Vimentin exhibits a complex pattern of tissue-specific and developmentally regulated expression, but the mechanisms underlying the complex transcriptional regulation remain poorly understood. Here we examined whether vimentin expression can be regulated by CpG methylation of the vimentin promoter. Two subclones of the rat C6 glioma cells were established with (C6vim+) and without (C6vim-) vimentin. Bisulfite genomic sequencing revealed that the vicinity of the transcription start site within the vimentin promoter is highly methylated in C6vim- cells but not in C6vim+ cells. Treatment of C6vim- cells with a demethylating agent, 5-aza-2'-deoxycytidine, restored vimentin expression, indicating that hypermethylation of the promoter region correlates with transcriptional silencing of the vimentin gene. Electrophoretic mobility shift assay (EMSA) and transient transfection experiments demonstrated that YY1 is a key transcriptional activator regulating vimentin expression and that CpG methylation is sufficient to prevent the binding of YY1 to the vimentin promoter. These data suggest that the inability of YY1 to access the hypermethylated promoter may be one of the mechanisms that mediate vimentin downregulation. (C) 2011 Elsevier Inc. All rights reserved.