Transcriptional Mechanisms Governing Beta Cell Differentiation

描述(申请人提供)：胰岛β细胞胰岛素分泌的绝对或相对不足是大多数形式的糖尿病发病的基础。有希望的治疗或治愈糖尿病的新方法将来自于概括非β细胞类型的β细胞基因表达模式的尝试。这项正在进行的赠款申请的长期目标是确定转录因子在发育中和成熟的β细胞中指导基因表达的生化机制。在这一应用中，我们建议扩展我们发表的关于β细胞转录因子Nkx6.1和PDX-1潜在机制的研究，从而将β细胞基因调控中的既定概念与转录复合体形成和染色质结构中令人兴奋的和新兴的主题相结合。Nkx6.1和PDX-1对于胚胎发育和β细胞的最终功能都是必需的。我们假设这些因子参与了关键的转录复合体，这些转录复合体负责染色质的重塑和随后基础转录机制的招募。这些效应导致选择性基因的激活或沉默，这些基因导致正常的β细胞发育和功能。为了验证这一假设，我们的具体目标是系统分析由PDX-1和Nkx6.1介导的转录复合体(目标1)，这些复合体对染色质结构的影响(目标2)，以及它们对基本转录机械的招募/激活的影响(目标3)。目的1：鉴定涉及PDX-1和Nkx6.1的转录复合体，并确定它们如何调节β细胞中靶基因的表达。目的2：确定PDX-1和Nkx6.1复合体在调控靶基因染色质结构中的作用。目的3：确定PDX-1和Nkx6.1复合体在基础转录机制招募中的作用。我们建议在细胞系和原代分离的胰岛中使用生化分析和活细胞成像技术的组合来解决这些目标。我们相信，拟议的研究将为阐明控制细胞发育和功能的分子事件提供框架。

DESCRIPTION (provided by applicant): The absolute or relative deficiency of insulin secretion by the pancreatic beta cell underlies the pathogenesis of most forms of diabetes mellitus. Promising new approaches to the treatment or cure of diabetes will come from attempts recapitulate beta cell gene expression patterns in non-beta cell types. The long-range objective of this ongoing grant application is to define the biochemical mechanisms by which transcription factors direct gene expression in the developing and mature beta cell. In this application, we propose to extend upon our published studies of the mechanisms underlying the beta cell transcription factors Nkx6.1 and Pdx-1, and thereby merge established concepts in beta cell gene regulation with exciting and emerging themes in transcriptional complex formation and chromatin structure. Nkx6.1 and Pdx-1 are necessary for both the embryonic development and eventual function of beta cells. We hypothesize that these factors participate in key transcriptional complexes that are responsible for the remodeling of chromatin and the subsequent recruitment of basal transcriptional machinery. These effects result in either the activation or silencing of selective genes that lead to normal beta cell development and function. To test this hypothesis, our specific aims are directed toward a systematic analysis of the transcriptional complexes mediated by Pdx-1 and Nkx6.1 (Aim 1), the consequences of these complexes on chromatin structure (Aim 2), and their effects on the recruitment/activation of basal transcriptional machinery (Aim 3). Aim 1: Characterize transcriptional complexes involving Pdx-1 and Nkx6.1 and determine how they regulate target gene expression in the beta cell. Aim 2: Determine the role of Pdx-1 and Nkx6.1 complexes in modulating chromatin structure at target genes. Aim 3: Determine the role of Pdx-1 and Nkx6.1 complexes in the recruitment of basal transcriptional machinery. We propose to use a combination of biochemical assays and live cell imaging techniques in both cell lines and primary isolated islets to address each of these aims. We believe that the proposed studies will provide the framework for elucidating the molecular events governing ¿ cell development and function.