Effects of chromatin decondensation on alternative NHEJ

• Alternative end-joining (A-NHEJ) is severely compromised in cells that enter the plateau-phase of growth but the mechanisms remain unknown.
• We study the effect on B-NHEJ A-NHEJ of chromatin decondensation mediated by treatment with 5′-aza-2′-dC, or incubation in hypotonic media.
• Both treatments have no detectable effects on C-NHEJ, but exert pronounced growth-state-dependent effects on A-NHEJ.
• In growing cells chromatin decondensation inhibits B-NHEJ, while in serum-deprived cells chromatin decondensation facilitates A-NHEJ.
• The results uncover for the first time a link between A-NHEJ and chromatin organization and hint to mechanisms regulating B-NHEJ.

In cells of higher eukaryotes, repair of DNA double strand breaks (DSBs) utilizes different forms of potentially error-prone non-homologous end joining (NHEJ): canonical DNA-PK-dependent (C-NHEJ) and alternative backup pathways (A-NHEJ). In contrast to C-NHEJ, A-NHEJ shows pronounced efficiency fluctuations throughout the cell cycle and is severely compromised as cells cease proliferating and enter the plateau phase (Windhofer et al., 2007 [23]). The molecular mechanisms underpinning this response remain unknown but changes in chromatin structure are prime candidate-A-NHEJ-modulators. Since parameters beyond chromatin acetylation appear to determine A-NHEJ efficiency (Manova et al., 2012 [42] and [76]), we study here the role of chromatin decondensation mediated either by treatment with 5′-aza-2′-deoxycytidine (AzadC) or growth in hypotonic conditions, on A-NHEJ. We report that both treatments have no detectable effect on C-NHEJ but provoke, specifically for A-NHEJ, cell-growth-dependent effects. These results uncover for the first time a link between A-NHEJ and chromatin organization and provide means for understanding the regulatory mechanisms underpinning the growth-state dependency of A-NHEJ. A-NHEJ is implicated in the formation of chromosomal translocations and in chromosome fusions that underlie genomic instability and carcinogenesis. The observations reported here may therefore contribute to the development of drug-based A-NHEJ suppression-strategies aiming at optimizing cancer treatment outcomes and possibly also at suppressing carcinogenesis.