The response of the tyrosine hydroxylase gene to cyclic AMP is mediated by two cyclic AMP-response elements

Tyrosine hydroxylase (TH) gene transcription rate is stimulated by cyclic AMP in cultured rat pheochromocytoma cells. This effect is at least partially due to the interaction of transcription factors with the canonical cyclic AMP-response element (CRE) at position -45 to -38 within the TH gene promoter. In this study we test whether a region of the TH gene promoter, which is adjacent to and upstream of the canonical TH CRE, also participates in the response of the promoter to cyclic AMP. Using electrophoretic mobility shift assays, we demonstrate that nuclear proteins from rat pheochromocytoma cell lines bind to the region of the TH gene from -102 to -73. A comparison of promoter sequences indicates that sequences within this region of the TH gene are highly homologous to proenkephalin promoter sequences (between -110 and -80) designated ENKCRE-1 and ENKCRE-2. We designated the TH gene sequence homologous to ENKCRE-1 as TH E1 and the sequence homologous to ENKCRE-2 as TH E2. Competition displacement binding assays suggest that protein binding to the -102/-73 region of the TH gene is critically dependent on the TH E1 sequence. Transient transfection assays using minimal promoter constructs demonstrate that this region acts as a cyclic AMP-responsive element. Mutagenesis of the TH E1 sequence within the normal context of the TH gene proximal promoter leads to a 50% decrease in the cyclic AMP inducibility of the promoter. These results support the hypothesis that the full response of the TH gene to cyclic AMP requires both the canonical TH CRE and this newly discovered element, which we term TH CRE2.