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Preferential accessibility to specific genomic loci for the repair of double-strand breaks in human cells.

ABSTRACT: The dynamic organization of the human genome in the nucleus is gaining recognition as a determining factor in its functional regulation. In order to be expressed, replicated or repaired, a genomic locus has to be present at the right place at the right time. In the present study, we have investigated the choice of a double-strand break (DSB) repair partner for a given genomic loci in an ATM-deficient human fibroblast cell line. We found that partner choice is restricted such that a given genomic locus preferentially uses certain sites in the genome to repair itself. These preferential sites can be in the vicinity of the damage site or megabases away or on other chromosomes entirely, while potential sites closer to the break along the length of the chromosome can be ignored. Moreover, there can be more than a 10-fold difference in usage between repair sites located only 10 kb apart. Interestingly, arms of a given chromosome are less accessible to one another than to other chromosomes. Altogether, these results indicate that the accessibility between genomic sites in the human genome during DSB repair is specific and conserved in a cell population.

SUBMITTER: D'Anjou H

PROVIDER: S-EPMC534631 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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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Preferential accessibility to specific genomic loci for the repair of double-strand breaks in human cells.

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