Histone H2AX phosphorylation in response to changes in chromatin structure induced by altered osmolarity

Department of Neurological Surgery, University of California, San Francisco, CA 94110, USA.
Mutagenesis (Impact Factor: 2.79). 03/2009; 24(2):161-7. DOI: 10.1093/mutage/gen064
Source: PubMed


DNA strand breaks trigger marked phosphorylation of histone H2AX (i.e. gamma-H2AX). While DNA double-strand breaks (DSBs) provide a strong stimulus for this event, the accompanying structural alterations in chromatin may represent the actual signal that elicits gamma-H2AX. Our data show that changes in chromatin structure are sufficient to elicit extensive gamma-H2AX formation in the relative absence of DNA strand breaks. Cells subjected to hypotonic (0.05 M) treatment exhibit gamma-H2AX levels that are equivalent to those found after the induction of 80-200 DNA DSBs (i.e. 2-5 Gy). Despite this significant increase in phosphorylation, cell survival remains relatively unaffected (<10% cytotoxicity), and there is no significant increase in apoptosis. Nuclear staining profiles indicate that gamma-H2AX-positive cells induced under altered tonicity exhibit variable levels of staining, ranging from uniform pan staining to discrete punctate foci more characteristic of DNA strand breakage. The capability to induce significant gamma-H2AX formation under altered tonicity in the relative absence of DNA strand breaks suggests that this histone modification evolved in response to changes in chromatin structure.

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Available from: Erich Giedzinski, Jan 24, 2014
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    • "After ultraviolet (UV) irradiation not only the S-phase-dependent induction of γH2AX was reported but also a weaker nuclear-wide γH2AX in G1 cells that is dependent on nucleotide excision repair (NER) (11). Moreover, changes in chromatin structure via hypotonic treatment led to the formation of pan-nuclear γH2AX (12). Also adeno-associated virus infection can induce a pan-nuclear γH2AX response (13,14). "
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    ABSTRACT: DNA double-strand breaks (DSB) are considered as the most deleterious DNA lesions, and their repair is further complicated by increasing damage complexity. However, the molecular effects of clustered lesions are yet not fully understood. As the locally restricted phosphorylation of H2AX to form γH2AX is a key step in facilitating efficient DSB repair, we investigated this process after localized induction of clustered damage by ionizing radiation. We show that in addition to foci at damaged sites, H2AX is also phosphorylated in undamaged chromatin over the whole-cell nucleus in human and rodent cells, but this is not related to apoptosis. This pan-nuclear γH2AX is mediated by the kinases ataxia telangiectasia mutated and DNA-dependent protein kinase (DNA-PK) that also phosphorylate H2AX at DSBs. The pan-nuclear response is dependent on the amount of DNA damage and is transient even under conditions of impaired DSB repair. Using fluorescence recovery after photobleaching (FRAP), we found that MDC1, but not 53BP1, binds to the nuclear-wide γH2AX. Consequently, the accumulation of MDC1 at DSBs is reduced. Altogether, we show that a transient dose-dependent activation of the kinases occurring on complex DNA lesions leads to their nuclear-wide distribution and H2AX phosphorylation, yet without eliciting a full pan-nuclear DNA damage response.
    Nucleic Acids Research 04/2013; 41(12). DOI:10.1093/nar/gkt304 · 9.11 Impact Factor
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    • "This breakage can trigger apoptosis, and errors in repair lead to mutations [33]. DSBs, however, do not play a role in heat- or hypertonicity-induced cell death [26,34]. In contrast, some EDSBs are derived from physiologic processes. "
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    ABSTRACT: Global hypomethylation and genomic instability are cardinal features of cancers. Recently, we established a method for the detection of DNA methylation levels at sites close to endogenous DNA double strand breaks (EDSBs), and found that those sites have a higher level of methylation than the rest of the genome. Interestingly, the most significant differences between EDSBs and genomes were observed when cells were cultured in the absence of serum. DNA methylation levels on each genomic location are different. Therefore, there are more replication-independent EDSBs (RIND-EDSBs) located in methylated genomic regions. Moreover, methylated and unmethylated RIND-EDSBs are differentially processed. Euchromatins respond rapidly to DSBs induced by irradiation with the phosphorylation of H2AX, gamma-H2AX, and these initiate the DSB repair process. During G0, most DSBs are repaired by non-homologous end-joining repair (NHEJ), mediated by at least two distinct pathways; the Ku-mediated and the ataxia telangiectasia-mutated (ATM)-mediated. The ATM-mediated pathway is more precise. Here we explored how cells process methylated RIND-EDSBs and if RIND-EDSBs play a role in global hypomethylation-induced genomic instability. We observed a significant number of methylated RIND-EDSBs that are retained within deacetylated chromatin and free from an immediate cellular response to DSBs, the gamma-H2AX. When cells were treated with tricostatin A (TSA) and the histones became hyperacetylated, the amount of gamma-H2AX-bound DNA increased and the retained RIND-EDSBs were rapidly repaired. When NHEJ was simultaneously inhibited in TSA-treated cells, more EDSBs were detected. Without TSA, a sporadic increase in unmethylated RIND-EDSBs could be observed when Ku-mediated NHEJ was inhibited. Finally, a remarkable increase in RIND-EDSB methylation levels was observed when cells were depleted of ATM, but not of Ku86 and RAD51. Methylated RIND-EDSBs are retained in non-acetylated heterochromatin because there is a prolonged time lag between RIND-EDSB production and repair. The rapid cellular responses to DSBs may be blocked by compact heterochromatin structure which then allows these breaks to be repaired by a more precise ATM-dependent pathway. In contrast, Ku-mediated NHEJ can repair euchromatin-associated EDSBs. Consequently, spontaneous mutations in hypomethylated genome are produced at faster rates because unmethylated EDSBs are unable to avoid the more error-prone NHEJ mechanisms.
    Molecular Cancer 03/2010; 9(1):70. DOI:10.1186/1476-4598-9-70 · 4.26 Impact Factor
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    ABSTRACT: We recently showed that histone H2AX phosphorylated on serine 139 (gamma-H2AX), a hallmark of DNA damage response (DDR), also forms early during apoptosis induced by death receptor activation. Here, we extend and discuss our findings on apoptotic gamma-H2AX, which differs from the well-established DDR with nuclear foci. During apoptosis induced by death receptors agonists (TRAIL and FasL) and staurosporine, gamma-H2AX is initiated in the nuclear periphery immediately inside the nuclear envelope while total H2AX remains distributed throughout the nucleus. This process is readily detectable by immunofluorescence microscopy and we refer to it as the "gamma-H2AX ring". It is conserved both in cancer and normal cells. The gamma-H2AX ring contains the activated checkpoints kinases, ATM, Chk2 and DNA-PK; the latter being the main effector for the apoptotic gamma-H2AX phosphorylation. Notably, we show here that the gamma-H2AX ring coincides with phosphorylated H2B on serine 14 (P(S14)-H2B), another histone modification associated with apoptosis. The coordinated phosphorylations of H2AX and H2B suggest a previously unrecognized histone phosphorylation signature for apoptosis consisting of gamma-H2AX together with P(S14)-H2B and possibly P(Y142)-H2AX. This signature ("phosphohistone 2 code") together with the gamma-H2AX ring provides a new feature to monitor and study apoptosis.
    Cell cycle (Georgetown, Tex.) 07/2009; 8(12):1853-9. DOI:10.4161/cc.8.12.8865 · 4.57 Impact Factor
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