Differential expression of globin genes has provided an interesting model system for better understanding commonly inherited diseases such as thalassemia. In the avian β-type globin cluster (5′-ρ-βH- βA-ε-3′), silencing of the embryonic ρ-globin gene occurs concomitantly with the activation of the adult βA-globin gene during embryonic development. DNA methylation is a dynamic process that regulates gene expression. We observed a progressive loss of methylation of βA-globin gene, during avian embryonic development that was concurrent with the expression of the gene. The promoter and exon 1 regions of the template strand were completely demethylated, whereas residual methylation was retained in exons 2 and 3. Using a modified methylation-sensitive single-nucleotide primer extension (MS-SNuPE) assay, we observed stage-specific demethylase activity in the nuclear extracts of chicken red cells; activity in 5-, 8-, and 11-day-old erythroid cell nuclear extracts was 6, 76, and 24%, respectively. The demethylase targeted both hemimethylated and fully methylated substrates. Our findings demonstrate stage-specific demethylase activity in nuclear extracts from primary chicken erythroid cells that could target the fully methylated promoter of a developmentally regulated native gene.
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