YAP repression of the WNT3 gene controls hESC differentiation along the cardiac mesoderm lineage

Abstract

Activin/SMAD signaling in human embryonic stem cells (hESCs) ensures NANOG expression and stem cell pluripotency. In the presence of Wnt ligand, the Activin/SMAD transcription network switches to cooperate with Wnt/β-catenin and induce mesendodermal (ME) differentiation genes. We show here that the Hippo effector YAP binds to the WNT3 gene enhancer and prevents the gene from being induced by Activin in proliferating hESCs. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data show that YAP impairs SMAD recruitment and the accumulation of P-TEFb-associated RNA polymerase II (RNAPII) C-terminal domain (CTD)-Ser7 phosphorylation at the WNT3 gene. CRISPR/CAS9 knockout of YAP in hESCs enables Activin to induce Wnt3 expression and stabilize β-catenin, which then synergizes with Activin-induced SMADs to activate a subset of ME genes that is required to form cardiac mesoderm. Interestingly, exposure of YAP^-/- hESCs to Activin induces cardiac mesoderm markers (BAF60c and HAND1) without activating Wnt-dependent cardiac inhibitor genes (CDX2 and MSX1). Moreover, canonical Wnt target genes are up-regulated only modestly, if at all, under these conditions. Consequently, YAP-null hESCs exposed to Activin differentiate precisely into beating cardiomyocytes without further treatment. We conclude that YAP maintains hESC pluripotency by preventing WNT3 expression in response to Activin, thereby blocking a direct route to embryonic cardiac mesoderm formation.

Keywords: YAP; cardiac mesoderm development; hESCs; stem cell differentiation.

Figure 1.

YAP selectively prevents hESC differentiation to mesendoderm in response to Activin. (A) Isolation of YAP knockout hESC clones. (Left) Immunoblot of two different YAP knockout clones. (Right) Quantitative PCR (qPCR) analysis of CTGF mRNA levels in wild-type and YAP knockout hESCs. Mean (SD). n = 3. (B) Phase contrast microscopy captures show colonies of wild-type and YAP knockout hESCs. OCT4 protein levels were analyzed by immunostaining. (C) Protocol for Activin- and Wnt-dependent hESC differentiation to ME cells. Activin and Wnt pathways synergize to induce optimal levels of ME gene transcription. YAP selectively blocks ME gene expression through an unknown mechanism. (D) Heat map display of RNA sequencing (RNA-seq) data from wild-type and YAP knockout hESCs treated with either 50 ng/mL Activin, GSK3 inhibitor (GSK3i; low concentration; 6 µM ChIR99021 [ChIR]), or Activin+GSK3i for 24 h. The heat map ranks the genes most strongly affected by Activin in YAP knockout cells. (E) Box plot showing the log fold change expression of pluripotency and ME differentiation genes in wild-type or YAP knockout hESCs after the indicated treatments. The adjusted P-value for multiple testing correction is shown above each box. (F) ME gene expression profiles (52 genes) projected onto the first two principal components. hESC lines and treatments are designated by colors and symbols. (G) Representative immunoblot of the indicated proteins in wild-type and YAP knockout cells treated for 24 h as indicated above each lane. n ≥ 3. (H) Immunostaining of wild-type and YAP knockout cells following 24 h of exposure to Activin.

Figure 2.

β-Catenin is required for Activin-induced differentiation of YAP knockout hESCs. (A) The efficiency of siRNA-mediated knockdown of the indicated proteins was analyzed by immunoblot. CDK2 was included as a loading control. The graph at the right shows the mRNA levels of the EOMES and MIXL1 genes in Activin-treated YAP knockout hESCs transfected with the indicated siRNAs. Mean (SD). n = 3. (B) Immunoblot analysis of the YAP and β-catenin double-knockout hESC lines. Analysis by qRT–PCR reveals that EOMES and MIXL1 are not induced by Activin in the YAP:β-catenin double-knockout (β^−/−Y^−/−) cells. Mean (SD). n = 3. (C) Heat map presentation of RNA-seq data ranking the genes most strongly affected by Activin treatment in YAP knockout cells. (D) The diagram depicts the number of genes that are induced by Activin signaling in YAP knockout cells but no longer induced in YAP/β-catenin knockout cells. (E) Gene ontology analysis of the genes down-regulated in the YAP:β-catenin knockout cells compared with YAP knockout cells. (F) The box plot shows the log fold change of YAP knockout and YAP:β-catenin knockout cell lines compared with wild-type hESCs for the set of genes involved in pluripotency and ME differentiation. The adjusted P-value for multiple testing correction is shown above each box.

