CCAAT displacement protein (CDP/cut) binds a negative regulatory element in the human tryptophan hydroxylase gene.
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.
Teerawatanasuk
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Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Skalnik
D G
DG
Carr
L G
LG
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D019175:DNA Methylation; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000502:physiology; D004214:Distamycins / Q000494:pharmacology; D042963:Electron Transport Complex II; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D012515:Mast-Cell Sarcoma; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D014365:Tryptophan Hydroxylase / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000201:enzymology
D000998:Antiviral Agents; C000624101:Cux1 protein, mouse; D015342:DNA Probes; D004268:DNA-Binding Proteins; D004214:Distamycins; D018398:Homeodomain Proteins; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D012097:Repressor Proteins; C030000:stallimycin; D010088:Oxidoreductases; D014365:Tryptophan Hydroxylase; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase; D016660:NAD(P)H Dehydrogenase (Quinone)