Functional characterization of the human TPH2 5' regulatory region: untranslated region and polymorphisms modulate gene expression in vitro.
Tryptophan hydroxylase-2 (TPH2) is a recently identified TPH isoform responsible for neuronal serotonin (5-HT) synthesis, and TPH2 polymorphisms are associated with a range of behavioral traits and psychiatric disorders. This study characterized cis-acting elements and three common polymorphisms (-703G/T, -473T/A, and 90A/G) in the 5' regulatory region of human TPH2 by using luciferase reporter assay, quantitative real-time PCR, and electrophoretic mobility shift assay (EMSA). The core promoter of human TPH2 was localized to the region between -107 and +7, and the segment of +8 to +53 within the 5'-UTR was found to exert a potent inhibitory effect on gene expression at both transcriptional and post-transcriptional levels. In both RN46A and HEK-293 cell lines, the TTA (-703T/-473T/90A) haplotype of the three polymorphisms showed the lowest gene expression compared with other haplotypes, and the -703G/T and -473T/A polymorphisms tended to exert a synergic effect on gene expression dependent upon the sequence of the 5'-UTR. In RN46A, the 90A/G polymorphism significantly increased luciferase activity and mRNA level irrespective of the other two polymorphisms, while in HEK-293 cells the effect of 90A/G was dependent on the alleles at loci -703 and -473. EMSA showed that all the three polymorphisms potentially alter DNA-protein interactions, while the 90A/G polymorphism predictably alters the 5'-UTR secondary structure of mRNA and influences RNA-protein interactions. In conclusion, our present study demonstrates that both the 5'-UTR and common polymorphisms (especially the 90A/G) in the 5' regulatory region of human TPH2 have a significant impact on gene expression.
Chen
Guo-Lin
GL
New England Primate Research Center, Division of Neurochemistry, Harvard Medical School, One Pine Hill Drive, Southborough, MA, 01772-9102, USA. gmiller@hms.harvard.edu
Vallender
Eric J
EJ
Miller
Gregory M
GM
D024506:5' Flanking Region; D020121:5' Untranslated Regions / Q000032:analysis / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004622:Embryo, Mammalian; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020641:Polymorphism, Single Nucleotide / Q000502:physiology*; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014365:Tryptophan Hydroxylase / Q000235:genetics*
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; C494784:TPH2 protein, human; D014365:Tryptophan Hydroxylase