METHYLATION OF THE MINIMAL PROMOTER OF AN EMBRYONIC GLOBIN GENE SILENCES TRANSCRIPTION IN PRIMARY ERYTHROID-CELLS

Methylation of cytosines in tile dinucleotide CpG has been shown to su ppress transcription of a number of tissue-specific genes, yet the pre cise mechanism is not fully understood, The vertebrate globin genes we re among the first examples in which an inverse correlation was shown between CPG methylation and transcription, We studied the methylation pattern of the 235-bp rho-globin gene promoter in genomic DNA from pri mary chicken erythroid cells using Ale sodium bisulfite conversion tec hnique and found all CpGs in the promoter to be methylated in erythroi d cells from adult chickens in which the rho-globin gene is silent but unmethylated in 5-day (primitive) embryonic red cells in which the ge ne is transcribed. To elucidate further the mechanism of methylation-i nduced silencing, an expression construct consisting of 235 bp of 5' p romoter sequence of the rho-globin gene along with a strong 5' erythro id enhancer driving a chloramphenicol acetyltransferase reporter gene, rho-CAT, was transfected into primary avian erythroid cells derived f rom 5-day embryos, Methylation of just the 235-bp rho-globin gene prom oter fragment at. every CPG resulted in a 20- to 30-fold inhibition of transcription, and this effect was not overridden by the presence of potent erythroid-specific enhancers. The ability of the 235-bp rho-glo bin gene promoter to bind to a DNA Methyl Cytosine binding Protein Com plex (MeCPC) was tested in electrophoretic mobility shift assays utili zing primary avian erythroid cell nuclear extract, The results mere th at fully methylated hut not unmethylated 235-bp rho-globin gene promot er fragment could compete efficiently for MeCPC binding, These results are a direct demonstration that site-specific methylation of a globin gene promoter-al the exact CpGs that are methylated in vivo can silen ce transcription in homologous primary erythroid cells, Further; these data implicate binding of MeCPC to the promoter in the mechanism of s ilencing.