Figure 8.

TBX20 promotes M/G/T genomic binding and activates cardiac enhancers. A, Top panels: heatmaps showing MEF2C, GATA4, and TBX5 CUT&Tag (cleavage under targets and tagmentation) signals on TBX20 binding sites in MGT + empty vector (EV) and MGT+TBX20 human induced cardiomyocytes (hiCMs). Bottom panels: density plots showing enhanced binding of MGT factors on TBX20 peaks. B, Western blot images and quantification (n=3 per group) showing expression of MEF2C, GATA4, and TBX5 in MGT+EV-induced or MGT+TBX20-induced hiCMs. C, Stacked bar plots showing the number of MEF2C, GATA4, and TBX5 binding sites and the proportion of co-binding sites by MEF2C, GATA4, or TBX5 in MGT+EV and MGT+TBX20 infected hiCMs. D, Percentage of TBX20 binding on the peaks occupied with 1, 2, or 3 factors of MGT. E, Bubble plot showing the Gene Ontology annotation for genes associated with peaks occupied with 1, 2, or 3 factors of MGT. F, Heatmaps and density plot showing TBX20 peaks in H9-derived fibroblasts (H9Fs) transduced with MGT+TBX20 or TBX20 alone. G, IGV browser tracks showing RNA sequencing signals and TBX20 peaks in H9Fs transduced with TBX20 alone or MGT+TBX20. H, IGV tracks showing read signals of RNA sequencing, CUT&Tag of MEF2C, GATA4, TBX5 and TBX20, ATAC-seq (assay for transposase-accessible chromatin with high-throughput sequencing), and CUT&RUN (cleavage under targets and release using nuclease) of H3K27ac, H3K4me1, and H3K27me3 at selective cardiac marker gene loci in MGT+EV or MGT+TBX20 infected hiCMs. The brown bars on the top of the graph indicate transcription enhancers (TE) used for reporter assay. I, Construct schematics for reporter assay. MYBPC3 and MYL4 reporter activation in 293T cells after cotransfection of reporter constructs together with empty vector, TBX20 alone, MGT+EV, or MGT+TBX20 (n = at least 3). pGL3-Basic served as negative controls. All data are expressed as mean ± SEM (B, Student t test; I, 2-way analysis of variance). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.