Regulation of tyrosine hydroxylase gene transcription by the cAMP-signalling pathway: Involvement of multiple transcription factors

The conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine by tyrosine h ydroxylase (TH) is the first and rate-limiting step in biosynthesis of cate cholamine neurotransmitters. TH gene expression is regulated in a cell type -specific and cAMP-dependent manner. Evidence from this laboratory and othe rs indicates that the cAMP response element (CRE), residing at -45 to -38 b p upstream of the transcription initiation site, is essential for both basa l and cAMP-inducible transcription of the TH gene. To understand the contro l mechanisms of TH gene transcription in greater detail, we sought to ident ify and characterize the transcription factors involved in recognition and activation of the CRE of the TH gene. Remarkably, electrophoretic mobility shift assay and antibody supershift experiments indicated that all three ma jor CRE-binding protein factors, i.e. CREB, ATF1, and CREM, may participate in forming specific DNA/protein complexes with the CRE of the TH gene. To address the transcriptional activation function of individual factors, we r eplaced the TH CRE with a GAL4-binding site and cotransfected this modified TH promoter-reporter gene with an effector plasmid that encodes GAL4-fused transcription factor. Our results indicate that CREB but not ATF1 can supp ort basal promoter activity while both can robustly induce the promoter act ivity in response to co-expression of the catalytic subunit of cAMP-depende nt protein kinase (PKA). We further show that the coactivator CBP up-regula tes PKA-mediated activation of the TH promoter and, if tethered to the TH p romoter by a GAL4-fusion, can robustly transactivate the TH promoter even i n the absence of PKA. Collectively, our results suggest that multiple CRE-b inding factors interact with the CRE and regulate, in conjunction with the coactivator CBP, the transcriptional activity of the TH gene.