The H2B ubiquitin ligase RNF40 cooperates with SUPT16H to induce dynamic changes in chromatin structure during DNA double-strand break repair

Abstract

Many anticancer therapies function largely by inducing DNA double-strand breaks (DSBs) or altering the ability of cancer cells to repair them. Proper and timely DNA repair requires dynamic changes in chromatin assembly and disassembly characterized by histone H3 lysine 56 acetylation (H3K56ac) and phosphorylation of the variant histone H2AX (γH2AX). Similarly, histone H2B monoubiquitination (H2Bub1) functions in DNA repair, but its role in controlling dynamic changes in chromatin structure following DSBs and the histone chaperone complexes involved remain unknown. Therefore, we investigated the role of the H2B ubiquitin ligase RNF40 in the DSB response. We show that RNF40 depletion results in sustained H2AX phosphorylation and a decrease in rapid cell cycle checkpoint activation. Furthermore, RNF40 knockdown resulted in decreased H3K56ac and decreased recruitment of the facilitates chromatin transcription (FACT) complex to chromatin following DSB. Knockdown of the FACT component suppressor of Ty homolog-16 (SUPT16H) phenocopied the effects of RNF40 knockdown on both γH2AX and H3K56ac following DSB induction. Consistently, both RNF40 and SUPT16H were required for proper DNA end resection and timely DNA repair, suggesting that H2Bub1 and FACT cooperate to increase chromatin dynamics, which facilitates proper checkpoint activation and timely DNA repair. These results provide important mechanistic insights into the tumor suppressor function of H2Bub1 and provide a rational basis for pursuing H2Bub1-based therapies in conjunction with traditional chemo- and radiotherapy.

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

“…that the knockdown of RNF40 affects SUPT16H accumulation after treatment with radiomimetics, possibly because of an indirect effect of the decreased transcription activity (Kari et al, 2011). However, we demonstrated here that FACT binds to RNF20 upon formation of DSBs through its C-terminal domain (Fig.…”

High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

“…In line with this hypothesis, a common characteristic between HMGA2 transgenic [41], ATM −/− [42], and H2AX −/− [43] mice is genomic instability. In addition, DNA repair-linked histone modifications, H3K56ac and H2Bub1 [44], have been related to transcription initiation [45]. Remarkably, HMGA2 has been shown to have a lyase activity that cleaves DNA containing apurinic/apyrimidinic sites, thereby inducing base excision repair [46].…”

Zinc finger protein 668 interacts with Tip60 to promote H2AX acetylation after DNA damage

“…Following the induction of DSBs, the H2B ubiquitin ligase RNF40 cooperates with the suppressor of Ty homolog-16 (SUPT16H) to induce dynamic change of chromatin structure, which facilitates proper checkpoint activation and timely DNA repair. 32 Recent studies have suggested a role of chromatin acetylation by histone acetyltransferase (HAT) complexes in DNAdamage detection and DNA repair. [33][34][35] The histone acetylation mediated by Tip60 acetyltransferase is required for the subsequent recruitment of repair proteins to the DSBs sites.…”