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

描述(申请人提供)：朗格汉斯人胰岛内的β细胞只负责分泌胰岛素，以维持适当的葡萄糖稳态。β细胞功能缺陷会导致糖尿病，这是一种影响全球数百万人的疾病。负责将胰岛素分泌的β细胞从祖细胞群体中分化出来的发育程序直到最近才变得更加清晰。已知的控制胰腺发育的因素大多来自对胰岛素和PDX-1启动子的研究。从这项工作中，由于其对胰岛素增强子的调节，MafA转录因子被分离出来。与任何其他已知的β细胞丰富转录因子相比，MafA的表达是独特的，始于发育后期，仅在注定要成为成年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，其中第3区在哺乳动物、鸟类和非洲爪哇中保留了最保守的区域。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，区域3至少部分受PDX-1、Nkx2.2、MafB和FOXA2控制，但这个保守结构域的大部分仍未确定。特定目标1将使用无偏倚突变方法和报告基因分析来定义区域3剩余的大部分保守顺式序列。具体目标2将使用包括芯片和凝胶位移分析在内的技术来确定结合新元件(S)的因素。这些研究可能会发现对β细胞功能至关重要的新因素。一些证据表明，在发育过程中和成人中，因子结合的时间变化直接导致独特的表达模式。具体目标3提出了一项关于MafA启动子上的结合因子和表观遗传标记随时间变化的研究。这将需要使用从不同发育和成年阶段的小鼠胰腺中分离出的染色质来制作芯片。与公共健康相关：糖尿病是一个主要的健康问题，它是由β细胞功能丧失引起的；目前的胰岛素替代治疗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前体细胞，使其成为具有功能的β细胞，用于移植治疗 越来越多的病人。旨在了解β细胞基因调控的研究，如这些与MafA相关的研究，可能会增加开发替代β细胞以改善糖尿病治疗的可能性。

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.