230-8 Li, B., S.G Pattenden, D Lee, J Gutierrez, J Chen, C Seidel, J Gerton, and J.L Workman 2005 116 Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling Proc Natl Acad Sci U S A 102:18385-90 Li, Y., G Chen, and W Liu 2010 Multiple metabolic signals influence GAL gene activation by modulating the interaction of Gal80p with the transcriptional activator Gal4p Mol Microbiol 78:41428 Liljelund, P., R Losson, B Kammerer, and F Lacroute 1984 Yeast regulatory gene PPR1 II Chromosomal localization, meiotic map, suppressibility, dominance/recessivity and dosage effect J Mol Biol 180:251-65 Lim, M.K., V Tang, A Le Saux, J Schuller, C Bongards, and N Lehming 2007 Gal11p dosagecompensates transcriptional activator deletions via Taf14p J Mol Biol 374:9-23 Liu, X., J Bowen, and M.A Gorovsky 1996 Either of the major H2A genes but not an evolutionarily conserved H2A.F/Z variant of Tetrahymena thermophila can function as the sole H2A gene in the yeast Saccharomyces cerevisiae Mol Cell Biol 16:2878-87 Lo, W.S., L Duggan, N.C Emre, R Belotserkovskya, W.S Lane, R Shiekhattar, and S.L Berger 2001 Snf1 a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription Science 293:1142-6 Lohr, D., and L Hereford 1979 Yeast chromatin is uniformly digested by DNase-I Proc Natl Acad Sci U S A 76:4285-8 Lohr, D., P Venkov, and J Zlatanova 1995 Transcriptional regulation in the yeast GAL gene family: a complex genetic network FASEB J 9:777-87 Loison, G., R Losson, and F Lacroute 1980 Constitutive mutants for orotidine phosphate decarboxylase and dihydroorotic acid dehydrogenase in Saccharomyces cerevisiae Current Genetics 117 2:39-44 Luger, K., A.W Mader, R.K Richmond, D.F Sargent, and T.J Richmond 1997 Crystal structure of the nucleosome core particle at 2.8 A resolution Nature 389:251-60 Malik, H.S., and S Henikoff 2003 Phylogenomics of the nucleosome Nat Struct Biol 10:882-91 Marques, M., L Laflamme, A.L Gervais, and L Gaudreau 2010 Reconciling the positive and negative roles of histone H2A.Z in gene transcription Epigenetics 5:267-72 Marzluff, W.F., and R.J Duronio 2002 Histone mRNA expression: multiple levels of cell cycle regulation and important developmental consequences Curr Opin Cell Biol 14:692-9 Massie, C.E., and I.G Mills 2008 ChIPping away at gene regulation EMBO Rep 9:337-43 Masumoto, H., D Hawke, R Kobayashi, and A Verreault 2005 A role for cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage response Nature 436:294-8 Matsubara, K., N Sano, T Umehara, and M Horikoshi 2007 Global analysis of functional surfaces of core histones with comprehensive point mutants Genes Cells 12:13-33 McGhee, J.D., and P.H von Hippel 1975 Formaldehyde as a probe of DNA structure I Reaction with exocyclic amino groups of DNA bases Biochemistry 14:1281-96 McKittrick, E., P.R Gafken, K Ahmad, and S Henikoff 2004 Histone H3.3 is enriched in covalent modifications associated with active chromatin Proc Natl Acad Sci U S A 101:1525-30 Meneghini, M.D., M Wu, and H.D Madhani 2003 Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin Cell 112:725-36 118 Meunier, J.R., and M Choder 1999 Saccharomyces cerevisiae colony growth and ageing: biphasic growth accompanied by changes in gene expression Yeast 15:1159-69 Michels, C.A 2002 Genetic techniques for biological research : a case study approach Wiley, Chichester [u.a.] Miller, M.J., and R.B Gennis 1983 The purification and characterization of the cytochrome d terminal oxidase complex of the Escherichia coli aerobic respiratory chain J Biol Chem 258:9159-65 Mizuguchi, G., H