Disease relevance of H2AFX

• Melanoma cells express elevated levels of phosphorylated histone H2AX foci [1].
• We demonstrate that the Haemophilus ducreyi CDT (HdCDT) induces phosphorylation of the histone H2AX as early as 1 h after intoxication and re-localization of the DNA repair complex Mre11 in HeLa cells with kinetics similar to those observed upon ionizing radiation [2].
• Typically, DNA breaks are reflected by an increase in ataxia and Rad-related kinase (ATR)-regulated phosphorylation of H2AX (gammaH2AX) and require replication fork movement [3].
• TSA-induced toxicity stimulates apoptosis and cell cycle checkpoint responses independent of p53, but does not increase phosphorylated histone H2AX (-H2AX) as compared with a clastogenic agent, camptothecin, indicating that the quantity of DSBs is not the primary cause of TSA-induced cell death [4].
• Mice deficient for both H2AX and p53 (H(delta/delta)P(-/-)) rapidly developed immature T and B lymphomas and solid tumors [5].

High impact information on H2AFX

• Although it is known that MSCI and MSUC are both dependent on histone H2A.X phosphorylation mediated by the kinase ATR, and cause repressive H3 Lys9 dimethylation, the mechanisms underlying silencing are largely unidentified [6].
• MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks [7].
• Histone variant H2AX phosphorylation in response to DNA damage is the major signal for recruitment of DNA-damage-response proteins to regions of damaged chromatin [7].
• Loss of H2AX causes radiosensitivity, genome instability, and DNA double-strand-break repair defects, yet the mechanisms underlying these phenotypes remain obscure [7].
• Moreover, H2AX haploinsufficiency caused genomic instability in normal cells and, on a p53-deficient background, early onset of various tumors including more mature B lymphomas [5].

Chemical compound and disease context of H2AFX

• Histone H2AX is a mediator of gastrointestinal stromal tumor cell apoptosis following treatment with imatinib mesylate [8].

Biological context of H2AFX

• Furthermore, this interaction is phosphorylation dependent as peptides containing the phosphorylated site on H2AX bind MDC1 in a phosphorylation-dependent manner [9].
• Here we identify a novel BRCA1 carboxy-terminal (BRCT) and forkhead-associated (FHA) domain-containing protein, MDC1 (mediator of DNA damage checkpoint protein 1), which works with H2AX to promote recruitment of repair proteins to the sites of DNA breaks and which, in addition, controls damage-induced cell-cycle arrest checkpoints [9].
• It has recently been shown that the histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage [9].
• Our findings suggest that, in addition to its role in the recognition and repair of double strand breaks, H2AX also participates in the surveillance of DNA replication [10].
• Thereby, the NDH II DEXH domain alone, i.e. its catalytic core, was able to support binding to gamma-H2AX [11].

Anatomical context of H2AFX

• DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (gammaH2AX) foci [12].
• ATM-deficient and p53-deficient fibroblasts also exhibited abnormally increased levels of phosphorylated H2AX (gamma-H2AX) at later intervals after irradiation [13].
• H2AX phosphorylation and Mre11 re-localization were induced also in HdCDT-treated, non-proliferating dendritic cells (DCs) in a differentiation dependent manner, and resulted in cell death [2].
• K562, an erythroid cell line, was found to contain a high concentration of the 1.6-kb polyadenylated H2A.X mRNA [14].
• Activation of ATM and/or H2AX phosphorylation in HL-60 or Jurkat cells treated with topotecan (Tpt) was detected immunocytochemically in relation to cell cycle phase, by multiparameter cytometry [15].

Associations of H2AFX with chemical compounds

• Mammalian ATR and ATM checkpoint kinases modulate chromatin structures near DNA breaks by phosphorylating a serine residue in the carboxy-terminal tail SQE motif of histone H2AX [16].
• Here we report that inhibition of DNA replication by hydroxyurea or ultraviolet irradiation also induces phosphorylation and foci formation of H2AX [10].
• Congruently, after actinomycin D treatment, NDH II accumulated in RNA-containing nuclear bodies that predominantly co-localized with gamma-H2AX foci [11].
• Here we show that gamma-H2AX foci were also formed when cells were incubated with 0.5 microg/ml DNA intercalating agent actinomycin D [11].
• Analysis of the radiation-induced H2AX phosphorylation revealed that BIBX, as well as the PI3K inhibitor LY294002, leads to a marked reduction of P-H2AX in K-RAS(mt)-A549 and MDA-MB-231 cells, but not in K-RAS(wt)-FaDu and HH4ded cells [17].

Physical interactions of H2AFX

• With this procedure we found that both HMG1 and HMG2 interact with H2A X H2B and also with (H3 X H4)2 [18].

Enzymatic interactions of H2AFX

• Rather, double mutant BLM protein induced the formation of DNA damage-induced foci (DDI) that contained BRCA1 protein and phosphorylated histone H2AX [19].
• Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks [20].

Co-localisations of H2AFX

• MDC1 forms foci that co-localize extensively with gamma-H2AX foci within minutes after exposure to ionizing radiation [9].
• At this time, a significant fraction of the gamma-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites [21].

Regulatory relationships of H2AFX

• Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells [22].
• Taken together, these results suggest that histone gamma-H2AX promotes binding of NDH II to transcriptionally stalled sites on chromosomal DNA [11].
• This study provides evidence for the importance of p21(CDKN1A) for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21(CDKN1A) and p53 enhance camptothecin-induced histone H2AX phosphorylation (gammaH2AX), a marker for DNA DSBs [23].
• In both cell lines, the highest degree of H2AX phosphorylation induced by UV was seen in S-phase cells, particularly during early portion of S [24].
• We further show that MDC1/NFBD1-gammaH2AX complex formation regulates H2AX phosphorylation and is required for normal radioresistance and efficient accumulation of DNA-damage-response proteins on damaged chromatin [7].
• TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage [25].
• The treatment of cytosine-beta-D-arabinofuranoside strikingly enhances the NER-dependent H2AX phosphorylation and induces the accumulation of replication protein A (RPA) and ATR-interacting protein (ATRIP) at locally UV-damaged subnuclear regions [26].

Other interactions of H2AFX

• Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention [27].
• In contrast, DNA damage-induced phosphorylation of the histone variant H2AX is independent of BRCA1 [28].
• Treatment of cells with doxorubicin led to the phosphorylation of histone H2AX on serine 139 with dependence on ATM for the initial response [29].
• DNA degradation diminished the amount of Ku70 associated with gamma-H2AX but not that of NDH II [11].
• Significantly, I3C does not increase the number of 53BP1 foci or H2AX phosphorylation, indicating that ATM is activated independent of DNA double-strand breaks [30].

Analytical, diagnostic and therapeutic context of H2AFX

• In eukaryotic cells, DNA double strand breaks (DSBs) cause the prompt phosphorylation of serine 139 at the carboxy terminus of histone H2AX to generate gamma-H2AX, detectable by Western blotting or immunofluorescence [31].
• METHODS: Phosphorylation of H2AX in MCF7 cells treated with AF was determined with immunofluorescence [32].
• Two forms of H2A.X mRNA, consistent with the two sets of processing signals were found in proliferating cell cultures [33].
• DSBs were not detected with the neutral comet assay, a less sensitive method for DSB assessment than H2AX phosphorylation [34].
• Chromosomal localization of the human histone H2A.X gene to 11q23.2-q23.3 by fluorescence in situ hybridization [14].

References