1. Stem Cells and Regenerative Medicine
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Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

  1. Lucy LeBlanc
  2. Bum-Kyu Lee
  3. Andy C Yu
  4. Mijeong Kim
  5. Aparna V Kambhampati
  6. Shannon M Dupont
  7. Davide Seruggia
  8. Byoung U Ryu
  9. Stuart H Orkin
  10. Jonghwan Kim  Is a corresponding author
  1. The University of Texas at Austin, United States
  2. Boston Children’s Hospital, United States
  3. Harvard Medical School, United States
  4. Dana-Farber Cancer Institute (DFCI), United States
  5. Howard Hughes Medical Institute, United States
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Cite this article as: eLife 2018;7:e40167 doi: 10.7554/eLife.40167

Abstract

Approximately, 30% of embryonic stem cells (ESCs) die after exiting self-renewal, but regulators of this process are not well known. Yap1 is a Hippo pathway transcriptional effector that plays numerous roles in development and cancer. However, its functions in ESC differentiation remain poorly characterized. We first reveal that ESCs lacking Yap1 experience massive cell death upon the exit from self-renewal. We subsequently show that Yap1 contextually protects differentiating, but not self-renewing, ESC from hyperactivation of the apoptotic cascade. Mechanistically, Yap1 strongly activates anti-apoptotic genes via cis-regulatory elements while mildly suppressing pro-apoptotic genes, which moderates the level of mitochondrial priming that occurs during differentiation. Individually modulating the expression of single apoptosis-related genes targeted by Yap1 is sufficient to augment or hinder survival during differentiation. Our demonstration of the context-dependent pro-survival functions of Yap1 during ESC differentiation contributes to our understanding of the balance between survival and death during cell fate changes.

https://doi.org/10.7554/eLife.40167.001

Introduction

Yap1 regulates genes involved in many cellular functions, including proliferation, organ size control, and tumorigenesis (Ehmer and Sage, 2016; Hansen et al., 2015; Huang et al., 2005). When Hippo signaling is active, kinases Lats1/2 phosphorylate Yap1, leading to cytoplasmic sequestration (Hao et al., 2008). When Hippo signaling is inactive, Yap1 translocates to the nucleus to co-activate or co-repress numerous target genes with interacting partner proteins such as Tead factors (Kim et al., 2015; Stein et al., 2015).

Previous research indicated that, in mouse embryonic stem cells (ESCs), nuclear translocation of Yap1 occurs shortly after withdrawal of leukemia inhibitory factor (LIF), a cytokine that maintains self-renewal, and that depletion of Yap1 inhibits differentiation, whereas overexpression (OE) of Yap1 stimulates differentiation (Chung et al., 2016). Deletion of Yap1 leads to embryonic lethality by E10.5 although the downstream mechanism remains poorly characterized (Morin-Kensicki et al., 2006). Additionally, whether Yap1 has any other roles during ESC differentiation and early development remains unclear.

Apoptosis influences numerous biological processes, including development, differentiation, and infection (Fuchs and Steller, 2011; Meier et al., 2000). A previous study has reported that withdrawal of LIF causes the death of 30% or more of ESCs (Bashamboo et al., 2006; Duval et al., 2000), and around 30% of human ESCs are also annexin V positive when they exit from self-renewal (Dravid et al., 2005). A proposed function of apoptosis during ESC differentiation is to cull cells that fail to exit self-renewal, thus promoting efficient differentiation (Wang et al., 2015). This process is not limited to ESCs, as defective cells are executed during human neural progenitor differentiation as well (Jaeger et al., 2015), and apoptosis eliminates self-reactive and non-reactive lymphocytes during T and B cell differentiation (Francelin, 2011; Nemazee, 2017; Opferman, 2008).

