Functional analysis of 5 upstream polymorphic variations of the human dopamine D1 receptor gene

We previously identified eight SNPs (single‐nucleotide polymorphisms) in the 5 regulatory region of the DRD1 gene.1 However, the function of the other 5 regulatory region (−2000 bp from the transcriptional start site) and the role of the different SNP loci have not been well charac‐ terized. In this study, luciferase reporter vectors containing a single SNP locus were constructed to explore the influence of the SNPs on the 5 promoter activity of the DRD1 gene. Moreover, the electrophoretic mo‐ bility shift assay (EMSA) and vectors containing serial deletion fragments were used to identify possible transcription factors and the DNA‐binding domain. In addition, we explored the role of three transcription factors (SP1, TFAP2B and CREB1) in the 5 promoter activity of the DRD1 gene and expression of endogenous DRD1 in the HEK293 cell line.


| BACKG ROU N D
We previously identified eight SNPs (single-nucleotide polymorphisms) in the 5 regulatory region of the DRD1 gene. 1

| SNP screening and construction of luciferasereceptor vectors
A total of nine vectors were constructed, which contained eight single SNP loci (Table S1). The vector M2 was subsequently used as the template to generate the 5 deletion vectors. The sequences of these primers are shown in Table S2.

| Cell culture and transient transfection
Human neuroblastoma cell line SK-N-SH and human embryonic kidney cell line HEK293 were used to test the luciferase activity of the generated vectors. 2 Both of the cells were seeded in 24-well plates (1 × 10 5 cells per well) and the pGL3 vectors (1.0 μg) were co-transfected with Renilla luciferase (100 ng) expression vector pRL-TK (Promega) using Lipofectamine 3000 reagent according to the manufacturer's protocol (Invitrogen, CA, USA).

| Luciferase assay
Here, 24 hours after transfection, cells were harvested. Firefly luciferase activity in the cell lysates was normalized to Renilla luciferase activity. Normalized activities of all the test clones were compared to those of the clones representing the reference haplotype. Each assay was performed in triplicate in two independent experiments.   (Table S3).

| Transcription factor-binding prediction
The prediction software Match was used to identify putative-binding sites for transcription factors at specific SNPs in the promoter region of the DRD1 gene (http://www.gene-regul ation.com/pub/ progr ams.html).

| Real-time PCR (RT-PCR)
However, β-actin was used as the reference gene. The mean value for the pEGFP-N1-basic vector control was used as a reference for statistical analysis.

| Statistics
Data are presented as the mean (χ) ± SD. Differences between the two groups were determined using the analysis of variance (ANOVA) or independent samples using the t test. P < 0.05 indicated statistically significant differences. SPSS 18.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis.

| Luciferase assays
In HEK293 cells, there was a significant increase in luciferase activity for clones M2, M3, M6 and M7 compared to WT with M2 demonstrating the highest activity (P < 0.05; Figure S1). The activity of M2 increased approximately 1.4 folds as compared with the activity of the WT. There was no significant difference between the clones and WT in the SK-N-SH cells ( Figure S2).

| Electrophoretic mobility shift assay
We conducted EMSA using specific probes to detect whether the rs10078866 locus could affect DNA-protein interactions because the M2 vector showed the highest luciferase activity in HEK293 cells.
As shown in Figure 1A-D, EMSA with SK-N-SH and HEK293 nuclear extracts revealed that two specific complexes were formed by the T and C alleles of the rs10078866 locus, respectively. However, the complexes could not be abolished by the unlabeled-specific competitors, which indicated that this DNA-protein binding was not specific for the T/C alleles. To ascertain the exact binding sequence in the DNA-protein complexes, four mutated oligonucleotides were used as non-specific competitors (Table S2). We observed the DNAprotein complex in the mut3 competitor in both the SK-N-SH and HEK293 nuclear extract ( Figure 1C and 1). The results indicated that the mutated sequence of the mut3 competitor (ACTTTGAGC, from −1153 to −1145 of the DRD1 gene) was the binding sequence of the complex. Prediction of the transcription factor binding-site alteration caused by the rs10078866 locus in the ACTTTGAGC oligonucleotide was performed ( Figure S3). Based on the prediction results, a supershift experiment was conducted using specific transcription factor antibodies ( Figure 1E and 1). However, no supershift bands were observed with the anti-MEI1, anti-SOX-10, anti-HOXA5, anti-NR4A2 and anti-ERRA antibodies.

| The role of three transcription factors in endogenous expression of DRD1
The mRNA expression of endogenous DRD1 decreased slightly 48 hours after pEGFP-N1-CREB1 transfection into HEK293 cells but increased significantly after pEGFP-N1-TFAP2B or pEGFP-N1-SP1 were transfected ( Figure S6A). The mRNA expression levels gradually returned to basal levels between 48 and 96 hours after the transfection ( Figure S6B and C).
The exogenous expression of the three transcription factors was detectable at 48 hours after transfection of HEK293 cells ( Figure 2E). There were no obvious changes in the endogenous DRD1 expression levels from 48 to 60 hours after transfection ( Figure 2A, 2, 2, and G). However, 72 hours after transfection, both CREB1 and SP1 downregulated, and TFAP2B upregulated endogenous DRD1 expression ( Figure 2C and H

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science  and 72 h after transfection. Normalized DRD1 protein levels following CREB1, TFAP2B and SP1 overexpressing were compared to the reference vector (pEGFP-N1-basic). The error bars represent the standard deviation of the mean. *P < 0.05; ***P < 0.001