Resumen de Contribution of histone modifications to double-strand break repair and aging
To efficiently maintain genome integrity cells have evolved an intricate signaling network termed as DNA damage response (DDR). A proper function of the DDR is required to repair deleterious DNA lesions, such as DNA double-strand breaks (DSBs). During in vitro and in vivo aging, a persistent accumulation of DSBs has been reported, suggesting a decline of the DNA repair mechanisms with age. Recent studies have highlighted the relevance of histone modifications in the regulation of the DDR. This is the case of the oxidation of H3 at lysine 4 (H3K4ox) and the acetylation of H4 at lysine 16 (H4K16Ac). The novel mark H3K4ox has been associated with the regulation of the DDR signaling. Regarding H4K16Ac, it has been related to DSB repair since its deacetylation is required for 53BP1 recruitment to DSB sites. Additionally, H4K16Ac varies during aging and cellular senescence. In this study we analyze the age-associated changes of these marks and their contribution to age-related DSB accumulation.
Although H3K4ox has been linked to heterochromatinization, our results showed a global reduction of H3K4ox in BJ fibroblasts displaying oncogene-induced senescence (OIS) and during in vitro aging of human dermal fibroblasts (HDFs). Accordingly, the expression levels of the oxidases LOXL2 and LOXL1 also suffered from a strong decrease in senescent fibroblasts while in in vitro aged HDFs only LOXL1 expression was reduced. Nonetheless, we described how, although global levels of H3K4ox were reduced after OIS, they specifically increased in senescence-associated heterochromatin foci (SAHF). Hence, our findings suggest a relationship between heterochromatin regions and H3K4ox, conferring it a potential role in SAHF assembly and gene silencing during senescence.
Similarly to H3K4ox, a global reduction of H4K16Ac was found in both senescent BJ fibroblasts and in vitro aged HDFs in comparison to the levels of their control counterparts. Our results indicated that the decrease of H4K16Ac was stablished through different molecular mechanisms in each process: while an increase in deacetylase activity was related to a higher expression of sirtuins in OIS, during in vitro aging the acetyltransferase activity was reduced due to decreased MOF expression.
Next, we analyzed the presence of 53BP1 at үH2AX foci along with H4K16Ac levels in the whole nucleus before and after DSB induction in young and aged HDFs. In untreated in vitro aged cells, the decrease in H4K16Ac levels correlated with a reduction in 53BP1 recruitment to basal үH2AX-labelled DSBs. Strikingly, following DSB induction, H4K16Ac slightly decreased in early passage HDFs while it increased in late passage HDFs. The same tendency was observed regarding 53BP1 recruitment to DSBs which prompted us to speculate that a relation might exist between H4K16Ac levels and efficient 53BP1 recruitment to DSBs. H4K16 hyperacetylation using both nicotinamide and trichostatin A failed to ameliorate the recruitment of 53BP1 to DSBs; instead, H4K16 hypoacetylation by MOF depletion impaired 53BP1 recruitment to γH2AX-labelled DSBs. Thus, our results suggest that a specific H4K16Ac level should be reached for proper 53BP1 recruitment to DSBs.
In summary, we report H3K4ox as a promising age-associated epigenetic modification. Indeed, its specific enrichment in SAHF indicates a prominent role for H3K4ox in senescence. Regarding H4K16Ac, our data suggest that following DSB induction, H4K16Ac reaches a specific level required for the recruitment of 53BP1 to DSB sites. In young cells, this level is rapidly reached by slightly lowering their basal levels of H4K16Ac. However, aged cells start from lower basal levels of H4K16Ac that might impede them to reach the optimum H4K16Ac level for proper 53BP1 recruitment. Consequently, H4K16Ac stands as a key mark in the regulation of DSB repair during aging.
