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CCAAT Displacement Protein (CDP/Cut) Binds a Negative Regulatory Element in the Human Tryptophan Hydroxylase Gene
Author(s) -
Teerawatanasuk gnit,
Skalnik David G,
Carr Lucinda G
Publication year - 1999
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1999.0720029.x
Subject(s) - tryptophan hydroxylase , microbiology and biotechnology , gene , electrophoretic mobility shift assay , reporter gene , biology , mutant , dna , oligonucleotide , binding site , transcription factor , gene expression , regulation of gene expression , regulatory sequence , genetics , serotonin , receptor , serotonergic
: Tryptophan hydroxylase (TPH) is the rate‐limiting enzymein the biosynthesis of serotonin, a neurotransmitter that has been implicatedin many psychiatric illnesses. The mechanism of transcriptional regulation ofthe human TPH gene is largely unknown. We have identified a negativeregulatory element located between nucleotides ‐310 and ‐220 in the human TPH (h TPH ) gene. Electromobility shift analyses performedwith the ‐310/‐220 h TPH probe and nuclear extract from P815‐HTR (aTPH‐expressing cell line) revealed two slow migrating protein‐DNA complexes,designated I and II. CCAAT displacement protein (CDP/Cut) is involved incomplex I formation as shown in electromobility shift analysis, usingconsensus oligonucleotide competitor and antibody. Mutations in the CDP/Cutbinding site not only disrupted the CDP‐DNA complex but also disrupted thesecond complex, suggesting that the core binding sequences of the two proteinsare overlapping. The functional importance of these protein‐DNA interactionswas assessed by transiently transfecting wild‐type and mutantp TPH/luciferase reporter constructs into P815‐HTR cells. Mutations inthe core CDP/Cut site resulted in an approximately fourfold increase inrelative luciferase activities. Because CDP/Cut has been shown to represstranscription of many target genes, we speculate that disruption of theCDP/Cut binding was responsible, at least in part, for the activation ofh TPH gene.