👤 Ashok Venkitaraman

The pathways that signal double-strand DNA breaks (DSBs) in mammalian cells are central to the maintenance of genome integrity. We have reported (Ayoub et al., Nature 2008; 453: 682-6) that the rapid mobilization of the heterochromatin protein, HP1beta, within seconds from DSB sites promotes chromatin changes like H2AX phosphorylation that trigger this response. Notably, this paper and a subsequent report (Ayoub et al., Cell Cycle 2009; 8: 1494-500), demonstrate that transient HP1beta mobilization is followed by its accumulation over time at DSB sites. Indeed, two recent papers (Luijsterburg et al., J Cell Biol 2009; 185:577-86 and Zarebski et al., Cytometry A May 2009) suggest that HP1 recruitment to damage sites, rather than its rapid mobilization, is the predominant behaviour exhibited by this protein. Here, we present new experimental analyses which corroborate that fluorophore-tagged HP1beta exhibits two distinct behaviours at DSB sites in living cells - rapid, transient mobilization, most evident in heterochromatic regions, followed by slower recruitment. Experimental methods allowing visualization of these behaviours are described. Interestingly, chemical inhibition of the DNA-damage responsive enzyme, casein kinase 2 (CK2), suppresses HP1beta mobilization while permitting recruitment. Our findings reconcile recent findings in a new model, wherein rapid HP1beta mobilization from DSBs mediated by its phosphorylation on Thr51 by CK2, is followed by, and may overlap with, its accumulation at these sites via the chromoshadow domain, independent of Thr51. Our analyses provide fresh insight into the earliest events that trigger the DNA damage response in mammalian cells. Show less

The dynamics of chromatin-associated proteins control the accessibility of DNA to essential biological transactions like transcription, replication, recombination and repair. Here, we briefly outline what is known about the chromatin changes that occur during the cellular response to DNA breakage, focusing on our recent findings revealing that the chromatin factor HP1beta is mobilized within seconds after DNA damage by an unrecognized signaling cascade mediated by casein kinase 2 (CK2) phosphorylation, paving the way for histone H2AX phosphorylation. We also show here that HP1beta mobilization is neither associated with histone H3 modification on Ser10, an alteration proposed to assist in HP1 ejection from chromatin, nor with evidence of a physical interaction between HP1beta and the CK2 regulatory subunit. Interestingly, following its rapid mobilization, we find that HP1beta gradually re-accumulates on damaged chromatin over a longer time period, suggesting that temporal changes in HP1beta dynamics and interaction with chromatin may assist in different stages of the cellular response to DNA breakage. Show less

Minutes after DNA damage, the variant histone H2AX is phosphorylated by protein kinases of the phosphoinositide kinase family, including ATM, ATR or DNA-PK. Phosphorylated (gamma)-H2AX-which recruits molecules that sense or signal the presence of DNA breaks, activating the response that leads to repair-is the earliest known marker of chromosomal DNA breakage. Here we identify a dynamic change in chromatin that promotes H2AX phosphorylation in mammalian cells. DNA breaks swiftly mobilize heterochromatin protein 1 (HP1)-beta (also called CBX1), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). Local changes in histone-tail modifications are not apparent. Instead, phosphorylation of HP1-beta on amino acid Thr 51 accompanies mobilization, releasing HP1-beta from chromatin by disrupting hydrogen bonds that fold its chromodomain around H3K9me. Inhibition of casein kinase 2 (CK2), an enzyme implicated in DNA damage sensing and repair, suppresses Thr 51 phosphorylation and HP1-beta mobilization in living cells. CK2 inhibition, or a constitutively chromatin-bound HP1-beta mutant, diminishes H2AX phosphorylation. Our findings reveal an unrecognized signalling cascade that helps to initiate the DNA damage response, altering chromatin by modifying a histone-code mediator protein, HP1, but not the code itself. Show less

The BRCA2 gene is mutated in familial breast and ovarian cancer, and its product is implicated in DNA repair and transcriptional regulation. Here we identify a protein, EMSY, which binds BRCA2 within a region (exon 3) deleted in cancer. EMSY is capable of silencing the activation potential of BRCA2 exon 3, associates with chromatin regulators HP1beta and BS69, and localizes to sites of repair following DNA damage. EMSY maps to chromosome 11q13.5, a region known to be involved in breast and ovarian cancer. We show that the EMSY gene is amplified almost exclusively in sporadic breast cancer (13%) and higher-grade ovarian cancer (17%). In addition, EMSY amplification is associated with worse survival, particularly in node-negative breast cancer, suggesting that it may be of prognostic value. The remarkable clinical overlap between sporadic EMSY amplification and familial BRCA2 deletion implicates a BRCA2 pathway in sporadic breast and ovarian cancer. Show less