Transcriptional Regulation of Beta-Cell-Specific Expression of the MafA Gene

描述（由申请人提供）：朗格汉斯胰岛内的β细胞仅负责分泌胰岛素以维持适当的葡萄糖稳态。β细胞功能缺陷导致糖尿病，这种疾病影响着全世界数百万人。负责胰岛素分泌β细胞从祖细胞群分化的发育程序直到最近才变得更加清楚。我们对控制胰腺发育的因素的了解大多来自于对胰岛素和Pdx-1启动子的研究。从这项工作中，MafA转录因子因其调节胰岛素增强因子而被分离出来。与其他已知的β细胞富集转录因子相比，MafA的表达是独特的，在发育后期开始，仅在注定成为成年β细胞的祖细胞中表达。由于这种不寻常的表达，我们研究了MafA的5'转录控制域。该启动子有6个保守区，其中3区在哺乳动物、鸟类和爪蟾中保存最多。区域3也是唯一能够驱动β细胞系特异性报告基因表达的区域。已发表的研究表明，第3区至少部分由Pdx-1、Nkx2.2、MafB和FoxA2控制，但该保守结构域的大部分仍未被表征。特异性目标1将使用无偏诱变方法和报告基因测定来定义3区大部分剩余的保守顺式序列。特异性目标2将使用包括ChIP和凝胶转移分析在内的技术确定结合新元件的因素。这些研究可能会发现对细胞功能至关重要的新因素。几条线索的证据表明，在发育期间和成人中，因子结合的直接独特表达模式的时间变化。特异性目标3提出了跨MafA启动子的结合因子和表观遗传标记作为时间函数的研究。这将需要ChIP使用从不同发育阶段和成年阶段的小鼠胰腺中分离的染色质。公共卫生相关性：糖尿病是由β细胞功能丧失引起的主要健康问题；目前的胰岛素替代并不能准确地概括内源性血糖控制。未来的治疗方法可能包括操纵细胞祖细胞成为移植治疗a的功能性β细胞

DESCRIPTION (provided by applicant): The beta cells within the pancreatic islets of Langerhans are solely responsible for secreting insulin for maintaining proper glucose homeostasis. Defects in beta cell function result in diabetes mellitus, a condition affecting millions of people worldwide. The developmental programs responsible for the differentiation of an insulin-secreting beta cell from progenitor populations have only recently become clearer. Much of what is known about the factors controlling pancreas development stem from studies of the Insulin and Pdx-1 promoters. From this work, the MafA transcription factor was isolated due to its regulation of the Insulin enhancer. MafA expression is unique as compared to any other known beta cell enriched transcription factor, beginning later in development and solely in progenitors fated to become adult beta cells. Because of this unusual expression, the 5' transcriptional control domains of MafA were studied. This promoter has six conserved regions, with Region 3 retaining the most conservation in mammals, birds and Xenopus. Region 3 is also the only domain able to drive beta cell-line specific reporter expression. Published studies demonstrate that Region 3 is at least partially controlled by Pdx-1, Nkx2.2, MafB, and FoxA2, however most of this conserved domain is still uncharacterized. Specific Aim 1 will define most of the remaining conserved cis-sequences of Region 3 using an unbiased mutagenesis approach and reporter gene assays. Specific Aim 2 will identify the factors that bind the novel element(s) using techniques including ChIP and gel-shift analyses. These studies may uncover new factors essential for beta cell function. Several lines of evidence suggest temporal changes in factor binding direct unique expression patterns during development and in adults. Specific Aim 3 proposes a study of binding factors and epigenetic marks across the MafA promoter as a function of time. This will require ChIP using chromatin isolated from mouse pancreata at different developmental and adult stages. PUBLIC HEALTH RELEVANCE: Diabetes is a major health concern that results from a loss of beta cell function; and current insulin replacement does not accurately recapitulate endogenous blood glucose control. Future therapies may involve the manipulation of cell progenitors to become functional beta cells for transplantation to treat a growing number of patients. Studies aimed at understanding the regulation of beta cell genes, like these with MafA, may increase the probability of developing replacement beta cells for improved diabetes treatment.