The Chromatin Structure and Function Research Group focuses on issues of genome integrity in eucaryotic systems. Understanding these issues is of paramount importance in preventing or ameliorating the effects of cancer, aging, and other degenerative diseases. One of the major issues in genome integrity concerns the DNA double-strand break (DSB), the Dr. Jekyl and Mr. Hyde of genome integrity. On the one hand intentional DSB formation is an essential aspect of several normal cellular processes. On the other accidental DSB formation is a potentially oncogenic or lethal event that requires a rapid cellular response. Our group is credited with the discovery that a """"""""first responder"""""""" to a nascent DSB is the phosphorylation of thousands of H2AX molecules in the chromatin flanking the break site. Work from our and other laboratories have shown that phosphorylated H2AX (gamma-H2AX) is key to the efficient repair of DSBs and maintenance of genome integrity. An antibody to gamma-H2AX revealed that these thousands of phosphorylated molecules form foci in cellular nuclei with each focus at a DSB site. It is accepted that every DSB site has a gamma-focus. The converse has not been proven untrue. Over the last five years, our research has had three major goals. The first is to elucidate how H2AX performs its functions in DSB recognition and resolution, to determine the components and steps involved in H2AX action. The second uses gamma-H2AX to help understand DSB behavior during various cellular metabolic processes. During these studies we have uncovered DSB involvement in several unexpected processes and have used this involvement to help elucidate these processes. The third goal is to investigate possible clinical, diagnostic, and epidemiological applications of gamma-H2AX formation including that of a biological dosimeter. 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 clonal nonreciprocal translocations and amplifications, including complex rearrangements that juxtapose the c-myc oncogene to antigen receptor loci.
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