FACTORS IMPORTANT IN BETA2 TARGETED ACTIVATION

Beta2是独立分离的，其特征是能够 在转基因的Beta细胞中激活胰岛素报告基因的转录 (称为Beta2)，以及在非洲爪哇异位表达时形成的轴突 胚胎(称为NeuroD1)。这种基本螺旋-环-螺旋(BHLH)转录 系数将称为Beta2。它在胰岛中表达 内分泌细胞，肠，脑下垂体和神经元的一个子集 中枢和外周神经系统。有趣的是， 表达胰岛素的Beta细胞在Beta2-/-小鼠中严重减少，并且 其余的内分泌细胞未能形成胰岛。这些动物发育成 早发性糖尿病和围产期死亡。神经系统看起来似乎 在Beta2-/-小鼠中正常发育，推测是由于存在 补偿系数(S)。总体而言，这些结果表明Beta2 在胰岛素基因转录中起着重要的调节作用 胰岛β细胞和胰岛必需基因(S)的表达 差异化。不幸的是，与肌源性或成脂性不同 系统中，不是可用于研究胰岛细胞的细胞系 差异化。然而，作为内分泌胰腺的祖细胞， 表达对神经外胚层和神经外胚层都至关重要的转录因子 和胰岛内分泌分化，包括ISL-1和PAX6，我们推测 非洲爪哇的神经发生分析可以提供对 β2在胰腺发育过程中的作用机制。作为一名 结果，我们一直在描述Beta2中的序列 是刺激胰岛素基因转录和神经发生所必需的 差异化。我们的研究结果已经证明，在进化过程中 跨越bHLH(氨基酸100-155)和C-末端的保守序列 (氨基酸156-355)区域对这两个过程都很重要。 对C-末端区域的缺失分析表明，调控的 激活由BETA2的两个独立和可分离的结构域介导， 它跨越156-251和252-355两个氨基酸。P300/CBP共激活子是 显示与猪瘟病毒156-251和300-355个氨基酸区域相互作用 Beta2.此外，我们最近还表明，Beta2：P300/CBP可以 激活编码细胞周期蛋白依赖性蛋白激酶基因的表达 抑制剂p21，提示这一关键细胞周期的上调 胰岛的正常进展可能需要调节剂 差异化计划。下面提出的实验将测试 假设Beta2介导的激活涉及招募 P300/CBP，一个增强重要调节器活性的因素 细胞的增殖和分化。

BETA2 was independently isolated and characterized by its ability to activate insulin reporter gene transcription in transfected Beta cells (termed BETA2), and neurite formation upon ectopic expression in Xenopus embryos (termed NeuroD1). This basic helix-loop-helix (bHLH) transcription factor will be referred to as BETA2. It is expressed in pancreatic islet endocrine cells, the intestine, the pituitary, and a subset of neurons in the central and peripheral nervous system. Interestingly, the number of insulin-expressing Beta cells was severely reduced in BETA2-/- mice, and the remaining endocrine cells failed to form islets. These animals develop early-onset diabetes and die perinatally. The nervous system appears to develop normally in BETA2-/- mice, presumably due to the presence of a compensatory factor(s). Collectively, these results suggested that BETA2 played an important regulatory role in transcription of the insulin gene in islet Beta cells, as well as for gene(s) required for pancreatic islet differentiation. Unfortunately, in contrast to the myogenic or adipogenic systems, the are not cell lines available to study islet cell differentiation. However, as progenitors cells of the endocrine pancreas express transcription factors that are essential for both neuroectodermal and islet endocrine differentiation, including ISL-1 and PAX6, we reasoned that the neurogenesis assay in Xenopus could provide insight into the mechanisms utilized by BETA2 during pancreatic development. As a consequence, we have been characterizing the sequences within BETA2 that are required for stimulating insulin gene transcription and neurogenic differentiation. Our results have demonstrated that evolutionarily conserved sequences spanning the bHLH (amino acids 100-155) and C-terminal (amino acids 156-355) regions are important for both of these processes. Deletional analysis of the C-terminal region indicate that regulated activation is mediated by two independent and separable domains of BETA2, which span amino acids 156-251 and 252-355. The p300/CBP co-activator was shown to interact with the 156-251 and 300-355 amino acid regions of BETA2. In addition, we have recently shown that BETA2:p300/CBP can activate expression of the gene-encoding the cyclin-dependent kinase inhibitor p21, suggesting that up-regulation of this key cell cycle regulator may be required for normal progression of the islet differentiation program. The experiments proposed below will test the hypothesis that BETA2-mediate activation involves the recruitment of p300/CBP, a factor that potentiates the activity of important regulators of cellular proliferation and differentiation.