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Diabetes 50:502-514, 2001
© 2001 by the American Diabetes Association, Inc.

Characterization of the Mouse Islet-Specific Glucose-6-Phosphatase Catalytic Subunit–Related Protein Gene Promoter by In Situ Footprinting

Correlation With Fusion Gene Expression in the Islet-Derived ßTC-3 and Hamster Insulinoma Tumor Cell Lines

Larry J. Bischof1, Cyrus C. Martin1, Christina A. Svitek1, Beth T. Stadelmaier1, Lauri A. Hornbuckle1, Joshua K. Goldman1, James K. Oeser1, John C. Hutton2, and Richard M. O’Brien1

1 Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee
2 Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, Colorado

Glucose-6-phosphatase (G6Pase) is a multicomponent system located in the endoplasmic reticulum comprising a catalytic subunit and transporters for glucose-6-phosphate, inorganic phosphate, and glucose. We have recently cloned a novel gene that encodes an islet-specific G6Pase catalytic subunit–related protein (IGRP) (Ebert et al., Diabetes 48:543–551, 1999). To begin to investigate the molecular basis for the islet-specific expression of the IGRP gene, a series of truncated IGRP–chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into the islet-derived mouse ßTC-3 and hamster insulinoma tumor cell lines. In both cell lines, basal fusion gene expression decreased upon progressive deletion of the IGRP promoter sequence between -306 and -66, indicating that multiple promoter regions are required for maximal IGRP-CAT expression. The ligation-mediated polymerase chain reaction footprinting technique was then used to compare trans-acting factor binding to the IGRP promoter in situ in ßTC-3 cells, which express the endogenous IGRP gene, and adrenocortical Y1 cells, which do not. Multiple trans-acting factor binding sites were selectively identified in ßTC-3 cells that correlate with regions of the IGRP promoter identified as being required for basal IGRP-CAT fusion gene expression. The data suggest that hepatocyte nuclear factor 3 may be important for basal IGRP gene expression, as it is for glucagon, GLUT2, and Pdx-1 gene expression. In addition, binding sites for several trans-acting factors not previously associated with islet gene expression, as well as binding sites for potentially novel proteins, were identified.

Abbreviations: CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; GSD, glycogen storage disease; G6P, glucose-6-phosphate; G6Pase, glucose-6-phosphatase; HNF, hepatocyte nuclear factor; IGRP, islet-specific G6Pase catalytic subunit–related protein; LMPCR, ligation-mediated polymerase chain reaction; MUT, mutant; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; TBE, 89 mmol/l Tris + 89 mmol/l boric acid + 2 mmol/1 EDTA; WT, wild type


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