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Title Page 2

ABSTRACT 5

Contents 7

ABBREVIATION 14

CHAPTER Ⅰ. Introduction 18

A. DNA damage response (DDR) 18

B. Mechanisms of DNA double-strand break repair 19

C. P53-mediated transcriptional regulation in the DNA damage response 23

D. The chromatin remodeling function of RSF1 27

E. 53BP1 function in DNA repair and transcription 29

F. Phase separation in the DNA damage response 33

G. Aims of this study 37

CHAPTER Ⅱ. RSF1 coordinates p300 and FACT to regulate p53-dependent transcription 39

A. Chapter Introduction 40

B. Materials and Methods 42

1. Cell culture 42

2. Plasmid and RNA interference 42

3. Western blot analysis 43

4. Antibodies and reagents 43

5. Flow cytometric (FACS) analysis 45

6. Whole-cell extraction 45

7. Immunofluorescence microscopy 45

8. Pull-down assay 46

9. Immunoprecipitation assay 46

10. Real-time PCR analysis 47

11. Reporter gene activity measurement using luciferase assay 47

12. Chromatin immunoprecipitation (ChIP) assay 47

13. Statistical analysis 49

C. Results 50

1. RSF1 promotes p53-dependent transcription and cell cycle arrest in response to DNA damage 50

2. RSF1 controls p53 transcriptional activity by regulating its acetylation at lysine 382 54

3. RSF1 facilitates p53-dependent transcription by coordinating p300 activity and pre-initiation complex assembly 58

4. RSF1 enhances p21 transcription by bridging the FACT complex and RNA polymerase Ⅱ to promoter regions 62

CHAPTER Ⅲ. RSF1 controls the transition from PAR-FUS to 53BP1 condensates within DNA damage compartments 66

A. Chapter Introduction 67

B. Materials and Methods 69

1. Cell culture 69

2. Cloning and plasmids 69

3. siRNA sequences, antibodies, and chemicals 70

4. OptoDroplet assay 72

5. Fluorescence recovery after photobleaching (FRAP) 73

6. Laser microirradiation and immunofluorescence 73

7. Immunoblotting 73

8. Recombinant protein expression and purification from Sf9 insect cells and E. coli 74

9. Poly-ADP-ribosylation in vitro assay 74

10. Quantitative real-time PCR 75

11. Reporter gene activity measurement using luciferase assay 75

12. Chromatin immunoprecipitation (ChIP) assay 76

13. Image quantification 76

14. Statistical analysis 77

C. Results 78

1. RSF1 predominantly comprises intrinsically disordered regions (IDRs) 78

2. RSF1 alone does not form liquid condensates 82

3. RSF1 is required for the 53BP1-mediated liquid condensate formation 86

4. The C-terminal IDR3 of RSF1 is required for 53BP1 condensate formation 90

5. RSF1 modulates 53BP1 condensate formation through PARylation dynamics 94

6. RSF1 is required for 53BP1 condensate formation but does not affect FUS-driven condensates 98

7. RSF1 facilitates timely PARG recruitment to DNA damage sites, independent of PARP1 accumulation 101

8. Delayed PARG recruitment underlies 53BP1 condensate defects in RSF1 C1-expressing cells 104

9. Disruption of phase separation attenuates p53 target gene transcription following DNA damage 107

10. RSF1-mediated condensates promote p53 recruitment to chromatin and transcriptional activation 110

CHAPTER Ⅳ. Discussion 113

CHAPER Ⅱ. RSF1 coordinates p300 and FACT to regulate p53-dependent transcription 113

CHAPTER Ⅲ. RSF1 controls the transition from PAR-FUS to 53BP1 condensates within DNA damage compartments 118

REFERENCES 123

국문요약 140

List of Tables 13

CHAPTER Ⅱ. RSF1 coordinates p300 and FACT to regulate p53-dependent transcription 13

Table 1. siRNA sequences 42

Table 2. Antibody list 43

Table 3. Chemical and reagent list 44

Table 4. RT PCR primer 47

Table 5. Chip primer 48

CHAPTER Ⅲ. RSF1 controls the transition from PAR-FUS to 53BP1 condensates within DNA damage compartments 13

Table 1. Plasmids information 69

Table 2. siRNA list 70

Table 3. Antibody list 70

Table 4. Chemical information 71

Table 5. RT PCR primer 75

Table 6. Chip primer 76

List of Figures 11

CHAPTER Ⅰ. Introduction 11

Figure 1. A schematic representation illustrating the cellular response to DNA damage... 21

Figure 2. Mechanistic overview of DDR signaling leading to p53-driven gene expression 25

Figure 3. DNA damage-induced p53 chromatin binding and transcriptional activation 26

Figure 4. Multifunctional roles of 53BP1 in DNA damage signaling, transcription and... 31

Figure 5. General principles of liquid-liquid phase separation (LLPS) 35

Figure 6. Phase separation dynamics during the DNA damage response 36

Figure 7. 'How RSF1 orchestrates transcriptional activation and repair factor... 38

CHAPTER Ⅱ. RSF1 coordinates p300 and FACT to regulate p53-dependent transcription 11

Figure 1. RSF1 regulates p53-dependent transcription in response to DNA damage 53

Figure 2. RSF1 promotes p53 acetylation at K382 and facilitates its transcriptional... 57

Figure 3. RSF1-p300 complex promotes p53 acetylation and pre-initiation complex... 61

Figure 4. RSF1 supports transcriptional activation by coordinating FACT and RNA... 65

CHAPTER Ⅲ. RSF1 controls the transition from PAR-FUS to 53BP1 condensates within DNA damage compartments 12

Figure 1. A large portion of RSF1 contains intrinsically disordered regions 81

Figure 2. RSF1 alone does not form phase separated droplets in optoDroplet assay 85

Figure 3. RSF1 is required for stable 53BP1 condensate formation and maintenance at... 89

Figure 4. IDR3 is required for 53BP1 condensate formation 93

Figure 5. RSF1 modulates PARylation dynamics to promote 53BP1 condensate... 97

Figure 6. RSF1 is required for 53BP1 condensate formation but not for FUS-driven... 100

Figure 7. RSF1 facilitates timely recruitment of PARG to DNA damage sites 103

Figure 8. Defective PARG recruitment by RSF1 C1 leads to failure in 53BP1... 106

Figure 9. Effect of LLPS inhibition on p53 pathway activation following upon DNA... 109

Figure 10. RSF1-dependent condensate formation enhances p53 chromatin binding... 112

CHAPER Ⅳ. Discussion 12

Figure 1. The schematic model illustrates the role of RSF1 in linking enhancer... 116

Figure 2. Schematic representation of the dynamic transition between early PAR-FUS... 122

초록보기

 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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