Regulation of IGRP Gene Expression

描述(申请人提供)：治疗I型糖尿病最有前途的方法之一是胰腺移植。然而，这一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，以及捐赠者供应有限。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，以期将它们转化为非免疫原性细胞，从而响应血糖浓度的变化而分泌胰岛素。识别丰富的胰岛转录因子对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这类细胞的研究人员面临的一个困难，即促进干细胞生长和分化的发育线索并不都是已知的。通过分析胰岛特异表达基因的启动子，可以最快地鉴定这些转录因子。 我们最近克隆了一个这样的基因，它编码胰岛特异性的葡萄糖-6-磷酸酶催化亚单位相关蛋白(IGRP)。我们已发表的研究表明，IGRP基因的最大转录需要多种顺式作用元件，并强烈暗示通过对IGRP启动子的研究将发现新的转录因子。此外，由于IGRP是人类1型糖尿病的一种自身抗原，了解IGRP基因表达的调控机制不仅有助于发现新的转录因子，而且这一信息也可能具有临床意义。这项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。这一目标将通过融合基因策略与组织培养细胞系和原代胰岛细胞的转基因相结合来实现。我们已经发现IGRP启动子中的另一个顺式作用元件与一个新的、胰岛丰富的转录因子结合，因此在目标2中，我们将克隆一个编码该蛋白的cDNA。我们的初步数据表明，在出生后胰岛的重塑过程中，IGRP基因表达所需的启动子元件的性质发生了变化。因此，-306到3IGRP启动子区域足以将IGRP-β半乳糖苷酶转基因表达引导到新生小鼠胰岛，但-911到3IGRP启动子区域是维持转基因在成年动物中表达所必需的。在目标3中，我们将通过原位足迹法对结合-911到-307 IGRP启动子区域的因子进行初步表征。在目的4中，我们将比较内源性IGRP基因和-911 IGRP-β半乳糖苷酶转基因的发育表达。

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.