The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation

Abstract

Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. YAP is phosphorylated and inhibited by the Lats kinase, a key component of the Hippo tumor suppressor pathway. Elevated YAP protein levels and gene amplification have been implicated in human cancer. In this study, we report that YAP is inactivated during embryonic stem (ES) cell differentiation, as indicated by decreased protein levels and increased phosphorylation. Consistently, YAP is elevated during induced pluripotent stem (iPS) cell reprogramming. YAP knockdown leads to a loss of ES cell pluripotency, while ectopic expression of YAP prevents ES cell differentiation in vitro and maintains stem cell phenotypes even under differentiation conditions. Moreover, YAP binds directly to promoters of a large number of genes known to be important for stem cells and stimulates their expression. Our observations establish a critical role of YAP in maintaining stem cell pluripotency.

Figure 1.

YAP is inactivated during mES cell differentiation. (A) YAP expression is decreased during differentiation. mES-D3 cell line was cultured in normal ES medium or under differentiation conditions described in the Materials and Methods. The differentiation of ES cells was verified by the loss of ALP staining and colony morphology (left panels), as well as multiple lineage markers using RT–PCR (middle panel). YAP protein levels as well as ES cell pluripotent marker proteins Oct4 and Sox2 were examined under normal ES cell culture conditions and differentiation conditions. Western blot analysis was performed and α-tubulin blot was included for equal sample loading. (Right panel) YAP expression was also confirmed by quantitative RT–PCR. (B) Differentiation by LIF withdrawal decreases YAP protein levels. mES-D3 was grown in the ES media (ES) or absence of LIF (d.ES). The cell lysates were harvested and analyzed for YAP protein level and ES marker Oct4. (C) Reduction of YAP protein level during the mES cell line R1 differentiation. mES-R1 cell line was cultured in normal and differentiation conditions as described in A. Western blot analysis was performed to detect YAP and ES cell markers. (D) ES cell differentiation induces the inhibitory phosphorylation of YAP. Lysates from control and differentiated ES cells (more lysate from differentiated cells) were loaded to achieve equal amount of YAP protein. Western blotting with phosphoYAP S127-specific antibody and YAP antibody was performed. Relative YAP phosphorylation was increased in differentiated ES cells. (E) Differentiation deprives YAP from the nucleus. Both control and differentiated ES cells were fractionated into cytosolic and nuclear fractions as indicated. The samples were blotted for YAP protein (YAP) and S127 phosphorylation level (pYAP). α-Tubulin (α-Tub) and histone 2AX (H2AX) were used as positive controls for both loading and cytosolic and nuclear proteins, respectively.

Figure 2.

YAP and TEAD are required for mES cell pluripotency. (A) YAP knockdown causes a loss of ES cell properties. ES cells were stably infected with lentiviral shRNA constructs targeting YAP as indicated. After selection, cells were grown in regular ES culture medium. Cell morphology (top panels) and ALP staining (bottom panels) are shown. (B) YAP knockdown results in ES cell differentiation. Cell lysates were prepared from control and YAP knockdown ES cells. (Left panel) Pluripotent ES markers Sox2 and Oct4 were examined by Western blotting. Protein level was normalized by α-tubulin. (Right panel) Several differentiation markers were determined by RT–PCR, including endoderm markers alphafetoprotein (AFP), CXC chemokine receptor 4 (CXCR4), Forkhead box a2 (Foxa2), and GATA4, and Mesoderm marker T-brachyury. (C) TEAD knockdown causes a loss of ES cell properties. Experiments were similar to those in A, except the shRNA targeting TEAD1/3/4 was used. (Left panel) Pluripotency was examined by ALP staining. (Middle panel) Expression of TEAD1, Oct4, and Sox2 was determined using specific antibodies along with the loading control GAPDH. (Right panel) Knockdown efficiencies of the shRNA were determined by quantitative RT–PCR. (D) TEAD2 overexpression does not rescue the loss of pluripotency in TEAD1/3/4 knockdown mES cells. Pluripotent ES markers Sox2 and Oct4 were examined by Western blotting along with loading control α-tubulin and the ectopically expressed TEAD2, which is tagged with calmodulin-binding protein (CBP).

Figure 3.

