Histone H2AX phosphorylation in response to changes in chromatin structure induced by altered osmolarity

Abstract

DNA strand breaks trigger marked phosphorylation of histone H2AX (i.e. γ-H2AX). While DNA double-strand breaks (DSBs) provide a strong stimulus for this event, the accompanying structural alterations in chromatin may represent the actual signal that elicits γ-H2AX. Our data show that changes in chromatin structure are sufficient to elicit extensive γ-H2AX formation in the relative absence of DNA strand breaks. Cells subjected to hypotonic (0.05 M) treatment exhibit γ-H2AX levels that are equivalent to those found after the induction of 80–200 DNA DSBs (i.e. 2–5 Gy). Despite this significant increase in phosphorylation, cell survival remains relatively unaffected (<10% cytotoxicity), and there is no significant increase in apoptosis. Nuclear staining profiles indicate that γ-H2AX-positive cells induced under altered tonicity exhibit variable levels of staining, ranging from uniform pan staining to discrete punctate foci more characteristic of DNA strand breakage. The capability to induce significant γ-H2AX formation under altered tonicity in the relative absence of DNA strand breaks suggests that this histone modification evolved in response to changes in chromatin structure.