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

Abstract

Background: Replication 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. Results: We 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. Conclusions: Our 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.

The NS1 protein of the parvovirus MVM Aids in the localization of the viral genome to cellular sites of DNA damage

“…Induction of DNA damage in these cells via an HU pulse led to the spread of γH2AX over wide areas (purple histogram and, cf. Fig 1D , panel 3), as previously characterized by others for γH2AX induction [ 26 , 31 ], and consistent with previously published findings during MVM infection [ 21 ]. NS1 broadly relocalized to the γH2AX domains in both analyses (blue histogram).…”

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.…”

Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide

“…It has been proposed that CFS instability results from fork barriers raised by sequences prone to form secondary structures 18,19,36 . At least four types of observations argue against this model: (i) analysis of the nucleotide sequence of several FISH-mapped CFSs has provided contrasting results regarding the presence of such roadblocks at a substantial proportion of the sites 37 .…”