Regulation of H2AX phosphorylation density around a defined double stranded DNA break in chromatin

One of the earliest events in cellular response to a nascent double stranded DNA break (DSB) is local induction of the phosphorylated histone H2AX (gammaH2AX). Phosphorylated by ATM and DNA-PK, gammaH2AX leads to retention of DNA damage response factors and facilitates the high fidelity DNA repair. In vivo, H2AX functions as a haploinsufficient tumor suppressor supressing genomic instability, thus the density of H2AX in chromatin appears to affect its function. Although gammaH2AX is localized in the region of chromatin harboring the break, localization with respect to the break site has remained largely unknown. In order to better understand the function of gammaH2AX and to address the question of its localization, I developed a system in which upon pharmacological stimulation, I can activate the Rag recombinase and induce double stranded breaks at the immunoglobulin kappa locus. Through a newly developed assisted chromatin immunoprecipitation assay, I found that gammaH2AX extends 400--500kb to one side of the break in chromatin. High density of gammaH2AX was found predominantly proximally to the break and was dependent on ATM anchoring to chromatin through MDC1. In the absence of either factor, gammaH2AX density was reduced five fold and the remaining gammaH2AX was induced solely through DNA-PK. In contrast, distal phosphorylation was mediated exclusively by ATM and was independent of MDC1. Inhibition of ATM-mediated phosphorylation of H2AX or the dephosphorylation of gammaH2AX through protein phosphatase 2A led to a marked decrease in gammaH2AX density. Notably, the levels of H2AX in chromatin also considerably affected the gammaH2AX density since 2--3 fold decrease in H2AX chromatin density results in a 10-fold decrease in gammaH2AX density. These data argue that there is a defined profile of gammaH2AX density around an unrepaired DSB. The maximal density of gammaH2AX depends on anchoring of ATM to chromatin and availability of the H2AX substrate for phosphorylation. Maximal distance is predominantly regulated by the nucleoplasmic pool of ATM and is independent of MDC1 anchoring. Notably, the observed phosphorylation profile is the result of a dynamic equilibrium between gammaH2AX induction and removal, potentially through gammaH2AX→H2AX histone exchange, creating a structure that is highly responsive to changes in ATM activity and potentially the repair status of the break.