Tryptophan hydroxylase catalyses the rate-limiting step in the biosynt
hesis of serotonin, a neurotransmitter which has been implicated in th
e etiologies of clinically important psychiatric illnesses. Tryptophan
hydroxylase is expressed in a tissue-specific manner, but little is k
nown about its transcriptional regulation. By analysing transcriptiona
l activities of a set of 5'-deletion constructs of promoter-reporter p
lasmids in P815-HTR mastocytoma cells, we found that transcription was
activated by sequences between nucleotides - 343 and - 21. DNase I fo
otprint analysis, using nuclear protein extracts from P815-HTR cells,
revealed a protein-DNA interaction between nucleotides - 77 and - 46.
A double stranded oligonucleotide, representing this binding site, spe
cifically bound nuclear protein in a gel shift assay. Methylation inte
rference analysis of this complex revealed that nuclear protein intera
cted with an inverted GGCCAAT element, which is a high-affinity bindin
g motif for the transcription factor NF-Y (also known as CP1 or CBF).
An NF-Y specific antibody abolished protein binding in a gel shift ass
ay. Mutagenesis of specific base pairs abolished protein binding in vi
tro, and mutagenesis of the same base pairs in a reporter gene constru
ct resulted in a 65% decrease in transcriptional activity. Our results
suggest that the transcription factor NF-Y binds to a GGCCAAT motif i
n the tph proximal promoter and activates transcription.