Genes encoding the heavy and light chains of antibody molecules are expressed only in B lymphocytes. This specificity arises, in part, due to the presence of regulatory DNA sequences known as enhancers. We have recently identified factors present in B lymphoid cells that recognize and bind to specific sequences within the mu and kappa enhancers. In this proposal I outline experiments to study the function of these proteins in B-cell- specific gene regulation.
The specific aims of the proposed experiments are: (i) to purify enhancer binding factors and subsequently clone the genes encoding them. Purifications will be carried out by a combination of ion exchange, gel filtration and affinity chromatographic steps and the purified factors will be used to generate specific antisera for cloning the genes. (ii) to develop a B-cell-specific in vitro transcription system to functionally characterize these factors. We will use enhancer mutations and biochemical reconstitutions to define the roles of specific factors in enhancer function. (iii) to further characterize the induction of the kappa enhancer binding factor, NF-kappa B, in non-B-cells and define the post- transcriptional modification leading to its generation. This will involve studying kappa enhancer function in appropriately stimulated non-B-cells using DNA transfection techniques. (iv) to study the role of the B-lymphocyte specific factors in specifying developmental activation of immunoglobulin genes by generating transgenic mice carrying hybrid enhancers. The combined biochemical and genetic approach is expected to provide a comprehensive view of the molecular mechanisms underlying B-lymphocyte-specific gene expression.
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