Regulation of IGRP Gene Expression

IGRP 基因表达的调控

• 批准号: 7053317
• 负责人: Richard M O'Brien
• 金额: $ 31.61万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7053317
• 关键词: DNA binding protein; DNA footprinting; autoantigens; beta galactosidase; developmental genetics; fusion gene; gel mobility shift assay; gene expression; genetic regulatory element; genetically modified animals; glucose 6 phosphatase; immunocytochemistry; insulin dependent diabetes mellitus; laboratory mouse; newborn animals; northern blottings; nucleic acid sequence; pancreatic islets; polymerase chain reaction; protein structure function; regulatory gene; site directed mutagenesis; tissue /cell culture; transcription factor; transfection

DESCRIPTION (provided by applicant): One of the most promising approaches to curing type I diabetes is pancreatic transplantation. However, major problems with this strategy are the need to use immunosuppressive drugs to prevent rejection and a limited donor supply. Therefore, as a variation on this approach, there is considerable interest in the growth, differentiation and modification of stem cells with a view to converting them into non-immunogenic cells that secrete insulin in response to changes in plasma glucose concentrations. The identification of islet-enriched transcription factors is critical to achieving this goal because the controlled expression of these transcription factors in stem cells may circumvent one of the difficulties facing investigators who are studying such cells, namely that the developmental cues that promote stem cell growth and differentiation are not all known. The identification of these transcription factors is most expeditiously achieved by analyzing the promoters of genes whose expression are islet-specific. We have recently cloned one such gene that encodes an islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Our published studies have demonstrated that multiple cis-acting elements are required for maximal IGRP gene transcription and strongly suggest that novel transcription factors will be identified through studying the IGRP promoter. In addition, since IGRP is an autoantigen in human type 1 diabetes, understanding the mechanisms that regulate IGRP gene expression should not only lead to the identification of novel transcription factors but this information may also have clinical significance. This application proposes four Specific Aims. In Aim 1 we will characterize five cis-acting elements and their associated trans-acting factors. This Aim will be achieved using a fusion gene strategy, in conjunction with the transfection of tissue culture cell lines and primary islet cells. We have already found that one other cis-acting element in the IGRP promoter binds a novel, islet-enriched transcription factor so in Aim 2 we will clone a cDNA that encodes this protein. Our preliminary data suggest that during the remodeling of the islet that occurs after birth there is a switch in the nature of the promoter elements that are required for IGRP gene expression. Thus, the -306 to +3 IGRP promoter region is sufficient to direct IGRP-beta galactosidase transgene expression to newborn mice islets but the -911 to +3 IGRP promoter region is required for the maintenance of transgene expression in adult animals. In Aim 3 we will perform an initial characterization of the factors binding the -911 to -307 IGRP promoter region by in situ foot-printing. And in Aim 4 we will compare the developmental expression of the endogenous IGRP gene with that of the -911 IGRP-betaa galactosidase transgene.

描述（由申请人提供）：治疗 I 型糖尿病最有希望的方法之一是胰腺移植。然而，该策略的主要问题是需要使用免疫抑制药物来防止排斥反应以及供体供应有限。因此，作为这种方法的一种变体，人们对干细胞的生长、分化和修饰非常感兴趣，以期将其转化为响应血浆葡萄糖浓度变化而分泌胰岛素的非免疫原性细胞。胰岛富集转录因子的鉴定对于实现这一目标至关重要，因为这些转录因子在干细胞中的受控表达可能会绕过研究此类细胞的研究人员所面临的困难之一，即促进干细胞生长和分化的发育线索尚不清楚。通过分析胰岛特异性表达的基因的启动子，可以最迅速地鉴定这些转录因子。 我们最近克隆了一个这样的基因，它编码胰岛特异性葡萄糖-6-磷酸酶催化亚基相关蛋白（IGRP）。我们发表的研究表明，IGRP 基因最大转录需要多个顺式作用元件，并强烈建议通过研究 IGRP 启动子来鉴定新的转录因子。此外，由于IGRP是人类1型糖尿病的自身抗原，了解调节IGRP基因表达的机制不仅可以识别新的转录因子，而且这些信息也可能具有临床意义。该申请提出了四个具体目标。在目标 1 中，我们将描述五种顺式作用元件及其相关的反式作用因子。该目标将通过使用融合基因策略，结合组织培养细胞系和原代胰岛细胞的转染来实现。我们已经发现 IGRP 启动子中的另一个顺式作用元件结合了一种新颖的富含胰岛的转录因子，因此在目标 2 中，我们将克隆编码该蛋白质的 cDNA。我们的初步数据表明，在出生后发生的胰岛重塑过程中，IGRP 基因表达所需的启动子元件的性质发生了转变。因此，-306至+3 IGRP启动子区域足以将IGRP-β半乳糖苷酶转基因表达引导至新生小鼠胰岛，但-911至+3 IGRP启动子区域对于维持成年动物中的转基因表达是必需的。在目标 3 中，我们将通过原位足迹对结合 -911 至 -307 IGRP 启动子区域的因子进行初步表征。在目标 4 中，我们将比较内源 IGRP 基因与 -911 IGRP-betaa 半乳糖苷酶转基因的发育表达。