Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX

Abstract

In mammalian cells, DNA double-strand breaks (DSBs) cause rapid phosphorylation of the H2AX core histone variant (to form ␥-H2AX) in megabase chromatin domains flanking sites of DNA damage. To investigate the role of H2AX in mammalian cells, we generated H2AX-deficient (H2AX ⌬/⌬ ) mouse embryonic stem (ES) cells. H2AX ⌬/⌬ 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 ⌬/⌬ ES cells support normal levels and fidelity of V(D)J recombination in transient assays and also support lymphocyte development in vivo. However, H2AX ⌬/⌬ ES cells exhibit altered IR-induced BRCA1 focus formation. Our findings indicate that H2AX function is essential for mammalian DNA repair and genomic stability. The DNA in eukaryotic cells is packaged into chromatin, the fundamental unit of which is the nucleosome. The nucleosome consists of DNA wrapped around an octamer of the four core histones-H2A, H2B, H3, and H4 (1). The H2A histones consist of several subfamilies that contain distinct, conserved amino-and carboxyl-terminal amino acid sequences (2). The H2AX subfamily contains a conserved carboxyl-terminal SerGln-Glu (SQE motif) amino acid sequence. This SQE motif represents the consensus in vitro phosphorylation site for members of the phosphoinositide 3-kinase related kinase (PIKK) family that includes the protein kinases DNA-dependent protein kinase catalytic subunit (DNA-PKcs), ataxia telangiectasia mutated (ATM), and ATM and Rad3-related (ATR) (3).The repair of spontaneous and induced DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In eukaryotic cells, the two major pathways of DSB repair are nonhomologous end-joining (NHEJ) and homologous recombination (HR; refs. 4 and 5). Covalent modifications of core histones via phosphorylation, acetylation, and methylation have been proposed to form a ''histone code'' that is read by cellular proteins to facilitate downstream molecular events (6). In response to DNA damage by agents that induce DNA doublestrand breaks, Mec1, the Saccharomyces cerevisiae homologue of ATR, phosphorylates the SQE motif of H2A (7). This phosphorylation event is required for the efficient repair of chromosomal DSBs by NHEJ but does not appear to be as important for homologous recombination (7). In mammalian cells, H2AX is rapidly phosphorylated on the induction of DSBs by ionizing radiation (IR) and DNA damaging agents (8, 9), resulting in formation of ␥-H2AX foci along megabase chromatin domains flanking DNA damage sites (9).Foci of ␥-H2AX also form at the immunoglobulin heavy chain locus during class switch recombination (CSR) in activated mature B cells (10). CSR occurs between large, highly repetitive S regions and also may be initiated by DSBs (10, 11) and completed by NHEJ factors (12)(13)(14)(15). Notably, CSR is significantly impaired in the absence of H2AX (10). Earlier during lymphocyte development...

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].…”

H2AX: the histone guardian of the genome

“…These nuclear micro-domains are thought to contain hundreds to thousands of molecules that accumulate in the vicinity of a DSB. Several lines of evidence led to the hypothesis that ␥-H2AX is essential for the the recruitment of repair/signaling proteins to DNA damage: (a) ␥-H2AX IRIF formation exhibits rapid kinetics that precedes repair factor assembly into IRIF [5,12]; (b) H2AX, and more precisely ␥-H2AX [37,38] is required for foci formation for numerous factors including 53BP1, NBS1, BRCA1 and MDC1 [12,35,39,40]; (c) ␥-H2AX physically interacts with NBS1, 53BP1 and MDC1 [35,38,41,42]. However, ␥-H2AX-mediated foci formation is not equivalent to factor recruitment [37].…”

Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier

“…15,20,46,47 10 mg of each Ter/HR reporter ROSA26 targeting plasmid was linearized by Kpn I digest and introduced by electroporation to 2 £ 10 7 cells. Cells were seeded on plates prepared previously with puromycin-resistant feeders.…”