title: Developmentally regulated demethylase activity targeting the betaA-globin gene in primary avian erythroid cells.
abstract: 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.
authors.affiliation: University of Miami, Miami, Florida 33136, USA.
authors.forename: Kavitha
authors.initials: K
authors.lastname: Ramachandran
authors.forename: Jane
authors.initials: J
authors.lastname: van Wert
authors.forename: Gopal
authors.initials: G
authors.lastname: Gopisetty
authors.forename: Rakesh
authors.initials: R
authors.lastname: Singal
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo / Q000254:growth & development; D018899:CpG Islands / Q000502:physiology; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000378:metabolism*; D041905:Erythroid Cells / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D011401:Promoter Regions, Genetic / Q000502:physiology
chemical_list: D005914:Globins; D015254:DNA Modification Methylases