Article

Dual recognition of phosphoserine and phosphotyrosine in histone variant H2A.X by DNA damage response protein MCPH1

Departments of Biochemistry and Molecular Biology, Laboratory Medicine and Pathology, and Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2012; 109(36):14381-6. DOI: 10.1073/pnas.1212366109
Source: PubMed
ABSTRACT
Tyr142, the C-terminal amino acid of histone variant H2A.X is phosphorylated by WSTF (Williams-Beuren syndrome transcription factor), a component of the WICH complex (WSTF-ISWI chromatin-remodeling complex), under basal conditions in the cell. In response to DNA double-strand breaks (DSBs), H2A.X is instantaneously phosphorylated at Ser139 by the kinases ATM and ATR and is progressively dephosphorylated at Tyr142 by the Eya1 and Eya3 tyrosine phosphatases, resulting in a temporal switch from a postulated diphosphorylated (pSer139, pTyr142) to monophosphorylated (pSer139) H2A.X state. How mediator proteins interpret these two signals remains a question of fundamental interest. We provide structural, biochemical, and cellular evidence that Microcephalin (MCPH1), an early DNA damage response protein, can read both modifications via its tandem BRCA1 C-terminal (BRCT) domains, thereby emerging as a versatile sensor of H2A.X phosphorylation marks. We show that MCPH1 recruitment to sites of DNA damage is linked to both states of H2A.X.

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    • "To date, BRCT 2 domains of three mammalian proteins— MCPH1, PTIP, and MDC1—have been found to specifically recognize the primary mark of DNA damage, gH2AX (Yan et al., 2011; Stucki et al., 2005; Singh et al., 2012). Our data show unambiguously that this is a property sharedFigure 3 . "
    [Show abstract] [Hide abstract] ABSTRACT: 53BP1 plays multiple roles in mammalian DNA damage repair, mediating pathway choice and facilitating DNA double-strand break repair in heterochromatin. Although it possesses a C-terminal BRCT2 domain, commonly involved in phospho-peptide binding in other proteins, initial recruitment of 53BP1 to sites of DNA damage depends on interaction with histone post-translational modifications—H4K20me2 and H2AK13/K15ub—downstream of the early γH2AX phosphorylation mark of DNA damage. We now show that, contrary to current models, the 53BP1-BRCT2 domain binds γH2AX directly, providing a third post-translational mark regulating 53BP1 function. We find that the interaction of 53BP1 with γH2AX is required for sustaining the 53BP1-dependent focal concentration of activated ATM that facilitates repair of DNA double-strand breaks in heterochromatin in G1.
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    • "during meiosis. These results are consistent with the notions that, in somatic cells, MCPH1 functions in an H2AX- dependent manner but through an MDC1-independent pathway (Wood et al., 2007), and that pTyr142 is incompatible with MDC1 binding (Singh et al., 2012). Thus, these results suggest that MCPH1 is associated with pTyr142 during meiosis. "
    [Show abstract] [Hide abstract] ABSTRACT: Meiosis is precisely regulated by the factors involved in DNA damage response in somatic cells. Among them, phosphorylation of H2AX on Serine 139 (γH2AX) is an essential signal for the silencing of unsynapsed sex chromosomes during male meiosis. However, it remains unknown how adjacent H2AX phosphorylation on Tyrosine 142 (pTyr142) is regulated in meiosis. Here we investigate the meiotic functions of BAZ1B (WSTF), the only known Tyr142 kinase in somatic cells, using mice possessing a conditional deletion of BAZ1B. Although BAZ1B deletion causes ectopic γH2AX signals on synapsed autosomes during the early pachytene stage, BAZ1B is dispensable for fertility and critical events during spermatogenesis. BAZ1B deletion does not alter events on unsynapsed axes and pericentric heterochromatin formation. Furthermore, BAZ1B is dispensable for localization of the ATP-dependent chromatin remodeling protein SMARCA5 (SNF2h) during spermatogenesis despite the complex formation between BAZ1B and SMARCA5, known as the WICH complex, in somatic cells. Notably, pTyr142 is regulated independently of BAZ1B and is dephosphorylated on the sex chromosomes during meiosis in contrast with the presence of adjacent γH2AX. Dephosphorylation of pTyr142 is regulated by MDC1, a binding partner of γH2AX. These results reveal the distinct regulation of two adjacent phosphorylation sites of H2AX during meiosis, and suggest that another kinase mediates Tyr142 phosphorylation. © 2015. Published by The Company of Biologists Ltd.
    Full-text · Article · May 2015 · Biology Open
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    • "It was demonstrated that MCPH1 has crucial functions in DNA damage response (e.g. Xu et al. 2004; Alderton et al. 2006; Singh et al. 2012). Although the patient cells do not show dramatic defects in response to DNA damage, we could show before that ionizing irradiation increases the fraction of cells with condensed chromatin in cultures of MCPH1- deficient cells (Gavvovidis et al. 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: Mutations in the MCPH1 gene result in primary microcephaly in combination with a unique cellular phenotype of defective chromosome condensation. MCPH1 patient cells display premature chromosome condensation in G2 phase of the cell cycle and delayed decondensation in early G1 phase, observable as an increased proportion of cells with prophase-like appearance. MCPH1 deficiency thus appears to uncouple the chromosome cycle from the coordinated series of events that take place during mitosis such as some phases of the centrosome cycle and nuclear envelope breakdown. Here, we provide a further characterization of the effects of MCPH1 loss-of-function on chromosome morphology. In comparison to healthy controls, chromosomes of MCPH1 patients are shorter and display a pronounced coiling of their central chromatid axes. In addition, a substantial fraction of metaphase chromosomes shows apparently unresolved chromatids with twisted appearance. The patient chromosomes also showed signs of defective centromeric cohesion, which become more apparent and pronounced after harsh hypotonic conditions. Taking together, the observed alterations indicate additional so far unknown functions of MCPH1 during chromosome shaping and dynamics.
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