Site specific demethylation in the promoter of human gamma-globin gene does not alleviate methylation mediated suppression

Human gamma A-globin genes with different patterns of methylation were synthesized in vitro. These methylated constructs were transfected into mouse L cells and their expression analyzed by S1-nuclease protection assays. We show that methylation downstream of +92 does not affect transcription while methylation downstream of -210 abolishes transcription. To test which of the six CpG dinucleotides between -210 and +92 plays a role in DNA methylation mediated suppression of transcription, we generated mutant gamma-globin genes which lack the three CpGs 5' to the cap site, the three CpGs 3' to the cap site, or all six CpGs. After in vitro methylation, we show that removal of methylated residues 5' or 3' to the cap site did not alleviate suppression, whereas removal of both 5' and 3' methylated residues restored partial expression. These results indicate that CpG methylation at different positions can cause suppression of expression and they are inconsistent with the hypothesis that site-specific demethylations in the promoter region can alleviate suppression. We propose that a minimum length of methylation-free DNA in the promoter area is the only requirement for expression of this promoter by ubiquitous transcription factors, and discuss these results with reference to methylation-free islands.