Targeting of somatic hypermutation and gene conversion

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DESCRIPTION (provided by applicant): Somatic hypermutation (SM) and gene conversion (GC) are mutagenic reactions responsible for the post-assembly diversification of immunoglobulin (Ig) variable regions. They play critical roles in B cell development and effective humoral immunity. SM and GC are mechanistically very similar, sharing a requirement for transcription and the activation induced deaminase (AID) and likely a common initiating DNA lesion. A fundamental gap in our understanding of SM and GC concerns the mechanism by which they are targeted to Ig genes. This issue has important clinical implications because mistargeting of SM has been linked to oncogene mutations and chromosomal translocations in B cell malignancies. Our long term goal is to determine the molecular mechanism that targets SM and GC to Ig genes. Two central hypotheses form the foundation for this proposal: first, that targeting of SM and GC relies on specific cis-acting DNA sequences (targeting elements) that reside in the Ig loci and second, that the targeting function of these elements is circumscribed, or bounded, so that adjacent genes are not affected. The specific aims of this proposal are to identify these targeting elements and to determine the boundaries of the chromosomal domain over which they act. We have chosen the Ig light chain locus (IgL) in the chicken B cell line DT40 as our experimental system for several reasons: 1) this locus is very compact and can undergo both SM and GC; 2) DT40 allows rapid and facile manipulation of the genome by homologous recombination; and, 3) we have created novel DT40 cell lines in which the normal IgL promoter has been replaced by a strong, enhancer-independent promoter (from the human elongation factor 1a gene). The targeted locus is transcribed at high levels and undergoes efficient GC, allowing us to distinguish the potential targeting activity of DNA elements in the IgL locus from their transcriptional regulatory activity. Using these new DT40 cell lines, we will systematically delete portions of the IgL locus to identify targeting elements. In addition, we will insert a mutational cassette at various positions in the wild type IgL locus to identify the boundaries of the mutationally active domain. The results of these experiments should set the stage for significant progress toward an understanding of the mechanism by which protein factors and DNA sequences collaborate to target SM and GC to Ig genes.