STABILITY OF PATCH METHYLATION AND ITS IMPACT IN REGIONS OF TRANSCRIPTIONAL INITIATION AND ELONGATION

CPG DNA methylation has previously been correlated with the suppressio n of transcription. The mechanism of this suppression is not understoo d, and many aspects of the temporal and positional relationships betwe en the region of methylation and transcription have not yet been defin ed. Here, 12-kb stable replicating episomes that can be maintained in human somatic cells for weeks to months were used, Such a system allow s more direct manipulation and is free from the positional effects att endant with the analysis of endogenous loci or integrated transgenes. By using these circular minichromosomes, patches of CpG methylation we re created to include or exclude the regions of transcriptional initia tion and elongation. I found that a 0.5-kb patch of methylation that c overed the promoter suppressed expression only 2-fold and that a 1.9-k b patch of methylation that covered the coding portion of the gene (bu t not the promoter) suppressed expression about 10-fold. In contrast, methylation of the entire minichromosome except for the promoter or th e coding portion suppressed transcription about 50- to 200-fold, I inf er the following. Methylation of the 0.5-kb promoter fragment does not significantly affect transcription at the level of transcription fact or binding or local chromatin structure, The dominant effect on transc ription occurs when the length of methylated DNA is long, with little disproportionate effect of methylation of specific regions, such as th at of initiation or elongation. I also found that the boundaries betwe en these methylated and unmethylated regions remained stable for the m any weeks that I monitored them.