Regulatory potential at type 2 diabetes-associated SNPs at the <i>CDC123/CAMK1D</i> locus.

<p>A) The 11 SNPs in high LD (r²≥.7, EUR) with GWAS index SNP rs12779790. Arrows indicate the two SNPs that overlap islet, liver, and HepG2 open chromatin and epigenomic marks and that are located near to HepG2 ChIP-seq peaks; these two SNPs were tested for allele-specific transcriptional activity. B) DNase hypersensitivity peaks identified in two pooled islet samples from the ENCODE Consortium. C) FAIRE peaks identified in one representative islet sample from the ENCODE Consortium. D) H3K4me1 histone modifications from the Roadmap Epigenomics Consortium. E) FOXA1 and FOXA2 ChIP-seq peaks and signal from ENCODE. Image is taken from the UCSC genome browser, February 2009 (GRCh37/hg19) assembly (<a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004633#pgen.1004633-Fujita1" target="_blank">[51]</a>. The 5′ end of <i>CAMK1D</i> begins after position 12,390,000.</p

rs11257655-T allele shows increased binding to FOXA1 and FOXA2 in human islets.

<p>FOXA1 (A) and FOXA2 (B) ChIP in human islets shows enrichment at rs11257655 compared to IgG control. Islets containing one copy of the rs11257655-T allele show 7.2-fold greater FOXA1 enrichment and 4.2-fold greater FOXA2 enrichment. rs11257655 CT heterozygotes are more significantly enriched than rs11257655 CC homozygotes for FOXA1 (one-sided <i>t</i>-test, <i>P</i> = .06) and FOXA2 (one-sided <i>t</i>-test, <i>P</i> = .026). A negative control region 28 kb downstream of rs11257655 was not enriched in FOXA1- and FOXA2- bound chromatin (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004633#pgen.1004633.s003" target="_blank">Figure S3A and S3B</a>). Error bars represent standard error of two to three islets for each represented genotype.</p

Alleles of rs11257655 differentially bind FOXA proteins in rat 832/13 insulinoma cells, mouse MIN6 insulinoma cells and human HepG2 hepatoma cells.

<p>EMSA using 832/13 (A), MIN6 (B) and HepG2 (C) nuclear extract shows differential protein-DNA binding of rs11257655 alleles. The probe containing risk allele rs11257655 -T shows allele-specific protein binding (arrows a–e) compared to the probe containing non-risk allele C. Excess unlabeled probe containing the T allele (T-comp) more efficiently competed away allele-specific bands than unlabeled probe for the C allele (C-comp). Incubation of 832/13 and HepG2, nuclear extract with FOXA1/FOXA2 antibodies disrupt the DNA-protein complex formed with T allele-containing DNA probe (arrow a, d, e) and result in band supershifts (asterisks). Incubation of MIN6 nuclear extract with FOXA2 antibody decreases the DNA-protein complex formed with T allele-containing DNA probe (arrow b) and results in a band supershift. To enhance visualization of protein complexes, free biotin-labeled probe is not shown. (D) DNA affinity-capture identified differential binding of FOXA2 at rs11257655 alleles in 832/13 cells. (E) The T allele of rs11257655 is predicted as a FOXA1 and FOXA2 consensus core-binding motif.</p

OxLDL induces changes in both open chromatin and enhancer status at a subset of sites with important regulatory function.

<p>(<b>A</b>) The oxLDL regulated change in open chromatin, at sites which also have changes in enhancer signal, is correlated with expression of the nearest gene (*** p < 0.0005). (<b>B</b>) The refined subset of sites with dynamic open chromatin and dynamic enhancer status shows enrichment for proximity to disease-associated gene sets linked with atherosclerosis. (<b>C</b>) The subset of sites with dynamic open chromatin and enhancer signal was analyzed for transcription factor motif enrichment and the top 25 motifs are shown with the p values for enrichment. Motifs were identified using SeqPos by comparison with known motifs from the Cistrome database and by identification of <i>de novo</i> motifs. SeqPos clusters similar motifs and the transcription factors listed are representative of each cluster. The position weight matrices for the <i>de novo</i> motifs are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005061#pgen.1005061.s019" target="_blank">S13 Table</a>.</p

OxLDL-induced changes in chromatin structure determined by FAIRE-seq.

<p>(<b>A</b>) Normalized FAIRE-seq signal in 2000bp windows centered on the TSS for gene sets grouped by their expression characteristics. OxLDL-induced changes in open chromatin were associated with concordant changes in gene expression (higher FAIRE signal indicates more open chromatin, *** p < 0.0005). (<b>B</b>) Profile view of FAIRE-seq signal around TSSs showing sharply delineated promoter open chromatin profiles and their relationship with expression.</p

Model showing the interplay between oxLDL, C/EBP beta and rs72664324 on expression of <i>PPAP2B</i>.

