REGULATION OF IMMUNOGLOBULIN GENE EXPRESSION IN B CELLS

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The immunoglobulin mu heavy chain gene enhancer activates transcription and recombination in differentiating B lymphocytes. Enhancer activity is mediated by several DNA binding proteins which assemble into a precise multiprotein complex on the enhancer. The long-term objectives of this program is to understand the molecular mechanism of enhancer function. Two aspects of enhancer function will be addressed in this application. First, the mechanisms of combinatorial transcription control will be examined (Specific Aim 1). Combinatorial control refers to a central characteristic of enhancers, whereby the properties of an enhancer are very different from the properties of individual DNA binding proteins that interact with the enhancer. Biochemical and genetic experiments are proposed to understand the mechanisms by which distal sites on the enhancer cooperate to activate transcription and recombination. Second, the mechanism of chromatin structure modulation by the enhancer will be studied in-vitro (Specific Aim 2) and in B lymphocytes (Specific Aim 3). In-vitro chromatin assembly will be used to examine the effects of individual, and combinations, of mu enhancer binding proteins. Structural consequences on nucleosome positioning and chromatin structure will be assessed by DNAase I and micrococcal nuclease digestion assays. In parallel, functional consequences of chromatin re-structuring will be evaluated by in-vitro transcription and recombination assays. Lastly, the chromatin structure of the unrearranged IgH locus in pre-B cells will be mapped by DNAase I, micrococcal nuclease and histone acetylation. The limits of locus accessibility will be defined and the contribution of important regulatory elements will be determined by analyzing cells in which the sequences have been deleted. Taken together, these studies will provide comprehensive insights into the function of the mu enhancer. In-depth understanding of tissue-specific gene regulation will be essential to develop approaches to selectively reduce aberrant gene expression in diseased states and, conversely, to enhance gene expression to overcome immunodeficiencies. The rules of combinatorial transcription activation will also allow the design of novel transcription regulatory sequences to direct therapeutic expression of exogenous genes in selected cells.