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Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG.

ABSTRACT: Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-β-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.

SUBMITTER: Scalera C

PROVIDER: S-EPMC8235791 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Accessibility within chromatin is an important factor in the prompt removal of UV-induced DNA damage by nucleotide excision repair (NER). Chromatin remodeling by the SWI/SNF complex has been shown to play an important modulating role in NER in vitro and yeast in vivo. Nevertheless, the molecular basis of cross-talk between SWI/SNF and NER in mammalian cells is not fully understood. Here, we show that knockdown of Brg1, the ATPase subunit of SWI/SNF, negatively affects the elimination of cyclobutane pyrimidine dimers (CPD), but not of pyrimidine (6, 4)pyrimidone photoproducts (6-4PP) following UV irradiation of mammalian cells. Brg1-deficient cells exhibit a lower chromatin relaxation as well as impaired recruitment of downstream NER factors, XPG and PCNA, to UV lesions. However, the assembly of upstream NER factors, DDB2 and XPC, at the damage site was unaffected by Brg1 knockdown. Interestingly, Brg1 interacts with XPC within chromatin and is recruited to UV-damaged sites in a DDB2- and XPC-dependent manner. Also, postirradiation decrease of XPC levels occurred more rapidly in Brg1-deficient than normal cells. Conversely, XPC transcription remained unaltered upon Brg1 knockdown indicating that Brg1 affects the stability of XPC protein following irradiation. Thus, Brg1 facilitates different stages of NER by initially modulating UV-induced chromatin relaxation and stabilizing XPC at the damage sites, and subsequently stimulating the recruitment of XPG and PCNA to successfully culminate the repair.

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