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 exte nding from nucleotide(nt) -1954 to +40 and found that the region betwe en nt -163 and +40 contains a regulatory element important for efficie nt transcription of the gene. DNase I footprint analyses, using P815-H TR and HeLa nuclear protein extracts, revealed a single prominent foot print between nt -78 and -44. A cis-acting element in the footprint re gion was identified as an inverted CCAAT box (-67 ATTGG -63) by gel sh ift assays. Two base pair (bp) mutations in the core CCAAT sequence el iminated protein binding in gel shift assays and reduced transcription al activity approximately 50% in transient transfection assays. Compet itive gel shift assays showed that the protein binding to the hTPH CCA AT box was effectively competed by an oligonucleotide (oligo) harborin g 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 supershi fted the protein-DNA complex formed between the -90/-50 oligo probe an d nuclear protein extracts. Our results indicate that the binding of C BF/NF-Y to the inverted CCAAT box is responsible for transcriptional a ctivation of the hTPH gene. (C) 1998 Elsevier Science B.V.