Transcriptional Mechanisms Governing Beta Cell Differentiation

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The absolute or relative deficiency of insulin secretion by the pancreatic beta cell underlies the pathogenesis of most forms of diabetes mellitus. Promising new approaches to the treatment or cure of diabetes will come from attempts recapitulate beta cell gene expression patterns in non-beta cell types. The long-range objective of this ongoing grant application is to define the biochemical mechanisms by which transcription factors direct gene expression in the developing and mature beta cell. In this application, we propose to extend upon our published studies of the mechanisms underlying the beta cell transcription factors Nkx6.1 and Pdx-1, and thereby merge established concepts in beta cell gene regulation with exciting and emerging themes in transcriptional complex formation and chromatin structure. Nkx6.1 and Pdx-1 are necessary for both the embryonic development and eventual function of beta cells. We hypothesize that these factors participate in key transcriptional complexes that are responsible for the remodeling of chromatin and the subsequent recruitment of basal transcriptional machinery. These effects result in either the activation or silencing of selective genes that lead to normal beta cell development and function. To test this hypothesis, our specific aims are directed toward a systematic analysis of the transcriptional complexes mediated by Pdx-1 and Nkx6.1 (Aim 1), the consequences of these complexes on chromatin structure (Aim 2), and their effects on the recruitment/activation of basal transcriptional machinery (Aim 3). Aim 1: Characterize transcriptional complexes involving Pdx-1 and Nkx6.1 and determine how they regulate target gene expression in the beta cell. Aim 2: Determine the role of Pdx-1 and Nkx6.1 complexes in modulating chromatin structure at target genes Aim 3. Determine the role of Pdx-1 and Nkx6.1 complexes in the recruitment of basal transcriptional machinery We propose to use a combination of biochemical assays and live cell imaging techniques in both cell lines and primary isolated islets to address each of these aims. We believe that the proposed studies will provide the frameworkfor elucidating the molecular events governing b cell development and function