Figure 3.

Activin induces binding of β-catenin genome-wide in YAP knockout hESCs. (A) Wild-type and YAP knockout hESCs were treated with Activin for 15 h prior to ChIP-seq analysis of SMAD2,3, β-catenin, and elongation-competent RNAPII CTD-Ser7P at target genes genome-wide. Captures show the distribution of immunoprecipitated proteins at the selected ME genes. (B) Venn diagrams depicting the number of SMAD and β-catenin peaks in Activin-treated wild-type and YAP knockout cells. Note that 531 new β-catenin peaks appear in YAP knockout cells, which are absent in wild-type hESCs. (C) ChIP-qPCR in YAP knockout cells transfected with siRNAs against SMAD2 or β-catenin, as indicated. (SBS) SMAD-binding site; (BBS) β-catenin-binding site; (NC) negative control. Mean (SD) n = 3.

Figure 4.

YAP binds to enhancers of developmental genes in hESCs. (A) Graph showing the number of YAP ChIP-seq peaks in wild-type and YAP knockout hESCs. (B) Venn diagram of overlapping of TEAD4 and YAP peaks. The bottom panel shows the top motif bound by YAP in hESCs. (C) Circle diagrams showing the top regulated genes ([blue] down-regulated; [red] up-regulated) that are differentially expressed in YAP knockout versus wild-type hESCs, based on the RNA-seq data. Genes that have a YAP peak within 50 kb from transcriptional start site and are transcriptionally regulated are indicated with small circles. A representative selection of these genes is shown in the columns. (D) Genome browser captures of ChIP-seq and RNA-seq experiments. The immunoprecipitated proteins are indicated at the left. The examples shown are from the YAP-activated CTGF gene and the directly repressed NODAL and WNT3 genes.

Figure 5.

YAP repression of WNT3 prevents premature differentiation in response to Activin. (A) Heat map showing expression levels of Wnt regulatory genes in wild-type and YAP knockout cells following treatment with Activin. (B) Diagram showing the mechanism of action of the Wnt inhibitors IWP2 and XAV and the Activin inhibitor A8301. The graphs show qPCR analysis of the indicated genes in YAP knockout hESCs treated with the specific compounds, as listed below the graphs. Mean (SD). n = 3. (C) YAP knockout hESCs were transfected with siRNAs against Wnt3 or SMAD2, and expression levels of the WNT3, EOMES, and MIXL1 genes were analyzed by qPCR. Mean (SD). n = 2. (D) Immunoblot showing expression levels of WNT3, EOMES, and T proteins after Activin treatment for the indicated times in wild-type and YAP knockout cells. CDK2 was used a loading control. (E) Genome browser captures of ChIP-seq and RNA-seq experiments at the WNT3 gene. Cell lines and immunoprecipitated proteins are indicated above each lane. (F) YAP knockout cells were transfected with the transposon-based vector PiggyBAC containing an intact Flag-YAP cDNA under a doxycycline promoter. The immunoblot shows YAP levels in cells exposed to different levels of doxycycline. The graphs illustrate the inverse correlation of WNT3 and YAP mRNAs in hESCs. Mean (SD). n = 2. (G) Genome browser capture of RNAPII CTD-Ser7P ChIP-seq in Activin-treated wild-type and PiggyYAP cell lines before and after doxycycline treatment. (H) A schematic depiction of the results of the figure. (S) SMAD2,3; (β) β-catenin; (Y) YAP; (enh) enhancer; (TSS) transcriptional start site.

Figure 6.

