臺灣博碩士論文加值系統

RNA轉錄與DNA複製衝突（TRCs）好發於活躍的轉錄基因組上。此過程不僅影響DNA複製叉的穩定性，也有可能造成基因突變。因RNA轉錄過程而產生的甲基化H3K4（H3K4me）組蛋白標記所在位置與TRCs熱區有一定關聯性，但是它們之間的相互作用尚且未知。本實驗發現H3K4me標記會加劇DNA複製期細胞週期檢查點缺失細胞內所發生的TRCs所導致的DNA複製機制之失能，並且發現該組蛋白標記具有減緩DNA複製叉的前進速率。細胞在應對DNA複製壓力時，完整的DNA複製期細胞週期檢查點功能與H3K4me是必要的，在TRCs發生頻率最高，且H3K4me被標記最多之高度表現的基因區域更顯得尤其重要。H3K4me透過降低DNA複製叉前進速率以緩解TRCs遭遇之強度，與在路況複雜的公路上設置減速丘迫使行進中的車輛將速度放緩以降低意外發生的機率如出一轍。此研究在活躍基因區域為何會被標記H3K4me的謎題上提出了新觀點，即此標記係細胞用以應對因TRCs所造成DNA複製壓力致基因不穩定之緩衝機制。

Transcription-replication conflicts (TRCs) occur when intensive transcriptional activity compromises replication fork stability, potentially leading to gene mutations. Transcription-deposited H3K4 methylation (H3K4me) is associated with regions that are susceptible to TRCs; however, the interplay between H3K4me and TRCs is unknown. Here we show that H3K4me aggravates TRC-induced replication failure in checkpoint-defective cells, and the presence of methylated H3K4 slows down ongoing replication. Both S-phase checkpoint activity and H3K4me are crucial for faithful DNA synthesis under replication stress, especially in highly transcribed regions where the presence of H3K4me is highest and TRCs most often occur. H3K4me mitigates TRCs by decelerating ongoing replication, analogous to how speed bumps slow down cars. These findings establish the novel concept that H3K4me defines the transcriptional status of a genomic region and defends the genome from TRC-mediated replication stress and instability.

中文摘要 I
Abstract II
Table of Contents III
Index of Figures VII
Index of Tables X

Chapter 1 1
Introduction 1
1.1 Context of this study 1
1.2 Chromatin and histone modifications 3
1.2.1 General Background 3
1.2.2 Histone crosstalk between H2B mono-ubiquitylation and H3K4/K79 methylation 4
1.2.3 Transcription and Set1C-mediated H3K4 methylation 5
1.2.4 The debate on biological function of H3K4 methylation with regard to transcription 6
1.2.5 Roles of H3K4 methylation in DNA replication and DNA repair 7
1.3 Replication fork stalling and genome instability 8
1.3.1 Genome instability and cancers 8
1.3.2 Genomic instability caused by stalled replication forks 9
1.3.3 Rad53 checkpoint kinase protects the integrity of stalled forks 11
1.4 Transcription-replication conflicts 13
1.4.1 Transcription effects on genome integrity 13
1.4.2 R-loops, transcription-replication conflicts and resolution 15
1.4.3 S-phase checkpoint functions regulate transcription-replication conflicts 16
1.5 Major findings in this study 18

Chapter 2 19
Materials and methods 19
2.1 Yeast culture medium and culture conditions 19
2.2 Yeast genomic DNA extraction 19
2.3 Yeast transformation 20
2.4 Yeast strain construction 20
2.5 Yeast spotting assay 21
2.6 Cell viability assay 21
2.7 Western blotting 22
2.8 Flow cytometry analysis 22
2.9 Chromatin immunoprecipitation (ChIP) - sequencing 23
2.10 ChIP-seq and ATAC-seq data analysis 24
2.11 ChIP-qPCR 25
2.12 Gene expression 25
2.13 2D gel electrophoresis 26
2.14 Southern blotting 27
2.15 32P hybridization 28
2.16 CAN1 mutation rate assay 29

Chapter 3 50
Genetic screening for modulators of the interaction between H3K4 methylation and Rad53 50
3.1 Introduction 50
3.2 Results 51
3.2.1 Loss of H2B-monoubiquitylation enhances HU-tolerance of Rad53 checkpoint-defective cells 51
3.2.2 H2B-K123R rescues HU-treated rad53-mutants through Set1-mediated pathway 51
3.2.3 Ablated H3K4 methylation rescues rad53 viability under HU stress 52
3.2.4 H3K4 methylation is necessary for the rad53-HU-sensitivity suppression effect 53
3.2.5 Loss of H3K4 methylation reduces fork stalling in HU-treated cells 54
3.2.6 The H3K4A rescue effect on HU-treated rad53 mutants is independent of Mec1-signaling 55
3.2.7 H3K4A HU-rescue effect is independent of R-loop formation 56
3.2.8 Ablation of the H3K4me HU-rescue effect is independent of known rad53-HU-sensitivity suppressors 56
3.3 Discussion 57

Chapter 4 69
The effect of H3K4 methylation on rad53 mutants during replication stress 69
4.1 Introduction 69
4.2 Results 70
4.2.1 Loss of H3K4 methylation restores DNA Pol2 binding in rad53 mutant under replication stress 70
4.2.2 H3K4 methylation prevents DNA Pol2 engagement with RNA Pol II on highly transcribed genes 71
4.2.3 Summary of the impact of H3K4 methylation on transcription and replication in HU-treated yeast 72
4.2.4 H3K4 methylation causes fork stalling in rad53-K227A cells following replication stress 73
4.2.5 H3K4 methylation alters replication progression independent of chromatin structure or dynamics 75
4.3 Discussion 76

Chapter 5 88
The effect of H3K4 methylation on transcription-replication conflicts in rad53 mutants 88
5.1 Introduction 88
5.2 Results 89
5.2.1 Characteristic signals in the 2D gels 89
5.2.2 Loss of H3K4 methylation prevents aberrant replication intermediates in HU-treated rad53 mutants 89
5.2.3 Construction of a transcription-replication conflict model for DNA 2D gel analysis 90
5.2.4 Loss of H3K4 methylation prevention of transcription mediates TRC-induced aberrant replication events 91
5.2.5 Construction of marker-free and orientated transcription-replication conflict model 92
5.2.6 H3K4 methylation aggravates TRCs in rad53 mutant independent of conflict orientations 93
5.2.7 H3K4 methylation promotes aberrant replication forks in HU-treated rad53 mutants 93
5.3 Discussion 95

Chapter 6 113
Speed bump model: Transcription-deposited H3K4 methylation protects genome integrity 113
6.1 Introduction 113
6.2 Results 114
6.2.1 Speed bump model 114
6.2.2 H3K4 methylation is a molecular decelerator for replication fork progression 114
6.2.3 H3K4me defends from TRC-mediated genome instability under HU-induced replication stress 115
6.3 Discussion 118

Chapter 7 128
General Discussion 128
7.1 Summary of results 128
7.2 H3K4 methylation, genome stability and cancer 130
7.3 Mechanisms for H3K4 methylation in TRCs 132
7.4 Interaction between H3K4 methylation and the S-phase checkpoint 133
7.5 Conclusion 134

Abbreviations 135
References 140
Appendix 157
Publication List (Journal papers) 157

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