Glutamate is the major excitatory neurotransmitter in the CNS that is cleared 
from the extracellular space by a family of high-affinity glutamate 
transporters. The astroglial glutamate transporter EAAT2 is thought to carry out 
the uptake of the vast quantity of glutamate, and dysregulation of EAAT2 
expression is involved in the pathogenesis of neurological disorders with marked 
excitotoxic components. Here, we present a novel epigenetic mechanism by which 
the human EAAT2 gene is kept in a silent state. Sequence inspection identified a 
classical CpG island at the EAAT2 promoter. Bisulfite analysis of the DNA 
methylation profile revealed that lack of EAAT2 expression in human glioma cell 
lines was associated with a densely methylated EAAT2 promoter. In contrast, 
EAAT2 positive normal human brain tissue used as reference displayed 
hypomethylation of the same promoter regions. In vitro methylation of EAAT2 
promoter sequences indeed altered the binding properties of nuclear factors to 
the respective DNA sites as illustrated by electrophoretic mobility shift assay. 
Moreover, we observed a reduced activity of a methylated EAAT2 promoter 
construct as compared to the unmethylated control, both in a human glioma cell 
line and rodent primary astrocytes. Further supporting a role of DNA methylation 
for EAAT2 silencing, inhibition of DNA methyltransferases robustly enhanced 
EAAT2 mRNA transcription in several cell lines tested. In conclusion, the idea 
is put forward of an epigenetic mode of EAAT2 regulation based on the 
differential methylation of the gene promoter. (c) 2007 Wiley-Liss, Inc.