Methylation of alpha-type embryonic globin gene alpha pi represses transcription in primary erythroid cells.
The inverse relationship between expression and methylation of beta-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian alpha-type globin genes. The embryonic alpha(pi)-globin promoter was unmethylated, and alpha(pi)-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the alpha(pi) promoter associated with loss of expression of alpha(pi)-globin gene was seen during development in primary erythroid cells. A 315-bp alpha(pi)-globin promoter region was cloned in an expression construct (alpha(pi)pGL3E) containing a luciferase reporter gene and SV40 enhancer. The alpha(pi)pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of alpha(pi)pGL3E plasmid and alpha(pi)-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp alpha(pi)-globin gene promoter fragment formed a methyl cytosine-binding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the alpha(pi)-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 alpha-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex.
Singal
Rakesh
R
Department of Medicine, Overton Brooks VA Medical Center and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport 71101, USA. rakeshsingal@hotmail.com
vanWert
Jane M
JM
Ferdinand
Larry
L
D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D005914:Globins / Q000235:genetics*; D006657:Histones / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D006657:Histones; D005914:Globins