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

Autor: Richard M. O'Brien, James K. Oeser, Cyrus C. Martin
Rok vydání: 2004
Předmět:
PAX6 Transcription Factor
Transcription
Genetic
Amino Acid Motifs
Response element
Oligonucleotides
medicine.disease_cause
Polymerase Chain Reaction
Biochemistry
Fusion gene
Mice
Catalytic Domain
Paired Box Transcription Factors
Luciferases
Promoter Regions
Genetic
Cells
Cultured
Mutation
Chromatin
Glucose-6-Phosphatase
Electrophoresis
Polyacrylamide Gel
Plasmids
Protein Binding
Subcellular Fractions
Chloramphenicol O-Acetyltransferase
endocrine system
Molecular Sequence Data
Biology
Methylation
Gene Expression Regulation
Enzymologic
Diabetes Mellitus
Experimental
Islets of Langerhans
medicine
Animals
Binding site
Eye Proteins
Molecular Biology
Transcription factor
Cell Nucleus
Homeodomain Proteins
Binding Sites
Base Sequence
Dose-Response Relationship
Drug
Promoter
DNA
Cell Biology
Precipitin Tests
Molecular biology
Footprinting
Protein Structure
Tertiary
Rats
Repressor Proteins
Disease Models
Animal
Mutagenesis
Site-Directed
Trans-Activators
Salts
Chromatin immunoprecipitation
Zdroj: Journal of Biological Chemistry. 279:34277-34289
ISSN: 0021-9258
DOI: 10.1074/jbc.m404830200
Popis: 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.
Databáze: OpenAIRE
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