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Multifaceted role of CTCF in DNA damage response: A comprehensive analysis of novel functions
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 이종수 | - |
| dc.contributor.author | 황순영 | - |
| dc.date.accessioned | 2025-01-25T01:36:11Z | - |
| dc.date.available | 2025-01-25T01:36:11Z | - |
| dc.date.issued | 2023-08 | - |
| dc.identifier.other | 32902 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/24723 | - |
| dc.description | 학위논문(박사)--생명과학과,2023. 8 | - |
| dc.description.tableofcontents | CHAPTER 1 1 <br> I. INTRODUCTION 2 <br> II. MATERIALS AND METHODS 5 <br> 1. Cells and reagents 5 <br> 2. Plasmid construction and transfection 5 <br> 3. Antibodies 6 <br> 4. Tandem affinity purification and mass spectrometry 6 <br> 5. Laser micro-irradiation 7 <br> 6. Immunofluorescence 7 <br> 7. Immunoprecipitation 8 <br> 8. ChIP assay 8 <br> 9. HR, canonical and alternative NHEJ, and single-strand annealing (SSA) DNA repair assays 9 <br> 10. Measurement of DNA end resection 9 <br> 11. siRNAs 10 <br> 12. Immunoblotting 10 <br> 13. Statistical analysis 10 <br> III. RESULTS 12 <br> 1. CTCF interacts with MRE11 upon DNA damage. 12 <br> 2. CTCF enrichment at sites of DNA damage depends on MRE11. 17 <br> 3. CTCF enrichment at damaged DNAs requires its N-terminal or zinc-finger domain. 22 <br> 4. CTCF is required for CtIP localization to sites of DNA damage. 25 <br> 5. CTCF enhances CtIP-mediated DNA end resection. 36 <br> 6. Essential role of CTCF in HR-mediated DSB repair. 44 <br> IV. DISCUSSION 48 <br>CHAPTER 2 51 <br> I. INTRODUCTION 52 <br> II. MATERIALS AND METHODS 57 <br> 14. Cells and reagents 57 <br> 15. Plasmid construction 57 <br> 16. Antibodies 57 <br> 17. Chemical agents 58 <br> 18. SiRNAs 58 <br> 19. Proximity Ligation Assay 58 <br> III. RESULTS 60 <br> 7. CTCF recruits BARD1-BRCA1 heterodimer to DSB at early time of repair process. 60 <br> 8. CTCF facilitates HP1γ localization at DNA lesions. 69 <br> 9. CTCF promotes transient heterochromatin through HP1 recruitment. 76 <br> 10. CTCF is required for localization of H3K9 methyl transferase complex to DNA lesions. 78 <br> 11. H3K4 demethylation contributes to translocate BARD1 and BRCA1 at DSB sites. 85 <br> 12. CTCF is required for extanded resection through recruiting BARD1-BRCA1 heterodimer. 93 <br> 13. Formation of transient heterochromatin is essential for HR repair. 99 <br> IV. DISCUSSION 105 <br>CHAPTER 3 109 <br> I. INTRODUCTION 110 <br> II. MATERIALS AND METHODS 113 <br> 20. Cells and reagents 113 <br> 21. Chemical agents 113 <br> 22. Fluorescence recovery after photobleaching (FRAP) 113 <br> 23. High resolution analysis of condensates 113 <br> 24. Super resolution analysis of condensates 114 <br> III. RESULTS 115 <br> 14. CTCF-mediated damage-induced long non-coding RNA transcription is required for 53BP1 phase separation. 115 <br> 15. Unrepaired DNA lesions are sequestered into 53BP1 condensates for DNA damage repair. 121 <br> 16. Along with DNA damage repair, transcriptional inhibition is also implicated within 53BP1 condensates. 125 <br> IV. DISCUSSION 130 <br>REFERENCES 132 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Multifaceted role of CTCF in DNA damage response: A comprehensive analysis of novel functions | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 대학원 | - |
| dc.contributor.alternativeName | Soonyoung Hwang | - |
| dc.contributor.department | 일반대학원 생명과학과 | - |
| dc.date.awarded | 2023-08 | - |
| dc.description.degree | Doctor | - |
| dc.identifier.localId | T000000032902 | - |
| dc.identifier.url | https://dcoll.ajou.ac.kr/dcollection/common/orgView/000000032902 | - |
| dc.subject.keyword | CTCF | - |
| dc.subject.keyword | DNA end resection | - |
| dc.subject.keyword | Homologous recombination | - |
| dc.subject.keyword | Liquid-liquid phase separation | - |
| dc.subject.keyword | heterochromatin | - |
| dc.description.alternativeAbstract | To maintain genomic stability, cells adopt a variety of repair mechanisms that depend on the type of DNA damage encountered. Above all, DNA double-strand breaks (DSBs) are one of the most hazardous types of DNA lesions that can cause deletions, chromosome rearrangements, and loss of heterozygosity, resulting in cell death or carcinogenesis. There are two canonical repair pathways for ensuring genome integrity in the presence of DSBs: error-free homologous recombination (HR) repair and error-prone non-homologous end joining (NHEJ) repair. A critical step in HR repair is DNA end resection, which generates a long 3' single-stranded DNA (ssDNA) tail that provides a platform