Bassing, C.H. et al. Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX. Proc. Natl. Acad. Sci. USA 99, 8173-8178

Howard Hughes Medical Institute, Department of Genetics, Children's Hospital, Boston, MA 02115, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2002; 99(12):8173-8. DOI: 10.1073/pnas.122228699
Source: PubMed


In mammalian cells, DNA double-strand breaks (DSBs) cause rapid phosphorylation of the H2AX core histone variant (to form gamma-H2AX) in megabase chromatin domains flanking sites of DNA damage. To investigate the role of H2AX in mammalian cells, we generated H2AX-deficient (H2AX(Delta)/Delta) mouse embryonic stem (ES) cells. H2AX(Delta)/Delta ES cells are viable. However, they are highly sensitive to ionizing radiation (IR) and exhibit elevated levels of spontaneous and IR-induced genomic instability. Notably, H2AX is not required for NHEJ per se because H2AX(Delta)/Delta ES cells support normal levels and fidelity of V(D)J recombination in transient assays and also support lymphocyte development in vivo. However, H2AX(Delta)/Delta ES cells exhibit altered IR-induced BRCA1 focus formation. Our findings indicate that H2AX function is essential for mammalian DNA repair and genomic stability.

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Available from: Ralph Scully, Sep 30, 2015
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    • "To test this hypothesis, we used RNA sequencing (RNA-seq) approaches to interrogate the transcriptomes of H2A.X KO ESCs and control ESCs (Figure 2A and Figure S2). To avoid potential long-term culture effects, we generated H2A.X KO ESCs from H2A.X floxed/floxed ESCs (Bassing et al., 2002) via Cre-mediated deletion and used them immediately (passage five to seven) for the ensuing analyses. The efficiency of the H2A.X allele deletion was demonstrated by PCR approaches (Figure S2A). "
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    ABSTRACT: For future application of induced pluripotent stem cell (iPSC) technology, the ability to assess the overall quality of iPSC clones will be an important issue. Here we show that the histone variant H2A.X is a functional marker that can distinguish the developmental potentials of mouse iPSC lines. We found that H2A.X is specifically targeted to and negatively regulates extraembryonic lineage gene expression in embryonic stem cells (ESCs) and prevents trophectoderm lineage differentiation. ESC-specific H2A.X deposition patterns are faithfully recapitulated in iPSCs that support the development of "all-iPS" animals via tetraploid complementation, the most stringent test available of iPSC quality. In contrast, iPSCs that fail to support all-iPS embryonic development show aberrant H2A.X deposition, upregulation of extraembryonic lineage genes, and a predisposition to extraembryonic differentiation. Thus, our work has highlighted an epigenetic mechanism for maintaining cell lineage commitment in ESCs and iPSCs that can be used to distinguish the quality of iPSC lines.
    Cell Stem Cell 09/2014; 15(3):281–294. DOI:10.1016/j.stem.2014.06.004 · 22.27 Impact Factor
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    • "The formation of γ-H2AX foci increases the local concentration of repair proteins and facilitates repair of DSBs [35]. Akt phosphorylation has been reported to mediate DNA damage repair, and treatment with an Akt inhibitor has been reported to suppress DNA-DSB repair through modulating the phosphorylation of the DNA-protein kinase catalytic subunit [36]. "
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    ABSTRACT: Tumor control of hepatocellular carcinoma by radiotherapy remains unsatisfactory. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in inhibiting cancer cell death. Elevated PI3K/Akt activity is associated with increased cellular resistance to irradiation. Our aim was to determine whether the inhibition of PI3K/Akt activity by a PI3K inhibitor, BKM120, contributes to the increased sensitivity of liver cancer cells to irradiation. The hepatocellular carcinoma cell lines (Huh7 and BNL) were used to evaluate the in vitro synergism between BKM120 and irradiation. Balb/c mice bearing ectopic BNL xenografts were treated with BKM120 and/or radiotherapy to assess the in vivo response. BKM120 increased cell killing by radiation, increased the expression of apoptotic markers, and suppressed the repair of radiation-induced DNA double-strand breaks. BKM120 pretreatment inhibited radiation-induced Akt phosphorylation and enhanced the tumor-suppressive effect and radiation-induced tumor cell apoptosis in ectopic xenografts. Inhibition of mTOR phosphorylation by rapamycin enhanced the radiosensitivity of BKM120-treated hepatocellular carcinoma cells. The synergism between BKM120 and irradiation likely inhibits the activation of Akt by radiation, leading to increased cell apoptosis and suppression of DNA-double-strand breaks repair in hepatocellular carcinoma cells. These data suggest that the BKM120/radiation combination may be a strategy worthy of clinical trials.
    Oncotarget 06/2014; 5(11):3662-3672. DOI:10.18632/oncotarget.1978 · 6.36 Impact Factor
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    • "Accordingly, we hypothesized that expression of RNF168-N221* may promote this ubiquitination event in cells. To test this, we expressed mutant forms of H2AX with several lysine to glutamine mutations at previously described ubiquitination sites (K13/15Q, K118/119Q, K13/15/118/119Q) (11,12), in H2ax−/− mouse embryonic stem cells (27), with or without expression of RNF168. These lysine residues are conserved between H2AX and H2A (11,12), but we have focused on H2AX due to the feasibility of examining mutants of this non-essential H2A variant using H2ax−/− cells. "
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    ABSTRACT: The E3 ubiquitin ligase RNF168 is a DNA damage response (DDR) factor that promotes monoubiquitination of H2A/H2AX at K13/15, facilitates recruitment of other DDR factors (e.g. 53BP1) to DNA damage, and inhibits homologous recombination (HR) in cells deficient in the tumor suppressor BRCA1. We have examined the domains of RNF168 important for these DDR events, including chromosomal HR that is induced by several nucleases (I-SceI, CAS9-WT and CAS9-D10A), since the inducing nuclease affects the relative frequency of distinct repair outcomes. We found that an N-terminal fragment of RNF168 (1-220/N221*) efficiently inhibits HR induced by each of these nucleases in BRCA1 depleted cells, and promotes recruitment of 53BP1 to DNA damage and H2AX monoubiquitination at K13/15. Each of these DDR events requires a charged residue in RNF168 (R57). Notably, RNF168-N221* fails to self-accumulate into ionizing radiation induced foci (IRIF). Furthermore, expression of RNF168 WT and N221* can significantly bypass the role of another E3 ubiquitin ligase, RNF8, for inhibition of HR in BRCA1 depleted cells, and for promotion of 53BP1 IRIF. We suggest that the ability for RNF168 to promote H2A/H2AX monoubiquitination and 53BP1 IRIF, but not RNF168 self-accumulation into IRIF, is important for inhibition of HR in BRCA1 deficient cells.
    Nucleic Acids Research 05/2014; 42(12). DOI:10.1093/nar/gku421 · 9.11 Impact Factor
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