Developmentally Regulated Demethylase Activity Targeting the β_A-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.

The social environment and the epigenome

“…A very recent Environmental and Molecular Mutagenesis. DOI 10.1002/em Social Environment and the Epigenome publication has used the assay described by Ramchanadni et al, to measure demethylase activity in white matter from multiple sclerosis patients [Mastronardi et al, 2007] and demethylase activity was also assayed recently in the nuclear extracts of chicken erythroid cells [Ramachandran et al, 2007]. Recent data support the view that DNA methylation is dynamic in postmitotic tissues in the brain [Weaver et al, 2004[Weaver et al, , 2005Miller and Sweatt, 2007] and in neurons in vitro [Levenson et al, 2006].…”

Serum GADD45a methylation is a useful biomarker to distinguish benign vs malignant prostate disease

“…Ms-SNuPE analysis was done with the primer spanning CpG 1: 5′-AAACTAATCTAC-3′. Ms-SNuPE reactions were carried out using 1 μ l of the PCR product in a 25 μ l reaction mixture containing 1 × PCR buffer (Sigma, St Louis, MO, USA), 1 μ M of Ms-SNuPE primer, 1 μ Ci of 32 P-labelled dATP or 32 P-labelled dGTP and 1 U of Taq Polymerase (Sigma) ( Gonzalgo and Jones, 1997 ; Ramachandran et al , 2007 , 2009 ). Percent methylation in each CpG was calculated as G/(G+A) × 100.…”