<p>OxLDL-induced C/EBP beta binding is greater to the A allele and leads to increased induction of <i>PPAP2B</i> in response to oxLDL compared to the G allele. <i>PPAP2B</i> encodes LPP3 whose enzymatic activity reduces pro-inflammatory signalling mediated by LPA.</p

OxLDL-regulated dynamic enhancer signals correlate with expression changes in local genes.

<p>(<b>A</b>) Open chromatin (FAIRE) and enhancer (H3K27ac) profile at the <i>IL6R</i> locus showing a dynamic chromatin cluster that overlaps a large, intronic dynamic enhancer site with increased signal in oxLDL-treated cells. (<b>B</b>) Correlation between fold change in gene expression for differentially expressed genes and mean fold change in enhancer signal for dynamic sites annotated to their nearest gene (r² = 0.45, Pearson r = 0.67). (C) The proportion of the genes in each quartile that are associated with a dynamic enhancer site is plotted. Differentially expressed genes were split into oxLDL up and down regulated genes and further split into quartiles based on fold change in expression (-/+ and—-/++++ indicate least and most differential quartiles, respectively). The proportion of differentially expressed genes in each quartile that are associated with a directionally concordant change in a dynamic enhancer site is correlated with the magnitude of gene expression change. (<b>D</b>) Enhancers in macrophages or foam cells were aggregated if within 12.5kb and plotted according to H3k27ac signal rank. The insets show gene expression (log2, normalized) for genes overlapping either enhancers within super enhancer domains or other enhancers.</p

OxLDL exposure induces <i>PPAP2B</i>-encoded LPP3 expression and activity in foam cells.

<p>(<b>A</b>) Western blotting demonstrates up-regulation of glycosylated and non-glycosylated LPP3 protein (as indicated) in macrophage-derived foam cells (data from 3 donors shown). (<b>B</b>) Human atherosclerotic plaque contains an abundance of LPP3-expressing foam cells (brown stain, nuclei counterstained blue with haematoxylin, scale bar indicates 50 um, inset shows close-up of foam cell—arrowed). All 5 cases immunostained showed similar LPP3 expression in foam cells. (<b>C and D</b>) OxLDL exposure induced increased specific activity of LPP3, measured by the degradation of receptor active species lysophosphatidic acid (LPA) to mono-acylglycerol (MG) (<b>C</b>) and sphingosine-1-phosphate (S1P) to sphingosine (<b>D</b>) (n = 3 donors, * p < 0.05). (<b>E</b>) OxLDL induced changes in the levels of LPP3 substrates and products (n = 3 donors).</p

An intronic SNP at the <i>PPAP2B</i> locus regulates enhancer activity and oxLDL-induced expression of <i>PPAP2B</i>.

<p>(<b>A</b>) Chromatin profile at the <i>PPAP2B</i> locus where rs72664324 is in a dynamic chromatin, enhancer and C/EBP beta site (red box). The region is part of an oxLDL-induced super enhancer. (<b>B</b>) Comparison of the human rs72664324 alleles and the corresponding mouse sequence with the CEBP motif aligned above. (<b>C</b>) EMSA demonstrating that only the A allele at rs72664324 binds nuclear protein, which is shown to be C/EBP beta by a super-shift in the presence of anti-C/EBP beta antibody. Also, only a cold probe with the A allele competes off binding to a C/EBP consensus probe. (<b>D</b>) Luciferase reporter assays in primary human macrophages and foam cells with a reporter element containing rs72664324 with either allele (* p < 0.05, ** p < 0.005). (<b>E</b>) Effect of C/EBP beta overexpression on luciferase reporter activity with either the A or G allele relative to empty vector (* p < 0.05). (<b>F</b>) Induction of <i>PPAP2B</i> expression by oxLDL in primary human macrophages from individuals with allele G or at least one copy of the A allele (long lines indicate mean, short lines indicate median, Mann-Whitney U test, p = 0.0013, GG n = 10, GA n = 2, AA n = 6).</p

C/EBP-beta binding is responsive to oxLDL.

<p>(<b>A</b>) Track view of a dynamic open chromatin and enhancer site which also overlaps a dynamic C/EBP-beta binding site with increased C/EBP-beta binding in foam cells (horizontal black bars indicate dynamic sites). (<b>B</b>) Enrichment of gene sets associated with different biological processes for dynamic C/EBP-beta binding sites.</p