Differential regulation of islet-specific glucose-6-phosphatase catalytic subunit-related protein gene transcription by Pax-6 and Pdx-1.
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.
Martin
Cyrus C
CC
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, TN 37232, USA.
Oeser
James K
JK
O'Brien
Richard M
RM
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins; D015971:Gene Expression Regulation, Enzymologic; D005952:Glucose-6-Phosphatase / Q000737:chemistry*; D018398:Homeodomain Proteins / Q000378:metabolism*; D007515:Islets of Langerhans / Q000201:enzymology*; D008156:Luciferases / Q000378:metabolism; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D012097:Repressor Proteins; D012492:Salts / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic
D002843:Chromatin; D005136:Eye Proteins; D018398:Homeodomain Proteins; D009841:Oligonucleotides; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; C000605733:Pax6 protein, rat; D012097:Repressor Proteins; D012492:Salts; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D004247:DNA; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D005952:Glucose-6-Phosphatase