PDX-1 REGULATION OF PANCREAS DEVELOPMENT/DIFFERENTIATION

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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.