Transcriptional co-regulators in pancreas development

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DESCRIPTION (provided by applicant): A hallmark of type 1 and type 2 diabetes is the failure of pancreatic beta cells to produce sufficient insulin to meet the body's needs. The molecular mechanisms regulating the development and maintenance of functional beta-cell mass are thus highly relevant to the development of effective preventive and therapeutic interventions for all forms of diabetes. PDX-1 is a Hox type homeodomain-containing transcription factor that is pivotally positioned in the transcriptional hierarchy governing beta cell development. Absence of PDX-1 causes pancreatic agenesis, and heterozygous PDX-1 gene mutations cause abnormal glucose tolerance and diabetes in mice as well as in humans (early [MODY4]- and late-onset type 2 diabetes). We have identified a novel nuclear POZ domain protein, PCIF1, which interacts with PDX- 1 and inhibits its ability to activate transcription. PCIF1 is expressed in the developing endocrine pancreas and is coexpressed with PDX-1 in adult islet beta cells. Over-expression of PCIF1 in beta cells diminishes insulin promoter activity, suggesting that PCIF1 expression level and/or its ability to interact with PDX-1 could regulate insulin gene transcription. Mapping studies indicate that inhibition by PCIF1 is mediated by a short conserved peptide sequence in the PDX-1 C-terminus and that the PCIF1 POZ domain is critically required. We hypothesize that PCIF1 interacts with a conserved peptide module in the PDX-1 C-terminus, possibly recruiting a repressor complex to the promoter of PDX-1 target genes, and thereby influencing beta cell development, growth and/or function. This overall hypothesis will be directly tested in the following Specific Aims: Aim 1: To determine the biological role(s) of PCIF1 in beta cells. We will determine (a) the embryonic and postnatal expression pattern of PCIF1 (b) whether PCIF1 regulates beta cell differentiation +/or growth using adenoviral vectors to deliver PCIF1, PCIF1 siRNA and dominant negative (DN) PCIF1 to MIN6 insulinoma beta cells (c) the role of PCIF1 in pancreas development and glucose homeostasis by creating null and conditional mouse PCIF1 alleles. Aim 2: To determine the biochemical basis and biological role for the binding of PDX-1 to PCIF1. We will (a) test the sufficiency of the conserved peptide module to confer PCIF1 interaction and inhibition and identify the key residues involved (b) examine the biological role of the conserved PDX-1 C-terminal module in zebrafish pancreas development and (c) characterize the zebrafish PCIF1 morphant. Aim 3: To determine the mechanism of PCIF1 inhibition of PDX-1. We will (a) identify the key residues for interaction with and inhibition of PDX-1 (b) determine whether PCIF1 inhibits PDX-1 transactivation through an effect(s) on DNA binding, nuclear localization, and/or co-activator/co-repressor recruitment (c) determine whether PCIF1 regulates histone acetylation at the insulin and other PDX-1 target promoters and (d) determine whether PCIF1 recruits co-repressors and/or histone deacetylases via its POZ domain. Together, the proposed studies will address major questions about the function of PDX-1 in beta cell biology. Insight gained from this could have important implications for novel treatments of diabetes focused on beta cell neogenesis from stem cells, transdifferentiation of beta cells from other cell types, and for gene therapy approaches to enhance beta cell number and survival.