The regulated expression of genes during mammalian development is coordinated, in large part, by communication between proximal promoters and one or more tissue-specific enhancers, which may be separated by hundreds of kilobases. A major challenge in studies of gene regulation is to identify collections of active cis elements in each cell type (its cistrome) and establish promoter-enhancer connections critical for gene regulation in health and disease. In this regard, antigen receptor (AgR) loci serve as excellent models for deciphering many facets of mammalian regulatory circuits because they harbor multiple promoters that are differentially expressed during lymphocyte development. Moreover, the segmented nature of each locus is ideal for studies of local, regional, and long-range control by promoter-enhancer communication. These regulatory circuits guide the stepwise assembly of AgR genes by V(D)J recombination, which yields the diverse repertoire of immunoglobulin (Ig) and T cell receptors (TCRs) required for mammalian immunity. Major enhancers governing the activity of initial, short-range recombination events at each AgR locus have been identified. However, elements that coordinate activation and inactivation of large V segment arrays for long- range recombination remain enigmatic. As such, the cistrome governing AgR gene assembly and expression during the early stages of lymphocyte development are incomplete. The applicants' laboratories recently used a combination of genome-wide chromatin and computational analyses in B cell precursors to identify novel enhancers in each of the three Ig loci. The current project aims to understand how yet unidentified cis elements activate then decommission regions within TcR loci during thymocyte development, generating a primary TCR repertoire. For this purpose, experiments are proposed to (i) collect and computationally decipher chromatin data, pinpointing new regulatory regions within all four TcR loci, (ii) characterize the regulatory function of such elements and their fluctuations during thymocyte development, and (iii) define the activity of two novel elements in the assembly and expression of TcR genes. This project will provide a valuable resource for immunologists to decipher gene circuits altered during a key developmental transition of T lymphocytes. Moreover, the studies will reveal a comprehensive cistrome for TcR loci that underlies spatial, genetic, and epigenetic mechanisms to sculpt diverse repertoires of antigen receptors. Based on these discoveries, future studies will provide a panel of animal models, each with distinct TCR repertoires, which will enable completely new approaches to understand how altered V segment usage impacts immune responses to pathogens.
The packaging and spatial organization of DNA in the nucleus are key determinants of gene function. Many aspects of these 'epigenetic' programs are directed by regulatory elements, termed enhancers, which alter gene expression in health, aging, and disease. In addition, epigenetic control is essential for the proper generation of antigen receptor genes; defects in this assembly process can cause chromosomal translocations that lead to lymphoid tumors. We propose studies to identify new tissue- and stage-specific regulatory elements that orchestrate epigenetic changes required for normal antigen receptor gene assembly during thymocyte development.
