A context-specific cardiac β-catenin and GATA4 interaction influences TCF7L2 occupancy and remodels chromatin driving disease progression in the adult heart

Abstract

Chromatin remodelling precedes transcriptional and structural changes in heart failure. A body of work suggests roles for the developmental Wnt signalling pathway in cardiac remodelling. Hitherto, there is no evidence supporting a direct role of Wnt nuclear components in regulating chromatin landscapes in this process. We show that transcriptionally active, nuclear, phosphorylated(p)Ser675-β-catenin and TCF7L2 are upregulated in diseased murine and human cardiac ventricles. We report that inducible cardiomyocytes (CM)-specific pSer675-β-catenin accumulation mimics the disease situation by triggering TCF7L2 expression. This enhances active chromatin, characterized by increased H3K27ac and TCF7L2 occupancies to cardiac developmental and remodelling genes in vivo. Accordingly, transcriptomic analysis of β-catenin stabilized hearts shows a strong recapitulation of cardiac developmental processes like cell cycling and cytoskeletal remodelling. Mechanistically, TCF7L2 co-occupies distal genomic regions with cardiac transcription factors NKX2-5 and GATA4 in stabilized-β-catenin hearts. Validation assays revealed a previously unrecognized function of GATA4 as a cardiac repressor of the TCF7L2/β-catenin complex in vivo, thereby defining a transcriptional switch controlling disease progression. Conversely, preventing β-catenin activation post-pressure-overload results in a downregulation of these novel TCF7L2-targets and rescues cardiac function. Thus, we present a novel role for TCF7L2/β-catenin in CMs-specific chromatin modulation, which could be exploited for manipulating the ubiquitous Wnt pathway.

Figure 1.

Nuclear phosphorylated-Ser675β-catenin triggers Wnt transcriptional reactivation upon cardiac pressure-overload in mice and humans. (A) Normalized transcript expression of murine and RPKMs of human TCF7L2 expression in foetal and adult hearts (n = 3/per group). (B) Relative transcript levels of Tcf7l2 in 8 weeks post-TAC heart tissue vs. sham control (n≥5). (C) Representative immunoblots of TCF7L2 and β-catenin expression 3 days and 8 weeks post-TAC in murine heart ventricles compared to sham (n≥4). (D) Immunofluorescence image representing increased TCF7L2 (magenta) in isolated cardiomyocytes (CM) from 3 days post-TAC murine hearts (n = 3/group). (E) Total β-catenin, TCF7L2 and pSer675-β-catenin in nuclear (TBX5-enriched) fraction, 6-weeks post-TAC. (F) Relative transcript levels of the classical Wnt target gene, Axin2, and CM hypertrophic marker, Natriuretic peptide b (Nppb) 3 days and 2 weeks post-TAC in murine ventricular tissue versus sham control (n ≥ 5). (G) Western blots showing total β-catenin, pSer675-β-catenin and TCF7L2 in cardiac ventricular biopsies from ischemic (ICM) and dilated cardiomyopathies (DCM) as compared to non-failing (NF) human hearts (NF: n = 2; DCM: n = 6; ICM: n = 6). (H) TCF7L2 and its target AXIN2 transcript levels in cardiac ventricular biopsies from DCM and ICM as compared to NF human hearts (NF: n = 7; DCM: n = 15; ICM: n = 11). (I) Immunoblot showing stabilized (70 kDa) β-catenin, pSer675-β-catenin and TCF7L2 protein in β-cat^Δex3 ventricles. Representative immunofluorescence images showing increased perinuclear/nuclear pSer675-β-catenin (lower panel) or (J) TCF7L2 (magenta) in isolated CM β-cat^Δex3 ventricles compared to Cre^pos along with corresponding plots below images representing fluorescence intensity profiles over the nucleus. ACTN2 is shown in green, DAPI nuclear staining in blue (n = 3/group). The profiles show DAPI (blue) signal overlapping with TCF7L2 (magenta) with a higher intensity in β-cat^Δex3 CM. (K) Relative transcripts of Axin2 after 3 days and 3 weeks of induction in β-cat^Δex3 ventricles vs. control Cre^pos/β-cat^wt (Cre^pos) hearts (n = 10; 3 and 5; respectively). TATA-binding protein (Tbp) (B, F) and GAPDH (H) were used for transcript normalization. GAPDH and TPT1 serve as protein loading control for whole cell lysate (C, G and I) and GAPDH for cytosolic fraction (E) and TBX5 for nuclear fraction in E. Data are mean ± SEM; t-test and ANOVA, Bonferroni's multiple comparison tests. Scale bar: I: 5 μm and D, J: 20 μm.

