Chromatin regulation is involved in DNA replication, transcriptional regulation and DNA damage repair and is a crucial step for stem cell self-renewal and function. We had previously shown that inactivation of the chromatin regulator MRG15 impairs proliferation of embryonic NSCs. In this report, we demonstrate that this occurs through activation of p53 and resulting increased expression of the cdk inhibitor p21. We have found increased expression of p21 and activated p53 in primary cell cultures of Mrg15 null NSCs but not wild-type. Focus formation of 53BP1, which indicates the presence of DNA damage, also co-localizes with p21 in Mrg15 null NSCs in growing culture condition, without any extrinsic insults. Focus formation of 53BP1 after γ-irradiation is delayed in Mrg15 null NSCs compared with wild-type NSCs. Our observations suggest that chromatin regulation and DNA damage repair through MRG15 complex(es) is essential to establish and maintain a functional NSC pool in mouse brain during development.
Maintenance of genomic integrity is important for stem cell function in various stem cells including NSCs [56
]. Perturbations in genes involved in DNA damage response signaling pathways and/or DNA repair are associated with neurological disorders such as neurodegeneration, microcephaly and brain tumors, suggesting that the inability to respond to DNA damage interferes with normal tissue homeostasis [58
]. DNA damage response and repair are critical for stem/progenitor cell amplification and ensure the establishment of a functional nervous system. Mice deficient for anyone of the many genes that play a role in the cellular response to DNA damage (Atm, Mre11, Nbs1) [61
] or genes actively involved in DNA repair (BRCA2 and Lig4) [65
] all share a phenotype of neurological failure due to defective DNA damage repair. These deletions affect NSC self-renewal as well as neuronal function.
MRG15 is involved in DNA damage repair [42
] in addition to transcriptional regulation of cell proliferation [47
]. 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 [42
] and knockdown of either dTip60 or dMrg15 in Drosophila
cells 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 [43
]. 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 leas to ubiquitination by DNA damage induced UBC13[45
]. 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 [44
]. Because the Tip60 complex is important for self-renewal of embryonic stem (ES) cells [68
], 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 [54
], although results from these reports are contradictory. Sy and co-workers 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 this cells. This suggests MRG15 inhibits homologous recombination through PALB2 interaction. On the other hand, Hayakawa and coworkers 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 [67
]. 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 [70
]. In the absence of p53, NSCs isolated from adult mice as well as mouse embryos exhibit a higher proliferation rate in culture [72
]. 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 [75
]. 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 [73
]. 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
Epigenetic mechanisms are essential for normal brain development and function and dysregulation in chromatin regulation results in neurodegenerative disorders, such as Alzheimer disease [78
]. It has been shown that expression of cell cycle-related proteins increases in the degenerating neurons in Alzheimer disease and re-entry into the cell cycle in neurons occurs aberrantly [79
]. Because MRG15 is involved in cell cycle regulation through chromatin regulation, dysregulation of MRG15 may contribute to the initiation and/or progression of such neurodegenerative disorders.
Our knowledge of the many functions of MRG15 continues to expand. We have determined that some of the major functions of MRG15 include transcriptional regulation via complexes involving both HATs and HDACs. Although we here demonstrate the importance of MRG15 in NSC proliferation through a DNA damage response, we speculate that other molecular mechanisms are also involved. MRG15 may directly modulate expression of genes that are important for cell cycle regulation. A small percentage of Mrg15
deficient NSCs did not show DNA damage foci, although p21 was overexpressed in these cells. MRG15 is a component of mSin3/HDAC1/Pf1 complex and it has been shown that this complex has a transcriptional repressor activity [29
]. Although this complex is mainly recruited into coding regions of actively expressing genes, which are marked by trimetylation of histone H3 at lysine 36, to prevent uncontrolled chromatin relaxation downstream of the transcriptional start sites [82
], it remains possible that this complex repressively modulates the promoter activity of specific genes such as p21. In fact, it has been shown that MRGX, MRG15 homolog, also has a transcriptional repressor activity depending on the cell-type analyzed [83
]. p21 expression may be repressed by a MRG15-containing repressor complex in wild-type NSCs under normal conditions and p21 accumulation in Mrg15
deficient NSCs may occur through de-repression of this promoter activity in a DNA damage independent manner. This possibility is currently being explored. It may also be possible that MRG15 negatively controls p21 expression via the Tip60/p400 complex, of which MRG15 is a component [84
]. Additional studies are required to determine the complexity of the molecular mechanisms involved.