Methylation of α-type embryonic globin gene απrepresses 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 π -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 π pGL3E) containing a luciferase reporter gene and SV40 enhancer. The α π pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of α π pGL3E 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 amethyl cytosine-binding proteincomplex (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.