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

• Published in: The Journal of biological chemistry Vol. 279; no. 33; pp. 34277 - 34289
• Main Authors: Martin, Cyrus C, Oeser, James K, O'Brien, Richard M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 13.08.2004
• Subjects: Amino Acid Motifs; Animals; Base Sequence; Binding Sites; Catalytic Domain; Cell Nucleus - metabolism; Cells, Cultured; Chloramphenicol O-Acetyltransferase - metabolism; Chromatin - metabolism; Diabetes Mellitus, Experimental; Disease Models, Animal; DNA - chemistry; DNA - metabolism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Eye Proteins; Gene Expression Regulation, Enzymologic; Glucose-6-Phosphatase - chemistry; Homeodomain Proteins - metabolism; Islets of Langerhans - enzymology; Luciferases - metabolism; Methylation; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Oligonucleotides - chemistry; Paired Box Transcription Factors; PAX6 Transcription Factor; Plasmids - metabolism; Polymerase Chain Reaction; Precipitin Tests; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Rats; Repressor Proteins; Salts - pharmacology; Subcellular Fractions - metabolism; Trans-Activators - metabolism; Transcription, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M404830200

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is selectively expressed in islet β 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 βTC-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 β cell-specific insulin gene expression, IGRP gene transcription appears to require Pax-6 but not Pdx-1.