The Single Nucleotide Substitution T → A rs2072580 Damages the CREB1 Binding Site in the Bidirectional SART3/ ISCU Promoter

Abstract

Background/objectives: The regulatory SNPs (rSNPs) that disturb the binding of transcription factors (TFs) and alter the transcription levels of genes play a paramount role in the formation of different traits and are associated with many pathologies. The search for allele-specific events in RNA-seq and ChIP-seq data is a powerful genome-wide approach to detect rSNPs. Using this approach, we have identified the T → A rs2072580 substitution in the bidirectional SART3/ISCU promoter as a potential rSNP and demonstrated its association with colorectal cancer, relying on International Cancer Genome Consortium data. The goal of this work was to identify the TF binding site that is affected by the T → A substitution and to study the effect of this substitution on reporter gene expression in different plasmid constructs.

Methods: Electrophoretic mobility shift assay (EMSA), cross-competition analysis and supershift assay, plasmid construction, and dual luciferase reporter assay.

Results: The T → A rs2072580 substitution is shown to damage the binding site for ubiquitous TF CREB1 and to significantly decrease the activity of the heterologous promoter carrying the cassettes of two or three repeated CREB binding sites inserted upstream of it. However, the substitution disturbing the CREB1 binding site within the bidirectional promoter shared by SART3 and ISCU inhibits the promoter activity of only the SART3 gene but has no effect on the activity of the ISCU promoter.

Conclusions: The performed comprehensive functional analysis of the T → A rs2072580 in the bidirectional SART3/ISCU promoter unambiguously implies it is an rSNP. These results form the background for further studies of this rSNP and its potential significance for various pathologies.

Keywords: CREB1; EMSA; luciferase reporter assay; regulatory SNPs; transcription factor binding sites.

Figure 1

Electrophoretic mobility shift assays (EMSAs) with the DNA probes containing either rs2072580-T (left) or rs2072580-A (right) using HepG2, MCF-7, and Caco-2 nuclear extracts; brace denotes free DNA probes and arrows, the DNA–protein complex is prevalently formed in the case of allele T.

Figure 2

Cross-competition analysis. Gel shift assays were performed with the DNA probes containing either rs2072580-T or rs2072580-A using (A) HepG2, (B) MCF-7, and (C) Caco-2 nuclear extracts: lanes 1–5, labeled DNA probe carrying allele T; lanes 6–10, labeled DNA probe carrying allele A; lanes 1 and 6, without competitor; and lanes 2–5 and 7–10, 10- and 25-fold excess of unlabeled oligonucleotide; arrow denotes the specific protein complex bound by the T allele.

Figure 3

Competition analysis in EMSA experiments using (A) HepG2 and (B) MCF-7 nuclear extracts. Lane 1, without competitor; lane 2, 25-fold excess of cold oligonucleotide containing T allele; and lanes 3–12, 25-fold and 100-fold excess of competitor oligonucleotides corresponding to TF binding sites; arrow denotes specific protein complex bound by the T allele of rs2072580.

Figure 4

Substitution T → A destroys the CREB1 binding site. (A) EMSA was conducted using anti-CREB1 antibodies. Red arrows denote the DNA probe–CREB1 complex and blue denotes supershift in the presence of antibodies. (B) CREB1 motif logo from the MotifbreakR aligned to the sequence of the identified CREB1 binding site; blue area highlights rs2072580 T/A.

Figure 5

The effect of the T → A rs2072580 substitution on the activities of SART3 and ISCU promoters. (A) Scheme of bidirectional promoter region shared by SART3 (NM_001410983.1, NCBI RefSeq genes) and ISCU (NM_001301141.1, NCBI RefSeq genes); angle arrows denote the transcription start sites (TSSs) and green rectangles denote exons (B–F). Schemes of the recombinant plasmids used in dual luciferase assays: bidirectional promoter constructs with (B) forward or (C) inverted orientations; constructs with (D) single, (E) double, and (F) triple oligonucleotide inserts. (G) Relative luciferase activity in the HepG2 and MCF-7 cells transfected with the constructs containing ISCU- and SART3-oriented promoter regions harboring alternative rs2072580 alleles (green column, allele T and red column, allele A); and the gray column shows the empty pGL3-basic vector. The data of three independent experiments are shown as mean values ± SD, * p < 0.05, significant difference between alleles; and *** p < 0.001, significant difference between the promoter-containing constructs and empty vector (Student’s t-test).

Figure 6

The effect of rs2072580 in the constructs containing single, double, and triple inserts of the CREB1 binding site when transfected into HepG2 cells: green columns show the T allele; red columns show the A allele; color intensity reflects the increasing number of inserts; and the blue column shows the empty pGL4.23 vector. All data were normalized to Renilla luciferase internal reference. The data from three independent experiments are shown as mean values ± SD; * p < 0.05, significant difference between the alleles (Student’s t-test).