Histone H2AX: DNA's BodyguardMy group is currently focusing on several ramifications of DNA double-strand break (DSB) formation in cellular metabolism. When a DSB is purposely or accidentally introduced into DNA, many molecules of histone H2AX become immediately phosphorylated, thus generating a highly amplified cellular response to DNA DSB formation. The response is easily monitored with the antibody we developed to the phosphorylated H2AX, which is named gamma-H2AX.We have begun in several recent studies to uncover critical roles for histone H2AX. In collaboration with Andre Nussenzweig's group, we targeted the H2AX gene in mice and successfully generated H2AX-null mice. Although viable, the H2AX-null mice are radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes are 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 specificDNA-repair complexes on damaged DNA. H2AX-null mice manifest a G2-M checkpoint defect close to that observed in ATM-null cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. Thus, H2AX-mediated concentration of 53BP1 at double-strand breaks appears to be essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.H2AX-null male mice are infertile. During meiotic prophase in male mammals, the X and Y chromosomes condense to form a macrochromatin body, termed the sex, or XY, body, within which X- and Y-linked genes are transcriptionally repressed. Gamma-H2AX accumulates in the sex body in a manner independent of meiotic recombination-associated double-strand breaks. In spermatocytes of H2AX-null mice the X and Y chromosomes fail to condense to form a sex body, fail to initiate meiotic sex chromosome inactivation, and exhibit severe defects in meiotic pairing. Moreover, other sex body proteins, including macroH2A1.2 and XMR, do not preferentially localize with the sex chromosomes in the absence of H2AX. Thus, H2AX is required for the chromatin remodeling and associated silencing in male meiosis.H2AX-null p53-null mice develop lymphomas with increased frequencies of clonalnonreciprocal translocations and amplifications, including complex rearrangements that juxtapose the c-myc oncogene to antigen receptor loci. When the H2AX-null mice contain a transgene encoding wild-type H2AX, genomic stability and radiation resistance are restored, but not when the H2AX transgene encodes alanine or glutaminic acid residues at the conserved serine phosphorylation sites. Mice containing only a single H2AX allele exhibit genomic instability and enhanced susceptibility to cancer in the absence of p53, establishing that as genomic caretaker, H2AX requires the function of both gene alleles for optimal protection against tumorigenesis. In collaboration with Carl Barrett's and Nickolai Popescu's laboratories, we showed that DSBs may have a causal role in mammalian ageing. Gamma-H2AX foci accumulate in senescing human cell cultures and in ageing mice, colocalizing with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of cryptogenic DSBs may have a causal role in mammalian ageing.Work with yeast has also provided insights into the role of H2AX. Cells maintain genomic stability by the coordination of DNA-damage repair and cell-cycle checkpoint controls. In replicating cells, DNA damage usually activates intra-S-phase checkpoint controls, which are characterized by delayed S-phase progression and increased Rad53 phosphorylation. Budding yeast lacking H2AX are hypersensitive to the topoisomerase I inhibitor camptothecin (CPT). The hypersensitivity was found to result from a failure of the intra-S-phase checkpoint controls permitting DNA synthesis to continue at normal rates. However, after replication there is a failure to process full-length chromosomal DNA molecules. H2AX is not epistatic to the RAD24 and RAD9 checkpoint genes, suggesting a non-checkpoint role for H2AX. Thus H2AX is an essential component for the efficient repair of DNA double-stranded breaks (DSBs) during replication in yeast.
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