Abbreviations used : CDP, CCAAT displacement protein ; Clox, Cutlike homeobox ; CR I, II, and III, Cut repeats I, II, and III, respectively ; Cux, Cut homeobox ; DBD, DNA binding domain ; DTT, dithiothreitol ; EMSA, electromobility shift analysis ; HD, homeodomain ; hTPH, human tryptophan hydroxylase gene ; poly(dI-dC), polydeoxyinosinic-deoxycytidylic acid ; RLA, relative luciferase activity ; TPH, tryptophan hydroxylase.
CCAAT Displacement Protein (CDP/Cut) Binds a Negative Regulatory Element in the Human Tryptophan Hydroxylase Gene
Article first published online: 18 JAN 2002
Journal of Neurochemistry
Volume 72, Issue 1, pages 29–39, January 1999
How to Cite
Teerawatanasuk, N., Skalnik, D. G. and Carr, L. G. (1999), CCAAT Displacement Protein (CDP/Cut) Binds a Negative Regulatory Element in the Human Tryptophan Hydroxylase Gene. Journal of Neurochemistry, 72: 29–39. doi: 10.1046/j.1471-4159.1999.0720029.x
- Issue published online: 18 JAN 2002
- Article first published online: 18 JAN 2002
- Tryptophan hydroxylase;
- CCAAT displacement protein;
Abstract : 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.