We have analyzed the distribution of γH2AX foci in relation to heterochromatin and euchromatin in the cell nucleus. γH2AX foci induced by IR were largely absent from nuclear regions containing the heterochromatin markers HP1α or H3K9Me3 in MCF7 cells. To our knowledge, this differential nuclear distribution of IR-induced γH2AX foci has not been reported previously, although re-examination of images presented in certain papers (for example 
) shows an apparently similar pattern in mouse cells, where heterochromatin can easily be recognized as bright DAPI staining regions. Also consistent with the findings reported here, Karagiannis et al 
reported that satellite 2 and alpha satellite-containing chromatin is resistant to the induction of γH2AX by ionizing radiation according to ChIP analysis 
. Notably, these satellite sequences are constituents of centromeric heterochromatin. In addition, this phenomenon appears to be conserved through evolution. Kim et al 
reported during the preparation of this manuscript, that in the budding yeast Saccharomyces cerevisiae
the heterochromatic silent HML
loci are resistant to γH2AX formation following the introduction a targeted DSB.
Several possible reasons can be postulated for the apparent preference of H2AX phosphorylation for the euchromatic fraction of the genome. (i) Fewer DSBs are generated in heterochromatin, (ii) histone H2AX is absent or at low abundance in heterochromatin, (iii) epigenetic or other features of heterochromatin prevent the phosphorylation of H2AX over a large enough chromatin domain to generate a detectable focus, or these features restricts access of ATM and DNA-PK, (iv) DSBs rapidly migrate to the periphery of heterochromatic regions or cause local decondensation and loss of heterochromatin features.
Starting with the first possibility, there is no consistent evidence that IR induces fewer DSBs in heterochromatin than in euchromatin. Since no intermediates other than free radicals generated following energy deposition and their interaction with the DNA molecule are involved 
, it appears theoretically unlikely that heterochromatin would be very refractory to DSB generation by IR. However, differences in free radical scavenging capacity between chromatin compartments could result in different sensitivities to IR. Notably, Warters and Lyons 
showed that decondensation of chromatin in isolated nuclei by hypotonic treatment resulted in a 4.5-fold increase in the sensitivity of DNA to DSB induction as estimated by gel electrophoresis. This was presumably due to reduced protection of DNA from radical damage in decondensed chromatin associated with a reduced local concentration of histones and other proteins and molecules that scavenge free radicals. A considerable body of published work exists that compares the frequencies of radiation induced CAs originating in heterochromatin versus euchromatin, (see for example 
and references within), but there is no consensus as to whether radiation induced CAs occur with higher or lower than expected frequencies in heterochromatin. Notably though, a recent study has shown no difference in the frequency of γ-radiation-induced chromosome breaks between the largest block of heterochromatin in the human genome (1cen-1q12) and a similarly sized euchromatic region 
. On balance, it seems unlikely that the lack of γH2AX foci in heterochromatin could be fully accounted for by a lower sensitivity to DSB induction in these regions.
If the abundance of the H2AX histone variant was markedly lower in heterochromatin, heterochromatic DSBs would not lead to a sufficient local concentration of phospho-H2AX molecules to generate γH2AX foci that are detectable by immunofluorescence. However, this does not appear to be the case, as exposure of cells to HU during replication leads to the appearance of abundant γH2AX in heterochromatin (see &). Other histone modifications such as histone H3 lysine 9 trimethylation, the presence of heterochromatin-specific proteins such as HP1α, or structural features of heterochromatin may prevent access of ATM and/or DNA-PK to H2AX molecules, or may limit the extent of the domain over which H2AX is phosphorylated. However, ATR, which is responsible for H2AX phosphorylation following replication inhibition 
, appears to have access to heterochromatin at least during S-phase. Thus, ongoing replication may leave heterochromatin more amenable to DSB-induced H2AX phosphorylation. In support of this notion, a greater number of nuclei exhibit at least some overlapping γH2AX and HP1α signals when cells were irradiated in late S phase compared to G1
(), suggesting that transient decondensation of heterochromatin or depletion of heterochromatin proteins during replication allows H2AX phosphorylation. Further support for the role of the condensed nature of heterochromatin or its specific epigenetic and protein binding complement in preventing H2AX phosphorylation following IR comes from the use of histone deacetylase inhibitors. Prolonged exposure to low concentrations of the histone deacetylase inhibitor TSA results in reorganization of heterochromatin, characterized by increased acetylation, loss of HP1 proteins from heterochromatin and the movement of pericentromeric heterochromatin regions to the nuclear periphery 
. Notably, Karagiannis et al 
reported an IR-induced increase α-satellite-derived γH2AX only when cells were first exposed to TSA (0.2 µM, 72 hr). The alternative hypothesis (iv above) that the occurrence of a DSB in a heterochromatic region does result in efficient H2AX phosphorylation, but that this is coupled to local decondensation and loss of heterochromatic features seems less likely, particularly considering the data reported by Karagiannis et al. However, this possibility cannot be completely discounted in the light of data showing local chromatin decondensation at the sites of DSBs 
Thus, we conclude that DSBs-inducing agents fail to efficiently generate γH2AX foci in heterochromatin. The evidence discussed above suggests that this is due to the epigenetic or packaging properties of heterochromatin, preventing efficient H2AX phosphorylation. Since γH2AX is regarded as forming a platform for the recruitment or retention of other DNA repair and signaling molecules at DSBs, this implies that the processing of DSBs in heterochromatin differs from that in euchromatic regions. The differential response 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.