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Exposure to DNA damaging agents triggers phosphorylation of histone variant H2AX (generating γH2AX) in large chromatin regions flanking DNA lesions, allowing their immunodetection as nuclear foci. Even though a predominance of γH2AX foci in euchromatin has been postulated, foci positioning when DNA insult occurs in replicating eu- or heterochromatin regions has not been extensively explored. Labeling of interphase nuclei with 5-ethynyl-2'-deoxyuridine (EdU) pulses has revealed that DNA replication is temporarily and spatially regulated: euchromatin replicates in early S (ES) and heterochromatin along mid and late S (MS/LS) phases. In order to map DNA damage with respect to replicating domains, the distribution of γH2AX foci induced by the radiomimetic agent bleomycin was analyzed in CHO9 interphase nuclei by delineating euchromatic (H3K4me3+) and replicating (EdU+) regions. Quantification of overlapping pixels and 3D inter-object overlap in binary masks revealed colocalization between γH2AX foci and EdU +  domains both in ES and MS/LS nuclei, indicating that primary damage distribution is modulated by DNA synthesis. Further, we verified that EdU incorporation by itself did not influence BLEO-induced γH2AX nuclear patterns. Our results also revealed a repeated localization of γH2AX foci in replicating/nonreplicating interfaces which could reflect short-range chromatin migration following DNA insult.

sion, correspond to features of facultative heterochromatin [7, 6]. The mammalian genome compartmentalization can be visualized in both banded metaphase chromosomes and stained interphase nuclei. Compartments Euchromatin Facultative heterochromatin Constitutive heterochromatin Chromatin types Euchromatin Tissue-specific Dosage compensation α-heterochromatin β-heterochromatin Location in metaphase chromosomes Light G-bands Dark G-bands Inactive X chromosome (Xi)

Through analysis of published data we positioned along human chromosome idiograms (850 bands) hyperacetylated H4 chromatin (H4^+a), regions of increased gene expression (RIDGEs), antiRIDGEs, ionizing radiation breakpoints, integration sites of highly expressed GFP reporter constructs and candidate genes differentially expressed in tumor tissues. Highly expressed regions of the human genome (especially RIDGEs) seem to be more sensitive to radiation damage. Comparatively, antiRIDGEs appear as radiation resistant. Tumor deregulated genes tend to cluster along and in the neighborhood of RIDGEs. We detected 35 clusters of genes differentially expressed in tumor tissues which colocalize with RIDGEs; 23 of these clusters also exhibit radiation damage. RIDGEs also accumulate highly expressed GFP reporter construct integration sites, evolutionary breakpoints as well as amplicons and/or deletion-prone chromosome segments in tumors. This could indicate that abnormal gene (up- or down-) regulation might require high-throughput transcription nuclear micro-environments to occur. Our results suggest that the human genome is a combination of regions with marked disparities regarding the topology of increased gene expression, ionizing radiation damage, evolutionary breakpoints, integration sites, and abnormal gene regulation.

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