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

Abstract

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.