Regulation of IGRP Gene Expression

描述（由申请人提供）：胰腺移植是治疗I型糖尿病最有前途的方法之一。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，以期将它们转化为分泌胰岛素以响应血浆葡萄糖浓度变化的非免疫原性细胞。鉴定富含胰岛的转录因子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规避研究此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完全清楚。通过分析胰岛特异性表达基因的启动子，可以最迅速地鉴定这些转录因子。

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.