An epigenomic investigation of atrial fibrillation in a matched left and right atrial human cohort

Figures

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  Fig. 1. H3K27ac profiling across right- and left- atrial samples in a cohort of atrial fibrillation patients and sinus rhythm controls. a H3K27ac enrichment differences across samples plotted on the first two principal components. Sample groups correspond to either left- (LA) or right- (RA) atrium samples from individuals with persistent atrial fibrillation (AF) or a normal heart rhythm (sinus rhythm, SR). Epigenomic differences more readily distinguish samples across anatomical side than disease status. b H3K27ac enrichment levels across samples are represented as pairwise Pearson correlations. Samples corresponding to the same anatomical side tend to show higher correlations. c Genome track examples of H3K27ac levels across the four sample groups for cardiac loci MYH6 and NPPA, and PITX2 and BMP10 as left and right atrium loci, respectively. Each track represents average fold ChIP enrichment over input across samples in each group (Methods). See also Figure S1 and Table S1

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  Fig. 2. H3K27ac epigenomic regions enriched across sample groups. a Venn diagram of epigenomic regions enriched in H3K27ac levels in each of the sample groups, as defined from pairwise comparisons in each category (Methods). Overlapping regions across sample groups were excluded from analysis (greyed colour shades). b Violin plots of H3K27ac normalised read coverage (FPKM) over epigenomic regions enriched in each sample group (coloured labels, top left of each plot). Read coverage is represented in samples corresponding to each group (x-axis in each plot). As expected, H3K27ac coverage shows an upward shift in the sample group enriched regions were identified in. p-values correspond to one-sided Wilcoxon rank-sum tests with Bonferroni correction for multiple testing. ***: p < 0.001. c Genome track examples of epigenomic regions enriched in each sample group, for NPPB (AF-LA), DNM1/GOLGA2 (AF-RA), NR2F1 (SR-LA) and KSR2 (SR-RA) loci. Tracks represent average H3K27ac fold enrichment over input across samples in each group, and the overlay of signals across the four groups (Merge). See also Figures S2–S3 and Tables S2–S3

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  Fig. 3. Association of H3K27ac enrichment with differential gene expression across sample groups and definition of candidate loci with altered gene regulation in atrial fibrillation. a Association of differentially-expressed genes with enriched H3K27ac regions for each sample group. Numbers correspond to the number of gene loci with corresponding changes in gene expression (x-axis) and H3K27ac enrichment (y-axis). Cells are coloured by statistical significance (-log10 p-value, Fisher’s exact test). b For each bolded comparison in a., Venn diagrams represent the overlap between differentially-expressed genes (cyan) and H3K27ac-enriched regions (violet). Overlapping gene loci (grey insets) correspond to candidate loci showing concordant gene expression and epigenomic changes in a particular sample group. c An AF-LA enriched epigenomic region at the promoter region in the LRRC32 locus (red bar), with gene expression levels being highest in this sample group (right handside, red points). n.s.: non-significant; ***: p < 0.001 (DESeq2 adjusted p-values). d Example of an AF-RA enriched epigenomic region proximal to the SCX gene (yellow bar). Consistent with promoter activity, SCX expression…

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  Fig. 4. Targeted validation of gene expression levels at candidate loci in an independent replication cohort. Locus-specific comparison of gene expression levels in AF and SR samples for selected candidate loci in the discovery cohort (top boxplots in each category, RNA-seq), and their validation in an independent replication set (bottom barplots in each category, RT-qPCR). RT-qPCR results are represented as relative expression levels over those of TBP as a reference gene. Gene expression differences across disease status were considered as validated in independent samples when the direction of change was maintained with a p-value < 0.2 (one-sided t-test). a Gene expression levels in the discovery cohort (top boxplots) and validation cohort (bottom barplots) across 8 AF-enriched candidate loci. b As in a. for five SR-enriched candidate loci. See also Figure S5 and Table S7

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  Fig. 5. Targeted validation of H3K27ac enrichment at candidate loci in an independent replication cohort. a–c Examples of ChIP-qPCR validation of H3K27ac enrichment for an AF-enriched region in the VASH1 locus (a, sienna bar), a SR-enriched region in the MTX1X locus (b, cyan bar), and AF- and AF-LA enriched regions in the NPPA/NPPB locus (c, sienna and red bars, respectively). For each example, genome tracks show H3K27ac levels in each sample group (AF-RA, AF-LA, SR-RA, SR-LA), the location of validated enriched regions (coloured bars) and single-cell ATAC-seq signals corresponding to atrial cardiomyocytes in the human heart. P followed by numbers and letters indicate primer pairs used for validation of each region (e.g. P2a and P2b in panel a.). ChIP-qPCR data for the best performing pair is shown in each barplot inset. Highlighted p-values correspond to one-sided t-test. For the P3 region in c, ANOVA p-value = 0.54. See also Figure S5 and Table S8

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  Fig. 6. Differentially-methylated regions in AF samples inform gene dysregulation mechanisms in candidate loci. a Heatmap of DMRs identified as hypermethylated (y-axis, top half) or hypomethylated (y-axis, bottom half) in AF samples. Heatmap scale corresponds to Z-score normalization of beta-values. Hierarchical clustering (x-axis, top; sienna for AF and cyan for SR) shows a subset of samples associates with DNA methylation differences across DMRs. b Examples of DMRs hypermethylated (DMR10, left) and hypomethylated (DMR4, right) in AF patients. Shaded trendlines correspond to local regression (LOESS method) with confidence interval, and p-values to statistical significance of CpGs within a DMR. c A hypomethylated DMR in SLC43A3 associates with H3K27ac enrichment and increased expression levels in AF patients. Representation as in a and previous Figures. d A hypomethylated DMR proximal to MYLK2 acts as a potential enhancer of FOXS1 expression and associates with H3K27ac enrichment and increased FOXS1 expression in AF patients. Representation as in a and previous Figures. FDR-corrected two-sided t-test for differentially-methylated CpGs; ***: p < 0.001; **: p < 0.01; *: p < 0.05). DESeq2…

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  Fig. 7. Transcription factor motif enrichment in AF differentially-methylated regions and examples of potential disruption of TF binding by DNA methylation. a De novo transcription factor motifs enriched in hypermethylated and hypomethylated DMRs. Circle plots in each category indicate percentage of DMRs harbouring sequence motifs (circle size) and statistical significance (circle shade; -log10 p-value, hypergeometric test with Benjamini–Hochberg correction for multiple comparisons). Each enriched motif was assigned to the indicated transcription factors based on motif similarity and cardiac expression (Methods). Heatmaps indicate median expression levels (log2(TPM + 1)) for each transcription factor in sinus rhythm controls (SR) or permanent AF patients (AF). Asterisks indicate differential gene expression (FDR p-value < 0.05; Methods). b Example of a hypermethylated DMR in the LRRC4B locus (DMR26), for which one of the differentially-methylated CpGs overlaps an ETV1 sequence motif. (Top tracks) Gene and DMR annotation, and promoter-enhancer interactions in this genomic locus. (Bottom middle inset) DNA methylation differences across the DMR, shown as beta-values for AF (sienna) and SR…