Systematic identification of fragile sites via genome-wide location analysis of γ-H2AX

Abstract

Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication fork stalling results in histone H2A phosphorylation to yield γ-H2A (yeast γ-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)- dependent manner. Here, we describe the genome-wide location analysis of γ-H2A as a strategy to identify loci prone to engage the Mec1 and Tel1 pathways. Remarkably, γ-H2A enrichment overlaps with loci prone to replication fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for γ-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of γ-H2A at these loci. Finally, our work indicates that high resolution mapping of γ-H2AX is a fruitful route to map fragile sites in eukaryotic genomes.

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

“…This is made possible by the advent of high-throughput technologies such as microarray and second-generation sequencing. In particular, chromatin immunoprecipitation (ChIP) followed by microarray (ChIP-chip) analyses of a commonly used doublestrand break (DSB) marker, phosphorylated histone H2A(X), have revealed in wild-type budding yeast and fission yeast cells the genome-wide distribution of sites prone to triggering phosphorylation of H2A(X) by primary DNA damage checkpoint kinases (Rozenzhak et al 2010;Szilard et al 2010). …”

Chromatin modification and NBS1: their relationship in DNA double-strand break repair

“…This phosphorylation is carried out by ATR (MEC1 in budding yeast). Genome-wide mapping of γ-H2A-rich loci using ChIP analysis clarified that γH2A accumulates at the sites of naturally stalled forks, including the ribosomal DNA locus, tRNA genes, telomeres and DNA replication origins in Saccharomyces cerevisiae (Szilard et al, 2010). The average size of the γ-H2A domain at these natural stall sites was 1255 bp, while HO endonuclease-induced DSBs caused accumulations of γ-H2A each spanning around 50 kb at DSB sites in S. cerevisiae, suggesting that γ-H2AX/ γ-H2A at stalled forks has a different role to that of γ-H2AX/γ-H2A at DSB sites that have been induced by IR.…”

Regulation of Minichromosome Maintenance (MCM) Helicase in Response to Replication Stress

“…γ-H2A or γ-H2AX is one of the well-characterized histone modifications that occur around DNA damage sites in the checkpoint kinase dependent manner (Downs et al, 2000). By using DNA microarray, recent studies mapped the localization of γ-H2A on budding or fission yeast genomes in unperturbed S phase (Rozenzhak et al, 2010;Szilard et al, 2010). In budding yeast, they found the accumulation of γ-H2A to occur in repressed genes and that is dependent on the activity of a histone deacetylase (HDAC).…”