Methylation of α-type embryonic globin gene α^π represses transcription in primary erythroid cells

The inverse relationship between expression and methylation of β-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian α-type globin genes. The embryonic α^π-globin promoter was unmethylated, and α-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the α^π promoter associated with loss of expression of α^π-globin gene was seen during development in primary erythroid cells. A 315-bp α^π-globin promoter region was cloned in an expression construct (α^πpGL3E) containing a luciferase reporter gene and SV40 enhancer. The α^πpGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of α^πpGL3E plasmid and α^π-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp α^π-globin gene promoter fragment formed a methyl cytosinebinding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the α^π-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian α-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.