Submitted February 12, 2002
Accepted July 15, 2002
Methylation of 
-type embryonic globin gene 
represses transcription in primary erythroid cells
Rakesh Singal*, Jane M vanWert, and Larry Ferdinand
* Corresponding author; email: rakeshsingal{at}hotmail.com.
The inverse relationship between expression and methylation of ß-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian -type globin genes. The embryonic -globin promoter was unmethylated and -globin RNA easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the -promoter associated with loss of expression of -globin gene was seen during development in primary erythroid cells. A 315 bp -globin promoter region was cloned in an expression construct (pGL3E) containing a luciferase reporter gene and SV40 enhancer. The pGL3E construct was transfected into primary erythroid cells derived from 5-day old chicken embryos. Methylation of pGL3E plasmid and -globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression respectively. The fully methylated but not the unmethylated 315-bp -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 -globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5 day when compared to 14 day erythroid cells. These results demonstrate that methylation can silence transcription of an avian -type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and a histone deacetylase complex.
