Characterization of a human 12/15-lipoxygenase promoter variant associated with atherosclerosis identifies vimentin as a promoter binding protein

Abstract

Background: Sequence variation in the human 12/15 lipoxygenase (ALOX15) has been associated with atherosclerotic disease. We functionally characterized an ALOX15 promoter polymorphism, rs2255888, previously associated with carotid plaque burden.

Methodology/principal findings: We demonstrate specific in vitro and in vivo binding of the cytoskeletal protein, vimentin, to the ALOX15 promoter. We show that the two promoter haplotypes carrying alternate alleles at rs2255888 exhibit significant differences in promoter activity by luciferase reporter assay in two cell lines. Differences in in-vitro vimentin-binding to and formation of DNA secondary structures in the polymorphic promoter sequence are also detected by electrophoretic mobility shift assay and biophysical analysis, respectively. We show regulation of ALOX15 protein by vimentin.

Conclusions/significance: This study suggests that vimentin binds the ALOX15 promoter and regulates its promoter activity and protein expression. Sequence variation that results in changes in DNA conformation and vimentin binding to the promoter may be relevant to ALOX15 gene regulation.

Figure 1. In vitro and In vivo Binding of Vimentin to Human ALOX15 Promoter Variants.

(A) Oligonucleotide sequences used for the gel shift assay experiments corresponding to promoters P1 and P2, which differ at rs2255888 (indicated by arrow). (B) Identification and analysis of Vimentin, from magnetic bead DNA-protein pull-down assay, by LC-MS/MS. Proteins were identified by database search of the fragment spectra against the National Center for Biotechnology Information nonredundant protein database (NCBInr) using Mascot (version 2.2, Matrix Science, London, UK). The sequence match for one of the assigned peptides is shown with the fragment ions identified as y(n), b(m) to indicate the y and b ions, respectively. The table shows the top-ranked peptide match and the scores for the next 9 for that spectrum. (C) Chromatin immunoprecipitation (ChIP) assay of P1 luciferase transfected NIH3T3 cells with anti-vimentin antibody (C20).

Figure 2. Promoter activities of ALOX15 variants in NIH3T3 cells and MCF-7 cells.

Vimentin binds to ALOX15 promoter (A) ALOX15 promoter sequence. Primers used for this study and alternative alleles of rs2255888 are shown in bold. (G/A) in bold denotes SNP rs2255888 position. (B) Qualitative measurement of human ALOX15 (upper panel) and GAPDH (lower panel) gene expression by RT-PCR in NIH3T3 cells. (C) Luciferase activities of P1 and P2 transfected NIH3T3 cells was measured after 24 h. *denotes p = 2×10⁻⁵ vs. P2. The results are the average of two independent transfections performed in triplicate ±S.E. (D) Human ALOX15 (upper panel) and GAPDH (lower panel) gene expression by RT-PCR in MCF-7 cells in presence of vimentin. (E) Luciferase activities of P1 and P2 transfected MCF-7 cells were measured after 24 h. The experiments were done in triplicates. **denotes p<0.0001 between the experimental sets. Transfection of NIH3T3 cells and MCF-7 cells with human ALOX15 cDNA for 24 hr was performed separately as control in gene expression experiments. pRL and pTK luciferase constructs were co-transfected in NIH3T3 and MCF-7 respectively. Luciferase activity was normalized using pRL and pTK luciferase activity in NIH3T3 and MCF-7 respectively. The results are the average of three independent transfections performed in triplicate ± S.E. (F) Western blot of vector (CMV) and vimentin cDNA transfected BPH-1 cells. The cells were harvested after 48 h. The cells were lysed with lysis buffer and loaded on to the SDS-PAGE Gel. The blot was probed with anti-ALOX15 antibody (1∶2000), anti vimentin antibody (1∶1000) and anti-β-actin antibody (1∶7000).

Figure 3. Specific in vitro Binding of Vimentin to Promoter Variants.

(A) Biotin-labeled duplex oligos and 10 µg NIH3T3 nuclear extract were used for the experiments. Arrow indicates protein binding to the oligos. (B) Determination of specificity of duplex 29G* by completion with unlabeled duplex oligos. (C) Specific binding of vimentin to 29G* and 29A* oligos was demonstrated with anti-vimentin antibody.

Figure 4. Higher-Order Structure in Duplex and Single Stranded Oligonucleotides.

(A) 15% non-denaturing polyacrylamide gel electrophoresis of duplex oligos in the presence of 40 mM NaCl. The oligos were incubated in binding buffer (Materials and Methods) for 40 min and 20 min at 4°C and 25°C respectively. 29G* (Lanes1 and 3), 29A* (Lanes 2 and 4) are at 4°C and 25°C respectively. (B) 1D proton NMR spectra (imino, amino and aromatic signal regions) of oligonucleotides 29G (top) and 29A (bottom) in the presence of 40 mM Na+, 10 mM phosphate, pH 7.0. (C) Denaturation profiles of single stranded oligos 29G (red) and 29A (blue) at 295 nm wavelength. Experimental conditions: 40 mM Na+, 10 mM phosphate, pH 7.0.

Figure 5. Salt Induced Folding of Single Strand Promoter Variant Oligos.

(A) Normalized differential absorbance spectra of 29G (red) and 29A (blue). (B) CD spectra of 29G and 29A in presence of 40 mM NaCl. (C) Relative fluorescence ratio values in the presence of monovalent cations (Na+, K+, Li+) for 29G (red) and 29A (blue). R = Fem, 585/Fem, 518 calculated from emission spectra, λex = 475 nm. PolyA (brown) used as a non-structured control.