Characterization of the 5′ Regulatory Region of the Human RGS4 Gene In Vitro

Background: In order to detect function of three SNPs (rs12041948, rs6678136 and rs7515900) in 5′ regulatory region of the human RGS4 gene, fragments of 5′ regulatory region of RGS4 (-1112–+365, TSS+1) was cloned into pGL3-Basic vector, and dual-luciferase reporter assay was conducted. We compared and analyzed the relative uorescence intensities of eight haplotype recombined vectors. Results: In HEK-293 and SK-N-SH cells, the relative uorescence intensities of haplotype 2 (ATA) was signicantly increased when it was compared with haplotype 3 (ACA), 5 (ACC), 7 (GCA), and 8 (GCC). Therefore, haplotypes with C of rs6678136 decreased expression than theses with T. However, no signicant difference was assessed in comparation among eight haplotypes, in the U87 cells. Conclusions: The mutant of T>C of rs6678136 might alter the binding of transcription factors to 5′ regulatory region of RGS4 gene, then change the expression. It was predicated that the rs6678136 might alter the binding region of GSX1, ALX3, BARHL1, and BARHL2. The binding is still worthy of further investigation.

2. Allele C of rs6678136 decreased the relative uorescence intensities than T. 3. It was predicated that the rs6678136 might alter the binding region of GSX1, ALX3, BARHL1, and BARHL2. The binding is still worthy of further investigation.

Background
Regulators of G-protein signaling (RGS) negatively modulate G-protein signaling by acting as GTPaseactivating proteins. RGS4 is one important RGS, and associated with many neurologic diseases, such as schizophrenia [1] and neuroglioma [2], addiction, seizure [3] and pain. RGS4 is reported to play an important role for neurotransmission, neuronal differentiation [4] and axonogenesis [5]. RGS4 gene is located in 1q21-22, which may support RGS4 gene as an inherent susceptibility factor for the psychiatric illness [6].
There are ve transcriptional initiation sites in the RGS4 gene, producing various protein subtypes [7]. The 5′ regulatory region affected the translation, localization and stability of the mRNA [8]. The 5′ regulatory region, a primary determinant of translation e ciency, regulated transcription and in uenced the expression of protein [9]. The longest variation (RGS4-1) was poorly studied. Association between SNPs of 5′ regulatory region in RGS4 gene and the risk of schizophrenia was widely studied, however, the association was controversial [10,11]. Our previous study suggested that genotype TT of rs12041948 and GG of rs6678136 and CC of rs7515900 could be risk factors for schizophrenia, in the northern Chinese Han population. These SNPs might change binding between transcription factors and 5′ regulatory region of gene [12]. Transcription factors are recognized as the master regulators of gene expression [13,14]. Functional studies of the three SNPs are required to con rm these ndings.

Samples
We selected DNA samples containing eight haplotypes (composed with rs12041948, rs6678136 and rs7515900) to construct the pGL3 recombinants. All samples were collected in accordance with the principle of informed consent. All patients and participants provided written informed consent prior to inclusion in this study. Specimens were obtained and analyzed with approval from the Ethics Committee of China Medical University.
Construction of haplotype recombinants in the 5′ regulatory region of RGS4 gene Fragments of 5′ regulatory region of RGS4 gene (-1112-+365, TSS + 1), containing haplotypes 1-8, were ampli ed with primers ( Table 1). The cleavage sites of NheI and Hind were introduced in the 5′ end of the sense and antisense primers, respectively. The target fragments were cloned into the pBM20S vector (Biomed, Beijing, China), and then re-cloned into the pGL3-Basic vector (Promega, Madison, WI), and then veri ed by DNA sequencing.

Statistical analysis
The relative uorescence intensity was showed as value of LUC normalized by TK. The relative uorescence intensity differences between each two recombinants were calculated with one-way analysis of variance and Bonferroni's multiple comparison test using Graphpad prism 7 [16]. A p-value less than 0.05 represented a statistically signi cant difference in relative chemiluminescence intensities.

