Figure 6.

TBX20 directly binds to enhancers of cardiac function genes. A, CUT&Tag (cleavage under targets and tagmentation) peak heatmap showing specific TBX20 binding loci by using 2 independent TBX20 antibodies. B, Pie chart showing the distribution of TBX20 peaks in different genomic regions as indicated. C, GREAT (Genomic Regions Enrichment of Annotations Tool) analysis of TBX20 peaks. D, De novo motif analysis identified from TBX20 peaks. E, Statistical analysis using Fisher exact test showed that significantly more enhancer regions were enriched in TBX20 peak sequences than randomly generated sequences. P<2.2×10⁻¹⁶ represents a P value approaching 0. F, Heatmap showing enrichment of TBX20 peaks in human tissue enhancers collected in EnhancerAtlas 2.0.³⁵ G, Density plots showing signals of ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing), H3K4me1, and H3K27ac at TBX20 binding sites. H, Venn diagram showing overlapping of genes bound and upregulated by TBX20. P values were calculated by Fisher exact test. Right panel, Gene Ontology terms enriched on TBX20 upregulated gene targets. I, IGV tracks showing RNA sequencing, TBX20 CUT&Tag, ATAC-seq, and CUT&RUN (cleavage under targets and release using nuclease) signals for H3K27ac, H3K4me1, and H3K27me3 at TNNI3 gene locus between MGT + empty vector (EV) and MGT+TBX20 human induced cardiomyocytes (hiCMs). J, Heatmap displaying class I and class II TBX20 upregulated targets and corresponding representative genes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment terms. Class I genes were defined as de novo upregulated genes by MGT+TBX20 and class II genes were defined as further activated by the addition of TBX20. K, Boxplot showing TBX20 binding peak distance to transcript start site for class I and class II genes. P value was calculated by a 2-sided Wilcoxon rank sum test. Box, median ± interquartile range. Whiskers, 1.5 × interquartile range. L, Schematic diagram showing the possible mechanism for the regulation of gene expression by TBX20. OR indicates odds ratio.