Antibody Production by B cells employs 3 separate types of genetic alterations. The variable region exon of immunoglobulin (Ig) heavy (IgH) and light (IgL) chain genes is assembled from germline V, D, and J segments during early B cell development. V(D)J recombination is initiated by the RAG-1/2 endonuclease, which introduces DNA double strand breaks (DSBs) at participating V, D, and J segments, and is completed by non-homologous end-joining (NHEJ) proteins that seal the RAG-generated DSBs. In response to antigen, mature B cells change the IgH constant region (CH) expressed with a particular variable region via a distinct recombination process termed class switch recombination (CSR). At this stage, the variable region coding sequence may be further altered by somatic hypermutation (SM). While the nature of the enzymes that initiate CSR or SM remains elusive, both processes, like V(D)J recombination, may involve DSBs and thus, employ NHEJ. We have shown that defective NHEJ during attempted V(D)J recombination in a p53 tumor-suppressor deficient background reproducibly leads to murine pro-B cell lymphomas that harbor translocations between the IgH and c-myc loci. We now propose to elucidate further the role of NHEJ proteins in CSR and SM, with a particular focus on how defects in these processes, and in V(D)J recombination, may lead to the generation of chromosomal translocations that contribute to the generation and maintenance of mature B cell lymphomas. To focus potential oncogenic mutations on mature, as opposed to progenitor B cells, we will employ several strategies, including conditional gene targeted mutation to inactivate NHEJ or activate specific oncogenes in mature B cells. Also, we will insert V(D)J recombination signal sequences, CSR target sequences (S regions), expressed variable region gene sequences, or meganuclease restriction enzyme (I-Sce 1) target sequences adjacent to the c-myc, bcl-2 or bcl-6 genes within various NHEJ or checkpoint-deficient ES cells to test for ability to stimulate translocations in appropriate B lineage cell populations. To eliminate large-scale breedings and facilitate rapid assays, we will introduce certain compound mutations into ES cells and assay effects on translocations and lymphomagenesis via Rag-2 Deficient Blastocyst Complementation. Once desired mature B cell lymphoma models are developed, we will employ gene-targeted mutations to determine the role of IgH expression, IgH enhancers, and particular oncogenes in the generation and maintenance of the transformed phenotype.
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