Developmentally regulated demethylase activity targeting the betaA-globin gene in primary avian erythroid cells

Differential expression of globin genes has provided an interesting model system for better understanding commonly inherited diseases such as thalassemia. In the avian beta-type globin cluster (5'-rho-betaH-betaA-epsilon-3'), silencing of the embryonic rho-globin gene occurs concomitantly with the activation of the adult betaA-globin gene during embryonic development. DNA methylation is a dynamic process that regulates gene expression. We observed a progressive loss of methylation of betaA-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.