Islet-Enriched Transcription Factors Synergistically Activate Transcription of the Gene Encoding the Autoantigen IGRP

Martin, Cyrus C.; Flemming, Brian P.; Wang, Yingda; Oeser, James K.; O'Brien, Richard M.
June 2007
Diabetes;Jun2007 Supplement 1, Vol. 56, pA430
Academic Journal
Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is selectively expressed in islet beta cells and is a major autoantigen in both mouse and human type I diabetes. It is possible that suppression oflGRP gene transcription may prevent or delay the progression of this disease. We have previously shown using chromatin immunoprecipitation (CHIP) assays that the IGRP promoter binds, directly or indirectly, the islet-enriched transcription factors Pdx- 1, Pax-6 and BETA2 in beta TC-3 cells in situ. We show here, again using ChIP assays, that the IGRP promoter also binds the islet-enriched transcription factors MafA and Foxa2 in beta TC-3 cells in situ. Single binding sites for these factors were identified in the proximal IGRP promoter mutation of which resulted in decreased IGRP fusion gene expression. Gel retardation studies demonstrate that MafA binds to the IGRP promoter with a five fold higher affinity than it binds the well-characterized C 1 element in the insulin promoter. Co-transfection experiments in HeLa ceils, in which neither these islet-enriched transcription factors nor the IGRP gene are endogenously expressed, were used to investigate the mechanism by which these factors activate IGRP gene expression. Individually Pdx-1, Pax-6, BETA2, MafA and Foxa2 have little effect on IGRP fusion gene expression in HeLa cells whereas combined expression of all five factors results in a synergistic induction of expression. An identical result is seen in HepG2 hepatoma cells in which Foxa2 is endogenously expressed. Interestingly in both HeLa and HepG2 cells the action of Foxa2 is biphasic in that it both stimulates and inhibits IGRP fusion gene expression through mechanisms that are DNA binding dependent and independent, respectively. The observation that Pdx-1 stimulates IGRP fusion gene expression in HeLa cells was surprising because mutation of the four Pdx-1 binding sites in the IGRP promoter has little effect on IGRP fusion gene expression in either HeLa or beta-TC-3 cells. This suggests that in both cell types Pdx-1 stimulates IGRP gene expression through a mechanism that is independent of DNA binding.


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