Normal B cell development depends on the proper assembly of immunoglobulin heavy- and light-chain genes from their component gene segments by the V(D)J recombinase. The process is regulated such that an individual mature B cell express a single antigen receptor specificity, a phenomenon known as allelic exclusion. Previous research has uncovered roles for transcriptional regulatory DNA sequences, chromatin structure, and the assembled Ig chains themselves in the regulation of V(D)J recombination. We propose a series of experiments aimed at understanding how the V(D)J recombinase is regulated during B cell development. RAG1 and RAG2 are the only two known essential lymphoid-specific components of the V(D)J recombinase. They are coordinately expressed only in B and T lymphocytes, where transcript levels vary at different stages of development. Inactivation of RAG gene expression is presumably essential for allelic exclusion and continued RAG gene expression is critical for receptor editing. We have identified key sequences and transcription factors necessary for activity of the murine RAG2 promoter. In addition, we have discovered a RAG locus transcriptional enhancer. We plan to determine the role of this novel enhancer in the developmental regulation of RAG gene expression, and identity additional DNA elements and transcription factors required for regulation of transcription of these important genes. Pre-B cells express high levels of V(D)J recombinase activity, yet they only rearrange one of their two allelic Ig kappa loci. We propose experiments to test the hypothesis that only a small traction of kappa alleles is accessible to the V(D)J recombinase in pre-B cells, making it very unlikely that a single pre-B cell would have two accessible alleles. Our experiments involve the analysis of a novel mouse model system that expresses a GFP cDNA from within a germline kappa transcript. In addition, we will use an experimental system in which overexpression of the transcription factor E47 activates germline kappa locus transcription and recombinase accessibility in a non-lymphoid cell to identity factors and target sequences responsible for the changes in kappa locus chromatin structure which lead to accessibility. In the final aim, we propose a series of preliminary experiments aimed at understanding the regulation of the VH-to-DJH rearrangement step in lymphoid development. We will a) scan the -90 kb interval between VHQ52 and DFL16.1 for DNA sequences involved in regulating VH-to-DJH rearrangement, b) determine whether a Pax-5 (BSAP)-expressing transgene can activate VH-to-DJH rearrangement in developing T cells and c) use a FISH assay to determine whether either of the two heavy-chain alleles in pro-B or pre-B cells is associated with heterochromatic regions of the nucleus
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