When Chromatin Decondensation Affects Nuclear γH2AX Foci Pattern and Kinetics and Biases the Assessment of DNA Double-Strand Breaks by Immunofluorescence

TL;DR

This paper shows how chromatin decondensation can alter the appearance of γH2AX foci, potentially leading to incorrect measurements of DNA damage.

Contribution

The study introduces a new model to explain how chromatin structure affects γH2AX foci patterns and their quantification.

Findings

Chromatin decondensation changes γH2AX foci size and brightness.

Minifoci from decondensed chromatin may mislead DSB quantification.

The 'Christmas light model' explains the variability in foci patterns.

Abstract

Immunofluorescence with antibodies against phosphorylated forms of H2AX (γH2AX) is revolutionizing our understanding of repair and signaling of DNA double-strand breaks (DSBs). Unfortunately, the pattern of γH2AX foci depends upon a number of parameters (nature of stress, number of foci, radiation dose, repair time, cell cycle phase, gene mutations, etc…) whose one of the common points is chromatin condensation/decondensation. Here, we endeavored to demonstrate how chromatin conformation affects γH2AX foci pattern and influences immunofluorescence signal. DSBs induced in non-transformed human fibroblasts were analyzed by γH2AX immunofluorescence with sodium butyrate treatment of chromatin applied after the irradiation that decondenses chromatin but does not induce DNA breaks. Our data showed that the pattern of γH2AX foci may drastically change with the experimental protocols in terms of size and brightness. Notably, some γH2AX minifoci resulting from the dispersion of the main signal due to chromatin decondensation may bias the quantification of the number of DSBs. We proposed a model called “Christmas light models” to tentatively explain this diversity of γH2AX foci pattern that may also be considered for any DNA damage marker that relocalizes as nuclear foci.

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