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Functional characterization of USP39 as a caretaker in maintaining genomic stability

  • 황이슬 (아주대학교 일반대학원)

초록/요약

Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non- homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. In addition, accumulated evidence showed post-translational modifications (PTMs) of histones are crucial for a wide range of biological processes such as replication, transcription, and the DDR. In particular, dynamic changes in histone modifications are critical for regulating double-strand break (DSB) repair in the DDR. PARP1 plays a key role as a major histone modifier that modulates chromatin structure for precise DNA repair. However, the process through which PARP1 controls the chromatin structure through histone modifications in DNA lesions remains unclear. In the present study, I identified Suppressor of variegation 3-9 homolog 1 (SUV39H1) as a direct PAR-mediated USP39 binding protein that catalyzes histone 3 lysine 9 trimethylation (H3K9me3) for heterochromatin formation. USP39 colocalizes with SUV39H1, and depletion of USP39 reduces SUV39H1 and H3K9me3 levels as well as heterochromatin formation, suggesting that USP39 participates in the maintenance of chromatin structure through SUV39H1-mediated H3K9me3. Our results also show that USP39 stimulates chromatin organization and activation of ataxia-telangiectasia mutated (ATM) around DNA break sites to facilitate DNA repair. These findings provide mechanistic insights into the process by which PAR chains precisely control DNA repair and chromatin dynamics in the DNA damage response, and indicate the functional role of USP39 in regulating the DDR to maintain genomic stability.

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목차

I. INTRODUCTION 1
II. MATERIALS AND METHODS 6
1. Cell lines and culture 6
2. DUBs library cloning and plasmids 6
3. siRNA sequences, antibodies and chemicals 13
4. Live cell imaging with laser micro-irradiation 17
5. Immunoblotting 18
6. Immunofluorescene 18
7. Analysis of ionizing radiation-induced foci formation 19
8. Micronucleus assay 20
9. Purification of recombinant protein from Sf9 cells or E. coli 20
10. PAR overlay assay 21
11. HR and NHEJ repair analysis 22
12. Neutral comet assay 22
13. Clonogenic survival assay 22
14. Immunoprecipitation 23
15. Chromatin immunoprecipitation (ChIP) 23
16. Cell synchronization 25
17. Cell cycle analysis 25
18. In vitro aggregation assay and Transmission electron microscopy analysis 26
19. Quantitative real-time PCR 26
20. Protein microarray 27
21. Image quantification 28
22. Statistical analysis 28
III. RESULTS 29
PART 1. USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing 29
A. Screening for DUBs localized to DNA lesion(s) in a PAR-dependent manner 29
B. USP39 is an essential DUB for the regulation of DSB repair 39
C. The tripartite RG motif of USP39 is crucial for its recruitment to DNA lesions during NHEJ repair 51
D. The tripartite RG motif of USP39 is crucial for PAR chain-mediated liquid demixing at DNA lesions 61
E. USP39 directly drives XRCC4/LIG4 dependent NHEJ repair. 69
F. The USP39 ZF domain is important for XRCC4/LIG4 complex recruitment 81
G. USP39 regulates HR repair in a spliceosome complex-dependent manner 87
PART 2. USP39 regulates the chromatin dynamics and ATM activation through the SUV39H1-mediated H3K9me3 in DDR 91
A. Identification of PAR-mediated USP39 binding protein using a human protein microarray 91
B. USP39 strongly interacts with SUV39H1 in a PAR-dependent manner and regulates the H3K9me3 levels 95
C. USP39 strongly binds to SUV39H1 and regulates chromatin dynamics upon DNA damage 100
D. USP39 leads to ATM activation by regulating SUV39H1-mediated H3K9me3 in the DDR 106
E. USP39 controls both H3K9me3 and H4K20me2 through interplay with SUV39H1 108
IV. DISCUSSION 114
REFERENCES 123

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