Figure 2.

Wnt activation promotes heart failure by triggering developmental reprogramming in the adult heart. Representative images (A) and quantification showing increased- (B) heart-to-body weight ratios (HW/BW, n ≥ 15/group), (C) CM cross-sectional area by WGA-FITC staining (n = 3; 150 cells/mouse) and (D) fibrosis by Sirius Red staining (n = 3/group) in β-cat^Δex3 versus control Cre^pos. (E) Echocardiographic analyses showing anterior and poster wall thickness diameters (AWTHd, PWTHd), left ventricular inner diameter (LVId) and fractional area shortening (FAS), 3 weeks post-TX induction in β-cat^Δex3 mice compared to Cre^pos and Cre^neg/β-cat^wt (Cre^neg) controls (n≥15/group). (F) Kaplan–Meier survival curve post-TX induction, n ≥ 21. (G) Half-times of intracellular calcium relaxation (RT_50%) of caffeine induced-Ca²⁺-transients along with SERCA2 activity in β-cat^Δex3 and Cre^pos CMs (n = 6/4–21 cells per mouse). Ventricular protein expression of Na⁺–Ca²⁺ exchanger (NCX) in β-cat^Δex3 and Cre^pos hearts, n = 4. GAPDH serves as loading control in G. (H) Heatmap representing row Z-scores of RPKM values of all 572 differentially expressed genes (DEGs) with a cut-off: log₂FC ± 0.5, P < 0.05 in Cre^pos and β-cat^Δex3 cardiac ventricles. Upregulated genes and downregulated genes are depicted in red and blue respectively (n = 3/group). (I) Volcano plot depicting the DEGs. Blue: DEGs with log₂FC≥0.9 and p≤0.05; red: log₂FC≤0.9 and P ≤ 0.05; black: unregulated genes. (J) Gene Ontology (GO) biological processes of upregulated (P≤ 0.05) genes. Data are mean ± SEM; t-test and ANOVA, Bonferroni's multiple comparison test. Scale bar in A: 20 μm.

Figure 3.

Wnt transcriptional activation results in increased CM cell cycling and cytoskeletal remodelling in the adult heart. (A) Heatmaps depicting row Z-scores of RPKMs of upregulated genes involved in heart morphogenesis, cell cycling and cytoskeleton organization in β-cat^Δex3 ventricles; with (B) corresponding qPCR validations (β-cat^Δex3n = 4; Cre^pos n = 5). (C) Confocal images of KI67 or EdU (magenta) immunostainings with quantification of double positive cTNT/KI67 and EdU cells (n = 3; >400 cells/mouse). White arrows indicate KI67 and EDU positive CM. (D) Immunostainings for Caveolin 3 (red), DAPI (blue) in single CM along with quantification of nuclei/CM (n = 3; ≥90 cells/CM per mouse). (E) qPCR validating increased cytoskeletal regulators Rock2 and Dstn; and immunoblot showing increased ROCK2 in β-cat^Δex3 ventricles (β-cat^Δex3n = 4; Cre^posn = 5). (F) Confocal images and (G) quantification of microtubule network density and complexity in single CMs (n = 3; 5–8 cells/mouse; i-ii represent higher magnification in F). Images are an overlay of α-TUBULIN images (green) with corresponding extracted skeletons (black). Tbp was used for normalization in B and E and GAPDH was loading control in F. Scale bar C: 20 μm, E, G: 10 μm. Confocal images were re-colored for color-safe combinations.

Figure 4.

