title: A methylation-responsive MDBP/RFX site is in the first exon of the collagen alpha2(I) promoter.
abstract: DNA methylation inhibits transcription driven by the collagen alpha2(I) promoter and the 5' end of the gene in transient transfection and in vitro transcription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory factor for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen alpha2(I) gene. Complex formation increased when the CpG site at +7 base pairs from the transcription start site was methylated. The identity of the protein was demonstrated by co-migration and cross-competition for a characteristic slowly migrating doublet complex formed on MDBP/RFX recognition sequences and the collagen sequences by band shift assays. A RFX1-specific antibody supershifted the collagen DNA-protein complexes. Furthermore, in vitro translated RFX1 protein formed a specific complex with the collagen sequence that was also supershifted with the RFX1 antibody. MDBP/RFX displayed a higher affinity binding to the collagen sequence if the CpG at +7 was mutated in a manner similar to TpG. This same mutation within reporter constructs inhibited transcription in transfection and in vitro transcription assay. These results support the hypothesis that DNA methylation-induced inactivation of collagen alpha2(I) gene transcription is mediated, in part, by increased binding of MDBP/RFX to the first exon in response to methylation in this region.
authors.affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
authors.forename: P K
authors.initials: PK
authors.lastname: Sengupta
authors.forename: M
authors.initials: M
authors.lastname: Ehrlich
authors.forename: B D
authors.initials: BD
authors.lastname: Smith
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics*; D024042:Collagen Type I; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005347:Fibroblasts; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D015533:Transcriptional Activation
chemical_list: D024042:Collagen Type I; D004268:DNA-Binding Proteins; C489324:Mbd1 protein, rat; D003094:Collagen