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Functional characterization of the human TPH2 5′ regulatory region: untranslated region and polymorphisms modulate gene expression in vitro

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Abstract

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.

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Acknowledgments

This study was supported by AA016194 (GMM), DA016606 (GMM), DA06303 (GMM), DA021180 (GMM), MH082507 (EJV) and RR00168.

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  1. New England Primate Research Center, Division of Neurochemistry, Harvard Medical School, One Pine Hill Drive, Southborough, MA, 01772–9102, USA

    Guo-Lin Chen, Eric J. Vallender & Gregory M. Miller

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Correspondence to Gregory M. Miller.

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Chen, GL., Vallender, E.J. & Miller, G.M. Functional characterization of the human TPH2 5′ regulatory region: untranslated region and polymorphisms modulate gene expression in vitro. Hum Genet 122, 645–657 (2008). https://doi.org/10.1007/s00439-007-0443-y

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