Epigenetic repression of cardiac progenitor gene expression by Ezh2 is required for postnatal cardiac homeostasis

Abstract

Adult-onset diseases can be associated with in utero events, but mechanisms for this remain unknown(1,2). The Polycomb histone methyltransferase Ezh2 stabilizes transcription by depositing repressive marks during development that persist into adulthood(3-9), but its function in postnatal organ homeostasis is unknown. We show that Ezh2 stabilizes cardiac gene expression and prevents cardiac pathology by repressing the homeodomain transcription factor gene Six1, which functions in cardiac progenitor cells but is stably silenced upon cardiac differentiation. Deletion of Ezh2 in cardiac progenitors caused postnatal myocardial pathology and destabilized cardiac gene expression with activation of Six1-dependent skeletal muscle genes. Six1 induced cardiomyocyte hypertrophy and skeletal muscle gene expression. Furthermore, genetically reducing Six1 levels rescued the pathology of Ezh2-deficient hearts. Thus, Ezh2-mediated repression of Six1 in differentiating cardiac progenitors is essential for stable gene expression and homeostasis in the postnatal heart. Our results suggest that epigenetic dysregulation in embryonic progenitor cells is a predisposing factor for adult disease and dysregulated stress responses.

Figure 1

Ezh2 limits cardiac growth and fibrosis. (a) Adult mice in which Ezh2 was deleted in second heart field progenitors (Ezh2^fl/fl; Mef2cAHF::Cre) present hypertrophied right ventricle, with increased (b) heart weight to tibia length ratio (mg/mm), as compared to controls (Ezh2^fl/fl). Ezh2^fl/fl (blue bars) and Ezh2^fl/fl; Mef2cAHF::Cre (red bars). (c) Wheat germ agglutinin (WGA) staining of heart sections in control (Ezh2^fl/fl) or Ezh2-deficient (Ezh2^fl/fl; Mef2cAHF::Cre) right ventricle (RV) and left ventricle (LV). (d) Cardiomyocyte cell surface area in Ezh2^fl/fl (blue bars) or Ezh2^fl/fl; Mef2cAHF::Cre (red bars) hearts. (e) Tissue disorganization and fibrosis, as revealed with Masson’s trichrome staining. In b and d, bars represent the mean +/− S.D. of measurements from at least three hearts per genotype. * indicates P < 0.05. (f) Vimentin staining of RV and LV of Ezh2^fl/fl or Ezh2^fl/fl; Mef2cAHF::Cre hearts. (g) Pecam staining showing invaginations of endocardium into myocardium (arrows). In f,g, the red arrow points to endocardial lining of the RV lumen. Reference bars in (a) and (e) (upper panel) = 0.5 cm; (e) (lower panel) = 100 µm; (c) = 25 µm; (f) and (g) = 100 µm.

Figure 2

Ezh2 represses expression of fetal genes, profibrosis factors and Six1. (a) QRTPCR showing relative expression of Nppa, Nppb and Myh7 mRNA. Ezh2^fl/fl (blue bars) and Ezh2^fl/fl; Mef2cAHF::Cre (red bars). (b) QRTPCR showing expression of Tgfβ3, Postn and Spp1. (c) Immunofluorescence showing low levels of perinuclear Tgfβ3 in control cardiomyocytes (arrows), and strong extracellular staining in the extracellular region in Ezh2^fl/fl; Mef2cAHF::Cre right ventricle (RV). Nuclei were counterstained with DAPI. (d) Chromatin immunoprecipitation for Suz12, H3K27me3, RNA PolII, and acetylated histone H3 (AcH3) on the Nppa core promoter in Ezh2-deficient Hearts (red bars), as compared with controls (blue bars). e, Heat map of 1132 upregulated genes identified by RNA-seq comparing Ezh2-deficient hearts to control hearts. Numbers at left indicate fold increase. Indicated genes were amongst the most upregulated. (f) Analysis of functional categories identified a high percentage of genes regulating skeletal muscle and tissue remodelling. (g) RNA-seq analyses showed a higher percentage of deregulated genes targeted by Six1, as compared to deregulated genes not targeted by Six1, in Ezh2-deficient hearts. Reference bars = 25 µm. Bars in panels (a), (b) and (d) represent the mean +/− S.D. from at least three biological replicates. * Indicates P < 0.05.