Comparison and analysis of relative uorescence intensities of eight haplotype recombined vectors
Fragments of 5′ regulatory region of RGS4 gene, containing haplotypes 1-8, were constructed into the pGL3-Basic vector, and successfully veri ed by sequencing. The relative uorescence intensities of eight haplotype recombined vectors were detected in HEK-293, SK-N-SH, and U87 cell lines (Fig. 1).
After analysis, it was detected that the relative uorescence intensities of haplotypes with C of rs6678136 were lower than these with T, in HEK-293 and SK-N-SH cells.

Results of bioinformatics platform prediction
Transcription factor predictions for the functional fragments were carried out using JASPAR (http://jaspar.genereg. net). The mutant of rs6678136 might in uence the binding of transcription factors, such as GSX1, ALX3, BARHL1, and BARHL2.

Discussion
RGS4 protein may negatively regulate G-protein coupled receptors to inhibit the interaction of glutamate, dopamine and other neurotransmitters, resulting in the disorder of above neurotransmitter system [17].
The dysfunction of these neurotransmitter systems is an important pathophysiological feature of psychiatric disorders [5]. Mirnics et.al found that only RGS4 gene expression was consistently altered among 70 genes mapped to the major schizophrenia susceptibility locus 1q21-22, on the microarrays [18]. SNPs in 5' regulatory region might change transcription and translation of gene [19]. Function of SNPs in 5' regulatory region of RGS4-1 was poorly studied. On base of our previous study [20], we conducted functional analysis of three SNPs (12041948, rs6678136 and rs7515900).
The relative uorescence intensities of eight haplotypes, composed with 12041948, rs6678136 and rs7515900, were detected in three cell lines (HEK-293, SK-N-SH and U87). In HEK-293 and SK-N-SH cells, the relative uorescence intensities of haplotypes with C of rs6678136 were lower than theses with T. Therefore, the transcriptional activity of the 5' regulatory region fragment carrying C of rs6678136 was the lower than that carrying T. The polymorphism of rs6678136 might in uence the binding of some transcription factors to the cis-regulatory elements within 5′ regulatory region of RGS4 gene, and then alter the transcription [21]. In the U87 cells, no signi cant difference was assessed in comparation among eight haplotypes. The inconsistent nding in the three cell lines was due to different cell microenvironment [22].
It was demonstrated that NF-YA and C/EBP binding to the RGS4-3 promoter [23],and altered the expression of RGS4 gene. However, there was few studies about character of the promoter of RGS4-1. In our study, it was predicted that this T > C mutation of rs6678136 might reduce ability of transcription factors binding, such as GSX1, ALX3, BARHL1, and BARHL2, and the induction of RGS4 transcription. It was studied GSX transcription factors controlled neuronal versus glial speci cation [24], promoted progenitor maturation and the acquisition of neuronal phenotypes [25]. BARHL1 was detected overexpressing in medulloblastoma and played an important role in neurogenesis. BARHL1 was downregulated in Alzheimer's Disease and other psychiatric disorders [26]. The changing binding of these transcription factors by mutation of rs6678136 might induced psychiatric disorders. Further studies were need to warrant the binding of transcription factors and 5′ regulatory region of RGS4 gene.

Conclusions
In HEK-293, SK-N-SH and U87 cells, the mutant of T > C of rs6678136 might alter the binding of transcription factors to 5′ regulatory region of RGS4 gene, then change the expression. It was predicated that the binding region of GSX1, ALX3, BARHL1, and BARHL2 in RGS4 gene might be altered by the SNP of rs6678136. The binding is still worthy of further investigation.

RGS
Regulators of G-protein signaling.

Declarations
Ethics approval and consent to participate All samples were collected in accordance with the principle of informed consent. All patients and participants provided written informed consent prior to inclusion in this study. Specimens were obtained and analyzed with approval from the Ethics Committee of China Medical University.

Consent for publication
All authors have read and approved the manuscript for submission.

Availability of data and materials
This was an evidence synthesis study; all data were available.