There is increasing evidence that epigenetic regulation of nucleosomes plays a key role in the response to DSBs. Alterations in the configuration of chromatin appear to be necessary for the initial recognition of DSBs (1
). Beyond the detection of the lesion, the ability to efficiently repair DSBs may also require chromatin-remodeling activities. For example, BRAF-35, a complex that interacts with the DNA repair factor Brca2 has the capacity to deacetylate histones (22
). A newly discovered Brca2-associated protein, EMSY, forms IRIF and interacts with the HP1β chromoshadow domain, which recognizes methylated lysine residues within histones (23
). Moreover, a histone deactylase HDAC4 also assembles in nuclear foci and is critical for irradiation-induced survival (24
). HDAC4, like γ-H2AX, is required for the formation of 53BP1 IRIF (24
), suggesting the possibility that chromatin configuration may be intimately linked to the cytological observation of foci. However, although HDACs normally act on acetylated lysines to induce chromatin compaction, it remains unclear which, if any, specific histone residues in the vicinity of DSBs are targeted by Brca2- or HDAC4-containing deacetylases. At present, the only known histone modification that is enriched at DSBs in mammalian cells is the phosphorylation of H2AX. Here we have uncovered an additional site of histone phosphorylation at DSBs that supports the involvement of histone tail modifications in the signaling/repair of DSBs.
The finding that H2B Ser-14 phosphorylation at DSBs is H2AX independent, whereas H2B-Ser14P
foci formation is γ-H2AX dependent is paradoxical. One interpretation for the absence of repair foci is that factors are not recruited sites of DNA damage. However, we have found that both events are independent and that despite a normal recruitment of factors to DSBs at early time points, factors may still not form IRIF (13
). There are at least two possible explanations for why H2B-Ser14P
foci formation is dependent on the phosphorylation marks on H2AX. First, γ-H2AX may be required to retain the H2B S-14 kinase after it's initial recruitment to DSBs. This could be mediated via weak interactions between the kinase and the SQ motif in the H2AX tail, thousands of which are modified by phosphorylation. However, such direct interactions appear to be unlikely given that H2AX is required for IRIF formation of almost all factors, some of which undoubtedly assemble at DSBs by independent pathways. To account for the universal role of H2AX in IRIF formation, we favor an alternative model that posits that H2AX phosphorylation has a direct effect on the chromatin structure surrounding a DSB (). In this scenario, the time-dependent increase of the intensity of IRIF may not be due to an increase in accumulation of factors at a DSB but rather to the condensed state of the DNA. As long as the break is not repaired, a higher amount of DNA will become condensed around the lesion, thereby leading to a higher concentration of factors that are associated with the damage (). These two possible functions of H2AX phosphorylation, the tethering of factors and chromatin compaction, are not mutually exclusive since the γ-H2AX mark may provide both a docking site and promote a change in chromatin folding.
Figure 3. The chromatin compaction model. (A) Intact DNA molecule showing the nucleosomes (blue) with protruding tail motifs (black). (B) Generation of a DSB. (C) Histone tails become specifically modified in the chromatin surrounding the lesion (i.e., γ-H2AX (more ...)
The known properties of γ-H2AX are consistent with a role in chromatin compaction. For example, H2AX is required for the chromatin condensation and transcription silencing of the sex chromosomes during spermatogenesis (25
). Moreover, H2AX regulates the long-distance synapsis of DNA ends during immunoglobulin class-switch recombination (26
). Interestingly, we have found that H2B-Ser14P
shows a similar staining pattern to that of γ-H2AX in mouse spermatocytes, being particularly enriched in the highly compacted XY chromosomes, also known as the sex body (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20032247/DC1
has already been reported to be associated with chromatin condensation both in vivo and in vitro (12
). The H2B peptide tail has the unique property of self-aggregating when phosphorylated at S-14, and therefore this modification could play a direct role in regulating chromatin condensation (12
). Thus, it is possible that H2B-Ser14P
can act in concert with H2AX to promote chromatin condensation at sites of DSBs ().
The chromatin compaction model predicts that H2AX would even be required for the foci formation of its own kinase. Indeed, we have found that the formation of ATMSer1981P
foci is abrogated in the absence of H2AX, whereas the recruitment of the ATMSer1981P
to DSBs is H2AX independent (unpublished data). A previous study indicated that DSB-induced chromatin relaxation might directly trigger ATM activation (1
). Such a chromatin alteration may also facilitate access of DNA repair/signaling machinery to DSBs and target enzymes that covalently modify histone tails. If the lesion persists, modifications on histone tails including phosphorylation of H2AX–Ser-136/139 and H2B-Ser14P (and possibly histone deacetylation) may cooperate to establish a heterochromatin-like state, which may prevent the premature separation of DNA ends. The presence of fragmented chromosomes associated with defective foci formation in H2AX−/−
) supports this model. Understanding how histone modification patterns dictate dynamic changes in chromatin topology at DNA damage sites is a major challenge for future research.