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 previously reported that 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) elicited apoptotic cell death as well as non-apoptotic programmed cell deaths (PCDs) with paraptotic and necroptotic characteristics, respectively. In the present study, we have further confirmed and extended these findings. Flow cytometric analyses of 1-NP-exposed/3NF-exposed Hepa1c1c7 cells revealed that caspase-3 was only activated in the subpopulation of cells corresponding to that with classic apoptotic morphology. Immunocytochemical analysis indicated that leucocyte elastase inhibitor-derived DNaseII (LEI/L-DNaseII), apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were more clearly translocated to the nucleus following 3-NF exposure than after 1-NP. These 3-NF-induced changes in AIF and EndoG translocation were reduced by necrostatin-1, an inhibitor of necroptotic cell death. Both compounds lead to accumulation of lipid droplets and induced DNA damage. Activation of checkpoint kinase (CHK) 1 and H2AX, but not ataxia telangiectasia mutated and CHK2, were observed. Furthermore, inhibition of p53 using pifithrin-alpha reduced the cell death induced by both compounds, suggesting a role of DNA damage/CHK1/p53 pathway in the death process. 1-NP-induced cell death was in addition characterized by increased oxidative damage and intracellular accumulation of Ca(2+). These findings further support the notion that 1-NP elicited apoptotic cell death and PCD with paraptotic characteristics, while 3-NF induced apoptosis and a PCD with necroptotic features.
    Mutagenesis 09/2009; 24(6):481-93. DOI:10.1093/mutage/gep032 · 2.79 Impact Factor
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