Methylation of the minimal promoter of an embryonic globin gene silences transcription in primary erythroid cells

Rakesh Singal, Richard Ferris, Jane A. Little, Shu Zhen Wang, Gordon D. Ginder

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

Methylation of cytosines in the dinucleotide CpG has been shown to suppress transcription of a number of tissue-specific genes, yet the precise mechanism is not fully understood. The vertebrate globin genes were 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 ρ-globin gene promoter in genomic DNA from primary chicken erythroid cells using the sodium bisulfite conversion technique and found all CpGs in the promoter to be methylated in erythroid cells from adult chickens in which the ρ-globin gene is silent but unmethylated in 5-day (primitive) embryonic red cells in which the gene is transcribed. To elucidate further the mechanism of methylation-induced silencing, an expression construct consisting of 235 bp of 5' promoter sequence of the ρ-globin gene along with a strong 5' erythroid enhancer driving a chloramphenicol acetyltransferase reporter gene, ρ-CAT, was transfected into primary avian erythroid cells derived from 5-day embryos. Methylation of just the 235-bp ρ-globin gene promoter 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 ρ-globin gene promoter to bind to a DNA Methyl Cytosine binding Protein Complex (MeCPC) was tested in electrophoretic mobility shift assays utilizing primary avian erythroid cell nuclear extract. The results were that fully methylated but not unmethylated 235-bp ρ-globin gene promoter fragment could compete efficiently for MeCPC binding. These results are a direct demonstration that site-specific methylation of a globin gene promoter at the exact CpGs that are methylated in vivo can silence transcription in homologous primary erythroid cells. Further, these data implicate binding of MeCPC to the promoter in the mechanism of silencing.

Original languageEnglish
Pages (from-to)13724-13729
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number25
DOIs
StatePublished - Dec 9 1997
Externally publishedYes

Fingerprint

Erythroid Cells
Globins
Methylation
Genes
Cytosine
Carrier Proteins
Chickens
Chloramphenicol O-Acetyltransferase
DNA
Electrophoretic Mobility Shift Assay
Cell Extracts
Reporter Genes
Protein Binding
Vertebrates
Embryonic Structures

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Methylation of the minimal promoter of an embryonic globin gene silences transcription in primary erythroid cells. / Singal, Rakesh; Ferris, Richard; Little, Jane A.; Wang, Shu Zhen; Ginder, Gordon D.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, No. 25, 09.12.1997, p. 13724-13729.

Research output: Contribution to journalArticle

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