CCAAT Displacement Protein (CDP/Cut) Binds a Negative Regulatory Element in the Human Tryptophan Hydroxylase Gene

• Published in: Journal of neurochemistry 1999-01, Vol.72 (1), p.29-39
• Main Authors: Teerawatanasuk, Nongnit, Skalnik, David G, Carr, Lucinda G
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antiviral Agents - pharmacology; CCAAT displacement protein; CDP/Cut; Distamycins - pharmacology; DNA Methylation; DNA Probes; DNA-Binding Proteins - physiology; Electron Transport Complex II; Gene Expression Regulation, Enzymologic - physiology; Gene Expression Regulation, Neoplastic - physiology; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Mast-Cell Sarcoma; Mice; Molecular Sequence Data; Multienzyme Complexes - metabolism; NAD(P)H Dehydrogenase (Quinone) - metabolism; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Oxidoreductases - metabolism; Protein Binding - drug effects; Protein Binding - genetics; Regulatory Sequences, Nucleic Acid; Repressor Proteins - genetics; Repressor Proteins - metabolism; Serotonin; Succinate Dehydrogenase - metabolism; Transcription; Transcription, Genetic - physiology; Tryptophan hydroxylase; Tryptophan Hydroxylase - genetics; Tryptophan Hydroxylase - metabolism; Tumor Cells, Cultured - chemistry; Tumor Cells, Cultured - enzymology
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.1999.0720029.x

: Tryptophan hydroxylase (TPH) is the rate‐limiting enzyme in the biosynthesis of serotonin, a neurotransmitter that has been implicated in many psychiatric illnesses. The mechanism of transcriptional regulation of the human TPH gene is largely unknown. We have identified a negative regulatory element located between nucleotides ‐310 and ‐220 in the human TPH (hTPH) gene. Electromobility shift analyses performed with the ‐310/‐220 hTPH probe and nuclear extract from P815‐HTR (a TPH‐expressing cell line) revealed two slow migrating protein‐DNA complexes, designated I and II. CCAAT displacement protein (CDP/Cut) is involved in complex I formation as shown in electromobility shift analysis, using consensus oligonucleotide competitor and antibody. Mutations in the CDP/Cut binding site not only disrupted the CDP‐DNA complex but also disrupted the second complex, suggesting that the core binding sequences of the two proteins are overlapping. The functional importance of these protein‐DNA interactions was assessed by transiently transfecting wild‐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 activation of hTPH gene.