title: Histone chaperone FACT regulates homologous recombination by chromatin remodeling through interaction with RNF20.
abstract: The E3 ubiquitin ligase RNF20 regulates chromatin structure through ubiquitylation 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 steps of HRR. Depletion of SUPT16H, a component of FACT, caused pronounced defects in accumulations of repair proteins and, 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 RING-finger domain in RNF20 abolished its interaction and accumulation, as well as that of RAD51 and BRCA1, at sites of DNA double-strand breaks (DSBs), whereas the localization of SUPT16H remained intact. Interestingly, PAF1, which has been 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 ubiquitylation 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, our results indicate a primary role of FACT in RNF20 recruitment and the resulting chromatin remodeling for initiation of HRR.
authors.affiliation: Division of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Yoshida-konoecho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Douglas V
authors.initials: DV
authors.lastname: Oliveira
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kato
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Nakamura
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Ikura
authors.forename: Masahiro
authors.initials: M
authors.lastname: Okada
authors.forename: Junya
authors.initials: J
authors.lastname: Kobayashi
authors.forename: Hiromi
authors.initials: H
authors.lastname: Yanagihara
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Saito
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tauchi
authors.forename: Kenshi
authors.initials: K
authors.lastname: Komatsu
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D042002:Chromatin Assembly and Disassembly; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000502:physiology*; D006609:High Mobility Group Proteins / Q000502:physiology*; D006657:Histones / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D054829:RING Finger Domains; D059767:Recombinational DNA Repair; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006609:High Mobility Group Proteins; D006657:Histones; D009687:Nuclear Proteins; C501972:PAF1 protein, human; C073537:SSRP1 protein, human; C489489:SUPT16H protein, human; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C506646:RNF20 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: Chromatin remodeling; H2B ubiquitylation; Homologous recombination; RNF20; SUPT16H