Demethylation of β_A-Globin Gene in Avian Primary Erythroid Cells Is Developmentally Regulated, Strand Asymmetrical and an Active Process.

Differential expression of globin genes has provided an interesting model system for the better understanding of commonly inherited diseases such as thalassemia. In the avian β-type globin cluster (5′- ρ-β_A- β_A - ε - 3′), silencing of the embryonic ρ-globin gene occurs concomitantly with the activation of adult β_A-globin gene during the embryonic development. The pattern of cytosine methylation plays an important role in gene regulation. We have previously shown that de novo methylation of the embryonic ρ-globin gene during development is strand-asymmetrical and spreads from the proximal transcribed region in the early definitive chicken erythroid cells. In the present study, we examined the methylation pattern of β_A- globin gene during development in primary chicken erythroid cells. Using bisulfite genomic sequencing we observed a progressive demethylation of β_A- globin gene during embryonic development. Maximum methylation is seen in the different regions of the β_A- globin gene on day 5, which reduces on day 8 by about 50 %, and on day 11 the degree of methylation is minimal. Further, strand and regional asymmetry was seen with respect to loss of methylation. The template strand gets demethylated first and demethylation of the coding strand lags behind. The promoter and exon 1 get completely demethylated by day 11 whereas residual methylation is retained on exons 2 and 3. To investigate the mechanism of loss of methylation, we examined the methylation pattern of multiple DNA strands derived from primitive and early definitive embryonic erythroid cells. The primitive embryonic erythroid cells demonstrated methylation of the β_A- globin promoter and exon 1 on most of the CpG dinucleotides. Discontinuous loss of methylation in early definitive erythroid cells suggested an active mechanism of demethylation since the DNA replication mediated passive demethylation is likely to be continuous. To investigate if an active process contributes to the demethylation of β_A- globin gene, we used a modified methylation sensitive single nucleotide primer extension assay (MS-SNuPE) assay for quantitation of demethylase activity. Demethylase assay was carried out using synthetic unmethylated, hemimethylated, and completely methylated oligonucleotides derived from the β_A-globin promoter region and nuclear extracts from embryonic chicken erythroid cells. The demethylase activity in day 5, 8 and 11 nuclear extracts was 6%, 76%, and 24% respectively. In contrast to the previously reported 5-methylcytosine glycosylase, which shows a significant substrate specificity for the hemimethylated DNA, we observed the demethylase activity in the embryonic chicken erythroid cells on both fully methylated and hemimethylated β_A-globin promoter sequences. Since human and chicken methyl binding domain 4 (MBD4) proteins exhibit 5-methylcytosine glycosylase activity, we determined the in vivo binding of MBD4 to β_A- globin promoter in chicken erythroid cells using Chromatin ImmunoPrecipitation (ChIP) followed by real time polymerase chain reaction. MBD4 binding to β_A- globin promoter was seen in day 8 but not day 5 chicken erythroid cells implicating a possible role of chicken MBD4 in mediating the active demethylation of the avian β_A- globin gene. Our findings demonstrate that demethylation of β_A globin gene in developing primary chicken erythroid cells shows strand and regional asymmetry and occurs at least in part by an active process.