Localized methylation in the key regulator gene endothelin-1 is associated with cell type-specific transcriptional silencing.

DNA methylation can contribute to the stable transcriptional silencing of mammalian genes. Often times, these genes are important developmental regulators, and their silencing in cell types where they are not supposed to be active is important for the phenotypic stability of the cells. To identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation, mouse dermal fibroblasts were demethylated with 5-aza-2'-deoxycytidine, and changes in gene expression monitored by microarray analysis. Endothelin-1 (Et1 or Edn1), which encodes a cytokine with diverse regulatory functions, was among the genes upregulated following demethylation. We found that CpG dinucleotides within a short region in intron 1 of the gene have dramatically higher levels of methylation in Et1-non-expressing fibroblasts and chondrocytes as compared to the Et1-expressing mouse cell line, mIMCD-3. Strong evolutionary conservation of this region implies its role in the cis-regulation of Et1 transcription. To confirm that should Et1 in dermal fibroblasts become aberrantly activated, it could indeed lead to the dysregulation of many downstream genes, we exposed fibroblasts to exogenous ET1 peptide and assayed for transcriptional changes by microarray. ET1 treatment resulted in significant expression changes - primarily downregulation - of a significant number of genes. In particular, Tgfbeta2 and Tgfbeta3 were among the downregulated genes, which in turn alter the expression status of their many target genes. These data suggest that the stable silencing of Et1 through its associated DNA methylation in intron 1 is critical for the developmental stability of dermal fibroblasts, and perhaps other cell types as well.