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

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 -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 -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 -globin gene along with a strong 5 erythroid enhancer driving a chloramphenicol acetyltransferase reporter gene, -CAT, was transfected into primary avian erythroid cells derived from 5-day embryos. Methylation of just the 235-bp -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 -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 -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.Methylation of cytosine residues in the dinucleotide CpG is the most common postsynthetic eukaryotic DNA modification. Since the reports of an inverse correlation between DNA methylation and expression of vertebrate ␤-type globin genes (1-3), a large body of evidence relating DNA methylation to gene expression has accumulated (4, 5). At the same time, the absence of detectable DNA methylation in some eukaryotes such as Drosophila (6) and Saccharomyces cerevisiae (7) has raised doubts about its role in normal development and tissue-specific gene expression. However a study by Li et al. (8) showing abnormal development and embryonic lethality in transgenic mice expressing decreased but not completely absent DNA methyl transferase activity following knockout of the DNA methyl transferase gene lends strong support to a critical role for DNA methylation in developmental gene regulation. Recently, a similar critical function of DNA methylation in plant development has been demonstrated by Ronemus et al. (9). However, a direct demonstration of the role of DNA methylation in suppressing transcription of a...

Expression of Human Histo-blood Group ABO Genes Is Dependent upon DNA Methylation of the Promoter Region

“…This differs with the observation that all CpGs were methylated in the 235-bp -globin gene promoter in genomic DNA from adult chicken erythroid cells (32). However, a high heterogeneity of the methylation patterns within the nonexpressor cells was reported in the genes encoding galectin-1, prolactin, and growth hormone (38 -39).…”

“…tissue-specific expression of various genes (19,(31)(32)(33). Furthermore, a 5Ј CpG island hypermethylation is well known to be associated with transcriptional inactivation of certain cancerrelated genes in neoplastic cells (20).…”

“…Since DNA methylation has been observed to determine the tissue-specific expression of a gene (19,(31)(32)(33), the cell lines derived from tissues originally expressing and not expressing the ABH antigens were investigated to address whether DNA methylation of the ABO promoter region is involved in regulation of tissue-specific expression of the ABO genes. The origins, the genotypes, and the enzyme levels of these cell lines are shown in Table I.…”

Tissue and site-specific methylation correlates with expression of the mouse lactoferrin gene

“…Experiments need to be carried out to determine if the methylation state of the lactoferrin gene in liver and other tissues is correlated with its expression during the course of development. DNA demethylation has also been linked to chromatin modification and subsequent transcriptional activation (Cooper et al 1993); conversely, methylation of a promoter leads to transcriptional silencing (Singal et al 1997). An hypothesis consistent with these results is that demethylation at a transcription factor binding element facilitates the binding of specific proteins for tissue-specific expression (Yokomori et al 1995a,b, Short et al 1996.…”

Identification of CCAAT Displacement Protein (CDP/cut) as a Locus-specific Repressor of Major Histocompatibility Complex Gene Expression in Human Tumor Cells

“…EMSA to detect methylcytosine-binding protein (MeCP1) binding activity in HeLa nuclear extracts was performed exactly as described (45).…”