cH2AX Foci Form Preferentially in Euchromatin after Ionising-Radiation Ian G. Cowell 1 *, Nicola J. Sunter 1 , Prim B. Singh 2 , Caroline A. Austin 1 , Barbara W. Durkacz 3 , Michael J. Tilby 3 1 Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom, 2 Division of Tumour Biology, Forschungszentrum Borstel, Borstel, Germany, 3 Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom Background. The 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 cH2AX 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 cH2AX foci following X-irradiation with regions of the nucleus devoid of foci. Methodology/Principle Findings. Using 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/Significance. We discus a number of possible mechanisms that could account for the observed pattern of H2AX phosphorylation. Since cH2AX 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. Citation: Cowell IG, Sunter NJ, Singh PB, Austin CA, Durkacz BW, et al (2007) cH2AX Foci Form Preferentially in Euchromatin after Ionising- Radiation. PLoS ONE 2(10): e1057. doi:10.1371/journal.pone.0001057 INTRODUCTION Up to 25% of the histone H2A complement in mammalian cells consists of the histone variant H2AX [1,2]. Compared to histone H2A1, this molecule has a unique C-terminal tail containing the phosphorylation target sequence for members of the phosphati- dylinositol 39-kinase like kinase (PIKK) family of serine/threonine protein kinases. This family includes ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR) and DNA- dependent protein kinase (DNA-PK)[3,4]. Histone H2AX is rapidly phosphorylated at Ser139 following treatments that induce DNA double-strand breaks (DSBs) or cause replication stress. At DSBs generated by ionizing radiation for example, H2AX becomes phosphorylated over megabase chromatin regions flanking the breaks [1]. This phosphorylation is dependent largely on ATM, with some redundancy with DNA-PK [5,6]. The resulting local concentrations of phosphorylated H2AX (cH2AX) can be detected at interphase by immunofluorescence microscopy, and are termed cH2AX foci. UV exposure or treatment with replication inhibitors such as hydroxyurea lead to ATR-dependent H2AX phosphorylation at sites of arrested replication forks [7]. Similarly, replication-dependent DSBs induced by topoisomerase I inhibitors lead to ATR-dependent H2AX phosphorylation [8]. cH2AX is believed to form a platform for the recruitment and/or retention of DNA repair and signaling molecules at sites of DNA damage. At least one of these components, MDC1, binds directly to the phosphorylated C-terminal tail of histone H2AX. The precise physiological role of H2AX phosphorylation is not yet fully understood, but cells derived from H2AX 2/2 mice display moderate radiosensitivity [9,10] and a G2/M checkpoint defect [11]. This is consistent with the notion that by concentrating signaling molecules at sites of damage, cH2AX amplifies the DNA damage signal. It has also been suggested that phosphorylation of H2AX helps anchor chromosomal ends together, reducing the chances of DSBs leading to illegitimate recombination events [12]. Phosphorylation of histone H2AX can be seen as one of a number of histone posttranslational modifications that delineate specific functions in particular segments of chromatin. Other such modifications include trimethylation of histone H3 lysine 9 and histone H4 lysine 20, that are characteristic of constitutive heterochromatin [13,14,15]. This compartment of the genome is gene-poor and remains condensed during interphase. It is composed largely of repeated elements found in centromeric and pericentromeric regions in most eukaryotes and in the short arms of the human acrocentric chromosomes. DNA replication occurs towards the end of S-phase in heterochromatic regions, whereas euchromatic regions generally replicate in early to mid S-phase. In addition, it is well established that heterochromatic regions are associated with the non-histone chromatin protein, HP1 Academic Editor: Beth Sullivan, Duke University, United States of America Received July 26, 2007; Accepted October 2, 2007; Published October 24, 2007 Copyright: ß 2007 Cowell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Breast Cancer Campaign (2002/343) and the Leukaemia Research fund (0361) and the Association for International Cancer Research (05-179). Competing Interests: The authors have declared that no competing interests exist. * To whom correspondence should be addressed. E-mail: i.g.cowell@ncl.ac.uk PLoS ONE | www.plosone.org 1 October 2007 | Issue 10 | e1057