Data describing the effect of DRD4 promoter polymorphisms on promoter activity

This data article tested whether polymorphisms within the dopamine D4 receptor (DRD4) gene promoter can lead to differences in the promoter activity. The variants, a 120-bp variable number tandem repeat (VNTR), −906 T/C, −809 G/A, −616G/C, and −521C/T, were introduced into the DRD4 promoter and the promoter activity was measured in a neural cell line using the luciferase assay. However, no differences were detected among the haplotypes investigated, and the in vitro data obtained from our protocol could not support the involvement of DRD4 promoter polymorphisms in heritable human traits.


a b s t r a c t
This data article tested whether polymorphisms within the dopamine D4 receptor (DRD4) gene promoter can lead to differences in the promoter activity. The variants, a 120-bp variable number tandem repeat (VNTR), À 906 T/C, À 809 G/A, À 616G/C, and À 521C/T, were introduced into the DRD4 promoter and the promoter activity was measured in a neural cell line using the luciferase assay. However, no differences were detected among the haplotypes investigated, and the in vitro data obtained from our protocol could not support the involvement of DRD4 promoter polymorphisms in heritable human traits.
& 2016 Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Biology More specific subject area

Molecular biology, Genetics
Type of data This data is useful for characterising the link between heritable mental traits and the polymorphisms.
Our data can provide insight into methodology and considerations for investigation of polymorphisms in non-coding regions.

Data
Endogenous dopamine D4 receptor (DRD4) gene expression in SH-SY5Y cells was detected by RT-PCR using cDNA derived from the cell line ( Fig. 1).
To test whether the polymorphisms within the promoter change the promoter activity, luciferase activity was measured under the influence of the DRD4 promoter into which polymorphisms were introduced ( Fig. 2 and Table 1). All of the reporter plasmids containing the DRD4 fragment exhibited significantly higher luciferase activity than the control pGL3-Basic, and although every possible combination of haplotypes was investigated, there were no activity differences among the introduced mutations in SH-SY5Y cells (Figs. 3 and 4).

Construction of reporter plasmid
A DNA fragment spanning À 1576 to À 1 of the DRD4 promoter region was amplified from human genomic DNA with TaKaRa LA Taq (TaKaRa) and inserted into pCR-Blunt (Life Technology). The cloned sequence was confirmed by Sanger sequencing and shown in Supplementary the VNTR; DNA ligation after NotI treatment converts a 2-repeat allele into a 1-repeat allele because one NotI recognition site is present within the repeat. The mutated insertion was subcloned into the XhoI and HindIII sites of pGL3-Promoter Vector (Promega) replacing the original SV40 promoter with the DRD4 promoter. The primer sequences used for construction are shown in Table 2.

Cell culture and transfection
Human neuroblastoma SH-SY5Y cells were cultured in Dulbecco's Modified Eagle's Medium (Sigma-Aldrich) supplemented with 10% foetal bovine serum (Invitrogen) at 37°C in a humidified atmosphere containing 5% CO 2 . Twenty-four hours before transfection, cells were plated at 4 Â 10 5 cells/well in 96-well plates.

Luciferase assay
Forty-eight hours after transfection, the luciferase activity was measured in quadruplicate with the Dual-Glo Luciferase assay System (Promega) using Centro LB960 (Berthold), following the manufacturer's instructions. Relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity.

Total RNA isolation and RT-PCR
Total RNA of SH-SY5Y was extracted with GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich). First-strand cDNA was synthesised from extracted RNA using Prime Script RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa). DRD4 mRNA expression was detected using TaKaRa LA Taq, as described [1]. To verify the procedure, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control. The primer sequences used for RT-PCR are shown in Table 2.  The DNA fragment cloned here corresponded to region À 1576 to À 1 relative to the translation start site, and was the longest among functional assays on mutated DRD4 promoter;À 591 to À 123 was cloned in Okuyama et al [2], À 1389 to À 1203 in D'Souza et al. [3], À 668 to À 389 in Kreszturi et al. [4] and À 1571 to À 389 in Kreszturi et al. [5].The putative silencer (dark grey boxes, À 1571 to À 800 and À 770 to À 678) and enhancer (light grey box, À 591 to À 123) regions are indicated in the DRD4 gene promoter (white arrow) [1,4]. The 120-bp VNTR is 1.2 kb upstream from the initial codon, and À 521C/T is a C/T SNP at À 521 in the promoter region (the description can be applied to the other SNPs). The promoter was cloned upstream of the firefly luciferase gene, so that luciferase expression was driven by the promoter. pGL3 promoter which contains SV40 promoter upstream luciferase gene was used as positive control.

Table 1
The constructed haplotypes consisted of 120-bp VNTR and four SNPs of the DRD4 gene.