Fig 2: Cbx1, PurB, and Sp3 regulate the PRC2 complex.A, endogenous Cbx1 and PurB was immunoprecipitated from cardiac fibroblast cell lysates. An isotype control antibody was used as a control. Immunoprecipitates were immunoblotted with a Cbx1, PurB, and a Sp3 antibody. The first lane contains cell extract (1/10th immunoprecipitation input). Representative immunoblots are shown from three independent experiments. B, cardiac fibroblasts were transfected with siRNAs targeting Cbx1, PurB, and Sp3. A nontargeting siRNA was used as a control. After 4 days, cell lysates were immunoblotted with H3K27me3 and H3 antibodies. Immunoblotting for Gapdh was used as a loading control. Representative immunoblots are shown from four independent experiments. C, endogenous Cbx1, PurB, and Sp3 was immunoprecipitated from cardiac fibroblast cell lysates. An isotype control antibody was used as a control. Immunoprecipitates were immunoblotted with an Eed antibody. The first lane contains cell extract (1/10th immunoprecipitation input). Representative immunoblots are shown from three independent experiments. D, cardiac fibroblasts were transfected with either a nontargeting control siRNA or a PurB targeting siRNA. After 3 days, endogenous Cbx1 was immunoprecipitated from cell lysates. An isotype control antibody was used as a control. Immunoprecipitates were immunoblotted with a Cbx1 and a Sp3 antibody. The first lane contains cell extract (1/10th immunoprecipitation input). Representative immunoblots are shown from three independent experiments. H3K27me3, trimethylated histone-H3 lysine-27.

Fig 3: Cbx1, PurB, and Sp3 regulate chromatin architecture. Cardiac fibroblasts were transfected with siRNAs targeting Cbx1, PurB, or Sp3. A nontargeting siRNA was used as a control. After 7 days, chromatin was isolated and digested with micrococcal nuclease (MNase). Following MNase digestion, the resulting undigested DNA was submitted for high-throughput sequencing (MNase-Seq) and mapped to the mouse genome. A, MNase digestion was optimized to give rise to one nucleosome. Read lengths were analyzed after sequencing and summed. As expected, the majority of read lengths were 1 nucleosome is size (~150 bp). B, MNase accessibility signals around transcription start sites (TSSs). The y-axis represents the read number for each 10 bp bin normalized to the effective genome size for the mouse. C, nucleosomes (black bars) were plotted on the cardiomyocyte-specific genes Ryr2 and Actn2. D, nucleosomes in noncardiomyocyte genes.

Fig 4: Loss of Cbx1, PurB, and Sp3 expression is associated with cardiomyocyte differentiation.A, cardiac fibroblasts were transfected with either a control nontargeting siRNA or a pool of siRNAs targeting Cbx1, PurB, and Sp3. After 14 days, expression of the indicated cardiomyocyte, fibroblast, endothelial, and neuronal-specific genes was determined by quantitative PCR (qPCR). For completeness, a number of general genes were included. Expression values were normalized to the control siRNA. N = 3. B, RNA was extracted from iPS cells and iPS-derived cardiomyocytes. Expression of the indicated genes was determined by qPCR, and expression values in iPS-derived cardiomyocytes were normalized to iPS cells. N = 4. Individual data points (open circles) and mean (horizontal bar) are shown. One-way ANOVA with Bonferroni post hoc tests was used to determine significance; ***p < 0.01. C, RNA-Seq of iPS differentiation into cardiomyocytes from the study by Churko et al. (20). Expression values of the indicated genes in iPS-derived cardiomyocytes were normalized to iPS cells. N = 1. D, RNA-Seq from the study by Pavlovic et al. (21). Cardiac tissue was isolated from 12 individuals and used to generate iPS cells. These iPS cells were then differentiated into cardiomyocytes. RNA-Seq was performed with the source cardiac tissue; undifferentiated iPS cells; as well as iPS-derived cardiomyocytes. N = 12. Read number is shown. Individual data points (open circles) and mean (horizontal bar) are shown. One-way ANOVA with Bonferroni post hoc tests was used to determine significance; **p < 0.01 and ***p < 0.001. iPS, inducible pluripotent stem.

Fig 5: Cbx1, PurB, and Sp3 are repressors of cardiomyocyte-specific genes.A, RNA-Seq read counts from the study by Hodgkinson et al. (18) were analyzed for the mRNA levels of the indicated genes in freshly isolated cardiomyocytes and noncardiomyocytes. Individual data points (open circles) and mean (horizontal bar) are shown. One-way ANOVA with Bonferroni post hoc tests was used to determine significance; ***p < 0.001. N = 3. B, cardiac fibroblasts were transfected with the direct cardiac reprogramming cocktail miR combo. A nontargeting miRNA (negmiR) was used as a control. Four days after transfection, expression of the indicated transcription factors was determined by quantitative PCR (qPCR). Expression values were normalized to negmiR. N = 5. Individual data points (open circles) and mean (horizontal bar) are shown. One-way ANOVA with Bonferroni post hoc tests was used to determine significance; ***p < 0.001. C, cardiac fibroblasts were transfected with siRNA targeting an individual putative repressor. A nontargeting siRNA was used as a control. After 4 days, expression was determined by qPCR. Expression values were normalized to the control siRNA. N = 3. Individual data points (open circles) and mean (horizontal bar) are shown. One-way ANOVA with Bonferroni post hoc tests was used to determine significance; ***p < 0.001. D, cardiac fibroblasts were transfected with siRNA targeting an individual putative repressor. A nontargeting siRNA was used as a control. After 14 days, expression of the indicated cardiomyocyte-specific genes was determined by qPCR. Expression values were normalized to the control siRNA. The heatmap summarizes the results of ten cardiomyocyte-specific genes. Increased expression of greater than twofold and with a significance <0.05 is shown in red. N = 3 to 5. One-way ANOVA with Bonferroni post hoc tests was used to determine significance; *p < 0.05. E, cardiac fibroblasts were transfected with siRNA targeting an individual putative repressor. A nontargeting siRNA was used as a control. After 14 days, the cells were incubated with antibodies to the cardiomyocyte-specific protein Actn2. Representative images are shown. N = 4. The scale bar represents 50 microns. F, higher resolution images of the cells shown in E are shown to display sarcomeres. The scale bar represents 50 microns. N = 4. Quantification of the percentage of Actn2+ cells displaying sarcomeres. Individual data points (open circles) and mean (horizontal bar) are shown. One-way ANOVA with Bonferroni post hoc tests was used to determine significance; **p < 0.01.