R. Singal et al., METHYLATION OF THE MINIMAL PROMOTER OF AN EMBRYONIC GLOBIN GENE SILENCES TRANSCRIPTION IN PRIMARY ERYTHROID-CELLS, Proceedings of the National Academy of Sciences of the United Statesof America, 94(25), 1997, pp. 13724-13729
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.