Genome-wide mapping and profiling of \u03b3H2AX binding hotspots in response to different replication stress inducers

Xinxing Lyu,Megan Chastain,Weihang Chai

doi:10.1186/s12864-019-5934-4

Xinxing Lyu, Megan Chastain + Show 1 more

Open Access

https://doi.org/10.1186/s12864-019-5934-4

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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.

Highlights

Replication stress (RS) gives rise to DNA damage that threatens genome stability
Significant enrichment of Short interspersed nuclear elements (SINE) is found in γH2AX sites in all treatments, indicating that SINEs may be a common barrier for replication polymerases
Mapping of γH2AX binding sites induced by APH, HU, methyl methanesulfonate (MMS) with ChIP-seq Prior to ChIP-seq, we tested the specificity of γH2AX antibody to ensure high specificity of ChIP (Additional file 1: Figure S1)

Summary

Introduction

Replication stress (RS) gives rise to DNA damage that threatens genome stability. RS can originate from different sources that stall replication by diverse mechanisms. Replication fork progression is constantly challenged and may be stalled by environmental insults and endogenous stress arising from normal cellular metabolism, leading to replication stress (RS) [1,2,3]. These challenges can arise from various genotoxic mechanisms, such as depletion of nucleotide pools, deficiency of replication complex, conflicts between replication and transcription, R-loop formation, DNA damage, and others (reviewed in [3]). ATR activation arrests cell cycle, promotes fork stability to prevent fork collapse, and regulates DNA repair pathways to rescue stalled forks. ΓH2AX marks stalled forks prior to DSB formation [6], presumably setting up a favorable chromatic environment that facilitates the recruitment of fork repair proteins to stalled sites. γH2AX accumulates at break sites after fork collapse [6,7,8], consistent with its

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