210 Chapter 8 Figure 1: Identification of differentially acetylated DNA regulation regions in PLN-R14del cardiomyopathy compared to the controls and other cardiomyopathies. A) An overview illustrating 487 the study design of comparing histone acetylation between PLN-R14del cardiomyopathy and the healthy controls using H3K27ac ChIP-seq. B) A workflow indicating the production of three main datasets obtained from identified histone acetylated regions: dataset 1) 2,107 differentially acetylated regions between PLN-R14del and controls; dataset 2) 863 genes annotated to differentially acetylated regions in the +/-5kb window from the gene’s transcription start site; dataset 3) 202 transcription factor binding motifs (TFBMs) enriched within the sequences of differentially acetylated regions. C) Heatmap showing top 100 differentially acetylated regions in cardiac tissue from PLN-R14del versus control hearts (dataset 1). D) Functional gene enrichment analysis using annotated genes in the vicinity of hypoacetylated regions pointed towards altered metabolism-related biological functions (dataset 2). E) PPARA and its interacting TFs involved in lipid metabolism obtained from TFBMs enriched in differentially acetylated regions (dataset 3). F) Immunofluorescence staining and quantification of nuclear PPARA signals (magenta) in PLN-R14del and control hearts. The nucleus was stained in blue and α-actinin in green. Confocal images were taken at 63x magnification. CMs: identified cardiomyocytes in the tissues; non-CMs: non-cardiomyocytes in the tissues. G) An overview showing additional biopsies from patients with ischemic cardiomyopathy and sarcomeric non-ischemic cardiomyopathy (non-PLN-related cardiomyopathies), besides PLN-R14del and control hearts as indicated in A). H) K-mean clustering analysis showing four PLN-R14del-specific clusters based on acetylation signals (AcS). I) Examples of genes involved in mitochondrial metabolism annotated to regions from PLN-R14dellowest clusters (cluster 3 and 4) shown in H).
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