Stability of patch methylation and its impact in regions of transcriptional initiation and elongation.

Abstract

CpG DNA methylation has previously been correlated with the suppression of transcription. The mechanism of this suppression is not understood, and many aspects of the temporal and positional relationships between the region of methylation and transcription have not yet been defined. Here, 12-kb stable replicating episomes that can be maintained in human somatic cells for weeks to months were used. Such a system allows more direct manipulation and is free from the positional effects attendant with the analysis of endogenous loci or integrated transgenes. By using these circular minichromosomes, patches of CpG methylation were created to include or exclude the regions of transcriptional initiation and elongation. I found that a 0.5-kb patch of methylation that covered the promoter suppressed expression only 2-fold and that a 1.9-kb patch of methylation that covered the coding portion of the gene (but not the promoter) suppressed expression about 10-fold. In contrast, methylation of the entire minichromosome except for the promoter or the coding portion suppressed transcription about 50- to 200-fold. I infer the following. Methylation of the 0.5-kb promoter fragment does not significantly affect transcription at the level of transcription factor binding or local chromatin structure. The dominant effect on transcription occurs when the length of methylated DNA is long, with little disproportionate effect of methylation of specific regions, such as that of initiation or elongation. I also found that the boundaries between these methylated and unmethylated regions remained stable for the many weeks that I monitored them.