Epigenetic Regulation of Replication-Dependent Histone mRNA 3’ End Processing Dissertation for the award of the degree “Doctor rerum naturalium (Dr. rer. nat.)” Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen submitted by Judith Pirngruber born in Oldenburg, Germany Göttingen, 2010 Thesis Supervisor: Prof. Dr. Steven A. Johnsen Doctoral Committee: Prof. Dr. Steven A. Johnsen (1 st Referee) Molecular Oncology, University of Göttingen Medical School, Göttingen Prof. Dr. Heidi Hahn (2 nd Referee) Molecular Developmental Genetics, University of Göttingen Medical School, Göttingen Prof. Dr. Holger Reichardt Cellular and Molecular Immunology, University of Göttingen Medical School, Göttingen Date of the oral examination: 28 th April 2010 Affidavit I hereby declare that the PhD thesis entitled “Epigenetic Regulation of Replication- Dependent Histone mRNA 3’ End Processing” has been written independently and with no other sources and aids than quoted. ____________________________________ Judith Pirngruber March, 2010 Göttingen, Germany Für Opa Bruno „Opa, ich denk’ an Dich, wenn ich das Gänseliesel hochkletter’.“ Table of contents Table of contents List of Abbreviations ...........................................................................................I List of Figures ..................................................................................................... V List of Tables ...................................................................................................VII Abstract............................................................................................................VIII 1 General Introduction ..................................................................................... 1 1.1 RNA Polymerase II-dependent transcription and mRNA processing ....... 1 1.1.1 Regulation of transcription by promoter-proximal pausing.................. 1 1.1.2 The positive transcription elongation factor b (P-TEFb)...................... 3 1.1.3 The “CTD-code” of RNAPII ................................................................ 4 1.1.4 Co-transcriptional mRNA processing................................................... 6 1.1.4.1 Mammalian 3’ end processing of polyadenylated mRNA..... 8 1.1.4.2 The importance of 3’ end processing..................................... 9 1.2 Chromatin structure and histone modifications .......................................... 9 1.2.1 Histone modifications and the “histone code”.................................... 10 1.2.1.1 Histone H2B monoubiquitination (H2Bub1)....................... 11 1.2.1.2 Histone H3 lysine 4 trimethylation (H3K4me3).................. 13 1.2.1.3 Histone H3 lysine 36 trimethylation (H3K36me3).............. 14 1.2.1.4 Histone modification crosstalk............................................. 15 1.2.2 Movement through chromatin is coupled to posttranslational histone modifications........................................... 16 1.2.3 Poised promoters show hallmarks of transcription initiation.............. 17 1.3 Coupling of histone synthesis with the cell cycle........................................ 18 1.3.1 Proteins that regulate the progression of the cell cycle....................... 18 1.3.1.1 Cyclins and cyclin-dependent kinases (CDKs).................... 19 1.3.1.2 The retinoblastoma protein (pRB) ....................................... 21 1.3.1.3 The E2F transcription factor family members ..................... 21 1.3.2 Histone synthesis is strictly coupled to the cell cycle......................... 23 1.3.2.1 Replication-dependent histone genes: organization and localization ............................................... 23 1.3.2.2 Replication-dependent histone mRNA 3’ end processing ... 24 1.3.2.3 Transport, translation and degradation of histone mRNA ... 26 1.3.2.4 Regulation of replication-dependent histone gene transcription .................................................... 27 1.3.2.5 The existence of polyadenylated replication-dependent histone mRNAs.................................................................... 28 1.3.3 The tumor suppressor p53 regulates cell cycle progression through p21 ..................................................... 29 1.3.3.1 The p53 regulator MDM2 and its inhibition by Nutlin-3a... 30 1.3.3.2 Does p53 regulation play a role in the expression of polyadenylated replication-dependent histone mRNAs?..... 31 Table of contents 2 Publications...................................................................................................33 2.1 Publication I: CDK9 directs H2B monoubiquitination and controls replication-dependent histone mRNA 3’ end processing ............................... 34 2.1.1 Supplementary Material...................................................................... 