γH2AX Foci Form Preferentially in Euchromatin after Ionising-Radiation

Abstract

BackgroundThe histone variant histone H2A.X comprises up to 25% of the H2A complement in mammalian cells. It is rapidly phosphorylated following exposure of cells to double-strand break (DSB) inducing agents such as ionising radiation. Within minutes of DSB generation, H2AX molecules are phosphorylated in large chromatin domains flanking DNA double-strand breaks (DSBs); these domains can be observed by immunofluorescence microscopy and are termed γH2AX foci. H2AX phosphorylation is believed to have a role mounting an efficient cellular response to DNA damage. Theoretical considerations suggest an essentially random chromosomal distribution of X-ray induced DSBs, and experimental evidence does not consistently indicate otherwise. However, we observed an apparently uneven distribution of γH2AX foci following X-irradiation with regions of the nucleus devoid of foci.Methodology/Principle FindingsUsing immunofluorescence microscopy, we show that focal phosphorylation of histone H2AX occurs preferentially in euchromatic regions of the genome following X-irradiation. H2AX phosphorylation has also been demonstrated previously to occur at stalled replication forks induced by UV radiation or exposure to agents such as hydroxyurea. In this study, treatment of S-phase cells with hydroxyurea lead to efficient H2AX phosphorylation in both euchromatin and heterochromatin at times when these chromatin compartments were undergoing replication. This suggests a block to H2AX phosphorylation in heterochromatin that is at least partially relieved by ongoing DNA replication.Conclusions/SignificanceWe discus a number of possible mechanisms that could account for the observed pattern of H2AX phosphorylation. Since γH2AX is regarded as forming a platform for the recruitment or retention of other DNA repair and signaling molecules, these findings imply that the processing of DSBs in heterochromatin differs from that in euchromatic regions. The differential responses of heterochromatic and euchromatic compartments of the genome to DSBs will have implications for understanding the processes of DNA repair in relation to nuclear and chromatin organization.

Imaging of the DNA damage‐induced dynamics of nuclear proteins via nonlinear photoperturbation

“…Since we cannot exclude the presence of less dense patches of DNA at the periphery of the nucleolus, this result highlights the sensitivity of our method with respect to chromatin structure. The observation is also consistent with the reduced susceptibility to damage and slower decondensation of highly compacted chromatin regions supporting the validity of our approach [25][26][27]. Our data are also in accordance with the slower kinetics and less efficient DNA repair in heterochromatin as compared to euchromatin [28].…”

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

“…6 and 7), TPX2 could regulate ␥-H2AX amplification via MDC1 and ATM. However, the accumulation of ␥-H2AX signals during the DNA damage response is also influenced by the chromatin structure (74,75). In light of the accumulation of TPX2 at ionizing radiation-induced foci (Figs.…”

Spontaneous and Bleomycin-Induced <i>γ</i>H2AX Signals in CHO9 Metaphase Chromosomes

“…Since chromatin features through homologous regions may be indistinguishable, the influence of the same chromatin structure in a particular chromosome region independently of the position in the karyotype could be put forward to explain this observation. [63]. Since it is expected that along homologous regions similar chromatin states are present (i e. early replicating G-light bands, late replicating G-dark bands, heterochromatic constitutive C band, interstitial telomeric sequences) [64] [65], a comparable response to DNA damage and H2AX 139 phosphorylation is envisaged.…”