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 global gene expression profiles for 67 human lung tumors representing 56 patients were examined by using 24,000-element cDNA microarrays. Subdivision of the tumors based on gene expression patterns faithfully recapitulated morphological classification of the tumors into squamous, large cell, small cell, and adenocarcinoma. The gene expression patterns made possible the subclassification of adenocarcinoma into subgroups that correlated with the degree of tumor differentiation as well as patient survival. Gene expression analysis thus promises to extend and refine standard pathologic analysis

Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (CH) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream CH genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction 1 . Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (γ-H2AX, also known as γ-H2afx), which facilitate DNA double-strand break (DSB) repair 2-4 , form nuclear foci at the CH region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX -/-mice. Localization of Nbs1 and γ-H2AX to the IgH locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (Sμ)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.Correspondence and requests for materials should be addressed to A.N. (andre_nussenzweig@nih.gov) or M.C.N. (nussen@mail.rockefeller.edu).. Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). Competing interests statementThe authors declare that they have no competing financial interests. To determine whether DNA repair factors associate with DSBs at the switch regions, we first examined the intracellular localization of γ-H2AX, Nbs1, Rad51 and Brca1 in activated B cells by immunofluorescence. Brca1 and Rad51 are required for homologous recombination, the Mre11-Rad50-Nbs1 complex has been implicated in both homologous recombination and non-homologous end-joining (NHEJ), and γ-H2AX is critical for recruiting these repair factors to DSBs 6 and facilitates NHEJ in Saccharomyces cerevisiae 4 . All four proteins showed diffuse nuclear staining in most of the resting B cells from C57BL/6 wild-type mice. High local concentrations of these factors (nuclear foci) were detected in a very small percentage of cells (<5%), which increased significantly when the cells were stimulated to undergo CSR in vitro with lipopolysaccharide (LPS) and interleukin (IL)-4 (Fig. 1a). After 3 days of stimulation, 37% of the B cells contained discrete Brca1 foci (12 ± 6 per cell) and 43% contained Rad51 foci (7 ± 3 per cell), consistent with previous results 7 ; the remaining cells exhibited a weak, diffuse pattern of nuclear staining (Fig. 1a). Many of the stimulated B cells also formed Nbs1 foci (32% contained, on average, 3 ± 2 per cell) and γ-H2AX foci (40% contained, on average, 4.5 ± 3 per cell). To determine which of these repair factors are co-localized in activated B cells, we performed two colour immunofluoresence experiments (Fig. 1b). Only 20% of the cells that contained Rad51 and Nbs1 (n = 687) or Brca1 and Nbs1 foci (n = 431) exhibited co-localization. In contrast, γ-H2AX foci co-localiz...

No abstract