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

<p>(<b>A</b>) Oligonucleotide sequences used for the gel shift assay experiments corresponding to promoters P1 and P2, which differ at rs2255888 (indicated by arrow). (<b>B</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. (<b>C</b>) Chromatin immunoprecipitation (ChIP) assay of P1 luciferase transfected NIH3T3 cells with anti-vimentin antibody (C20).</p

Higher-Order Structure in Duplex and Single Stranded Oligonucleotides.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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.</p

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

<p>Vimentin binds to ALOX15 promoter (<b>A</b>) 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>B</b>) Qualitative measurement of human ALOX15 (upper panel) and GAPDH (lower panel) gene expression by RT-PCR in NIH3T3 cells. (<b>C</b>) 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. (<b>D</b>) Human ALOX15 (upper panel) and GAPDH (lower panel) gene expression by RT-PCR in MCF-7 cells in presence of vimentin. (<b>E</b>) 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. (<b>F</b>) 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).</p

Characterization of a Human 12/15-Lipoxygenase Promoter Variant Associated with Atherosclerosis Identifies Vimentin as a Promoter Binding Protein

<div><h3>Background</h3><p>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.</p> <h3>Methodology/Principal Findings</h3><p>We demonstrate specific <em>in vitro</em> and <em>in vivo</em> 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 i<em>n-vitro</em> 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.</p> <h3>Conclusions/Significance</h3><p>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.</p> </div

Specific <i>in vitro</i> Binding of Vimentin to Promoter Variants.

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

Additional file 1: of Exosome miR-371b-5p promotes proliferation of lung alveolar progenitor type II cells by using PTEN to orchestrate the PI3K/Akt signaling

Selection and characterization of hiPSC-, hESC-, and mESC-derived ATIICs. (PDF 482 kb

Whole exome sequence-based association analyses of plasma amyloid-β in African and European Americans; the Atherosclerosis Risk in Communities-Neurocognitive Study

<div><p>Objective</p><p>We performed single-variant and gene-based association analyses of plasma amyloid-β (aβ) concentrations using whole exome sequence from 1,414 African and European Americans. Our goal was to identify genes that influence plasma aβ₄₂ concentrations and aβ₄₂:aβ₄₀ ratios in late middle age (mean = 59 years), old age (mean = 77 years), or change over time (mean = 18 years).</p><p>Methods</p><p>Plasma aβ measures were linearly regressed onto age, gender, <i>APOE</i> ε4 carrier status, and time elapsed between visits (fold-changes only) separately by race. Following inverse normal transformation of the residuals, seqMeta was used to conduct race-specific single-variant and gene-based association tests while adjusting for population structure. Linear regression models were fit on autosomal variants with minor allele frequencies (MAF)≥1%. T5 burden and Sequence Kernel Association (SKAT) gene-based tests assessed functional variants with MAF≤5%. Cross-race fixed effects meta-analyses were Bonferroni-corrected for the number of variants or genes tested.</p><p>Results</p><p>Seven genes were associated with aβ in late middle age or change over time; no associations were identified in old age. Single variants in <i>KLKB1</i> (rs3733402; p = 4.33x10^-10) and <i>F12</i> (rs1801020; p = 3.89x10^-8) were significantly associated with midlife aβ₄₂ levels through cross-race meta-analysis; the <i>KLKB1</i> variant replicated internally using 1,014 additional participants with exome chip. <i>ITPRIP</i>, <i>PLIN2</i>, and <i>TSPAN18</i> were associated with the midlife aβ₄₂:aβ₄₀ ratio via the T5 test; <i>TSPAN18</i> was significant via the cross-race meta-analysis, whereas <i>ITPRIP</i> and <i>PLIN2</i> were European American-specific. <i>NCOA1</i> and <i>NT5C3B</i> were associated with the midlife aβ₄₂:aβ₄₀ ratio and the fold-change in aβ₄₂, respectively, via SKAT in African Americans. No associations replicated externally (N = 725).</p><p>Conclusion</p><p>We discovered age-dependent genetic effects, established associations between vascular-related genes (<i>KLKB1</i>, <i>F12</i>, <i>PLIN2</i>) and midlife plasma aβ levels, and identified a plausible Alzheimer’s Disease candidate gene (<i>ITPRIP</i>) influencing cell death. Plasma aβ concentrations may have dynamic biological determinants across the lifespan; plasma aβ study designs or analyses must consider age.</p></div

Additional file 1: of Cerebral white matter hyperintensities on MRI and acceleration of epigenetic aging: the atherosclerosis risk in communities study

Top 100 CpGs from the epigenome-wide association study (EWAS) of WMH burden in ARIC African-Americans. Association P-value, effect size, and standard error for each probe are shown as well as location(chromosome, position) and probe type. (PDF 195 kb

Effects of carrying a minor allele in <i>TSPAN18</i>, <i>ITPRIP</i>, and <i>PLIN2</i> on the third visit aβ₄₂: aβ₄₀ ratio in EAs.

<p>The plotted values are the inverse normal transformed ratios adjusted for age, gender, and <i>APOE ε</i>4 carriage status.</p

Descriptive statistics of sequenced ARIC participants.

<p>Descriptive statistics of sequenced ARIC participants.</p