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 betaTC-3 and hamster insulinoma tumor cell lines

• Published in: Diabetes (New York, N.Y.) Vol. 50; no. 3; pp. 502 - 514
• Main Authors: Bischof, L J, Martin, C C, Svitek, C A, Stadelmaier, B T, Hornbuckle, L A, Goldman, J K, Oeser, J K, Hutton, J C, O'Brien, R M
• Format: Journal Article
• Language: English
• Published: United States 01.03.2001
• Subjects: Animals; Artificial Gene Fusion; Base Sequence - genetics; Cell Line; Chloramphenicol O-Acetyltransferase - genetics; Cricetinae; DNA-Binding Proteins - metabolism; Gene Expression; Genes, Reporter; Glucose-6-Phosphatase; Hepatocyte Nuclear Factor 3-alpha; Hepatocyte Nuclear Factor 3-beta; Insulinoma - genetics; Insulinoma - pathology; Islets of Langerhans - cytology; Islets of Langerhans - physiology; Mice; Molecular Sequence Data; Nuclear Proteins - metabolism; Peptide Fragments - physiology; Promoter Regions, Genetic - genetics; Promoter Regions, Genetic - physiology; Protein Footprinting; Proteins - chemistry; Proteins - genetics; Stereoisomerism; Transcription Factors
• ISSN: 0012-1797

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 betaTC-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 betaTC-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 betaTC-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.