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DNA 손상 신호 체계와 손상으로 인한 DNA 전사 조절 과정에서 크로마틴 리모델링 단백질인 RSF1의 역할

The role of RSF1 in DNA damage signaling pathway and DSB-induced transcriptional regulation

초록/요약

Chromatin remodeling factors are known as a key determinant of chromatin modification in DNA replication, transcription, and double strand break (DSB) repair. As a member of imitation switch (ISWI) family in ATP-dependent chromatin remodeling factors, the remodeling and spacing factor (RSF) complex consists of two subunits, SNF2h ATPase and RSF1. Recent studies suggest that the function of chromatin remodeling factor is to temporally regulate the chromatin modification in the crosstalk between DSB signaling pathway and transcriptional regulation for the efficient DSB repair. Although it has been reported that SNF2h ATPase is recruited to DSB sites in poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner, the function of RSF1 is still elusive. Here various cellular analyses confirmed that RSF1 is recruited and accumulated at DSBs in ATM-dependent manner, and the putative pSQ motifs of RSF1 by ATM are required for its accumulation at DSBs. In addition, depletion of RSF1 attenuates the activation of DNA damage checkpoint signals upon DNA damage. This defect is rescued by Trichostatin (TSA) treatment via chromatin relaxation. Thus, chromatin relaxation by RSF1 as chromatin remodeling factor is required for the propagation of ɣH2AX signaling pathway. As a result, RSF1 promotes homologous recombination repair (HRR) by recruiting HR factors. Although RSF1 propagates ɣH2AX signal pathway for the efficient repair as one of chromatin remodeling factors, the function of RSF1 in crosstalk between transcription and DDR is still elusive. Here inducible transcription system at DSB sites showed that RSF1 promotes DSB-induced transcriptional silencing, while SNF2h is dispensable for transcriptional silencing at DSB sites. The major determinant of DSB-induced transcriptional silencing, ATM signaling, is also impaired in RSF1 depleted cells. To determine the molecular mechanism of DSB-induced transcriptional silencing regulated by RSF1, the proteins in RSF1 mass spectrometry were screened by microirradiation. On the basis of the screening results from mass spectrometry analysis, the recruitment of transcriptional repressors at DSB sites in RSF1-dependent manner promotes DSB-induced transcriptional silencing. In addition, RSF1 interacts with polycomb repressive complex (PRC) at transcriptionally active site, and Swi3, Ada2, N-Cor, and TFIIIB (SANT) domain of enhancer of zeste homolog 2 (EZH2) is required for its recruitment and its interaction with RSF1. The impaired recruitment of EZH2 at DSB sites also leads to the defects in recruitment of RING1B and its substrate, H2AK119 ubiquitination at DSB sites. In addition, the defect in deacetylation of H2AK118 at DSB sites by HDAC1 in RSF1 depleted cells reduces the level of H2AK119 ubiquitination at DSB sites and eventually leads to the failure in DSB-induced transcriptional silencing. Finally, transcriptome analysis by RNA-sequencing reveals that transcriptome in cells upon DNA damage is changed in RSF1 depleted cells, and as a result, in RSF1 depleted cells, cell death is remarkably reduced upon the continuous DNA damage. Altogether, these data reveal that RSF1 is recruited at DSB sites and regulates ATM-dependent checkpoint signaling pathway by chromatin relaxation. In parallel, RSF1 also regulates DSB-induced transcription silencing through PRC complex and HDAC1 and the crosstalk between the histone modifications by these histone modifiers.

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목차

I. INTRODUCTION 1
A. DNA damage response 1
B. DSB repair pathways 2
C. Remodeling and spacing factor 1 in ISWI family 3
D. Chromatin remodeling factor in DNA damage response 3
E. DNA damage response in the context of chromatin 4
F. DSB-induced transcriptional silencing 6
G. Aim of this study 7

II. MATERIALS AND METHOD 10
1. Cell culture, reagents, and treatment 10
2. Plasmids and RNA interference 10
3. Mutagenesis 11
4. Laser micro-irradiation 11
5. Immunofluorescence microscopy 12
6. Antibodies 12
7. Purification of recombinant protein in vitro 13
8. Immunoblotting and protein membrane overlay assay 13
9. Immunoprecipitation and chromatin fractionation 14
10. MNase assay 14
11. Homologous recombination (HR) and Non-homologous end joining (NHEJ) repair assay 15
12. Cell cycle analysis 15
13. Chromatin Immunoprecipitation (ChIP) 16
14. FokI assays 18
15. RNA isolation and RNA sequencing 18
16. Reverse transcription and quantitative RT-PCR 18
17. ATM retention assay 19
18. Nucleosome stability assay 20
19. Statistical analysis 20

III. RESULTS 21
PART I. ATM-dependent chromatin remodeler RSF1 facilitates DNA damage checkpoints and homologous recombination repair 21
1. RSF1 is recruited at DNA double-strand break sites 21
2. ATM-dependent accumulation of RSF1 at DSBs 25
3. The putative motifs pSQ of RSF1 by ATM is required for its accumulation at DSBs 29
4. Depletion of RSF1 attenuates DNA damage checkpoint signals 32
5. Depletion of RSF1 caused the failure in chromatin relaxation upon DNA damage 37
6. Depletion of RSF1 decreased histone H2A and H2B exchange at the sites of DSB 42
7. RSF1 facilitates homologous recombination repair by recruiting resection factors 45

PART II. RSF1 recruits EZH2 and HDAC1 for efficient ubiquitination of H2A to promote DSB-induced transcriptional silencing at DSB sites 48
1. RSF1 leads to DSB-induced transcriptional silencing at DNA lesions 48
2. SNF2h is dispensable to DSB-induced transcriptional silencing at DSB lesions 52
3. RSF1 promotes DSB-induced transcriptional silencing by regulating ATM activity 55
4. Screening RSF1-interacting proteins identified that RSF1 recruits transcriptional repressors at DSB sites 58
5. Transcriptional repressors, recruited by RSF1, are involved in DSB-induced transcriptional silencing at DSB sites 61
6. RSF1 depletion impaired EZH2 recruitment at DSB sites 66
7. SANT domain of EZH2, interacting with RSF1, is important to its recruitment at DSB sites 69
8. RSF1 depletion induces DSB-induced transcriptional silencing by the reduction in H2A ubiquitination at transcriptionally active region upon DNA damage 73
9. RSF1 depletion impaired HDAC1 recruitment at DSB sites. 76
10. H2AK118 deacetylation by HDAC1 is required for the efficient monoubiquitination of H2A at K119 for transcriptional silencing at DSB sites 82
11. Failure of local transcriptional regulation resulted in inefficient repair of DNA double strand breaks 85
12. RSF1 regulates p53 signaling pathway, resulting in the reduction of cell death upon DNA damage 88
13. Acetylation of p53 at K392 is reduced in RSF1 depleted cells 93

IV. DISCUSSION 96

REFERENCE 103

국문요약 109

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