Genome-wide mapping and profiling of γH2AX binding hotspots in response to different replication stress inducers

Abstract

BackgroundReplication stress (RS) gives rise to DNA damage that threatens genome stability. RS can originate from different sources that stall replication by diverse mechanisms. However, the mechanism underlying how different types of RS contribute to genome instability is unclear, in part due to the poor understanding of the distribution and characteristics of damage sites induced by different RS mechanisms.ResultsWe use ChIP-seq to map γH2AX binding sites genome-wide caused by aphidicolin (APH), hydroxyurea (HU), and methyl methanesulfonate (MMS) treatments in human lymphocyte cells. Mapping of γH2AX ChIP-seq reveals that APH, HU, and MMS treatments induce non-random γH2AX chromatin binding at discrete regions, suggesting that there are γH2AX binding hotspots in the genome. Characterization of the distribution and sequence/epigenetic features of γH2AX binding sites reveals that the three treatments induce γH2AX binding at largely non-overlapping regions, suggesting that RS may cause damage at specific genomic loci in a manner dependent on the fork stalling mechanism. Nonetheless, γH2AX binding sites induced by the three treatments share common features including compact chromatin, coinciding with larger-than-average genes, and depletion of CpG islands and transcription start sites. Moreover, we observe significant enrichment of SINEs in γH2AX sites in all treatments, indicating that SINEs may be a common barrier for replication polymerases.ConclusionsOur results identify the location and common features of genome instability hotspots induced by different types of RS, and help in deciphering the mechanisms underlying RS-induced genetic diseases and carcinogenesis.

Unscheduled origin building in S-phase upon tight CDK1 inhibition suppresses CFS instability

“…We therefore asked whether this concentration of HU destabilizes CFSs. In agreement with previous reports 51, 52 , we observed breaks in metaphase chromosomes, part of them at CFSs. However, we found that only a subset of the Aph-sensitive CFSs displays breaks in HU, and at far lower frequency than in Aph.…”

Histone dynamics during DNA replication stress

“…In addition, the importance of γH2A.X in rescuing stalled replication has been suggested by experiments in the yeast model [ 123 ]. Recently, ChIP-seq to map the γH2A.X distribution genome-wide caused by distinct fork stalling mechanisms in a human lymphocyte cell line revealed that different treatments can induce non-random γH2A.X chromatin binding at discrete regions [ 124 ]. Characterization of the γH2A.X distribution showed two consistent epigenetic features: (1) different treatments induce γH2A.X loading at largely non-overlapping regions, and (2) γH2A.X loading hotspots are depleted at CpG islands and transcription start sites but are enriched at compact chromatin regions.…”

“…Characterization of the γH2A.X distribution showed two consistent epigenetic features: (1) different treatments induce γH2A.X loading at largely non-overlapping regions, and (2) γH2A.X loading hotspots are depleted at CpG islands and transcription start sites but are enriched at compact chromatin regions. The γH2A.X histone variant may therefore coordinate with different protein molecules and repair pathways to rescue forks stalled at different types of fragile sequences [ 124 ].…”