Spatiotemporal control of recombination by the RAG proteins and ATM

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DESCRIPTION (provided by applicant): Lymphoid neoplasms are among the most common malignancies in humans, and, for reasons that remain obscure; their incidence has been increasing over the past two decades. Although these disorders arise from diverse etiologies, chromosomal translocations involving the antigen receptor loci are a common underlying mechanism. B and T lymphocyte development is driven by V (D)J recombination, a process by which gene segments at the antigen receptor loci are repeatedly rearranged to create a vast repertoire of antigen receptor genes. Because V(D)J recombination entails the cleavage and joining of widely dispersed gene segments many millions of times each day, even a miniscule error rate still carries considerable risk of translocation. This risk is further increased in B cells, which undergo yet another genomic rearrangement process known as class switch recombination (CSR) to change the effector function of the immunoglobulin (Ig) molecule. Given the deleterious consequences of aberrantly repaired double strand breaks (DSBs), both V (D)J recombination and CSR must tightly regulate accessibility of substrates for cleavage and the activities of the DNA damage response and repair machineries. Although detailed genetic and biochemical studies have identified the factors involved in these processes, these "bulk techniques" cannot provide much insight into their spatiotemporal workings; only recently have microscopic techniques allowed us to view these processes as they occur in specific cells. Using 3D FISH and other techniques, my lab recently demonstrated that homologous Ig alleles physically pair up in a stage-specific manner that parallels the sequential stages of recombination of these loci. Surprisingly, this interallelic association is mediated by the V(D)J recombinase and the DNA damage checkpoint protein ATM: introduction of a double-strand break at one Ig allele induces ATM-dependent repositioning of the other allele to pericentromeric heterochromatin to prevent further cleavage (Nature Immunology, in press). Notably, ATM-deficient mice and humans are prone to certain recurrent oncogenic translocations, by mechanisms that have remained unclear; our work sheds light on the spatiotemporal aspects of ATM function. We propose that homologous pairing of alleles undergoing recombination protects genomic stability by ensuring that broken ends are aligned with homologous alleles rather than in contact with other loci. We want to delve more deeply into this mechanism, investigating how ATM and the RAG proteins exert control over locus conformation and nuclear location. PUBLIC HEALTH RELEVANCE: Chromosomal translocations underlie a number of tumors of the lymphoid lineage. Recent advances in microscopy and FISH techniques allows us to visualize DNA breaks and interactions between chromosomes in individual cells, tracing their movements in and out of euchromatic regions in the nucleus. We recently discovered that the V (D)J recombinase and ATM mediate pairing of homologous Ig alleles and movement of one allele to pericentromeric heterocrhomatin as control points to preserve genomic stability during recombination. We propose to further these studies to understand precisely how these factors achieve these unexpected effects.