The Regulation of Antigen Receptor Gene Rearrangement and Lymphocyte Development. Immunoglobulin (Ig) and T cell receptor (TCR) genes are assembled during lymphocyte development from their component gene segments by a novel series of highly regulated DNA rearrangement reactions known as V(D)J recombination. (Figure 1) Understanding the regulation of gene rearrangement is important because its products (Ig and TCR molecules) control the development and function of B and T cells and because errors in the process can result in leukemia, lymphoma, or immunodeficiency disease. (Figure 2) A single enzyme complex known as the V(D)J recombinase is required for the rearrangement of each of the seven complex genetic loci encoding antigen receptor chains. The recombinase recognizes a highly conserved DNA sequence, the RSS, and introduces a dsDNA break. The DNA break repair machinery expressed in all cells then generates various molecular joints. (Figure 3) Our data suggests that transcription or transcription factor binding targets the recombination reaction to a particular genetic locus at the appropriate stage of development. My lab is interested in determining the molecular basis of this link between transcription and gene rearrangement during B cell development and in understanding how a common recombinase recognizing a conserved DNA element can result in highly regulated patterns of gene rearrangement. (Figure 4) In addition, we are interested in the reaction mechanism of the V(D)J recombinase.
Using bone marrow, thymus and fetal liver from wild-type or recombination-deficient Ig or TCR transgenic mice, we can purify primary lymphoid cells "frozen" at different stages of development. In addition, we can use gene targeting technologies in embryonic stem cells to introduce specific mutations into key genes. We are analyzing signal transduction pathways, patterns of transcription factor activity and chromosomal DNA-protein interactions to determine the critical receptors, protein factors and DNA sequences involved in the regulation of B and T cell development and Ig and TCR gene rearrangement. We have also devised a series of novel assays which detect recombination reaction intermediates and are using them to study both the developmental regulation and reaction mechanism of the V(D)J recombinase. (Figure 5) Recently, we devised an in vitro assay system which recapitulates the developmental regulation of V(D)J recombination and are engaged in the dissection of its molecular components. (Figure 6)
Active projects in the lab include 1) an analysis of the influence of chromatin structure on the targeting of V(D)J recombination; 2) reconstitution of regulated V(D)J recombination in vitro using purified recombinase components and long DNA templates; 3) promoter and enhancer activities involved in the transcriptional regulation of genes encoding the V(D)J recombinase (RAG1 and RAG2); 4) the use of gene targeting to determine the function of various domains of RAG1 and RAG2; 5) identifying transcription factors and DNA sequences involved in the regulated activation of rearrangement at the Ig kappa locus and TCR beta locus; 6) determining the role of transcriptional enhancer sequences in the targeting of recombination and in coding joint formation; 7) elucidating the rules governing receptor editing; 8) studying the mechanism and determining the frequency of transposition events catalyzed by the recombinase during lymphocyte development; and 9) determining the role of the c-Abl protein tyrosine kinase in lymphocyte development and cellular transformation