YAP overexpression promotes ES cell self-renewal and suppresses differentiation. (A) Ectopic YAP expression maintains ES cell properties even under differentiation conditions. mES cells were infected with vector control (VEC), YAP wild-type (WT), or the constitutive active YAP-5SA (5SA) viruses. Stable pools were selected and maintained in normal ES cell medium. (Top panels) Cells were induced to the differentiation and their morphology and ALP activity (Supplemental Fig. S1A) were examined. (Bottom panel) In addition, cell lysates were analyzed for YAP, Oct4, and Sox2 protein levels by Western blotting. (B) YAP overexpression retards neuronal differentiation of ES cells. Vector or YAP-5SA-overexpressing ES cells were induced to neuronal-specific differentiation for 5 d after EB formation. Pluripotency was determined by ALP staining (ALP panel). Immunostaining with β-tubulin III (Tubb III panel), a neuronal marker protein, was used to determine the progress of neuronal differentiation. (C) Forced YAP expression rescued YAP knockdown-induced differentiation. ES cells were infected with a retrovirus harboring a gene encoding constitutively active YAP-5SA, which is resistant to the YAP knockdown shRNA construct targeting the YAP 3′UTR region. The YAP-5SA-expressing ES cells were infected with scramble or the YAP knockdown shRNA construct (YAPKD). Knockdown of endogenous YAP protein was confirmed by Western blot (note that the epitope-tagged YAP-5SA migrated more slowly than the endogenous YAP). Pluripotency of the ES cells was determined by Oct4 and Sox2 protein levels (top panel) as well as positive ALP activity (bottom panel). (D) Teratoma formation from YAP-WT-overexpressed ES cells. ES cells were grown under differentiation conditions and then injected subcutaneously into the nude mice. Teratomas were observed in mice injected with YAP-WT ES cells (right side, indicated by a red arrow) but not in the control (vector-only ES cells, left side). Shown is a representative of five injected mice. Teratoma were harvested and processed by H&E staining. Cells resembling characteristics of three germ layers were observed including, cartilage (mesoderm) (panel 1), muscle (mesoderm) (panel 2), retinal epithilium (ectoderm) (panel 3), squamous (ectoderm) (panel 4), neuroepithelium (ectoderm) (panel 5), epithelium (ectoderm) (panel 6), and cilia (endoderm) (panel 7). (E) Teratoma formation from YAP-5SA-overexpressed ES cells. Experiments were similar to D except YAP-5SA-expressing ES cells were used. (Panel 1) Cartilage (mesoderm). (Panel 2) Muscle (mesoderm). (Panel 3) Epidermal (ectoderm). (Panel 4) Goblet (endoderm). (Panel 5) Neuroepithelium (ectoderm). (Panel 6) Epithelial (ectoderm). (Panel 7) Epithelium squamous cilia (ecdoderm).

Figure 4.

YAP is induced during iPS reprogramming and enhances iPS induction. (A) YAP expression is elevated during iPS reprogramming. Human fibroblasts were infected with OSK and c-Myc to produce iPS as documented. The iPS properties were characterized by cell morphology and staining for pluripotent stem cell markers TRA-1-81 (red) and Nanog (green). (B) Karyotype of iPS cells generated from human fibroblast (Fibro-27). (C) YAP expression in the parental fibroblasts and iPS cells were determined by Western blotting. (D) iPS cells generated from human fibroblasts (Fibro-27) can be differentiated successfully to neural stem cells (NSCs) stained by Nestin (green, left panel) and neurons stained by βIII-tubulin (red, right panel), MAP-2 (green), and DAPI (blue). (E) YAP increases iPS induction. Mouse embryonic fibroblasts (MEFs) were infected by three factors (OSK) in the presence or absence of YAP coinfection. (Top panel) Nineteen days after viral infection, cells were stained for ALP. (Bottom panel) The Oct4-GFP reporter expression was examined to verify iPS induction (note the high magnification to show GFP-positive colonies). Expression of ALP and GFP were quantified by ALP-positive (ALP⁺) and GFP-positive (Oct4-GFP⁺) colony numbers.

Figure 5.

YAP binds to and induces genes important for ES cell functions. (A) An enrichment network linking YAP1-bound genes to gene sets regulated in ES cells. YAP1-bound genes (hollow node with black ring) were derived from ChIP-seq analysis of YAP in undifferentiated ES cells, and were compared against all other concepts available in the MCM in Oncomine for significant overlap. Each node represents one molecular concept or gene set with node size proportional to the number of genes. Each edge represents a statistically significant overlap (P < 1 × 10⁻¹⁰) of genes in the two linked nodes. Significance of overlap between two concepts was determined by Fisher's exact test comparing the chance and expected overlap (Rhodes et al. 2007). The odds ratio and P-value of such tests are shown in the table. The P-values for pairs between YAP and all concepts in this network are <3.8 × 10⁻³⁴ (Supplemental Table S3). (B) YAP binds preferentially to gene promoters containing TEAD sites. The intensity of YAP binding as determined by the number of sequence hits is grouped and plotted on the X-axis. The Y-axis indicates the percentage of genes containing TEAD sites in a given intensity group. The data indicate a positive correlation between the YAP-binding intensity and the presence of TEAD sites (R = 0.92 for control mES cells [light-gray bars] and R = 0.66 for differentiated mES cells [dark-gray bars]). (C) YAP regulates a large set of ES cell important genes. mRNAs were isolated from undifferentiated, differentiated, YAP-5SA-expressing, and YAP knockdown ES cells. Expression of YAP ChIP-positive genes were determined by quantitative RT–PCR. The top panel shows gene expression between control and differentiated ES cells. The middle panel shows expression of corresponding genes in YAP knockdown cells. The bottom panel shows gene expression in YAP-5SA ES cells under differentiation conditions. Quantitative PCR data were normalized to GAPDH control then scaled proportionally in the Y-axis. Samples to the left of the dashed line follow the scale on the left, while samples to the right of the dashed lines follow the scale on the right Y-axis, respectively.