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. 2017 Oct 10;27(1):199–210. doi: 10.1093/hmg/ddx375

Figure 3.

Figure 3.

The BP-associated variant rs1173771 affects chromatin status and nuclear protein binding. In this figure, ‘E’ indicates BP-elevating allele, ‘L’ denotes BP-lowering allele. (A) Formaldehyde-assisted isolation of regulatory elements followed by allelic imbalance assay by Sanger sequencing. Left panel: Representative images showing different peak heights of the E and L alleles of rs1173771 in chromatograms derived from reference (Ref.) DNA and FAIRE DNA, respectively. Right panel: values in the bar chart are mean±SEM of n=6 heterozygous samples, *P <0.05. (B) Representative result of an electrophoretic mobility shift assay with the use of biotin-labelled double-stranded oligonucleotide probes and unlabelled competitors of the E and L alleles of rs11737711, and nuclear protein extracts from VSMCs. (C) Chromatin immunoprecipitation with an anti-H3K27ac antibody and IgG (the negative control) in heterozygous VSMCs. Upper left panel: PCR amplified DNA containing the rs1173771 site derived from anti-H3K27ac precipitated DNA, anti-H3K4me1 precipitated DNA and input DNA, respectively. Lower left panel: representative chromatographs showing the peak heights of the E and L alleles of rs1173771 from Input and precipitated DNA, respectively. Right panel: values in the bar chart are mean±SEM of seven heterozygous samples, **P <0.01. (D) Relative activity of the rs1173771 E and L alleles, respectively, in driving firefly luciferase reporter gene expression in transfected VSMCs, standardized for transfection efficiency. Mean±SEM, **P <0.01, ***P <0.001.