Differential regulation of islet-specific glucose-6-phosphatase catalytic subunit-related protein gene transcription by Pax-6 and Pdx-1.

Martin CC, Oeser JK, O'Brien RM
J Biol Chem. 2004 279 (33): 34277-89

PMID: 15180990 · DOI:10.1074/jbc.M404830200

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

MeSH Terms (40)

Amino Acid Motifs Animals Base Sequence Binding Sites Catalytic Domain Cell Nucleus Cells, Cultured Chloramphenicol O-Acetyltransferase Chromatin Diabetes Mellitus, Experimental Disease Models, Animal DNA Dose-Response Relationship, Drug Electrophoresis, Polyacrylamide Gel Eye Proteins Gene Expression Regulation, Enzymologic Glucose-6-Phosphatase Homeodomain Proteins Islets of Langerhans Luciferases Methylation Mice Molecular Sequence Data Mutagenesis, Site-Directed Mutation Oligonucleotides Paired Box Transcription Factors PAX6 Transcription Factor Plasmids Polymerase Chain Reaction Precipitin Tests Promoter Regions, Genetic Protein Binding Protein Structure, Tertiary Rats Repressor Proteins Salts Subcellular Fractions Trans-Activators Transcription, Genetic

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