Abstract
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 signaling pathway. A region of 2,117 base pairs upstream of the transcription initiation site of the hTPH gene efficiently directed the transcription 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 promoter region from -73 to +2 is sufficient to direct cAMP-dependent transcription, although it does not contain a motif exhibiting a significant 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 inducibility of the hTPH promoter. This sequence between -73 and -51 alone allowed cAMP enhancement of transcription when fused to a heterologous promoter. Additionally, electrophoretic mobility shift assays showed that a specific protein-DNA complex is formed between an oligonucleotide corresponding to the inverted CCAAT box motif and nuclear proteins from pinealocytes treated or not treated with cAMP. Thus cAMP responsiveness of hTPH gene expression is mediated by a cis-acting element, which shares strong identity with an inverted CCAAT box and which binds to a constitutively produced nuclear factor.
Highlights
From the Laboratoire de Genetique Moleculaire, de la Neurotransmission, et des Processus Neurodegeneratifs, C.N.R.S., F91198 Gif-sur-Yuette Cedex, France
We have studied the genomic organization of the human TPH (hTPH) gene and shown that a single transcriptional initiation site produces a large diversity of tryptophan hydroxylase (TPH) mRNAs
We show that the 2-kb region upstream of the single human TPH mRNA cap site exhibits the characteristic features of a promoter and is able to drive the expression of a luciferase reporter gene in primary cultures of rat pinealocytes and in PC12 cells
Summary
TRANSCRIPTIONAL REGULATION BY cAMP REQUIRES A NEW MOTIF DISTINCT FROM THE cAMP-RESPONSIVE ELEMENT*. Following site-directed mutagenesis of the region between -73 and -51, an inverted CCAAT box motif was identified as essential for cAMP inducibility ofthe hTPH promoter This sequence between -73 and -51 alone allowed cAMP enhancement of transcription when fused to a heterologous promoter. We show that the 2-kb region upstream of the single human TPH mRNA cap site exhibits the characteristic features of a promoter and is able to drive the expression of a luciferase reporter gene in primary cultures of rat pinealocytes and in PC12 cells. Human Tryptophan Hydroxylase Gene Promoter cAMP treatment in the two cell types, and we identify by site-directed mutagenesis a cAMP regulatory motif very similar to an inverted CCAAT box and which confers cAMP responsiveness to a heterologous promoter
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