TISSUE-SPECIFIC METHYLATION OCCURS IN THE ESSENTIAL PROMOTER ELEMENT OF THE TYROSINE-HYDROXYLASE GENE

Expression of tyrosine hydroxylase (TH) is regulated in a tissue-speci fic manner by multiple mechanisms. In catecholaminergic cells, the exp ression of TH-mRNA is up-regulated by forskolin (FK) and is suppressed by retinoic acid (RA). We have previously provided evidence that, in N-18 cells, the expression of TH-mRNA is suppressed by DNA methylation of the TH gene itself. In the present study, using a catecholaminergi c cell line, N1E-115, we performed deletional and mutational analyses on the 5'-flanking region of the mouse TH gene. The results indicate t hat a cAMP response element (CRE) mediates constitutive transcription of the TH gene, as well as responsiveness to FK and RA. Using bisulfit e sequencing methods, we analyzed the methylation status of the TH gen e 5'-flanking region in various cell lines and rat tissues. We found t hat three cytosine residues in the domain surrounding the CRE of the T H gene promoter were specifically methylated in N-18 cells and TH non- expressing rat tissues. In contrast, these cytosines were undermethyla ted in TH expressing cell lines and tissues. The inverse correlation b etween the frequency of cytosine methylation at these specific sites a nd the levels of TH expression supports a role for DNA methylation in the regulation of tissue-specific gene expression.