CHARACTERIZATION OF THE HUMAN TRYPTOPHAN-HYDROXYLASE GENE PROMOTER - TRANSCRIPTIONAL REGULATION BY CAMP REQUIRES A NEW MOTIF DISTINCT FROM THE CAMP-RESPONSIVE ELEMENT

We isolated and sequenced 2,117 nucleotides of the promoter region of the human tryptophan hydroxylase (TPH) gene. Transient transfection in pinealocyte cultures and PC12 cells was used to investigate the human TPH (hTPH) gene promoter activity and its regulation by the cAMP sign aling pathway. A region of 2,117 base pairs upstream of the transcript ion initiation site of the hTPH gene efficiently directed the transcri ption of a luciferase reporter gene but not in a cell-specific manner. The hTPH promoter activity was significantly enhanced by a cyclic AMP analog in the two cell types. Deletion analysis showed that the promo ter region from -73 to +2 is sufficient to direct cAMP-dependent trans cription, although it does not contain a motif exhibiting a significan t identity to the cAMP-responsive element (CRE) or AP-2 binding site, Following site-directed mutagenesis of the region between -73 and -51, an inverted CCAAT box motif was identified as essential for cAMP indu cibility of the hTPH promoter. This sequence between -73 and -51 alone allowed cAMP enhancement of transcription when fused to a heterologou s promoter. Additionally, electrophoretic mobility shift assays showed that a specific protein-DNA complex is formed between an oligonucleot ide corresponding to the inverted CCAAT box motif and nuclear proteins from pinealocytes treated or not treated with cAMP. Thus cAMP respons iveness of hTPH gene expression is mediated by a cis-acting element, w hich shares strong identitiy with an inverted CCAAT box and which bind s to a constitutively produced nuclear factor.