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.