A methylation-responsive MDBP/RFX site is in the first exon of the collagen alpha 2(I) promoter

DNA methylation inhibits transcription driven by the collagen alpha 2(I) pr omoter and the 5' end of the gene in transient transfection and in vitro tr anscription assays. DNA-binding proteins in a unique family of ubiquitously expressed proteins, methylated DNA-binding protein (MDBP)/regulatory facto r for X box (RFX), form specific complexes with a sequence overlapping the transcription start site of the collagen alpha 2(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-mig ration 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 D NA-protein complexes. Furthermore, in vitro translated REX1 protein formed a specific complex with the collagen sequence that was also supershifted wi th the RFX1 antibody, MDBP/RFX displayed a higher affinity binding to the c ollagen sequence if the CPG at +7 was mutated in a manner similar to TpG. T his same mutation within reporter constructs inhibited transcription in tra nsfection and in vitro transcription assay. These results support the hypot hesis that DNA methylation-induced inactivation of collagen alpha 2(I) gene transcription is mediated, in part, by increased binding of MDBP/RFX to th e first exon in response to methylation in this region.