Journal: bioRxiv

Article Title: Coronary Artery Disease Risk Variant rs6903956 Links to Endothelial Dysfunction via PHACTR1 Regulation

doi: 10.1101/2025.05.11.653298

Figure Lengend Snippet: ChIP-TaqMan qPCR, siRNA Knockdown, and Dual Luciferase Assays to Validate the Enhancer Role of rs6903956. (A) Schematic of the ChIP-qPCR assay design targeting the proposed rs6903956 enhancer region. Chromatin immunoprecipitation (ChIP) was performed on wild-type (WT) AA, unedited (UNΔ) AA, and edited (Δ) GG endothelial cells using anti-HOXA9 and anti-MEIS1/2 antibodies. TaqMan qPCR was conducted to amplify a 147-bp region flanking rs6903956. (B) Bar graph showing the percentage of chromatin input for HOXA9 and MEIS1 binding in WT AA, UNΔ AA, and Δ GG ECs, relative to WT AA aECs. Data are presented as means ± S.D. (1-2 donor cell lines, n=3 technical replicates/ donor). One-way ANOVA with post-hoc Tukey’s tests determines statistical significance among three experimental groups within each TF, ** p < 0.01, *** p < 0.001. (C) Quantitative RT-PCR analysis of PHACTR1 expression following siRNA-mediated knockdown of HOXA4 and MEIS1 in UNΔ (AA) ECs and Δ (GG) ECs. Expression levels were normalized to its own scrambled siRNA controls. Bar graphs show means ± S.D. (n = 3 biological replicates). One-way ANOVA with post-hoc Tukey’s tests determines statistical significance relative to its own scrambled, ** p < 0.01, **** p < 0.0001. (D) Schematic of the pGL3 plasmid constructs used in the dual-luciferase assays. The pGL3 control plasmid contains a 519-bp region flanking rs6903956 cloned under the SV40 promoter, while the pGL3 basic plasmid includes the 519-bp region and the PHACTR1 promoter. These constructs were employed to assess enhancer activity. (E) Relative luciferase activity of pGL3 control plasmids, with and without the 519-bp region flanking rs6903956 ‘A’, transfected into human umbilical vein endothelial cells. The effect of the enhancer region on firefly luciferase activity, normalized to Renilla luciferase activity (internal control), is shown. Data are presented as means ± S.D. (n = 3 biological replicates). Statistical significance between the two groups was determined by two-tailed t-test, ** p < 0.01. (F) Relative luciferase activity of pGL3 basic plasmids containing the 519-bp region flanking rs6903956 (A and G alleles) and the PHACTR1 promoter. The plasmids were transfected into human umbilical vein endothelial cells with single or co-transfections of HOXA4 and MEIS1. The far-right bar represents the pGL3 basic plasmid control without the enhancer or promoter. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are presented as means ± S.D. (n = 3 biological replicates). Statistical significance between the two groups was determined by two-tailed t-test and one-way anova with post hoc tukey test, * p < 0.05; ns, non-significant. (G) Relative luciferase activity of pGL3 control plasmids with the 519-bp region flanking rs6903956 ‘A’ cloned under SV40 or PHACTR1 promoter control. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are presented as means ± S.D. (n = 3 biological replicates). Statistical significance was determined by two-tailed t-tests, *p ≤ 0.05.

Article Snippet: Additionally, the same 519 bp region was cloned into the pGL3 Basic Vector (Addgene, Plasmid #212936) using XhoI and HindIII.

Techniques: Knockdown, Luciferase, ChIP-qPCR, Chromatin Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Expressing, Plasmid Preparation, Construct, Control, Clone Assay, Activity Assay, Transfection, Two Tailed Test