A common polymorphism in the promoter region of the TNFSF4 gene is associated with lower allele-specific expression and risk of myocardial infarction

Abstract

Background: The TNFSF4/TNFRSF4 system, along with several other receptor-ligand pairs, is involved in the recruitment and activation of T-cells and is therefore tentatively implicated in atherosclerosis and acute coronary syndromes. We have previously shown that genetic variants in TNFSF4 are associated with myocardial infarction (MI) in women. This prompted functional studies of TNFSF4 expression.

Methods and results: Based on a screening of the TNFSF4 genomic region, a promoter polymorphism (rs45454293) and a haplotype were identified, conceivably involved in gene regulation. The rs45454293T-allele, in agreement with the linked rs3850641G-allele, proved to be associated with increased risk of MI in women. Haplotype-specific chromatin immunoprecipitation of activated polymerase II, as a measure of transcriptional activity in vivo, suggested that the haplotype including the rs45454293 and rs3850641 polymorphisms is functionally important, the rs45454293T- and rs3850641G-alleles being associated with lower transcriptional activity in cells heterozygous for both polymorphisms. The functional role of rs45454293 on transcriptional levels of TNFSF4 was clarified by luciferase reporter assays, where the rs45454293T-allele decreased gene expression when compared with the rs45454293C-allele, while the rs3850641 SNP did not have any effect on TNFSF4 promoter activity. Electromobility shift assay showed that the rs45454293 polymorphism, but not rs3850641, affects the binding of nuclear factors, thus suggesting that the lower transcriptional activity is attributed to binding of one or more transcriptional repressor(s) to the T-allele.

Conclusions: Our data indicate that the TNFSF4 rs45454293T-allele is associated with lower TNFSF4 expression and increased risk of MI.

Figure 1. The TNFSF4 gene as represented in the NCBI database.

Vertical arrows denote SNPs tested in human subjects. Horizontal arrow indicates direction of transcription. Exons are depicted by boxes: filled-in regions indicate translated portions of the gene and untranslated regions are depicted by open boxes; introns are indicated as solid lines between boxes. LD: Linkage disequlibrium.

Figure 2. Allele-specific loading of phosphorylated Pol II in vivo at rs45454293 (A-D) and rs3850641 (F-I) sites.

To quantify the relative levels of abundance of allele-specific fragments, pyrosequencing was used to analyze input chromatin used in ChIP reactions (A, F); products of ChIP using specific antibodies to total Pol II as positive control (B, G); to phosphorylated serine residues of Pol II CTD (C, H); or to SV40 T antigen as mock antibody control (D, I). Graphs show input nucleotide sequence along the x axis and intensity of signal along the y axis. (E/L) The ratios between the C and T alleles of SNP rs45454293 (E) and the A and G alleles of SNP rs3850641 (L) of phosphorylated Pol II loading compared with input chromatin used in ChIP reactions are shown. Data are expressed as mean (95% c.i.) of two independent immunoprecipitation reactions, with each immunoprecipitation analyzed by PCR in up to three replicates.

Figure 3. Transcriptional regulatory activity on TNFSF4 polymorphisms in HEK293T cells.

Relative activity was calculated by taking the relative luciferase activity of the empty vector to be 1. Data show the relative activity (mean ± s.e.m) from three experiments done in duplicate. *P = 0.0005.

Figure 4. (A) TNFSF4 region across the transcription start site.

Conservation profile between human and mouse obtained using the RAVEN software. Area above the gray area indicates a degree of homology >70%; arrows represent tested SNPs. The TNFSF4 gene was retrieved from the NCBI database (GenBank accession number D90224). (B) Sequence of the TNFSF4 promoter between positions –931 and –911 (upper) and of intron 1 between positions +474 and +494 (lower), indicating the polymorphic sites. Potential binding sites for different transcription factors are indicated for both alleles. Allelic variants for each polymorphism are indicated in bold, mismatches between TNFSF4 sequence and transcription factor binding sites are indicated in lower case italic.

Figure 5. Representative EMSAs of nuclear extract derived from U937 cells bound to the rs45454293 region and the rs3850641 region.

(A) EMSA of a 25 bp DNA fragment containing either the rs45454293C (lanes 1-4) or the rs45454293T site (lanes 5-8). Lanes 1 and 5, no extract; lanes 2 and 6, 0.5 µg of extract; lanes 3 and 7, 1 µg of extract; lanes 4 and 8, 2 µg of extract. Arrow denotes the specific DNA-protein complexes associated with the polymorphic sites. (B) Competition experiment demonstrating specific binding to the rs45454293T allele: lanes 1 and 5, without competitors; lanes 2 and 6, rs45454293C and rs45454293T probe, respectively, with a 100-fold excess of rs45454293C probe as competitor; lanes 3 and 7, rs45454293C and rs45454293T probe, respectively, with a 100-fold excess of rs45454293T probe as competitor; lanes 4 and 8, rs45454293C and rs45454293T probe, respectively, with a 100-fold excess of non-specific (X) competitor. (C) EMSA of a 25 bp DNA fragment containing either the rs3850641A (lanes 1–4) or the rs3850641G site (lanes 5-8).