Regulation of IGRP Gene Expression

IGRP 基因表达的调控

• 批准号: 7214150
• 负责人: Richard M O'Brien
• 金额: $ 30.69万
• 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 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7214150
• 关键词: Address; Adult; Applications Grants; Autoantigens; Beta Cell; Binding; Binding Sites; Biological; Birth; Catalytic Domain; Cell Line; Cells; Characteristics; Cloning; Complementary DNA; Cues; DNA; DNA Sequence Analysis; Data; Development; Differentiation and Growth; Elements; Fibrinogen; Funding; Galactosidase; Gel; Gene Expression; Genes; Genetic Transcription; Glucose Plasma Concentration; Goals; Grant; Human; Immunosuppressive Agents; In Situ; Indium; Insulin; Insulin-Dependent Diabetes Mellitus; Islet Cell; Islets of Langerhans; Laboratories; Lead; Ligation; Location; Maintenance; Maps; Mediating; Modification; Molecular; Mus; Nature; Neonatal; Newborn Infant; Pancreas; Pharmaceutical Preparations; Physiology; Play; Polymerase Chain Reaction; Printing; Promoter Regions; Protein Binding; Proteins; Publishing; Regulation; Research Personnel; Role; Stem cells; Suggestion; Techniques; Trans-Activators; Transfection; Transgenes; Transgenic Mice; Transplantation; Variant; base; beta-Galactosidase; cell growth; cell type; cis acting element; clinically significant; foot; fusion gene; glucose-6-phosphatase; immunogenic; interest; islet; mature animal; novel; prevent; promoter; research study; response; tissue culture; transcription factor; transgene expression

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，这种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和有限的供体供应。因此，作为该方法的一种变化，对干细胞的生长、分化和修饰有相当大的兴趣，目的是将它们转化为响应血糖浓度变化而分泌胰岛素的非免疫原性细胞。富含胰岛的转录因子的鉴定对于实现这一目标是至关重要的，因为这些转录因子在干细胞中的受控表达可以规避研究人员研究这些细胞所面临的困难之一，即促进干细胞生长和分化的发育线索并不完全已知。这些转录因子的鉴定最迅速地通过分析其表达是胰岛特异性的基因的启动子来实现。 我们最近克隆了一个这样的基因，编码胰岛特异性葡萄糖-6-磷酸酶催化亚基相关蛋白（IGRP）。我们已发表的研究表明，多个顺式作用元件是所需的最大的IGRP基因转录，并强烈建议，新的转录因子将通过研究的IGRP启动子确定。此外，由于在人类1型糖尿病的自身抗原，了解的机制，规范的IGRP基因表达不仅导致新的转录因子的识别，但这一信息也可能具有临床意义。本申请提出了四个具体目标。在目标1中，我们将描述五个顺式作用元件及其相关的反式作用因子。这一目标将使用融合基因策略，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，一个其他的顺式作用元件在IGRP启动子结合一个新的，胰岛富集的转录因子，因此在目标2中，我们将克隆一个cDNA编码这种蛋白质。我们的初步数据表明，在出生后发生的胰岛重塑过程中，有一个开关的启动子元件的性质，所需的IGRP基因表达。因此，-306至+3IGRP启动子区足以将IGRP-β半乳糖苷酶转基因表达引导至新生小鼠胰岛，但-911至+3IGRP启动子区是维持成年动物中的转基因表达所必需的。在目标3中，我们将通过原位足迹法对结合-911至-307 IGRP启动子区的因子进行初步表征。在目的4中，我们将比较内源性IGRP基因与-911 IGRP-β a半乳糖苷酶转基因的发育表达。