Transcriptional co-regulators in pancreas development

描述（由申请人提供）：1型和2型糖尿病的一个标志是胰腺β细胞不能产生足够的胰岛素来满足身体的需要。因此，调节功能性β细胞群的发育和维持的分子机制与开发针对所有形式的糖尿病的有效预防和治疗干预措施高度相关。PDX-1是一种含有Hox型同源结构域的转录因子，其在调控β细胞发育的转录层次中枢转定位。PDX-1缺失导致胰腺发育不全，杂合PDX-1基因突变导致小鼠和人类的糖耐量异常和糖尿病（早期[MODY 4]和迟发性2型糖尿病）。我们已经确定了一种新的核POZ结构域蛋白，PCIF 1，它与PDX- 1相互作用，并抑制其激活转录的能力。PCIF 1在发育中的内分泌胰腺中表达，并在成人胰岛β细胞中与PDX-1共表达。β细胞中PCIF 1的过表达降低了胰岛素启动子活性，表明PCIF 1表达水平和/或其与PDX-1相互作用的能力可以调节胰岛素基因转录。图谱研究表明，PCIF 1的抑制作用是由PDX-1 C-末端的一个短的保守肽序列介导的，并且PCIF 1 POZ结构域是关键所需的。我们假设PCIF 1与PDX-1 C-末端的保守肽模块相互作用，可能将阻遏物复合物募集到PDX-1靶基因的启动子，从而影响β细胞发育、生长和/或功能。这一总体假设将在以下具体目标中直接进行检验：目标1：确定PCIF 1在β细胞中的生物学作用。我们将确定（a）PCIF 1的胚胎和出生后表达模式（B）PCIF 1是否调节β细胞分化+/或生长，使用腺病毒载体将PCIF 1、PCIF 1 siRNA和显性阴性（DN）PCIF 1递送至MIN 6胰岛素瘤β细胞（c）通过产生无效和条件性小鼠PCIF 1等位基因，PCIF 1在胰腺发育和葡萄糖稳态中的作用。目的2：探讨PDX-1与PCIF 1结合的生物学基础和生物学作用。我们将（a）测试保守肽模块赋予PCIF 1相互作用和抑制的充分性，并鉴定所涉及的关键残基（B）检查保守PDX-1 C-末端模块在斑马鱼胰腺发育中的生物学作用，以及（c）表征斑马鱼PCIF 1 morphant。目的3：探讨PCIF 1抑制PDX-1的作用机制。我们将（a）鉴定与PDX-1相互作用和抑制PDX-1的关键残基（B）确定PCIF 1是否通过对DNA结合、核定位、和/或共激活物/共阻遏物募集（c）确定PCIF 1是否调节胰岛素和其它PDX-1靶启动子处的组蛋白乙酰化和（d）确定PCIF 1是否募集共阻遏物和/或共激活物/共阻遏物募集。或通过其POZ结构域的组蛋白脱乙酰酶。总之，拟议的研究将解决有关PDX-1在β细胞生物学中功能的主要问题。由此获得的见解可能对糖尿病的新型治疗具有重要意义，这些治疗专注于干细胞的β细胞新生，β细胞从其他细胞类型的转分化，以及提高β细胞数量和存活率的基因治疗方法。

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.