MRG is involved in DNA damage repair

MRG15 is involved in DNA damage repair (Kusch et al., 2004; Garcia et al., 2007) in addition to transcriptional regulation of cell proliferation (Tominaga et al., 2005). Thus there are two possibilities to explain the molecular mechanism by which MRG15 could be involved in the proliferative defects in Mrg15 null NSCs that we have observed. These are via the Tip60 complex or the PALB2/BRCA2 interaction involving MRG15. In Drosophila, the Tip60 complex acetylates nucleosomal phospho-H2Av, a Drosophila H2AX homolog, in response to ionizing radiation and exchanges it with an unmodified H2Av (Kusch et al., 2004) and knockdown of either dTip60 or dMrg15 in Drosophila adenosine deaminase impaired this acetylation and exchange of H2Av following irradiation. In mammalian cells, depletion of either Tip60 or TRRAP, other components of the Tip60 complex, results in impairment of recruitment of DNA-repair proteins such as 53BP1 to damage sites (Murr et al., 2006). The Tip60/TRRAP complex acetylates histone H2A and H2AX at DNA damage sites and thereby maintains open chromatin and facilitates access of DNA repair machinery to DNA strand break sites. Ikura et al. showed that H2AX acetylated by Tip60 after ionizing radiation leads to ubiquitination by DNA damage induced UBC13 (Ikura et al., 2007). Tip60 promotes the acetylation-dependent ubiquitination of H2AX by UBC13, causing H2AX release from chromatin and thereby facilitates chromatin reorganization following DNA damage. We have also shown that acetylation of histone H2A, in response to ionizing radiation (IR), is impaired and recruitment of DNA repair proteins delayed in Mrg15 null MEFs (Garcia et al., 2007). Because the Tip60 complex is important for self-renewal of embryonic stem (ES) cells (Fazzio et al., 2008a, 2008b), the role of MRG15 in proliferation defects of NSC may also occur via the Tip60 complex. Another possible connection between MRG15 and DNA damage is PALB2. PALB2 was originally identified as an interacting partner of BRCA2 which is a tumor suppressor for breast and ovarian cancers and is required for the loading of the BRCA2–RAD51 repair complex onto DNA. More recently, it was shown that PALB2 can also bind to BRCA1 and that it is an integral component of the BRCA1–BRCA2–RAD51 axis, which is critical for the maintenance of genomic stability via recombinational repair. Two groups have shown that MRG15 can bind directly to PALB2 and that knockdown of MRG15 affects homology-directed DNA repair (Sy et al., 2009; Hayakawa et al., 2010), although results from these reports are contradictory. Sy et al. showed that PALB2-deficient EUF1341F cells reconstituted with MRG15-binding defective PALB2 mutant exhibited increased gene conversion rates although damage-induced RAD51 foci formation and mitomycin C sensitivity returned to normal in these cells. This suggests that MRG15 inhibits homologous recombination through PALB2 interaction. On the other hand, Hayakawa et al. demonstrated that MRG15 deficient cells showed reduced efficiency for homology-directed DNA repair and hypersensitivity to DNA interstrand cross-linking agents similar to PALB2 or BRCA2 deficient cells. They also showed that MRG15 knockdown diminished the recruitment of PALB2, BRCA2, and RAD51 to DNA damage sites. Although we do understand this discrepancy, our previous and current findings support the fact that MRG15 is an essential factor for DNA damage repair in somatic cells as well as stem cells. Deletion of Brca2 in the entire nervous system in mice leads to microcephaly and defects in neurogenesis (Frappart et al., 2007). p53 contributes to these phenotypes because simultaneous inactivation of p53 improves Brca2 depletion phenotypes in mouse brains. p53 is responsible for both cell growth defect and apoptosis in Brca2-deficient NSCs. MRG15 may therefore also function through a BRCA2 pathway in NSCs. The tumor suppressor p53 is an important regulator of cell cycle and apoptosis in both developing and adult brain and plays an important role in maintaining a proper balance of neural stem/progenitor pools. It is known that one of the p53 downstream target genes, p21, is also important for maintaining NSC self-renewal during the lifespan of an organism (Kippin et al., 2005; Pechnick et al., 2008). In the absence of p53, NSCs isolated from adult mice as well as mouse embryos exhibit a higher proliferation rate in culture (Gil-Perotin et al., 2006; Meletis et al., 2006; Armesilla-Diaz et al., 2009). On the other hand, p44Tg mice, in which p53 is constitutively activated, exhibit premature aging without increased tumor risk, indicating that constitutive activation of p53 limits NSC self-renewal following constitutive expression of p21 (Medrano and Scrable, 2005; Medrano et al., 2009). Therefore, it is possible that p53 activation following increased p21 expression may limit self-renewal potential in Mrg15 deficient NSCs. This is supported by the fact that knockdown of p53 levels results in decreased p21 expression and an increase in BrdU-positive cycling cells in both wild-type and Mrg15 null cells, as shown in this study. It is known that p53 also inhibits neuronal differentiation because p53 deficient NSCs differentiate into neuronal lineage in higher rate (Meletis et al., 2006; Ferron et al., 2009). We have previously shown Mrg15 deficient NSCs had a defect in neuronal differentiation. This defect may also be explained by upregulation of p53 activity in Mrg15 deficient NSCs.