Xiao, J Wisniewski, M.M Smith, and C Wu 2007 Nonhistone Scm3 and histones CenH3-H4 assemble the core of centromere-specific nucleosomes Cell 129:1153-64 Moore, S.D., S.R Herrick, T.A Ince, M.S Kleinman, P Dal Cin, C.C Morton, and B.J Quade 2004 Uterine leiomyomata with t(10;17) disrupt the histone acetyltransferase MORF Cancer Res 64:5570-7 Morrison, A.J., and X Shen 2005 DNA repair in the context of chromatin Cell Cycle 4:568-71 Muthurajan, U.M., Y Bao, L.J Forsberg, R.S Edayathumangalam, P.N Dyer, C.L White, and K Luger 2004 Crystal structures of histone Sin mutant nucleosomes reveal altered protein-DNA interactions EMBO J 23:260-71 Nakanishi, S., B.W Sanderson, K.M Delventhal, W.D Bradford, K Staehling-Hampton, and A Shilatifard 2008 A comprehensive library of histone mutants identifies nucleosomal residues required for H3K4 methylation Nat Struct Mol Biol 15:881-8 Nakanishi, T., A Nakano, K Nomura, K Sekimizu, and S Natori 1992 Purification, gene cloning, and gene disruption of the transcription elongation factor S-II in Saccharomyces cerevisiae J Biol Chem 267:13200-4 Nasmyth, K.A., and S.I Reed 1980 Isolation of genes by complementation in yeast: molecular 119 cloning of a cell-cycle gene Proc Natl Acad Sci U S A 77:2119-23 Nehlin, J.O., M Carlberg, and H Ronne 1991 Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response EMBO J 10:3373-7 Ng, H.H., R.M Xu, Y Zhang, and K Struhl 2002 Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79 J Biol Chem 277:34655-7 Nowak, S.J., and V.G Corces 2004 Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation Trends Genet 20:214-20 Orlando, V 2000 Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation Trends Biochem Sci 25:99-104 Ozkaynak, E., D Finley, M.J Solomon, and A Varshavsky 1987 The yeast ubiquitin genes: a family of natural gene fusions EMBO J 6:1429-39 Palmer, D.K., K O'Day, H.L Trong, H Charbonneau, and R.L Margolis 1991 Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone Proc Natl Acad Sci U S A 88:3734-8 Parent, S.A., C.M Fenimore, and K.A Bostian 1985 Vector systems for the expression, analysis and cloning of DNA sequences in S cerevisiae Yeast 1:83-138 Peng, G., and J.E Hopper 2000 Evidence for Gal3p's cytoplasmic location and Gal80p's dual cytoplasmic-nuclear location implicates new mechanisms for controlling Gal4p activity in Saccharomyces cerevisiae Mol Cell Biol 20:5140-8 Peng, G., and J.E Hopper 2002 Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein Proc Natl Acad Sci U S A 99:8548-53 120 Ploger, R., J Zhang, D Bassett, R Reeves, P Hieter, M Boguski, and F Spencer 2000 XREFdb: cross-referencing the genetics and genes of mammals and model organisms Nucleic Acids Res 28:1202 Pusarla, R.H., and P Bhargava 2005 Histones in functional diversification Core histone variants FEBS J 272:5149-68 Raisner, R.M., P.D Hartley, M.D Meneghini, M.Z Bao, C.L Liu, S.L Schreiber, O.J Rando, and H.D Madhani 2005 Histone variant H2A.Z marks the 5' ends of both active and inactive genes in euchromatin Cell 123:233-48 Rando, O.J., and F Winston 2012 Chromatin and transcription in yeast Genetics 190:351-87 Ransom, M., B.K Dennehey, and J.K Tyler Chaperoning histones during DNA replication and repair Cell 140:183-95 Redon, C., D Pilch, E Rogakou, O Sedelnikova, K Newrock, and W Bonner 2002 Histone H2A variants H2AX and H2AZ Curr Opin Genet Dev 12:162-9 Rine, J., W Hansen, E Hardeman, and R.W Davis 1983 Targeted selection of recombinant clones through gene dosage effects Proc Natl Acad Sci U S A 80:6750-4 Ropero, S., M.F