of Work The somatic assembly of antigen receptor (AgR) genes by V(D)J recombination creates the diverse antigen receptor repertoires of T and B lymphocytes and is fundamental to adaptive immunity in jawed vertebrates. AgR genes are contained within large and complex genetic loci that must be regulated at multiple levels to achieve essential outcomes of early lymphocyte development. Recombination must be developmentally ordered to allow the production and selection of lymphocyte subsets with appropriate characteristics and in appropriate numbers. Recombination may be subject to allelic regulation to ensure that each lymphocyte expresses a single receptor with unique antigen specificity. Recombination must be subject to safeguards that ensure genomic integrity. Recombination must also be sufficiently stochastic to generate AgR repertoires characterized by extensive combinatorial diversity. RAG recombinase activity is targeted to AgR loci by multiple chromatin-based mechanisms, including local modifications to AgR chromatin structure, large-scale conformational features and long-distance DNA contacts, and subnuclear compartmentalization of AgR loci. Our studies aim to reveal fundamental molecular mechanisms underpinning the developmental regulation of V(D)J recombination at T cell receptor (TCR) loci in developing thymocytes in vivo. Among the various AgR loci, the Tcra-Tcrd locus is unrivaled in its complexity, because it contains two sets of gene segments that undergo recombination at different stages of T cell development and contribute to the formation of TCR chains expressed by distinct subsets of T cells. Notably, a single allele can undergo multiple cycles of rearrangement, with initial Tcrd rearrangement followed by a succession of primary and secondary Tcra rearrangements, with each deletional rearrangement replacing the one prior. We have developed substantial insights into how this recombination program is enforced at the level of chromatin, and now seek to understand two major aspects of this program. First, what role does Tcrd recombination play in the development of a combinatorially diverse Tcra repertoire? Second, what is the role of the DNA damage response in pacing the succession of Tcra rearrangements? Temporal regulation is essential to allow thymocyte selection based on expression of TCR proteins before the rearranged Tcra gene is deleted by a subsequent Tcra recombination event. Among the TCR loci, only Tcrb is subject to allelic exclusion. This locus is also highly unusual in that it associates with the nuclear lamina (NL), a compartment generally considered repressive for transcription and V(D)J recombination. Yet the locus associates with the NL during the developmental stage when the locus is initially transcribed and undergoes rearrangement. Our recent work provides both a map and mechanistic insight into Tcrb-NL interactions. We now plan to leverage this knowledge to determine how association with the NL regulates Tcrb rearrangement and Tcrb allelic exclusion in developing thymocytes in vivo. We will address these questions by generation and analysis of novel strains of genetically manipulated mice.
V(D)J recombination is essential for the development of a functional adaptive immune system. Failure of V(D)J recombination can cause immunodeficiency and dysregulated V(D)J recombination can result in autoimmunity or chromosomal translocations that contribute to tumorigenesis. These studies will elucidate basic mechanisms that contribute to normal immune system development as well as pathologies such as immunodeficiency, autoimmunity and cancer.
