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DNA 손상 신호 체계와 손상으로 인한 DNA 전사 조절 과정에서 크로마틴 리모델링 단백질인 RSF1의 역할
- Alternative Title
- The role of RSF1 in DNA damage signaling pathway and DSB-induced transcriptional regulation
- Author(s)
- 민선우
- Alternative Author(s)
- Sunwoo Min
- Advisor
- 조혜성
- Department
- 일반대학원 의생명과학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2018-02
- Language
- eng
- Keyword
- DNA damage response; DNA repair; Chromatin remodeling factor; DSB-induced transcriptional silencing
- Alternative Abstract
- 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.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Biomedical Sciences > 4. Theses(Ph.D)
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