Methylation of promoter proximal-transcribed sequences of an embryonic globin gene inhibits transcription in primary erythroid cells and promotes formation of a cell type-specific methyl cytosine binding complex.
The methylation pattern of a 248-base pair proximal transcribed region (rho248) of the avian embryonic rho-globin gene was found to correlate inversely with stage-specific expression in avian erythroid cells. In vitro methylation of the rho248 segment alone (in the absence of promoter methylation) resulted in a 5-fold inhibition of transcription in a transient transfection assay in primary erythroid cells in which the transfected gene is packaged into nucleosomal chromatin. This effect was observed if the rho248 segment was positioned adjacent to the promoter but not when it was located 2.7 kilobases downstream. Fully methylated but not unmethylated rho248 formed a novel cell type-specific methyl cytosine-binding protein complex (MeCPC) that contained methyl binding domain protein-2 (MBD-2) and histone deacetylase 1 proteins but differed from MeCP-1. The histone deacetylase inhibitor trichostatin A failed to relieve methylation-mediated repression of transcription from the rho-gene promoter, supporting the notion of the dominance of methylation over histone deacetylation in silencing through CpG-rich sequences at this locus. These data demonstrate that site-specific methylation of a vertebrate gene 5'-transcribed region alone at the exact CpGs that are methylated in vivo can suppress transcription in homologous primary cells and facilitate binding to a cell type-specific MeCPC.
Singal
Rakesh
R
Massey Cancer Center and Departments of Internal Medicine and Human Genetics, Virginia Commonwealth University, Richmond, Virginia 23298-0037, USA.
Wang
Shou Zhen
SZ
Sargent
Thanh
T
Zhu
Sheng Zu
SZ
Ginder
Gordon D
GD
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D003596:Cytosine / Q000378:metabolism*; D019175:DNA Methylation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D005914:Globins / Q000235:genetics*; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection
D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; C012589:trichostatin A; D003596:Cytosine; D005914:Globins