Induced TCF7L2 and H3K27ac recruitment to disease-associated enhancers defines the β-cat^Δexon3 cardiac epigenome. (A) Genomic distribution of TCF7L2 occupancy in β-cat^Δex3 ventricles in number of regions and as distance from TSS (arrow) in kb. (B) Heatmaps of H3K27ac in β-cat^Δex3 ventricles and high sensitivity DNAse-sequencing (DHS), -RNAPII, -H3K4me1 occupancy in normal adult heart, on TCF7L2 occupied regions in β-cat^Δex3 ventricles, aligned according to their maximum signal of TCF7L2 occupancy. Scale depicts normalized RPKM values for heatmaps. (C) Exemplary occupancy profiles of TCF7L2, H3K27ac and DHS on an identified distal enhancer of Adamts19. (D) Total number of H3K27ac bound-regions as well as (E) average signal profiles of H3K27ac on TCF7L2 occupied loci in Cre^pos and β-cat^Δex3 ventricles. (F) GO disease ontologies of TCF7L2 bound regions in β-cat^Δex3 ventricles. (G) Binding affinity plot of 25,563 differential ChIPseq-H3K27ac enriched regions (pink dots) in the Wnt normal (3,040 regions, log FC > +0.5, n = 3) and in β-cat^Δex3 (Wnt activated) (22,523 regions, log FC < –0.5, n = 2) hearts. (H) Motif analysis on differentially enriched distal H3K27ac regions (5,748 from 22,523) in ‘Wnt activated’ and control ‘Wnt normal’. TCF7L2 motif occurrence is highlighted in an orange box. (I) Disease Ontologies are for the regions in H. For heatmaps regions ± 5 kb are shown.

Figure 5.

TCF7L2 elicits tissue-specific gene regulation in pathological heart remodelling. (A) Venn diagram of genes bound by TCF7L2 (green, 977) and upregulated (red, 376; log₂FC ≥ 0.5, P ≤ 0.05) in β-catΔex3 ventricles. GO biological processes annotation of the 68 common genes at the intersection. Hand2, Tbx20, Rock2 and Dstn are part of these common genes. (B) Occupancy profiles of TCF7L2 and H3K27ac in β-catΔex3 and Cre^pos ventricles on the identified putative enhancers upstream of Hand2, Tbx20, Rock2 and Dstn. (C) Hand2, Tbx20, Rock2 and Dstn relative transcript levels in ventricles upon TAC-induced hypertrophy and sham controls, n ≥ 5. (D) Heatmaps showing row Z-scores of RPKMs of dysregulated genes in human ventricular tissue from non-failing (NF), hypertrophic and failing hearts (HF). (E) Luciferase reporter assays for Hand2 and Tbx20 identified enhancers upon Wnt activation by β-catenin stabilization in HEK293 cells normalized to empty vector (Renilla luciferase was used as transfection control, n = 3/independent experiments). Tbp was normalization control in C. Data are mean ± SEM; t-test.

Figure 6.

TCF7L2 cooperates with cardiac-TFs to enable heart-specific gene regulation. (A) Comparison of TCF7L2-bound regions in in vivo models of β-catenin stabilization (β-cat^Δex3) in CMs and hepatocytes. GO biological processes for unique regions in CM (magenta) and hepatocytes (blue). (B) TCF7L2 occupancy profiles on identified Hand2, Tbx20, Rock2 and Dstn enhancers and common Wnt targets Axin2 and Lef1 in β-catenin stabilized CM (magenta) and hepatocytes (blue). (C) Table enlisting the transcription factors (TFs) enriched on TCF7L2-bound regions in β-cat^Δex3 ventricles by de-novo motif search using MEME-SpaMo. (D) Heatmap depicting the occupancy of cardiac-TFs GATA4 and NKX2.5 in the normal heart on regions occupied by TCF7L2 in β-cat^Δex3 hearts. Regions ± 5 kb are shown. (E) Average profiles of GATA4 and NKX2.5 occupancy on TCF7L2-bound liver and heart-specific regions. Data are mean ± SEM; t-test.

Figure 7.

