THE RESPONSE OF THE TYROSINE-HYDROXYLASE GENE TO CYCLIC-AMP IS MEDIATED BY 2 CYCLIC-AMP-RESPONSE ELEMENTS

Tyrosine hydroxylase (TH) gene transcription rate is stimulated by cyc lic AMP in cultured rat pheochromocytoma cells. This effect is at leas t partially due to the interaction of transcription factors with the c anonical cyclic AMP-response element (CRE) at position -45 to -38 with in 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 AM P. Using electrophoretic mobility shift assays, we demonstrate that nu clear proteins from rat pheochromocytoma cell lines bind to the region of the TH gene from -102 to -73. A comparison of promoter sequences i ndicates that sequences within this region of the TH gene are highly h omologous to proenkephalin promoter sequences (between -110 and -80) d esignated ENKCRE-1 and ENKCRE-2. We designated the TH gene sequence ho mologous 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 pr omoter constructs demonstrate that this region acts as a cyclic AMP-re sponsive element, Mutagenesis of the TH E1 sequence within the normal context of the TH gene proximal promoter leads to a 50% decrease in th e cyclic AMP inducibility of the promoter. These results support the h ypothesis 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.