Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis

Transient overexpression of H2AX was found to lead to a detectable fraction of soluble H2AX and was associated with increased apoptosis. This effect was enhanced by the induction of DNA replication stress using the DNA polymerase α inhibitor aphidicolin. Cells manipulated to stably express H2AX did not contain soluble H2AX, however, short-term treatment with aphidicolin (1 h) resulted in detectable amounts of H2AX in the soluble nuclear fraction and enhanced apoptosis. Similarly, soluble endogenous H2AX was detected under these conditions. We found that excessive soluble H2AX causes chromatin aggregation and inhibition of ongoing gene transcription as evidenced by the redistribution and/or loss of active RNA polymerase II as well as the transcriptional co-activators CBP and p300.Taken together, these results show that DNA replication stress rapidly leads to increased soluble H2AX and that non-chromatin-associated H2AX can sensitize cells to undergo apoptosis. Our findings encourage further studies to explore H2AX and the cellular pathways that control its expression as anti-cancer drug targets.Chromatin-associated histone H2AX is a key regulator of the cellular response to genotoxic stress and DNA double-strand breaks [1-6]. H2AX is a core histone H2A variant that is randomly incorporated into nucleosomes during DNA replication. It differs from histone H2A by a unique C-terminal tail that contains a highly conserved SQE motif with a serine residue at position 139 [7]. Serine 139 becomes rapidly phosphorylated in response to DNA double strand breaks by protein kinases of the phosphatidylinositol 3-OH-kinase-related kinase (PI3KK) family including ATM, ATR or DNA-PK [8-10]. The phosphorylated form of H2AX has been referred to as γ-H2AX [2]. Several lines of evidence suggest that the main function of histone H2AX is to orchestrate the DNA damage response through interactions with BRCT repeat-containing proteins that bind phosphorylated H2AX such as MDC1 [11-13]. Howeve