for the recruitment of HR repair factors that can invade the homologous DNA strand. DNA end resection for HR is accomplished through a two-step process in which the MRE11 and CtIP proteins engage in the initiation step, and the EXOI and DNA2 nucleases are involved in the extension step. Recent studies show that the pleiotropic CCCTC-binding factor (CTCF), a multifunctional factor involved in genomic imprinting, transcription, and chromatin organization, plays an important role in HR repair of DSBs. However, the precise mechanistic role of CTCF in HR remains largely unclear. Here, it is shown that CTCF functions in DNA end resection for HR through promoting both the initial and extensive resection steps. <br>Firstly, proteomic analysis of the CTCF interactome reveals that CTCF physically interacts with MRE11, which is a subunit of the MRE11/RAD50/NBS1 (MRN) complex and resects the 5' DNA end of DSBs. Additionally, CTCF interacts with CtIP, known as a co-factor of MRE11 regulating its catalytic activity, and facilitates CtIP recruitment at DSB sites. Subsequently, CTCF promotes the initiation of DNA end resection to ensure HR, in conjunction with MRE11-CtIP. Notably, the zinc finger domain of CTCF is responsible for bridging CtIP to MRE11, leading to DNA end resection initiation for HR repair. Overall, this suggests that CTCF plays a significant role in DNA end resection initiation through CtIP recruitment to facilitate HR. <br>Secondly, it is discovered that CTCF is involved in the extensive resection step through EXOI and DNA2 recruitment. Depletion of CTCF considerably abrogates the early recruitment of the BARD1-BRCA1 heterodimer, which is a crucial mediator of HR repair and participates in HR throughout the whole HR process at sites of DNA damage. Additionally, CTCF interacts with heterochromatin protein 1 γ (HP1γ), which is required for early BARD1 recruitment to DNA lesions and facilitates the recruitment of HP1γ to DSBs. CTCF boosts transient heterochromatin formation at DSBs through the methylation of histone 3 Lys 9 (H3K9) and demethylation of histone 3 Lys 4 (H3K4) via recruiting the H3K9 methyltransferases SUV39H1 and SETDB1, and the H3K4 demethylase KDM5a, respectively. Subsequently, the localized BARD1-BRCA1 at DSBs ubiquitinates Lys 127 of histone H2A and promotes the recruitment of the chromatin remodeling factor SMARCAD1, known for recognizing the ubiquitination of H2A at Lys 127. Chromatin remodeling by SMARCAD1 fosters an environment for the recruitment of EXOI and DNA2, which anchor and generate extensive 3' single-stranded DNA. In summary, these findings suggest that CTCF engages in the formation of heterochromatin and BARD1-BRCA1 recruitment for the promotion of extensive DNA end resection. Overall, in parallel with the initialization of DNA end resection at DSBs, CTCF takes part in the extension of end resection, which is a key step for the decision point of deploying HR. <br>Finally, it is found that CTCF is essential for the DNA damage-induced liquid-liquid phase separation (LLPS) of DNA damage response (DDR) factors, including 53BP1 and BRCA1-BARD1, into foci. The foci assemble to form LLPS condensates over a considerable time after DNA damage, However, the physiological functions of DNA damage-induced condensates remain unelucidated. To define the cellular functions of DNA damage-induced condensates, this research is performed by focusing on the analysis of biological and biochemical properties of 53BP1 condensates. Depletion of CTCF abolishes the transcription of DNA damage-induced long non-coding RNAs (dilncRNAs) at sites of DSB, which is required for the development of DDR condensates and acts as seeds for DNA damage-induced LLPS. Furthermore, knockdown of CTCF suppresses the phase separation of the DNA damage response factor 53BP1, indicating that CTCF-mediated transcription of dilncRNA is essential for the formation of 53BP1 damage-induced condensates. To define the cellular functions of 53BP1 condensates, the composition of the condensate is analyzed, revealing the compartmentalization of the DNA damage marker γH2AX foci within the condensates. Also, a variety of repair factors, including ATM and the MRN complex, are sequestrated in the 53BP1 condensate. Interestingly, the ongoing RNA polymerases are paused, and thus transcription is repressed within the condensates. In summary, these results suggest that CTCF regulates a post-repair mechanism by sequestering unrepaired DNA damage sites into 53BP1 condensates, accompanied by transcriptional repression at damaged DNAs. | - |
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