B cells develop from bone-marrow derived precursors via discrete intermediates that have been characterized by the expression of genes encoding immunoglobulins (Ig) and other cell-surface markers. Early expression of the Ig heavy chain gene in pre-B cells is governed primarily by a transcription regulatory element - the mu enhancer. Our long-term goals are (i) to identify and study the regulation of critical factors that activate this enhancer in pre-B cells, as a means of investigating B cell differentiation prior to this stage and (ii) to study the multiple protein DNA interactions that occur on an enhancer, in order to elucidate the mechanisms of enhancer function. Two lymphoid-specific proteins, ets-1 and PU.1 are likely to be critical determinants of mu enhancer activity. These two factors will be over- expressed in bacteria, purified and used for in-vitro binding studies to analyze protein-DNA and protein-protein interactions on a minimal mu enhancer fragment. In addition, to gain insights into early B cell differentiation, regulation of the PU.1 gene will be studied by a combination of DNAase 1 hypersensitive site mapping, transfection analysis and in-vitro binding assays. The transcription factor NF-kappaB, which is necessary for the constitutive expression of Ig kappa genes as well as inducible expression of several other immune system genes, belong to a family of proteins that are homologous to the proto-oncogene c-rel. The function of induced NF-kappaB- like factors during cellular activation is likely to be important in understanding the role of altered c-rel protein leading to transformation. To study the normal functions of these factors, the subunit compositions of NF-kappaB-like proteins induced by various stimuli will be assessed by immunoprecipitation assays using an antiserum we have produced. Furthermore, the functional properties of the different factors will be studied by transfection experiments into lymphoid and non-lymphoid cell lines. The combined biochemical and genetic experiments are expected to generate valuable insights into processes involved in immunodifferentiation and oncogenesis by rel and ets related proteins.
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