Introduction The role of chromatin status

Introduction The role of chromatin status on DNA damage response (DDR) and DNA double-strand break (DSB) repair has been extensively investigated in differentiated non-stem and cancer cells. Chromatin structure and remodeling plays an important role in the recruitment/assembly of repair factors and allows their access to DNA lesions (Price and D\'Andrea, 2013). Activation of DNA DSB repair machinery is promoted not only by local decondensation of chromatin but also by the binding of heterochromatin-associated proteins and the transient compaction of chromatin (Burgess et al., 2014; Lemaître and Soutoglou, 2014). However, chromatin of differentiated/non-stem and cancer cells is starkly different from the stem/progenitor cells in tissue niches in vivo, such as neural stem cells in the subgranular zone of brain and neural stem cells (NS) cells in culture, intestinal crypt stem cells, testicular stem cells, and embryonic stem cells (ES) in vivo and in culture. These “normal” stem cells are characterized by elevated levels of histone marks associated with active chromatin and lower levels of repressive chromatin (Chen and Dent, 2014). Previously, we have delineated the epigenetic influence of H3K56 acetylation on attenuation of DDR in murine embryonic ES and NS cells, as well as in several tissue niches in vivo, demonstrating H3K56ac as an epigenetic suppressor of DDR that promotes ionizing radiation (IR) hypersensitivity in normal stem cells (Jacobs et al., 2016). Ataxia telangiectasia mutated (ATM) kinase is involved in multiple DDR processes that include phosphorylation of several histone and non-histone proteins, amplification of the DNA damage signal, repair factor activation, and initiation of the cell-cycle arrest (Shiloh and Ziv, 2013). ATM activation is promoted by its acetylation through Tip60 (Sun et al., 2007). This activation requires the binding of Tip60 to tri-methylated histone H3 lysine 9 (H3K9me3) (Sun et al., 2009), a modification associated with repressive heterochromatin (Grewal and Jia, 2007). H3K9me3 is locally and transiently upregulated around the DNA damage site by the methyltransferase Suv39h1, improving ATM activation via Tip60 (Ayrapetov et al., 2014). Recruitment of Suv39h1 enabling H3K9me3 and assembly of binding partners HP1 and KAP1 is rapid and transient, observed for few minutes after DSB induction. Interestingly, acetylation on the same amino oxyntomodulin residue (H3K9ac) is locally downregulated at the DNA damage sites (Bártová et al., 2011). We compared karyotypically normal, early-passage, radiosensitive murine ES and NS with the isogenic radioresistant non-stem cells (ED and ND), proliferating at identical rates in culture; to investigate the differential regulation of DNA damage and apoptotic responses of radiosensitive stem and radioresistant non-stem cells (Jacobs et al., 2016). In this study, we demonstrate constitutively high H3K9ac but low H3K9me3 levels in ES that fails to downregulate H3K9ac at the DSB sites (microirradiation tracks), unlike the ED. Suppression of the H3K9 acetyltransferase monocytic leukemia zinc finger protein (MOZ) reduced H3K9ac in ES, protected them from IR-induced apoptosis, and improved ES survival after irradiation. On the contrary, knockdown of MOZ did not protect non-stem or cancer cells from IR-induced cell death. Besides ES, reduction of IR-induced apoptosis after MOZ knockdown in human neuroprogenitor cells suggests that a transient suppression of MOZ may very likely be a useful therapeutic strategy for radioprotection of human neuroprogenitor cells.
Results
Discussion H3K9 modifications have been previously analyzed in ESCs (Lee et al., 2004; Meshorer et al., 2006; Azuara et al., 2006; Loh et al., 2007). This study supports previous observations and additionally demonstrates an increased constitutive H3K9ac along with distinctly reduced H3K9me3 in stem cells, in contrast to the isogenic non-stem ED cells in culture as well as in vivo. Local downregulation of H3K9ac has been reported in ESCs, accompanying recruitment of the transcription factor OCT4 to DNA lesions (Bártová et al., 2011). In the same study, a downregulation of H3K9 acetylation was not observed in one-third of the analyzed stem cells, which is in agreement with our finding of a local decrease of H3K9ac in 40% of the stem cells in culture. Moreover, our work compares the local H3K9 deacetylation at or around DSBs in ES directly with the frequency of H3K9ac downregulation in non-stem ED cells, showing a less efficient deacetylation response in ES cells. While the function of the local H3K9ac downregulation in the DDR is not clear, the regulation of ATM activation by H3K9me3 through Suv39h1 has been extensively studied (Ayrapetov et al., 2014; Sun et al., 2009). We hypothesize that deacetylation at H3K9 is a prerequisite to allow tri-methylation at this same amino acid residue for efficient ATM activation. In accordance, we observed that while ED cells show an increase in H3K9me3 at the DSB tracks, ES did not display such chromatin modification. The lower percentage of ED positive for H3K9me3 compared with deacetylated H3K9 can be explained by the very transient/temporal nature of the process: in fact we observed that the high percentage of ED showing tri-methylation can be observed only within 2 min after the DNA damage induction, and this percentage reduced drastically at 10 min after DSB induction. We thus provide evidence that differential ATM activation through Suv39h1 is influenced by the characteristic chromatin profiles. Suppression of Suv39h1 methyltransferase alone had a limited effect on ATM activation or IR-induced apoptosis of stem cells. This is in accord with our hypothesis that H3K9 deacetylation is required before Suv39h1-mediated methylation of H3K9, and therefore Suv39h1 activity is restricted in stem cells with consistently elevated H3K9ac.