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The Role of RNF40 Mediated H2B Monoubiquitination in Transcription

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The Role of RNF40 Mediated H2B Monoubiquitination in Transcription The Role of RNF40 mediated H2B monoubiquitination in transcription Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen Submitted by Wanhua Xie born in Chongqing, China Göttingen, 2016 Thesis Supervisor: Prof. Dr. Steven A. Johnsen Thesis Committee: Prof. Dr. Steven A. Johnsen (reviewer) Clinic for General, Visceral and Pediatric Surgery University Medical Center Göttingen Prof. Dr. Heidi Hahn (reviewer) Molecular Developmental Genetics Group Institute for Human genetics University of Göttingen Prof. Dr. Matthias Dobbelstein Institute of Molecular Oncology University Medical Center Göttingen Date of oral examination: 16th of August 2016 Affidavit I hereby declare that the PhD thesis entitled “The Role of RNF40 mediated H2B monoubiquitination in transcription” has been written independently and with no other sources and aids than quoted. Wanhua Xie June, 2016 Göttingen Table of Contents Abstract ........................................................................................................................... I Acknowledgements ....................................................................................................... II Abbreviations ............................................................................................................... IV 1. Introduction ............................................................................................................... 1 1.1 Nucleosome: How to organize the DNA double helix ............................................. 1 1.2 Histone modifications and gene expression ........................................................... 2 1.3 Histone H2B monoubiquitination ............................................................................ 4 1.3.1 Biochemical pathway for the monoubiquitination of H2B ................................. 4 1.3.2 H2Bub1 facilitates transcription elongation ...................................................... 5 1.3.3 H2Bub1 also functions as a transcription suppressor .................................... 15 1.3.4 H2Bub1 crosstalk with H3 methylation .......................................................... 16 1.4 Polycomb group proteins (PcG) ........................................................................... 18 1.4.1 PcG in gene regulation .................................................................................. 18 1.4.2 H2Bub1 and Bivalency .................................................................................. 20 1.4.3 Another poised chromatin signature: the dynamic equilibrium between H3K27me3 and H3K27ac ....................................................................................... 21 1.5 Enhancers and transcription ................................................................................ 23 1.5.1 Enhancer-promoter looping activates transcription ........................................ 23 1.5.2 Enhancer associated histone modifications ................................................... 25 1.6 Aims of this project ............................................................................................... 26 2. Materials ................................................................................................................... 28 2.1 Equipment ............................................................................................................ 28 2.1.1 Technical equipment ...................................................................................... 28 2.1.2 Bioinformatic software ................................................................................... 30 2.2 Consumable materials ......................................................................................... 30 2.3 Chemicals ............................................................................................................ 31 2.4 Kits and reagents ................................................................................................. 35 2.5 Nucleic acids ........................................................................................................ 36 2.5.1 Primers for PCR ............................................................................................. 36 2.5.2 Plasmid and primers for cloning ..................................................................... 38 2.5.3 Primers for genome typing mouse embryos .................................................. 38 2.5.4 siRNA Oligonucleotides ................................................................................. 38 2.6 Proteins ................................................................................................................ 39 2.6.1 Molecular weight standards ........................................................................... 39 2.6.2 Antibodies ...................................................................................................... 39 2.6.3 Enzymes ........................................................................................................ 40 2.7 Animal and Cells .................................................................................................. 40 2.7.1 Bacterial Cells ................................................................................................ 40 2.7.2 Mice ............................................................................................................... 40 2.7.3 Mouse embryonic fibroblast cells (MEFs) ...................................................... 40 2.8 Buffers and cell culture medium ........................................................................... 40 2.8.1 Buffers for PCR .............................................................................................. 40 2.8.2 Buffers for western blot .................................................................................. 41 2.8.3 Buffers for ChIP ............................................................................................. 43 2.8.4 Cell culture medium ....................................................................................... 45 3. Methods ................................................................................................................... 46 3.1 Conditional Rnf40 knockout mouse model ........................................................... 46 3.2 Cell culture ........................................................................................................... 47 3.2.1 Isolation and culturing of primary MEFs ......................................................... 47 3.2.2 Inducing Rnf40 knockout and inhibiting EZH2 enzymatic activity in MEFs .... 47 3.2.3 siRNA transfection in Rnf40+/+, Rnf40-/-, and EZH2 inhibited MEFs ............... 48 3.3 Molecular biology ................................................................................................. 48 3.3.1 Genome typing for mouse embryos ............................................................... 48 3.3.2 RNA isolation ................................................................................................. 49 3.3.3 First-Strand Synthesis of cDNA ..................................................................... 50 3.3.4 High throughout RNA sequencing (RNA-Seq) ............................................... 50 3.3.5 Chromatin immunoprecipitation (ChIP) .......................................................... 51 3.3.6 Quantitative real-time PCR (qPCR) ............................................................... 53 3.3.7 ChIP-Seq library preparation ......................................................................... 53 3.3.8 Protein level analysis ..................................................................................... 54 3.3.9 Data analysis ................................................................................................. 55 4. Results ..................................................................................................................... 59 4.1 H2Bub1 and transcription activation .................................................................... 59 4.1.1 H2Bub1 is specially enriched transcribed regions, and gradually decreases toward the 3’ end ................................................................................... 59 4.1.2 H2Bub1 is tightly correlated with gene transcription and active histone modifications ........................................................................................................... 61 4.1.3 Inducible knockout of RNF40 globally affects active histone modifications ... 63 4.1.4 Loss of RNF40 selectively affects low or moderate H2Bub1 targeted gene transcription ............................................................................................................ 65 4.1.5 Transcription dependency on H2Bub1 is not associated with gene length in RNF40 deleted cells ............................................................................................ 68 4.1.6 Three classified states of promoters ............................................................. 69 4.1.7 RNF40-dependent genes were enriched with H3K4me3, while RNF40- suppressed genes were enriched with H3K27me3 ................................................. 74 4.1.8 The downregulation of RNF40-dependent genes in Rnf40-/- MEFs is associated to the widespread narrowing of H3K4me3 peaks ................................. 76 4.1.9 Broadest H3K4me3 is associated with cell development .............................. 79 4.1.10 H2Bub1-dependent differentiation
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