Fraga, E Ballestar, R Hamelin, H Yamamoto, M Boix-Chornet, R Caballero, M Alaminos, F Setien, M.F Paz, M Herranz, J Palacios, D Arango, T.F Orntoft, L.A Aaltonen, S Schwartz, Jr., and M Esteller 2006 A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition Nat Genet 38:566-9 Rumore, P.M., and C.R Steinman 1990 Endogenous circulating DNA in systemic lupus erythematosus Occurrence as multimeric complexes bound to histone J Clin Invest 86:69-74 121 Sakamoto, M., S Noguchi, S Kawashima, Y Okada, T Enomoto, M Seki, and M Horikoshi 2009 Global analysis of mutual interaction surfaces of nucleosomes with comprehensive point mutants Genes Cells 14:1271-330 Santisteban, M.S., T Kalashnikova, and M.M Smith 2000 Histone H2A.Z regulats transcription and is partially redundant with nucleosome remodeling complexes Cell 103:411-22 Santos-Rosa, H., R Schneider, A.J Bannister, J Sherriff, B.E Bernstein, N.C Emre, S.L Schreiber, J Mellor, and T Kouzarides 2002 Active genes are tri-methylated at K4 of histone H3 Nature 419:40711 Sarma, K., and D Reinberg 2005 Histone variants meet their match Nat Rev Mol Cell Biol 6:139-49 Schmid, M., G Arib, C Laemmli, J Nishikawa, T Durussel, and U.K Laemmli 2006 Nup-PI: the nucleopore-promoter interaction of genes in yeast Mol Cell 21:379-91 Schones, D.E., and K Zhao 2008 Genome-wide approaches to studying chromatin modifications Nat Rev Genet 9:179-91 Schwartz, B.E., and K Ahmad 2005 Transcriptional activation triggers deposition and removal of the histone variant H3.3 Genes Dev 19:804-14 Sellick, C.A., R.N Campbell, and R.J Reece 2008 Galactose metabolism in yeast-structure and regulation of the leloir pathway enzymes and the genes encoding them Int Rev Cell Mol Biol 269:11150 Sherman, F 2002 Getting started with yeast Methods Enzymol 350:3-41 Sikorski, R.S., and J.D Boeke 1991 In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast Methods Enzymol 194:302-18 122 Smith, M 1985 In vitro mutagenesis Annu Rev Genet 19:423-62 Solomon, M.J., P.L Larsen, and A Varshavsky 1988 Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene Cell 53:937-47 Spingola, M., L Grate, D Haussler, and M Ares, Jr 1999 Genome-wide bioinformatic and molecular analysis of introns in Saccharomyces cerevisiae RNA 5:221-34 St John, T.P., and R.W Davis 1981 The organization and transcription of the galactose gene cluster of Saccharomyces J Mol Biol 152:285-315 Sterner, D.E., and S.L Berger 2000 Acetylation of histones and transcription-related factors Microbiol Mol Biol Rev 64:435-59 Strahl, B.D., and C.D Allis 2000 The language of covalent histone modifications Nature 403:41-5 Struhl, K 1998 Histone acetylation and transcriptional regulatory mechanisms Genes Dev 12:599606 Struhl, K., D.T Stinchcomb, S Scherer, and R.W Davis 1979 High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules Proc Natl Acad Sci U S A 76:1035-9 Sullivan, K.F., M Hechenberger, and K Masri 1994 Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere J Cell Biol 127:581-92 Suto, R.K., M.J Clarkson, D.J Tremethick, and K Luger 2000 Crystal structure of a nucleosome core particle containing the variant histone H2A.Z Nat Struct Biol 7:1121-4 Tang, Y., X.D Gao, Y Wang, B.F Yuan, and Y.Q Feng 2012 Widespread existence of cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry Anal Chem 84:7249- 123 55 Thatcher, T.H., and M.A Gorovsky 1994 Phylogenetic analysis of the core histones H2A, H2B, H3, and H4 Nucleic Acids Res 22:174-9 Tollefsbol, T.O 2011 Handbook of epigenetics the new molecular and medical genetics