Ultimate goals of our research are to understand the molecular mechanisms underlying B lymphocyte development with particular emphasis on the role of specific transcription factors in the regulation of immunoglobulin heavy chain (IgH) gene expression. The primary objective of our research is to study the biological function of proteins that regulate B cell-specific gene expression during early stages of B cell development. Our studies currently focus on transcription factors binding to two IgH enhancer elements, muB and pi, whose activation we have implicated to be directly linked to B cell-specific expression of the IgH gene and of other Beta cell-specific genes. Particularly striking is the cell-and developmental-stage specificity of these two enhancer elements. In contrast to muB which is active throughout B cell development, pi enhances transcription only at early stages of B cell development prior to immunoglobulin kappa gene expression and is turned off upon maturation of B cells. The IgH gene serves as a well characterized marker for B cell differentiation. We are now purifying the transcription factor NF-pi which interacts with the pi enhancer element and we will clone the gene coding for this factor. Thus, the specific aims are to: 1) Characterize and purify the transcription factor NF-pi binding to the pi enhancer element. A. Purify NF-pi from B cells. B. Determine the molecular weight. C. Determine which other genes contain binding sites for NF-pi. D. Determine the consensus DNA-binding sequence for NF-pi. E. Examine whether purified NF-pi is able to induce transcription in vitro. 2) Isolate and characterize the murine gene encoding NF-pi. A. isolate the Nf-pi gene. B. Determine the functional DNA-binding, transactivation and potential dimerization domains of NF-pi by site-directed mutagenesis. C. Determine, if NF-pi binds as monomer or dimer to DNA. 3) Study the regulation of NF-pi activity. A. Analyze the tissue-and developmental-stage-distribution of NF-pi. B. Determine, if NF-pi is regulated at the transcriptional, translational or post-translational level. 4). Determine the role of NF-pi in the regulation of B cell specific gene expression and B cell differentiation. A. Test directly the role of NF-pi in IgH gene regulation by co- transfection experiments. B. Determine if NF-pi is involved in the transcriptional control of a whole set of B cell-specific genes. C. Test directly if NF-pi is involved in the transcriptional control of a whole set of B cell-specific genes by co-transfection experiments. D. Overexpress mutant NF-pi protein. To explore whether aberrant expression of NF-pi might lead to deregulated B cell differentiation we plan eventually to investigate the expression of NF-pi in a variety of clinical conditions involving deregulated B cell differentiation including B cell lymphomas, leukemias and inherited immunodeficiency syndromes. These experiments will help us 1) to understand the regulation of B cell development at the molecular level, and 2) to elucidate defects in B cell development which lead to deficient B cell differentiation and B cell tumorigenesis.
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