IDENTIFICATION/CHARACTERIZATION OF RIPE3B1

位于-340至-91碱基对（bp）之间的5'侧区序列介导胰腺β细胞特异性和葡萄糖调节的胰岛素基因转录。C2 （-317/-311 bp）、A3 （-301 ~ - 196 bp）、C1 （-118 ~ -107 bp）和E1 （-100 ~ -91 bp）是控制中必不可少的元素。胰岛素C2-、A3-和E1-补体刺激转录的激活因子分别是PAX6、PDX-1和BETA2。引人注目的是，进行的基因消融实验也清楚地确定了这些富含胰岛的转录因子在胰腺发育过程中的重要功能。此外，Pdx-1和BETA2基因的杂合突变有助于2型糖尿病的β细胞功能障碍。这些发现说明了胰岛富集产物（如胰岛素）的关键转录调节因子的鉴定和表征对于我们理解影响β细胞的疾病过程至关重要。然而，关于参与C1元件刺激的蛋白知之甚少。在凝胶迁移转移实验中，与这些元素形成的RIPE3b1因子DNA复合物在β细胞提取物中被唯一检测到，并且在葡萄糖处理的β细胞中，与胰岛素基因转录的变化同时被选择性调节。这些结果强烈暗示RIPE3b1复合体含有在胰岛素转录的生理调节中重要的dna结合蛋白。包含C1元件的序列与任何已知转录因子结合的序列无关，强烈表明RIPE3b1将是一种新的dna结合转录。我们的第一个目标将是分离和表征RIPE3b1蛋白的激活特性。我们的第二个目标是研究缺乏RIPE3b1的小鼠的胰腺发育和胰岛β细胞功能是如何受到影响的。这些实验将确定RIPE3b1蛋白在细胞特异性和葡萄糖调节胰岛素基因转录和胰腺发生过程中的作用。

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.