The Effect of DNA Methylation on Transcription From the SV40 Early Promoter

[摘要] The effect of DNA methylation on transcription from the SV40 early promoter was studied. The plasmid pVHC1 containing the SV40 promoter in the early direction linked to the chloramphenicol acetyltransferase (CAT) gene was methylated in vitro with mouse Krebs II ascites cell DNA methylase. This resulted in methylation of 10-25% of CpG dinucleotides. Comparison of this DNA with unmethylated plasmid in a transient expression assay indicated a 30-40% inhibition of CAT expression by methylation. At this level of methylation an average of only 1 to 2 of the CpGs contained within the promoter region will be methylated and the enhancer sequence which does not contain any CpG dinucleotides will remain unmodified. Therefore it is unlikely that this effect is caused by the direct inhibition of transcription factor binding, it is most likely due to the formation of inactive chromatin structures induced by the presence of methyl groups throughout the plasmid. This theory is supported by the observation that binding of the transcription factor Sp1 to GC box motifs is unaffected by methylation. In experiments where mouse cells were cotransfected with pVHC1 and oligonucleotides containing methylated or unmethylated GC boxes, the level of CAT expression was greatly reduced regardless of the methylation state of the GC box, indicating that methylation had no effect on Sp1 binding. Similarly in gel retardation assays, methylated and unmethylated GC box-containing oligonucleotides competed equally well with a labelled restriction fragment containing the SV40 promoter for Sp1 binding. These findings indicate that the SV40 promoter is methylation sensitive in a methylcytosine rich environment where these residues are most likely acting in an indirect manner, causing the formation of inactive chromatin and hence limiting transcription.

[发布机构] University:University of Glasgow

[关键词] Biochemistry