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HP1 regulates the localization of FANCJ at sites of DNA double-strand breaks.

ABSTRACT: The breast and ovarian cancer predisposition protein BRCA1 forms three mutually exclusive complexes with Fanconi anemia group J protein (FANCJ, also called BACH1 or BRIP1), CtIP, and Abraxas/RAP80 through its BRCA1 C terminus (BRCT) domains, while its RING domain binds to BRCA1-associated RING domain 1 (BARD1). We recently found that the interaction between heterochromatin protein 1 (HP1) and BARD1 is required for the accumulation of BRCA1 and CtIP at sites of DNA double-strand breaks. Here, we investigated the importance of HP1 and BARD1-HP1 interaction in the localization of FANCJ together with the other BRCA1-BRCT binding proteins to clarify the separate role of the HP1-mediated pathway from the RNF8/RNF168-induced ubiquitin-mediated pathway for BRCA1 function. FANCJ interacts with HP1? in a BARD1-dependent manner, and this interaction was enhanced by ionizing radiation or irinotecan hydrochloride treatment. Simultaneous depletion of all three HP1 isoforms with shRNAs disrupts the accumulation of FANCJ and CtIP, but not RAP80, at double-strand break sites. Replacement of endogenous BARD1 with a mutant BARD1 that is incapable of binding to HP1 also disrupts the accumulation of FANCJ and CtIP, but not RAP80. In contrast, RNF168 depletion disrupts the accumulation of only RAP80, but not FANCJ or CtIP. Consequently, the accumulation of conjugated ubiquitin was only inhibited by RNF168 depletion, whereas the accumulation of RAD51 and sister chromatid exchange were only inhibited by HP1 depletion or disruption of the BARD1-HP1 interaction. Taken together, the results suggest that the BRCA1-FANCJ and BRCA1-CtIP complexes are not downstream of the RNF8/RNF168/ubiquitin pathway, but are instead regulated by the HP1 pathway that precedes homologous recombination DNA repair.

SUBMITTER: Wu W

PROVIDER: S-EPMC5084677 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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HP1 regulates the localization of FANCJ at sites of DNA double-strand breaks.

Wu Wenwen W Togashi Yukiko Y Johmura Yoshikazu Y Miyoshi Yasuo Y Nobuoka Sachihiko S Nakanishi Makoto M Ohta Tomohiko T

Cancer science 20160901 10

The breast and ovarian cancer predisposition protein BRCA1 forms three mutually exclusive complexes with Fanconi anemia group J protein (FANCJ, also called BACH1 or BRIP1), CtIP, and Abraxas/RAP80 through its BRCA1 C terminus (BRCT) domains, while its RING domain binds to BRCA1-associated RING domain 1 (BARD1). We recently found that the interaction between heterochromatin protein 1 (HP1) and BARD1 is required for the accumulation of BRCA1 and CtIP at sites of DNA double-strand breaks. Here, we ...[more]

PMID: 27399284

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Project description:Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 ?M, 30 min) or ? irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or ? irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.

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HP1 regulates the localization of FANCJ at sites of DNA double-strand breaks.

Publications

HP1 regulates the localization of FANCJ at sites of DNA double-strand breaks.

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