GATA4 interacts with β-catenin and fine-tunes the molecular switch driving adult heart disease progression in vivo. (A) Spearman's correlation plot of TCF7L2 co-occupancy with GATA4, NKX2.5 and TBX3, highlighting highest correlation with GATA4 (black box). (B) Venn diagram of genes bound by TCF7L2 (orange) with upregulated (violet) or downregulated (green) genes with log₂FC≥0.5, p≤0.05 in β-cat^Δex3 ventricles and corresponding motif enrichment of the intersections. (C) Venn diagram showing commonly bound genes (319) between TCF7L2 in β-cat^Δex3 hearts and GATA4 in normal hearts. (D) Immunoblot of GATA4 with β-catenin co-immunoprecipitation in WT, β-cat^Δex3 and 6 weeks post-TAC hearts. Input represents the total, sheared chromatin-protein complexes before immunoprecipitation, (*) protein ladder. (E) Immunoblot of total β-catenin and active pSer675 β-catenin in the nuclear fractions of control, β-cat^Δex3 and 6 weeks post-TAC hearts. TBX5 and GAPDH were used to detect nuclear and cytosolic enrichments respectively. (F) IGV binding profiles for TCF7L2 occupancy in β-cat^Δex3 hearts along with GATA4 co-occupancy in normal hearts on Hand2 enhancer locus; ChIP-qPCR for GATA4 binding on Hand2 enhancer in normal (WT), 6 weeks post-TAC (WT TAC) and β-cat^Δex3 hearts. Relative fold enrichment was calculated to IgG control, normalized to 10% input chromatin (n = 3 hearts/ChIP) (G) Profiles of enhancers with GATA4 and TCF7L2 overlapping occupancy (Hand2) and with only TCF7L2 occupancy (Tbx20). Luciferase reporter assay for Hand2 enhancer (-enh) and Tbx20-enh upon β-catenin stabilization, GATA4 overexpression or both normalized to empty vector (EV). (Renilla luciferase was the transfection control, n = 3/independent experiments). Data are mean ± SEM; t-test and ANOVA, Bonferroni's multiple comparison test. (H) Spearman's correlation plot depicting high correlations between GATA4 and repressive elements KLF15, H3K27me3 and CTCF in normal hearts specifically on TCF7L2-bound regions in β-cat^Δex3 hearts.

Figure 8.

β-catenin loss of function in CM rescues Wnt-dependent pathological gene regulation in vivo. (A) Representative examples of M-mode echocardiograms, and quantification of fractional shortening (FAS) by echocardiographic analysis of 3 weeks TAC-induced Cre^pos control and β-cat^Δex2–6 mice, n ≥ 7. (B) Relative transcript levels of hypertrophic marker Nppb in β-cat^Δex2–6 and controls Cre^pos; sham and TAC (n≥8/per group). (C) Immunoblot depicting total β-catenin and pSer675-β-catenin upon TAC in β-catenin loss of function (β-cat^Δex2–6). GAPDH was protein-loading control (n = 2/group). Relative transcript levels of (D) classical Wnt target Axin2 and (E) newly identified cardiac Wnt targets Tbx20 and Dstn, 6 weeks post-TAC in β-cat^Δex2–6 and controls, n≥7. Data are mean ± SEM; ANOVA, Bonferroni's multiple comparison test. Tbp was used for transcript normalization. (F) ChIP-qPCR for GATA4 binding on Hand2 enhancer in normal (WT), 6 weeks post-TAC (WT TAC) and β-cat^Δex2–6 TAC hearts. Relative fold enrichment was calculated to IgG control, normalized to 10% input chromatin (n = 3 hearts/ChIP). (G) Schematic representation of the findings of this study. In the healthy adult heart, β-catenin/TCF7L2-dependent loci are inactive, inaccessible or bound by transcriptional repressors, resulting in low transcription. On a subset of these loci, GATA4 binds to transcriptionally inactive β-catenin, fine-tuning Wnt-dependent transcription. This chromatin state guaranties normal homeostasis in the adult heart. Pathological stimuli leading to active pSer675-β-catenin accumulation activates Wnt signalling and the epigenetic state switches on to ‘active’, replaced by transcriptionally active pSer675-β-catenin bound to TCF7L2, leading to enriched H3K27ac occupancy and a high Wnt transcriptional activity. This results in the expression of disease-associated genes leading to adverse remodelling and heart failure.