|dc.description.abstracteng||Open chromatin is induced locally during the repair of DNA double-strand breaks when a cascade of protein recruitments and modifications is triggered. This "ATM cascade" includes components like the Mrn complex, ATM kinase, phosphorylated histone variant γH2AX, MDC1, E3 ligases RNF 8 and RNF8, 53BP1, and Rif1. Recent investigations have shown that also the Mad2l2 protein is a downstream effector of the ATM cascade during DNA repair. It was first described as an accessory subunit during translesion DNA repair, and more recently as a key factor inhibiting the resection of DNA 5`ends, thus promoting non-homologous end joining, and repressing homologous recombination.
Naive embryonic stem cells (ESCs) have a globally open chromatin. A preliminary study from this laboratory has demonstrated that ESCs require the presence of the Mad2l2 protein for the maintenance of their transcriptional and epigenetic profiles, and thus for the stability of pluripotency (Pirouz et al., Cell Cycle 14, 1596, 2015). The aim of the present study was to correlate Mad2l2 with the ATM cascade and the chromatin status in ESCs.
Mad2l2, as well as several other components of the ATM cascade, are expressed at extraordinarily high levels in the euchromatin of ESCs. High-resolution microscopy revealed that the localization of heterochromatin markers (H3K9me2 and H3K27me3) and Mad2l2 were mutually exclusive. In the absence of Mad2l2, the methylation of DNA and the amount of heterochromatin increased significantly. Comparative gene expression profiling indicated a striking, activating effect of Mad2l2 on the expression of Dppa3, a common marker of naive ESCs and primordial germ cells. Mass spectrometry identified Mad2l2 partners as a direct interactor of ATM cascade proteins, and DNA methyltransferase I (Dnmt1) in the ESC chromatin. Mad2l2 interaction with Dnmt1 had a substantial impact on the expression of imprinted genes in ESCs, which were downregulated in mutant ESCs. Inhibition of ATM, ATR, and DNA-PK kinases supported the position of Mad2l2 as a downstream effector in the ATM cascade, just upstream of a DNA-Pk mediated Dppa3 activity. The suppression of Dppa3 in the absence of Mad2l2 was at least partially achieved by an occupation of the promoter with H3K9me2. Further epigenetic effects related to Mad2l2 were the loss of the mutually exclusive localization of 53BP1 and H3K27me and the necessity of Mad2l2 for the epigenetic reprogramming of ESCs into germ cells in vitro.
In conclusion, this study provides evidence for the function of Mad2l2 as a downstream effector in the constitutive ATM cascade in ESCs. This implies that the importance of Mad2l2 for DNA repair, germ cell development, and ESC stability lies in the epigenetic regulation of the chromatin status.||de