NF-Y activates mouse tryptophan hydroxylase transcription.
Tryptophan hydroxylase catalyses the rate-limiting step in the biosynthesis of serotonin, a neurotransmitter which has been implicated in the etiologies of clinically important psychiatric illnesses. Tryptophan hydroxylase is expressed in a tissue-specific manner, but little is known about its transcriptional regulation. By analysing transcriptional activities of a set 5'-deletion constructs of promoter-reporter plasmids in P815-HTR mastocytoma cells, we found that transcription was activated by sequences between nucleotides -343 and -21. DNase I footprint 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, specifically bound nuclear protein in a gel shift assay. Methylation interference analysis of this complex revealed that nuclear protein interacted with an inverted GGCCAAT element, which is a high-affinity binding 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 assay. Mutagenesis of specific base pairs abolished protein binding in vitro, and mutagenesis of the same base pairs in a reporter gene construct resulted in a 65% decrease in transcriptional activity. Our results suggest that the transcription factor NF-Y binds to a GGCCAAT motif in the tph proximal promoter and activates transcription.
Reed
G E
GE
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Kirchner
J E
JE
Carr
L G
LG
D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry / Q000187:drug effects; D022762:CCAAT-Enhancer-Binding Proteins; D002467:Cell Nucleus / Q000378:metabolism; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014365:Tryptophan Hydroxylase / Q000096:biosynthesis* / Q000235:genetics; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D008156:Luciferases; D014365:Tryptophan Hydroxylase