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J. Biol. Chem., Vol. 279, Issue 33, 34277-34289, August 13, 2004

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Differential Regulation of Islet-specific Glucose-6-phosphatase Catalytic Subunit-related Protein Gene Transcription by Pax-6 and Pdx-1^*

Cyrus C. Martin, James K. Oeser, and Richard M. O'Brien

From the Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is selectively expressed in islet cells and is a major autoantigen in a mouse model of type I diabetes. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion gene expression through transient transfection of islet-derived TC-3 cells revealed that a promoter region, located between –273 and –254, is essential for high IGRP-CAT fusion gene expression. The sequence of this promoter region does not match that for any known islet-enriched transcription factor. However, data derived from gel retardation assays, a modified ligation-mediated polymerase chain reaction in situ footprinting technique and a SDS-polyacrylamide separation/renaturation procedure led to the hypothesis that this protein might be Pax-6, a conclusion that was confirmed by gel supershift assays. Additional experiments revealed a second non-consensus Pax-6 binding site in the –306/–274 IGRP promoter region. Pax-6 binding to these elements is unusual in that it appears to require both its homeo and paired domains. Interestingly, loss of Pax-6 binding to the –273/ –246 element is compensated by Pax-6 binding to the –306/–274 element and vice versa. Gel retardation assays revealed that another islet-enriched transcription factor, namely Pdx-1, binds four non-consensus elements in the IGRP promoter. However, mutation of these elements has little effect on IGRP fusion gene expression. Although chromatin immunoprecipitation assays show that both Pax-6 and Pdx-1 bind to the IGRP promoter within intact cells, in contrast to the critical role of these factors in cell-specific insulin gene expression, IGRP gene transcription appears to require Pax-6 but not Pdx-1.

Received for publication, April 30, 2004

^* This work was supported in part by National Institutes of Health (NIH) Grant DK61645 (to R. M. O.) and by NIH Grant P60-DK20593, which funds the Vanderbilt Diabetes Center Core Laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by the Vanderbilt Viruses, Nucleic Acids, and Cancer Training Program (Grant 5T32-CA09385-17).

To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, 761 PRB (MRB II), Vanderbilt University Medical School, Nashville, TN 37232-0615. Tel.: 615-936-1503; Fax: 615-322-7236; E-mail: richard.obrien{at}vanderbilt.edu.

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