TRANSCRIPTIONAL REPRESSION BY METHYLATION - COOPERATIVITY BETWEEN A CPG CLUSTER IN THE PROMOTER AND REMOTE CPG-RICH REGIONS

Cytosine methylation of binding sites for transcription factors is a s traightforward mechanism to prevent transcription, while data on an in direct mechanism, by methylation outside of the factor binding sites, are still scarce, We have studied the latter effect using a model prom oter construct. For this, a 69 bp G + C rich DNA segment with a cluste r of 14 CpG sites was inserted between upstream lexA sites and the TAT A box, Transcription was measured in transient transfection assays wit h lexA-VP16 as an activating factor. When the entire plasmid was methy lated at all CpGs before transfection, transcription was blocked (to 3 % residual activity), whereas transcription was only mildly inhibited (to 60%) by methylation of a control plasmid that lacked the 69 bp CpG cluster, However, the effect could not simply be attributed to methyl ation of the CpG cluster: neither a methylated CpG cluster in an other wise methylation-free reporter gene plasmid, nor the methylated plasmi d with an unmethylated CpG cluster,inhibited transcription considerabl y (69% and 44% remaining activity, respectively), The data presented h ere suggest that a minimal length of methylated DNA in the promoter is required for repression, and imply that concomitant methylation of Cp Gs in the promoter region and in remote sequences can cooperatively bl ock transcription, without the need to methylate any binding sites for transcription factors. We also note that the cooperation for a negati ve effect described here bears an analogy to transcriptional activatio n, where a promoter often cooperates with a remote enhancer.