The investigation of transcription related protein protein interactions of histones in saccharomyces cerevisiae

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 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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