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.
