NF-Y ACTIVATES MOUSE TRYPTOPHAN-HYDROXYLASE TRANSCRIPTION

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.