Elsevier/Academic Press, Amsterdam ; Boston Treitel, M.A., and M Carlson 1995 Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein Proc Natl Acad Sci U S A 92:3132-6 Tuan, R.S 1997 Recombinant gene expression protocols Humana Press, Totowa, NJ Varshavsky, A 1997 The N-end rule pathway of protein degradation Genes Cells 2:13-28 Waddington, C.H The epigenotype 1942 Int J Epidemiol 41:10-3 Wahi, M., K Komachi, and A.D Johnson 1998 Gene regulation by the yeast Ssn6-Tup1 corepressor Cold Spring Harb Symp Quant Biol 63:447-57 Wan, Y., R.A Saleem, A.V Ratushny, O Roda, J.J Smith, C.H Lin, J.H Chiang, and J.D Aitchison 2009 Role of the histone variant H2A.Z/Htz1p in TBP recruitment, chromatin dynamics, and regulated expression of oleate-responsive genes Mol Cell Biol 29:2346-58 White, C.L., R.K Suto, and K Luger 2001 Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions EMBO J 20:5207-18 Workman, J.L., and R.E Kingston 1998 Alteration of nucleosome structure as a mechanism of transcriptional regulation Annu Rev Biochem 67:545-79 124 Xiao, W 2006 Yeast protocols Humana Press, Totowa, N.J Xu, F., K Zhang, and M Grunstein 2005 Acetylation in histone H3 globular domain regulates gene expression in yeast Cell 121:375-85 Ye, J., X Ai, E.E Eugeni, L Zhang, L.R Carpenter, M.A Jelinek, M.A Freitas, and M.R Parthun 2005 Histone H4 lysine 91 acetylation a core domain modification associated with chromatin assembly Mol Cell 18:123-30 Zacharioudakis, I., T Gligoris, and D Tzamarias 2007 A yeast catabolic enzyme controls transcriptional memory Curr Biol 17:2041-6 Zanton, S.J., and B.F Pugh 2006 Full and partial genome-wide assembly and disassembly of the yeast transcription machinery in response to heat shock Genes Dev 20:2250-65 Zhang, H., D.N Roberts, and B.R Cairns 2005 Genome-wide dynamics of Htz1, a histone H2A variant that poises repressed/basal promoters for activation through histone loss Cell 123:219-31 Zhang, L., E.E Eugeni, M.R Parthun, and M.A Freitas 2003 Identification of novel histone posttranslational modifications by peptide mass fingerprinting Chromosoma 112:77-86 Zhao, J., W.L Siew, W Sun, and N Lehming 2011 The histone variant H2A.Z interconverts two stable epigenetic chromatin states Biochem J 439:487-495 Zhou, Z., H Feng, D.F Hansen, H Kato, E Luk, D.I Freedberg, L.E Kay, C Wu, and Y Bai 2008 NMR structure of chaperone Chz1 complexed with histones H2A.Z-H2B Nat Struct Mol Biol 15:8689 Zhu, P., E Martin, J Mengwasser, P Schlag, K.P Janssen, and M Gottlicher 2004 Induction of HDAC2 expression upon loss of APC in colorectal tumorigenesis Cancer Cell 5:455-63 125 LIST OF PUBLICATIONS [1] Zhao, J., W.L Siew, W Sun, and N Lehming 2011 The histone variant H2A.Z interconverts two stable epigenetic chromatin states Biochem J 439:487-495 [2] Lim, M.K., W.L Siew, J Zhao, Y.C Tay, E Ang, and N Lehming 2011 Galactose induction of the GAL1 gene requires conditional degradation of the Mig2 repressor Biochem J 435:641-9 126 .. .THE INVESTIGATION OF TRANSCRIPTION- RELATED PROTEIN- PROTEIN INTERACTIONS OF HISTONES IN SACCHAROMYCES CEREVISIAE ZHAO JIN (M Sc (Molecular Engineering of Biological and Chemical... Aim of the study To investigate the effects of single point mutation of histones on transcription regulation and the relevant protein- protein interactions in Saccharomyces cerevisiae To study the. .. number of interactions detected in larger screens Meanwhile as the design of the split-ubiquitin system poses steric constraints on the two fusion proteins, almost all protein- protein interactions