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Role of RSF1 in the regulation of 53BP1 phase separation and p53-mediated transcriptional activation in the DNA damage response

DNA 손상 반응 과정에서 53BP1의 상분리와 p53 매개 전사 활성 조절에서의 RSF1의 역할

  • 허윤경 (아주대학교 일반대학원)

초록/요약

DNA double-strand breaks (DSBs) are among the most detrimental forms of genomic injury, threatening chromosome stability and cellular viability. If left unrepaired, such breaks may trigger apoptosis, chromosomal rearrangements, or malignant transformation. To counteract this, eukaryotic cells activate the DNA damage response (DDR), a signaling network that detects DNA lesions, initiates repair signaling, and determines cellular outcomes such as apoptosis or repair. A key regulator of the DDR is p53, a tumor suppressor protein that governs transcriptional programs involved in cell fate determination under genotoxic stress. While acetylation at lysine 382 (K382) is known to enhance p53 transcriptional activity, the precise chromatin-based mechanisms underlying this regulation remain incompletely defined. In this study, I identified RSF1 (Remodeling and Spacing Factor 1) as a pivotal chromatin remodeler involved in p53-driven transcriptional responses to DNA damage. RSF1, in complex with SNF2H, modulates nucleosome positioning and chromatin accessibility. Loss of RSF1 in U2OS cells led to diminished p53 acetylation at K382 and impaired activation of downstream genes, including CDKN1A (p21). Mechanistically, RSF1 facilitates the recruitment of the histone acetyltransferase p300 to p53-responsive enhancers and promotes chromatin looping between enhancers and promoters. In addition to its remodeling function, RSF1 enhances transcriptional output by stabilizing the pre-initiation complex and enabling the engagement of FACT and RNA polymerase II at stress-inducible promoters. Furthermore, RSF1 is involved in organizing DNA damage-induced condensates through liquid-liquid phase separation (LLPS). Although RSF1 does not undergo phase separation itself, it is required for the proper formation and maintenance of 53BP1 condensates at damage sites. Domain-mapping studies identified a disordered region at the RSF1 C-terminus as essential for this scaffolding role. RSF1 depletion disrupted the maturation of 53BP1 foci and impaired downstream signaling. Furthermore, RSF1 facilitated the recruitment of PARG, thereby promoting the transition from early PARP1-mediated condensates to stable 53BP1 assemblies. This spatial reorganization enhanced p53 recruitment to damaged chromatin and amplified transcriptional activation of its target genes. Taken together, the results suggest that RSF1 serves two distinct functions in the DNA damage response: it facilitates p53-driven transcription through chromatin remodeling, while also supporting the spatial assembly of 53BP1 repair condensates via liquid-liquid phase separation. RSF1 links epigenetic regulation to phase separation-mediated repair architecture, ensuring coordinated transcriptional and structural responses to genotoxic stress.

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목차

CHAPTER I : Introduction 1
A. DNA damage response (DDR) 1
B. Mechanisms of DNA double-strand break repair 2
C. P53-mediated transcriptional regulation in the DNA damage response 6
D. The chromatin remodeling function of RSF1 10
E. 53BP1 function in DNA repair and transcription 12
F. Phase separation in the DNA damage response 16
G. Aims of this study 20
CHAPER II : RSF1 coordinates p300 and FACT to regulate p53-dependent transcription 22
A. Chapter Introduction 23
B. Materials and Methods 25
1. Cell culture 25
2. Plasmid and RNA interference 25
3. Western blot analysis 26
4. Antibodies and reagents 26
5. Flow cytometric (FACS) analysis 28
6. Whole-cell extraction 28
7. Immunofluorescence microscopy 28
8. Pull-down assay 29
9. Immunoprecipitation assay 29
10. Real-time PCR analysis 30
11. Reporter gene activity measurement using luciferase assay 30
12. Chromatin immunoprecipitation (ChIP) assay 30
13. Statistical analysis 32
C. Results 33
1. RSF1 promotes p53-dependent transcription and cell cycle arrest in response to DNA damage 33
2. RSF1 controls p53 transcriptional activity by regulating its acetylation at lysine 382 37
3. RSF1 facilitates p53-dependent transcription by coordinating p300 activity and pre-initiation complex assembly 41
4. RSF1 enhances p21 transcription by bridging the FACT complex and RNA polymerase II to promoter regions 45
CHAPER III : RSF1 supports 53BP1 repair condensate formation through chromatin remodeling 49
A. Chapter Introduction 50
B. Materials and Methods 52
1. Cell culture 52
2. Cloning and plasmids 52
3. siRNA sequences, antibodies, and chemicals 53
4. OptoDroplet assay 55
5. Fluorescence recovery after photobleaching (FRAP) 56
6. Laser microirradiation and immunofluorescence 56
7. Immunoblotting 56
8. Recombinant Protein Expression and Purification from Sf9 Insect Cells and E. coli 57
9. Poly-ADP-ribosylation in vitro assay 57
10. Quantitative real-time PCR 58
11. Reporter gene activity measurement using luciferase assay 58
12. Chromatin immunoprecipitation (ChIP) assay 59
13. Image quantification 59
14. Statistical analysis 60
C. Results 61
1. RSF1 predominantly comprises intrinsically disordered regions (IDRs) 61
2. RSF1 alone does not form liquid condensates 65
3. RSF1 is required for the 53BP1-mediated liquid condensate formation 69
4. The C-terminal IDR3 of RSF1 is required for 53BP1 condensate formation 73
5. RSF1 modulates 53BP1 condensate formation through PARylation dynamics 77
6. RSF1 is required for 53BP1 condensate formation but does not affect FUS-driven condensates 81
7. RSF1 facilitates timely PARG recruitment to DNA damage sites, independent of PARP1 accumulation 84
8. Delayed PARG recruitment underlies 53BP1 condensate defects in RSF1 C1-expressing cells 87
9. Disruption of phase separation attenuates p53 target gene transcription following DNA damage 90
10. RSF1-mediated condensates promote p53 recruitment to chromatin and transcriptional activation 93
CHAPER IV : Discussion 96
REFERENCES 106
국문요약 123

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