Figure 3

Six1 is epigenetically repressed by PRC2. (a) QRTPCR showing expression of Six1 and Eya1 in embryonic and adult myocardium. (b) Upper panel: In situ hybridization showing Six1 mRNA in E13.5 Ezh2-deficient (Ezh2^fl/fl; Mef2cAHF::Cre) right ventricle (RV). Reference bar = 200 µm. Lower panel. Immunofluorescence for Eya1. Cardiomyocytes were co-stained with an anti tropomyosin (Tpm1) antibody, and nuclei with DAPI. (c) Immunofluorescence for Six1 and alpha-actinin 3 (Actn3) in control (Ezh2^fl/fl) and Ezh2^fl/fl; Mef2cAHF::Cre adult hearts. Only Ezh2-defficient right ventricular (RV) cardiomyocytes co-expressed Six1 and Actn3. Arrows point to Six1-possitive cardiomyocyte nuclei. LV: left ventricle. Reference bar = 25 µm. (d) Chromatin immunoprecipitation for Suz12, H3K27me3, RNA PolII, and acetylated histone H3 (AcH3) on the Six1 core promoter in Ezh2-deficient Hearts (red bars), and controls (blue bars). Bars in panels (a) and (d) represent the mean +/− S.D. of at least three biological replicates. * Indicates P < 0.05.

Figure 4

Six1 has differentiated myocardium expression potential. (a) rVista plot of the percentage of conservation between the mouse (Mm) and chicken (Gg) Six1 upstream regulatory region. The indicated peak represents a highly conserved element including potential Nkx2-5, Mef2c and Gata binding motifs. (b) A LacZ reporter driven by the Hsp68 promoter and a 80bp DNA fragment including such a conserved element was injected into mouse blastocysts. (c) Lateral and frontal views of whole mount transient transgenic Six1-hsp68-lacZ embryos at E11.5 that were stained for β-galactosidase (β-gal) activity. (d) Close up of the heart from the same embryo. (e) Section from heart in g showing b-gal staining in ventricular myocardium. RA, LA, RV and LV: right and left atria, and ventricles, respectively.

Figure 5

Six1 induces cardiac hypertrophy and skeletal muscle gene expression in cardiomyocytes. (a) Cardiac myocytes infected with adenoviruses (ad) expressing control cDNA (lacZ), Six1, or Eya1, or treated with Endothelin-1. Expression of LacZ, Six1 and Eya, was confirmed with staining for β-galactosidase (β -gal), and V5-tagged Six1 and Eya1. Cardiomyocytes were stained for tropomyosin, and nuclei counterstained with DAPI. Graph shows average cell surface area. Bars represent the mean +/− S.D. from at least 100 myocytes measured. Reference bars = 10 µm. (b) QRTPCR of skeletal muscle genes on RNA from infected cardiomyocytes. Bars in (a,b) represent the mean +/− S.D. from at least three independent infections. * Indicates P < 0.05. LV: left ventricle

Figure 6

Ezh2 limits cardiac hypertrophy and fibrosis by repressing Six1. (a) Whole heart images and heart mass, as represented by heart weight to tibia length ratio of Ezh2 floxed (Ezh2^fl/fl), Six1 heterozygous (Ezh2^fl/fl; Six1^LacZ/+), Ezh2-deficient mice (Ezh2^fl/fl; Mef2c::Cre), and Ezh2-deficient mice with decreased levels of Six1 (Ezh2^fl/fl; Mef2c::Cre;Six1^LacZ/+) (b), Heart sections from mice with the same genotypes as in (a) stained with wheat germ agglutinin (WGA) and (c) vimentin. Graphs show the respective quantitations. (d) QRTPCR for Six1, Myh7, Myh8, Myl1, Myl4 and Actn3. Reference bars = 10 µm. Bars represent the mean +/− S.D. from at least three hearts per genotype. * indicates P < 0.05.