Blood, 1 December 2002, Vol. 100, No. 12, pp. 4217-4222
RED CELLS
Methylation of 
-type embryonic globin gene 
represses transcription in primary erythroid cells
Rakesh Singal,
Jane M. vanWert, and
Larry Ferdinand Jr
From the Department of Medicine, Overton Brooks VA
Medical Center and Feist-Weiller Cancer Center, Louisiana State
University Health Sciences Center, Shreveport.
The inverse relationship between expression and methylation of
-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 was 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 with 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 histone deacetylase complex.
