CCAAT displacement protein (CDP/Cut) binds a negative regulatory element in the human Tryptophan hydroxylase gene

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesi s of serotonin, a neurotransmitter that has been implicated in many psychia tric illnesses. The mechanism of transcriptional regulation of the human TP H gene is largely unknown. We have identified a negative regulatory element located between nucleotides -310 and -220 in the human TPH (hTPH) gene. El ectromobility shift analyses performed with the -310/-220 hTPH probe and nu clear extract from P815-HTR (a TPH-expressing cell line) revealed two slow migrating protein-DNA complexes, designated I and II. CCAAT displacement pr otein (CDP/Cut) is involved in complex I formation as shown in electromobil ity shift analysis, using consensus oligonucleotide competitor and antibody . Mutations in the CDP/Cut binding site not only disrupted the CDP-DNA comp lex but also disrupted the second complex, suggesting that the core binding sequences of the two proteins are overlapping. The functional importance o f these protein-DNA interactions was assessed by transiently transfecting w ild-type and mutant pTPH/luciferase reporter constructs into P815-HTR cells . Mutations in the core CDP/Cut site resulted in an approximately fourfold increase in relative luciferase activities. Because CDP/Cut has been shown to repress transcription of many target genes, we speculate that disruption of the CDP/Cut binding was responsible, at least in part, for the activati on of hTPH gene.