Human Tryptophan Hydroxylase Research Articles

CBF/NF-Y activates transcription of the human tryptophan hydroxylase gene through an inverted CCAAT box

The human tryptophan hydroxylase gene (h TPH) encodes the rate-limiting enzyme in the biosynthesis of serotonin, a neurotransmitter which has been implicated in a number of psychiatric illnesses. This enzyme is expressed in a tissue-specific manner. We examined the transcriptional activity of a series of 5′ deletion promoter–reporter constructs extending from nucleotide (nt) −1954 to +40 and found that the region between nt −163 and +40 contains a regulatory element important for efficient transcription of the gene. DNase I footprint analyses, using P815-HTR and HeLa nuclear protein extracts, revealed a single prominent footprint between nt −78 and −44. A cis-acting element in the footprint region was identified as an inverted CCAAT box (−67 ATTGG −63) by gel shift assays. Two base pair (bp) mutations in the core CCAAT sequence eliminated protein binding in gel shift assays and reduced transcriptional activity approximately 50% in transient transfection assays. Competitive gel shift assays showed that the protein binding to the h TPH CCAAT box was effectively competed by an oligonucleotide (oligo) harboring a binding site for CCAAT box binding factor (CBF)/nuclear factor-Y (NF-Y). A selective antibody against the B subunit of CBF/NF-Y supershifted the protein-DNA complex formed between the −90/−50 oligo probe and nuclear protein extracts. Our results indicate that the binding of CBF/NF-Y to the inverted CCAAT box is responsible for transcriptional activation of the h TPH gene.

Sequence, splice site and population frequency distribution analyses of the polymorphic human tryptophan hydroxylase intron 7.

A human tryptophan hydroxylase intron seven polymorphism previously associated with low CSF 5-HIAA and suicidal behavior was sequenced and characterized for its potential role in TPH pre-mRNA splicing. Two polymorphic sites were identified: A218C and A779C. The 779A allelic frequency in various populations ranged from 0.43 to 0.61 and was in strong linkage disequilibrium with the A218C site. A218C provides a site for restriction fragment length polymorphism analysis. TPH mRNA was reverse-transcribed and sequenced. No aberrant splice products from the 779A or 779G TPH genes were detected nor were any other polymorphic nucleotides found.

The Human Tryptophan Hydroxylase Gene: AN UNUSUAL SPLICING COMPLEXITY IN THE 5′-UNTRANSLATED REGION

We report the isolation and the organization of the gene encoding human tryptophan hydroxylase (TPH) and an analysis of the corresponding mRNAs. The gene spans a region of 29 kilobases, which contains at least 11 exons and a variably spliced 5'-untranslated region (5'-UTR). The sequence of the coding region and the majority of the positions of the intron-exon boundaries of human TPH gene are very similar to those encoding human tyrosine hydroxylase and phenylalanine hydroxylase, the other members of the aromatic amino acid hydroxylase family. Phylogenetic analysis evidences the early divergence and the independent evolution of the three hydroxylase types. TPH cDNA cloning and anchored polymerase chain reaction revealed a diversity of the TPH mRNA, which is restricted to the 5'-UTR. Four TPH mRNA species were detected by Northern blot with pineal gland and carcinoid tumor RNAs. These messengers are transcribed from a single transcriptional initiation site, and their diversity results from differential splicing of three intron-like regions and of three exons located in the 5'-UTR. Analysis by S1 nuclease protection revealed that the intron-like regions in the 5'-UTR are mostly unspliced and that TPH mRNA species where the three intron-like regions are eliminated are present at low level in pineal gland and not detectable in carcinoid tumors.

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 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.

High-level expression and deletion mutagenesis of human tryptophan hydroxylase.

Human tryptophan hydroxylase has been expressed as a soluble and active form in Escherichia coli by fusion with an affinity tag, maltose-binding protein. The fusion protein has been purified to near homogeneity by affinity chromatography on crosslinked amylose resin. The purified fusion protein has a specific activity of 86 nmol of 5-hydroxytryptophan per min per mg of fusion protein. A series of truncation mutants have also been made to explore the domain organization of tryptophan hydroxylase. All deletion mutants were subject to affinity purification and kinetic characterization. While removal of the N-terminal 164 amino acids completely inactivates the enzyme, deletion of the first 91 residues results in a 7-fold reduction in specific activity. From the C terminus, deletion of 36, 55, or 112 amino acids abolishes the activity, whereas deletion of 19 residues decreases the specific activity by approximately 11-fold. These results are consistent with a model for tryptophan hydroxylase in which the enzyme consists of an N-terminal regulatory domain, a catalytic core, and a small C-terminal region of uncertain but important function.

Correlation of a human tryptophan hydroxylase polymorphism to phenotypes related to serotonergic function : David A. Nielsen, David Goldman, Matti Virkkunen, Riitta Tokola, Robert Rawlings, Markku Linnoila National Institute of Alcohol Abuse and Alcoholism, Bethesda MD 20892 and University of Helsinki, Helsinki, Finland

Correlation of a human tryptophan hydroxylase polymorphism to phenotypes related to serotonergic function : David A. Nielsen, David Goldman, Matti Virkkunen, Riitta Tokola, Robert Rawlings, Markku Linnoila National Institute of Alcohol Abuse and Alcoholism, Bethesda MD 20892 and University of Helsinki, Helsinki, Finland

Localization of human tryptophan hydroxylase (TPH) to chromosome 11p15.3→p14 by in situ hybridization

The human gene for tryptophan hydroxylase has been previously assigned to chromosome 11 by analysis of a panel of somatic cell hybrids. We report here on the refinement of this localization by in situ hybridization.

Complete coding sequence of human tryptophan hydroxylase

Complete coding sequence of human tryptophan hydroxylase

Assignment of human tryptophan hydroxylase locus to chromosome 11: gene duplication and translocation in evolution of aromatic amino acid hydroxylases.

A cDNA clone for rabbit tryptophan hydroxylase was used as a probe to identify human tryptophan hydroxylase gene fragments in a panel of hamster-human somatic cell hybrids and determine its chromosomal location in man. A single locus was identified for tryptophan hydroxylase on chromosome 11. Tryptophan hydroxylase is a member of the superfamily of pterin-dependent aromatic amino acid hydroxylases which includes tyrosine hydroxylase, located at 11p15.5-p15, and phenylalanine hydroxylase, located at 12q22-q24.1 in human. The locations of these genes and the evolutionary distance between their sequences suggest that at least three distinct genetic events have occurred during the evolution of the aromatic amino acid hydroxylase superfamily: two sequential gene duplications giving rise to the three distinct hydroxylase loci, and a translocation which separated the tryptophan and tyrosine hydroxylase loci on chromosome 11 from the phenylalanine hydroxylase locus on chromosome 12.