Genomic Instability in Mice Lacking Histone H2AX

Abstract

Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX −/− mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNArepair complexes on damaged DNA.The first 120 amino acids of the H2AX and the H2A1/2 bulk isoprotein species exhibit a high degree of similarity, but H2AX carries a unique COOH-terminal tail that contains the * To whom correspondence should be addressed. andre_nussenzweig@nih.gov. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript consensus phosphatidyl inositol 3-kinase (PI-3 kinase) motif that is activated by DSBs (1, 2). Phosphorylation of H2AX (γ-H2AX) is induced by external genotoxic agents (2, 3) and is activated at physiological sites of recombination in lymphocytes (4, 5) and germ cells (6). Several essential DNA-repair factors implicated in homologous recombination (HR) (e.g., Brca1, Brca2, and Rad51) or that participate in both HR and nonhomologous end-joining (NHEJ) (e.g., Rad50, Mre11, Nbs1) form immunofluorescent foci that colocalize with γ-H2AX (7). However, the precise relation between focus formation and DNA repair is not understood.To determine the physiological role of H2AX in mammalian cells, we produced a targeted disruption of mouse H2AX (Web fig. 1A) (5,8). H2AX −/− mice were born at the expected frequency, and absence of H2AX protein was confirmed by two-dimensional gel electrophoresis and Western blotting (Web fig. 1, B to E) (8). Despite the loss of H2AX, treatment with γ-irradiation resulted in normal phosphorylation of Nbs1 (Web fig. 1E) (8).We conclude that H2AX is not essential for survival, or for irradiation-induced phosphorylation of Nbs1.H2AX −/− mice were growth retarded (Web fig. 2) (8), and H2AX −/− mouse embryo fibroblasts (MEFs) proliferated poorly in vitro (Fig. 1A). The difference in the growth of MEFs was partly due to a decrease in the number of dividing cells in H2AX −/− cultures as determined by incorporation of bromodeoxyuridine (BrdU) into DNA. During a 24-hour labeling period, only 44% of passage 1 H2AX −/− MEFs were actively cycling, compared with 72% for the controls, and the mitotic index of H2AX −/− MEFs was at least 50% lower than in wild-type cultures (see below; Fig. 1, D and F). By passage 4, H2AX −/− MEFS accumulated nondividing giant cells, suggesting premature entry into senescence. With continual passage, both H2AX −/− and wild-type MEFs went through crisis, after wh...

The γ‐H2A.X: Is it just a surrogate marker of double‐strand breaks or much more?

“…Based on its very early appearance, it was assumed that g-H2A.X is a very early response to DSBs and, hence, required for the recruitment of DSB signaling and repair proteins to the sites of DSBs. This hypothesis was supported by data showing that the accumulation in DSB repair foci of numerous repair proteins including 53BP1, BRCA1, and MDC1 is impaired in H2A.X null cells [Bassing et al, 2002;Celeste et al, 2002;Fernandez-Capetillo et al, 2002;Stewart et al, 2003]. In contrast, another study from the Nussenzweig lab showed that H2A.X is dispensable for the initial recruitment of these proteins to the sites of DSBs in MEFs lacking H2A.X expression [Celeste et al, 2003b].…”

DNA double-strand break repair and development

“…A major function of g-H2AX is recruitment of DNA damage response factors to the sites of DNA DSBs to enhance the fidelity of DNA repair Fernandez-Capetillo et al, 2004). While H2AX is not strictly required for development in the mouse, its loss predisposes to genomic instability (Celeste et al, 2002(Celeste et al, , 2003. H2AX phosphorylation results in recruitment of mediator of DNA damage checkpoint protein 1 to the DNA break (Goldberg et al, 2003;Lou et al, 2003;Stewart et al, 2003), via binding to the g-H2AX C terminus, and this interaction is required for signal amplification and effective modulation of downstream ATM signaling (Bekker-Jensen et al, 2005;Stucki et al, 2005;Lou et al, 2006;Stucki and Jackson, 2006).…”

ERK activity facilitates activation of the S-phase DNA damage checkpoint by modulating ATR function

“…These foci are essential in facilitating the assembly of repair factors, including Brca1 and the MRE11-RAD50-NBS1 complex, on damaged DNA (Paull et al, 2000;Celeste et al, 2002), and also aid in the transduction of DNA damage signals by binding to 53BP1 and MDC1 (FernandezCapetillo et al, 2002;Stewart et al, 2003). gH2AX foci also form at sites of physiological DSBs in lymphocytes and germ cells (Chen et al, 2000;Mahadevaiah et al, 2001;Petersen et al, 2001;Fernandez-Capetillo et al, 2003).…”