PDX-1 REGULATION OF PANCREAS DEVELOPMENT/DIFFERENTIATION

1型和2型糖尿病的一个特征是胰岛β细胞群不能产生足够的 胰岛素以满足新陈代谢需求。PDX-1(胰腺十二指肠同源盒-1)是一个同源结构域 转录因子：在控制β蛋白发育的转录层次中起关键作用的转录因子 细胞团。虽然PDX-1的纯合子突变会导致小鼠和人类的胰腺发育不全， PDX-1杂合基因突变损害了β细胞的功能和存活，导致糖耐量异常和 老鼠和人类的糖尿病(早期[MODY4]和迟发性2型糖尿病)。PDX-1单倍体不足 显著损害几种胰岛素抵抗小鼠模型的胰岛代偿反应，原因是 胰岛素分泌和/或β细胞质量扩张和β细胞存活受损。还有很多东西需要学习 关于PDX-1调控基因转录的分子机制及相关的PDX-1 调节其在β细胞上的关键作用的靶点。我们假设PDX-1C终端，也就是 家族性人类2型糖尿病突变是胎儿晚期/早期正常BEA细胞增殖所必需的 新生儿期。此外，我们假设，在成人β细胞中识别PDX-1靶标将给出 2型糖尿病患者胰岛代偿机制的研究。这些假设将是 测试如下：目标1：确定PDX-1C末端的体内和分子作用：我们将 PDX-1C-/-小鼠过早表达缺失C-末端的PDX-1的特征 通过评估谱系规范、贝塔细胞增殖和存活、贝塔细胞身份的关键标记， 和胰岛素分泌。生物化学实验将解决C末端在蛋白质周转中的作用， 亚细胞定位和靶向启动子占有率。C末端的点突变和一种快速的 将对斑马鱼的选定突变进行生物筛选。目的2：鉴定PDX-1转录 成体β细胞中的靶点。我们将鉴定一种位于基因上游的新型PDX-1增强子 与PDX-1的出生后功能高度相关，我们将探讨其在介导β细胞营养中的作用 GLP-1和In在胰岛素抵抗中的代偿作用此外，我们还将进行“芯片上的芯片” 用成年小鼠胰岛染色质进行分析。一种互补的生物信息学方法将集中于 受PDX-1：PBX异源二聚体调控的PDX-1增殖相关靶基因的子集。 总之，拟议的研究将解决有关PDX-1在β细胞中的功能的重要问题 生物学，尤其与其在胰岛素抵抗的β细胞补偿中的作用有关。

A hallmark of both type 1 and type 2 diabetes is the failure of pancreatic beta cell mass to produce sufficient insulin to meet metabolic demands. PDX-1 (Pancreas Duodenal homeoboX-1) is a homeodomain transcription factor that is pivotally positioned in the transcriptional hierarchy governing the development of beta cell mass. Whereas homozygous mutation of PDX-1 causes pancreatic agenesis in both mice and humans, heterozygous PDX-1 gene mutations impair beta-cell function and survival, leading to glucose intolerance and diabetes in mice and in humans (early [MODY4]- and late-onset type 2 diabetes). PDX-1 haploinsufficiency markedly impairs the islet compensatory response in several insulin resistance mouse models, due to impaired insulin secretion and/or beta cell mass expansion and beta cell survival. Much remains to be learned about the molecular mechanisms whereby PDX-1 regulates gene transcription and about the relevant PDX-1 targets that mediate its critical actions on the beta cell. We hypothesize that the PDX-1 C terminus, which is mutated in familial human type 2 diabetes, is required for normal bea cell expansion in the late fetal/early neonatal period. Further, we hypothesize that the identification of PDX-1 targets in the adult beta cell will give insight into mechanisms of islet compensation in patients with type 2 diabetes. These hypotheses will be tested as follows: Aim 1: Determine the in vivo and molecular role of the PDX-1 C-terminus: We will characterize PDX-1 Cterm-/- mice that express a prematurely truncated PDX-1 lacking the C-terminal domain by assessing lineage specification, beta cell proliferation and survival, critical markers of beta cell identity, and insulin secretion. Biochhemical experiments will address the role of the C-terminus in protein turnover, subcellular localization and target promoter occupancy. Point mutagenesis of the C-terminus and a rapid biological screen of selected mutations in zebrafish will be carried out. Aim 2: Identify PDX-1 transcriptional targets in the adult beta cell. We will characterize a novel PDX-1 enhancer located upstream of a gene with high relevance to the postnatal functions of PDX-1, and we will explore its role in mediating the beta cell trophic effects of GLP-1 and in islet compensation for insulin resistance. Further, we will perform a "ChIP on chip" analysis using adult mouse pancreatic islet chromatin. A complementary bioinformatics approach will focus on a subset of PDX-1 proliferation related target genes that are regulated by PDX-1: PBX heterodimers. Together, the proposed studies will address important questions about the functions of PDX-1 in beta cell biology, with particular relevance to its role in beta cell compensation for insulin resistance.