Effects of changes in chromatin structure on DNA double-strand break repair with emphasis on alt-EJ
Our results reveal that chromatin compaction in non-cycling /G0 cells is higher than that of EG cells which is in line with previous observations. Furthermore we showed that global changes in chromatin structure of G0 cells, mediated by hypotonic or hypertonic treatment resulted in severe consequences for DNA DSB repair. Specifically global condensation of chromatin by hypertonic treatment was refractory for c-NHEJ and alt-EJ repair, while global relaxation of chromatin by hypotonic treatment had milder effects on both pathways, showing only a small reduction in their efficiency. According to these findings global changes in chromatin structure in either way, do not promote DSB repair, on the contrary they become a barrier to repair. On the other hand, the use of a molecular HKMT inhibitor named chaetocin, recovered the inhibited repair of alt-EJ in G0 cells by changes in specific chromatin modifications. These changes resulted in a significant increase in acetylation levels of H3K9 but did not affect H3K9me3 levels. We suspect that the unchanged levels of H3K9me3 were due to the absence of SUV39H1 in G0 cells; therefore chaetocin could not influence its activity further. These results suggest that relaxation of chromatin at specific sites improves alt-EJ in G0 cells. In addition the chaetocin-mediated repair showed to be ATM-dependent. At last the absence of Suv39h1 and Suv39h2 in EG MEFs did not affect DSB repair by c-NHEJ or alt-EJ. On the other hand in G0 cells the absence of both Suv39h showed great reduction of c-NHEJ efficiency and even more for alt-EJ. Further experiments will help to further elucidate the response of these cells as they show particular characteristics. It is understood from our results that chromatin is a major contributor to DSBs repair and the different ways of changing the chromatin structure, whether globally or more locally, can result in different influence on the DSB repair.
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