The 5'-flanking region sequences located between-340 to -91 base pairs (bp) mediate pancreatic beta cell-specific and glucose-regulated transcription of the insulin gene. The C2 (-317/-311 bp)), A3 (-301 to - 196 bp), C1 (-118 and -107 bp), and E1 (-100 to -91 bp) elements are essential in control. The activators of insulin C2-, A3-, and E1- complement stimulated transcriptions are PAX6, PDX-1 and BETA2, respectively. Strikingly, gene ablation experiments performed have also clearly established an essential function in mice for each of these islet- enriched transcription factor during pancreatic development. In addition, heterozygous mutations in the Pdx-1 and BETA2 genes contribute to beta cell dysfunction in type 2 diabetes. These findings illustrate how the identification and characterization of the critical transcription regulators of islet-enriched products, like insulin, are essential for our understanding of the disease process affecting the beta cell. However, little is known about the protein(s) involved in C1 element stimulation. The RIPE3b1 factor DNA complex formed with these element in gel mobility shift assays is uniquely detected in beta cells extracts, and is selectively regulated in parallel with changes in insulin gene transcription in glucose- treated beta cells. These results strongly imply that the RIPE3b1 complex contains a DNA-binding protein(s) important in the physiological regulation of insulin transcription. The sequences comprising the C1 element are unrelated to those bound by any known transcription factor, strongly suggesting that RIPE3b1 will be a novel DNA-binding transcription. Our first objective will be to isolate and characterize the activation properties of the RIPE3b1 protein(s). Our second objective will be to examine how pancreatic development and islet beta cell function is affected in mice lacking RIPE3b1. These experiments will determine the role of the RIPE3b1 protein(s) in both cell-specific and glucose- modulated regulation of insulin gene transcription and during pancreaogenesis.
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