Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter.

Cytosine methylation within CpG dinucleotides has been implicated in the regulation of gene expression in vertebrates and, in some cases, has been shown to be causative in repression of transcription. We have examined whether methylation of CpG dinucleotides located within the binding site for a specific transcription factor, MLTF or USF, affects its binding to DNA. This HeLa cell factor binds to the adenovirus major late promoter (AdMLP), as well as endogenous cellular genes, and stimulates transcription in an in vitro assay. Synthetic oligonucleotides in which 5-methylcytosine replaces cytosine at specific sites were used to generate duplex DNAs, and the formation of complexes of these oligomers with MLTF was studied using a gel retardation assay. Methylation at a CpG site centrally located within the binding site strongly inhibited complex formation, whereas methylation at a site 6 bases away had no demonstrable effect. Methylation at the central site was also shown to inhibit specific transcription in vitro from the AdMLP. Methylation at the central site on only one strand caused a partial inhibition of binding, the effect being greater when the noncoding strand was methylated. The results indicate that in some cases, site-specific methylation may inhibit gene expression directly by blocking binding to DNA of factors required for optimal transcription. Along with other recent findings, they suggest an interplay between DNA methylation and transcription factors in the regulation of gene expression.