Effect of in vitro DNA methylation on beta-globin gene expression.

When the human beta-globin gene was methylated at every cytosine residue and was inserted into mouse fibroblasts by DNA-mediated gene transfer, the transcription of the gene was strongly inhibited. This methylation also prevented expression and induction of the gene in mouse erythroleukemia cells. By using partially methylated hybrid molecules, it was shown that methylation-sensitive negative regulatory elements are located in both the 5' and 3' ends of the beta-globin gene but not in the 90-base-pair region usually associated with promoter activity. To further investigate the role of DNA methylation in the regulation of the beta-globin gene, 50-base-pair poly(dG-dC) tracts were introduced into various sites in a mouse-human hybrid gene, and these inserts were methylated by means of the Hha I methylase. Heavy methylation of these artificially added sites had no effect on either transcription initiation or elongation, suggesting that DNA modification operates through fixed endogenous sites in the gene domain.