CBF/NF-Y activates transcription of the human tryptophan hydroxylase gene through an inverted CCAAT box.
The human tryptophan hydroxylase gene (hTPH) 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 hTPH 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 nTPH gene.
Teerawatanasuk
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Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Carr
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D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D004790:Enzyme Induction; D017930:Genes, Reporter; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D012515:Mast-Cell Sarcoma / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012701:Serotonin / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014365:Tryptophan Hydroxylase / Q000096:biosynthesis / Q000235:genetics*; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D012701:Serotonin; D008156:Luciferases; D014365:Tryptophan Hydroxylase