Histone chaperone FACT regulates homologous recombination by chromatin remodeling through interaction with RNF20

Abstract

The E3 ubiquitin ligase RNF20 regulates chromatin structure through ubiquitination of histone H2B, so that early homologous recombination repair (HRR) proteins can access the DNA in eukaryotes during repair. However, it remains unresolved how RNF20 itself approaches the DNA in the presence of chromatin structure. Here, we identified the histone chaperone FACT as a key protein in the early step of HRR. Depletion of SUPT16H, a component of FACT, caused pronounced defects in accumulations of repair proteins, consequently decreased HRR activity. This led to enhanced sensitivity to ionizing radiation (IR) and mitomycin-C in a fashion similar to RNF20-deficient cells, indicating that SUPT16H is essential for RNF20-mediated pathway. Indeed, SUPT16H directly bound to RNF20 in vivo, and mutation at the RNF20's RING-finger domain abolished its interaction and accumulation, as well as RAD51 and BRCA1 at sites of DSBs, whereas the localization of SUPT16H remained intact. Interestingly, PAF1, implicated in transcription as a mediator of FACT and RNF20 association, was dispensable for DNA damage-induced interaction of RNF20 with SUPT16H. Furthermore, depletion of SUPT16H caused pronounced defects in RNF20-mediated H2B ubiquitination and thereby, impaired accumulation of the chromatin remodeling factor SNF2h. Consistent with this observation, the defective phenotypes of SUPT16H were effectively counteracted by enforced nucleosome relaxation. Taken together, present results indicate a primary role of FACT in RNF20 recruitment and the resulting chromatin remodeling for initiation of HRR.