46 2.2 Publication II: Insights into the function of the human P-TEFb component CDK9 in the regulation of chromatin modifications and co-transcriptional mRNA processing................................ 53 2.3 Publication III: Induced G 1 cell cycle arrest controls replication-dependent histone mRNA 3’ end processing through p21, NPAT and CDK9 ................ 66 2.3.1 Supplementary Material...................................................................... 85 3 General Discussion ....................................................................................... 95 3.1 Chromatin structure, histone modifications and the CTD of RNAPII.... 95 3.1.1 A CTD-independent way for monoubiquitination of human histone H2B? ...................................... 95 3.1.2 The role of H2Bub1 in chromatin assembly and disassembly............ 98 3.1.3 Chromatin structure versus CTD-code: What gives the signal for productive transcription? ......................... 100 3.1.4 A role for pausing in mRNA 3’ end formation................................. 101 3.2 Polyadenylated replication-dependent histone transcripts: formation and possible functions............................................................... 103 3.2.1 Is polyadenylation of replication-dependent histone transcripts a result of splicing? ............................................. 104 3.2.2 Function and regulation of NPAT in replication-dependent histone mRNA 3’ end processing ................. 105 3.2.3 G0/G 1 cell cycle arrest but not a G 2 arrest seems to be relevant for the production of polyadenylated histone mRNAs ..................... 108 3.2.4 The diverse mechanisms by which CDK9 controls histone mRNA 3’ end processing........................................ 109 3.2.5 What is the molecular mechanism which controls the mode of replication-dependent histone mRNA 3’ end processing? ........... 111 3.2.6 Possible physiological functions of polyadenylated replication-dependent histone mRNA transcripts............................. 113 4 Summary and Conclusions........................................................................ 115 5 Reference List ............................................................................................. 118 6 Acknowledgements..................................................................................... 149 7 Curriculum Vitae ....................................................................................... 150 List of Abbreviations List of Abbreviations (NH 4)2SO 4 ammonium sulphate 36B4 mouse homolog of human RPLP0 gene 5-FU 5-Fluorouracil 7-AAD 7-amino-actinomycin D 7SK snRNA 7SK small nuclear RNA ActD Actinomycin D ADP adenosine diphosphate APC/C anaphase-promoting complex/cyclosome Asf1 anti-silencing factor 1 ASH2L absent, small, or homeotic-like protein ATM ataxia telangiectasia mutated ATP adenosine triphosphate BGP beta-glycerophosphate bp base pair BRD4 bromodomain containing 4 protein BrdU 5-bromodeoxyuridine Bre1p yeast brefeldin A sensitive protein 1 Bur1, 2 bypass upstream activating sequence requirement protein 1, 2 CAK cyclin-dependent kinase activating kinase CB cajal body CBC cap binding complex CBP80 cap binding protein 80 CCND1 cyclin D1 CDC cell division cycle CDK cyclin-dependent kinase cDNA complementary DNA CE capping enzyme CF cleavage factor CHD1 chromodomain protein 1 ChIP cromatin immunoprecipitation Cip cyclin inhibitor protein CKI cyclin-dependent kinase inhibitor COMPASS complex of proteins associated with Set1 Cont. control CPSF cleavage and polyadenylation specifity factor CSTF cleavage stimulating factor CTD carboxy-terminal domain Ctr9p yeast cyclin three requiring 9 protein CUL1 cullin 1 DCP1, 2 decapping protein 1, 2 DHFR dihydrofolate reductase DISC death-inducing signaling complex dMes-4 Drosophila maternal effect sterile family member 4 DMSO dimethyl sulfoxide DNA deoxyribonuleic acid DOT1L disruptor of telomeric silencing 1-like protein DP1, 2 dimerization partner 1, 2 DRB 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole DSE downstream sequence element DSIF DRB sensitivity-inducing factor E(z) enhancer of zeste E1 ubiquitin activating enzyme E2 ubiquitin conjugating enzyme E2F E2 promoter binding factor E3 ubiquitin-protein-isopeptide ligase EDTA ethylenediaminetetraacetic acid EIF4G eukaryotic translation initiation factor 4-γ ER α estrogen receptor α I List of Abbreviations EST expressed sequence tags et al. et alii (and others) F forward f. and the following page FACT facilitates chromatin transcription FBS fetal bovine serum FCP1 TFIIF-associated CTD phosphatase 1 FCS fetal calf serum ff. and the following pages Fig Figure FIP1 factor interacting with poly(A) polymerase 1 protein FLASH FADD-like IL-1β-converting enzyme associated