Activin selectively promotes differentiation to cardiac mesoderm in YAP knockout hESCs. (A) Heat map plot showing the lateral mesoderm (LM) and early cardiac genes up-regulated in YAP knockout versus wild-type hESCs at day 3 following an initial 24 h of exposure to Activin. (B) Analysis of HAND1, GATA4, and BAF60c proteins by immunofluorescence in wild-type and PiggyYAP hESCs before and after doxycycline treatment. (C) Schematic showing the developmental pathway induced by Activin-treated YAP knockout cells. (D) Graphs showing the expression of cardiac precursor markers NKX2.5 and TBX5 in response to different treatments, as indicated below the graph; Activin or GSK3i treatments were added from day 0 to day 1, and XAV, IWP2, or A83-01 inhibitors were added from day 1 to day 2. Cells were collected at day 5 for analysis. For example, lanes 2 and 3 indicate 24 h of Activin treatment followed by A83-01 exposure for an additional 24 h (lane 3) or without further treatment (lane 2). (Lane 1) For comparison, wild-type hESCs were treated following the GiWi protocol, as indicated by the pink bar. Mean (SD). n = 3. (E) Scheme showing the GiWi protocol for hESC differentiation to cardiomyocytes (CM) (Lian et al. 2013). The main cardiac regulators that express sequentially are listed at the left. Proteins required for cardiomyocyte development are shown in green, and cardiac inhibitors are shown in red. (F) RNA-seq was performed in wild-type and YAP knockout cells treated with the specified cytokines for 30 h. Note that the GSK3i concentration used was equivalent to that used for the cardiomyocyte induction protocol (50 nM XV). The heat map shows mRNA levels of genes induced by Activin and GSK3i in wild-type and YAP knockout cells (top) and genes induced by only GSK3i treatment in wild-type and YAP knockout cells (bottom). (G) qPCR analysis shows mRNA levels of CDX2 in wild-type and YAP knockout hESCs after different concentrations of GSK3i (50–5 nM XV) and Activin (100–5 ng/mL) treatment for 24 h. The graph shows the average of two representative experiments of at least four independent replicates. Mean (SEM). n = 2. (H) qPCR analysis shows mRNA levels of CDX2 in wild-type and YAP knockout hESCs after treatment with GSK3i or Activin in the presence or absence of 1 µM Activin inhibitor A8301. The graphs show the average of two representative experiments of at least four independent replicates. Mean (SEM). n = 2. (I) Genome browser captures show β-catenin and CTD-Ser7P-RNAPII distribution on CDX2 gene in hESCs at day 0 and at days 1, 3, and 5 after initial differentiation following the GiWi protocol (see also Fig. 6E). (J) ChIP-qPCR analysis of β-catenin, Smad2,3, and RNAPII-Ser5P binding to the CDX2 enhancer (+3.5 kb) after treatment with GSK3i in the presence or absence of Activin inhibitor in YAP knockout hESCs. (NC) Negative control region. Mean (SEM). n = 2. (K) A schematic depiction to summarize the results of the figure.

Figure 7.

Human cardiomyocyte differentiation using a one-step protocol. (A) The schematic outline of the one-step protocol used to differentiate YAP knockout hESCs into beating cardiomyocytes in comparison with the GiWi protocol. (B) Wild-type and YAP knockout cells were differentiated using the conditions indicated at the left of the graph. Around day 22 following the initial treatment, cells were stained with the cardiac marker CTNT and subjected to FACS analysis. Mean (SD). n ≥ 3. (C) Immunostaining of wild-type- and YAP knockout-derived cardiomyocytes using the protocol indicated below the images. (D) The Venn diagram shows up-regulated genes in wild-type and YAP knockout cardiomyocytes compared with hESCs. (Below) The heat map shows a list of up-regulated cardiac markers in wild-type and YAP^−/− cardiomyocytes. (E) Immunoblot analysis of cardiac markers CTNT, NKX2.5, and MCL2a in wild-type and PiggyYAP cells at day 22 after initial differentiation according to the Activin one-step protocol. Doxycycline was added at different time points (not added, day 0, day 1, day 3, or day 5). Note that the reintroduction of YAP at day 0 and day 1 impaired Activin-mediated differentiation, whereas expression of YAP in later stages (day 3 and day 5) did not affect differentiation, as indicated in the bottom illustration. (F) A schematic diagram summarizing the results of the figure. (CM) Cardiomyocyte.