The post-translational modification (PTM) of histones regulates many biological processes, including gene transcription, DNA replication, and chromosome segregation. Histone PTMs function, in part, by modulating chromatin structure to render associated genes more or less accessible to nucleoproteins that mediate these biological processes. Histone PTMs also target the association of nuclear factors to specific regions of chromatin via interactions with diverse protein domains. One essential aspect of DNA metabolism in which the function of histone PTMs remains a mystery is the repair of DNA double-strand breaks (DSBs). Prior studies have shown that the histone variant H2AX is phosphorylated exclusively in chromatin flanking DSBs, generating a form called 3-H2AX. In yeast, more general changes in histone PTMs are induced at chromatin flanking DSBs, producing patterns that are reminiscent of those found at accessible, transcribed genes. However, in mammalian cells almost nothing is known about DSB-induced changes in histone PTMs due to the lack of experimental approaches for efficiently introducing these lesions at defined locations in the genome. For this purpose, we have innovated cell culture-based approaches that take advantage of primary and transformed lymphocytes in which the V(D)J recombinase (RAG) targets persistent DSBs at precise locations in the genome, allowing us to monitor the cellular response to DNA breaks. Using this novel cell system, we have shows that 3-H2AX is generated over extended distances (>100 kb) in chromatin flanking these RAG- DSBs. We now propose to define the constellation of histone PTMs that respond to RAG-DSBs in neighboring chromatin with the goal of elucidating their function in coordinating DSB repair. To achieve this goal, we will initially screen RAG-DSB chromatin for changes in a broad panel of histone PTMs using chromatin immunoprecipitation. Guided by the initial screens, we will generate high-resolution maps of DSB-induced changes to histone PTMs spanning several Mb using a custom microarray that contains all lymphocyte antigen receptor loci. These studies will define both time- and distant-dependent changes to the epigenetic landscape surrounding physiologic DSBs (Aim 1). The epigenetic maps generated in Aim 1 will be incorporated into strategies to (i) elucidate DNA damage response pathways that are critical for the revision of specific histone PTMs, (ii) determine which histone modifying enzymes are responsible for stamping/erasing DSB-induced changes in chromatin, and (iii) determine whether DSBs mediate a permanent change in regional chromatin. These pioneering studies will provide a foundational set of epigenetic data to reveal mechanisms of crosstalk between histone PTMs and DNA repair pathways that cooperate to maintain genomic integrity. Such mechanisms are particularly important in lymphocytes, where efficient repair must be directed to programmed DSBs in antigen receptor loci to avoid chromosomal translocations that promote cancer.
DNA double-strand breaks (DSBs) are generated upon exposure to genotoxic agents, such as ionizing radiation, and are intermediates during several important physiologic processes, including lymphocyte antigen receptor gene assembly. It is critical that DSBs are appropriately repaired rather than accessing alternative pathways that potentially result in oncogenic chromosomal translocations. We now propose to identify histone modifications that function as intermediates to coordinate the normal repair of DNA-DSBs in mammalian cells.
| Sikes, Michael L; Oltz, Eugene M (2012) Genetic and epigenetic regulation of Tcrb gene assembly. Curr Top Microbiol Immunol 356:91-116 |
| Helmink, Beth A; Tubbs, Anthony T; Dorsett, Yair et al. (2011) H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes. Nature 469:245-9 |