This process must be finely tuned to ensure efficient changes in cell identity without excessive loss of cell viability. However, mechanisms that regulate the balance between survival and death during ESC differentiation remain insufficiently characterized. Here, we find that Yap1 attenuates mitochondrial apoptosis during ESC differentiation, primarily by upregulating anti-apoptotic factors, such as Bcl-2, Bcl-xL (Bcl2l1), and Mcl-1, through direct transcriptional regulation. Mouse ESCs lacking Yap1 have no defect in survival in self-renewing conditions. However, just after the exit from self-renewal, we find that Yap1 knockout (KO) cells develop a high degree of mitochondrial priming that precedes elevated rates of apoptosis. OE of anti-apoptotic factors or repression of pro-apoptotic factors in Yap1 KO cells rescues this enhanced rate of cell death during differentiation. This collectively suggests that Yap1 is critical for ESC survival in a context-dependent manner, advancing our understanding of regulation of cell death during changes in cell identity.

Results

Genetic ablation of Yap1 intensifies caspase-dependent cell death during ESC differentiation

To determine context-specific roles of Yap1, we attempted to differentiate J1 ESCs in which Yap1 had been deleted via CRISPR/Cas9 in KO clones established in our previous publication (Figure 1—figure supplement 1A). While ~30% cell death was observed from wild-type (WT) cells as previously reported (Bashamboo et al., 2006), cell death was dramatically higher (up to >70%) in Yap1 KO cells 72 hr after LIF withdrawal (Figure 1A and Figure 1—figure supplement 1B). In both cases, cell death was substantially reduced after supplementation with Z-VAD-FMK (zVAD), a pan-caspase inhibitor, but not with necrostatin-1, which blocks necroptosis. Undifferentiated cells had extremely low rates of cell death regardless of genotype (Figure 1A). An additional J1 Yap1 KO clone as well as Yap1 KO clones established in the CJ7 and E14 ESC lines (Figure 1—figure supplement 1C) also experienced drastically heightened cell death during differentiation, but not self-renewal (Figure 1B). Furthermore, depletion of Yap1 using shRNA-mediated knockdown (KD) dose-dependently increased cell death during differentiation (Figure 1—figure supplement 1D and E). Finally, measuring cell death in stable Yap1 OE cell lines (Figure 1-figure supplement F) reduced cell death to a mere ~10% during differentiation (Figure 1C). Thus, Yap1 is key for survival during ESC differentiation, and ablation of Yap1 specifically exacerbates apoptosis.

Figure 1 with 1 supplement see all
Loss of Yap1 substantially increases apoptosis during ESC differentiation.

(A) Lactate dehydrogenase (LDH) assay of WT and Yap1 KO ESCs in ±LIF. Cells were treated with either Z-VAD-FMK (Z-VAD), necrostatin-1, DMSO, or no treatment. Values were normalized to wells that had been lysed completely. (B) LDH assay measuring cell death after Yap1 KO in three different ESC lines during differentiation (72 hr) or self-renewal. (C) LDH assay measuring cell death in Yap1 KO, WT, and three different stable FLAG-Bio (FB) Yap1 overexpression cell lines during differentiation (72 hr). (D) Representative brightfield and fluorescence microscopy images of WT and Yap1 KO ESCs incubated with NucView 488 Casp3 substrate at the indicated times after LIF withdrawal. (E) Representative flow cytometry density plots of WT and Yap1 KO ESCs detecting fluorescent signal from annexin-V (conjugated to CF594) and NucView 488 reagent during differentiation (60 hr). (F) Fold enrichment of annexin-V and active Casp3-positive Yap1 KO vs. WT ESCs according to flow cytometry. (G) Immunoblot of Casp9, Casp8, Casp3, cleaved Casp3, and cleaved Parp1 in WT and Yap1 KO cells during differentiation. β-actin was used as a loading control. (H) Luminescent assay of caspase activity in Yap1 KO vs. WT ESCs in ±LIF media. (I) LDH assay of WT and Yap1 KO cells ± KD of Casp9 during differentiation (72 hr). All data are expressed as mean ±standard deviation (n = 4 independent samples for LDH assays and n = 3 for other experiments). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.002

Loss of Yap1 leads to caspase hyperactivation during differentiation

During apoptosis, initiator caspases 8 (Casp8) and 9 (Casp9) are activated first, either by death receptors or mitochondrial outer membrane permeabilization, respectively (Bao and Shi, 2007). They then cleave executioner caspases such as caspase-3 (Casp3), which then cleave hundreds of downstream targets in the cell that result in its death, including Parp1 (Fischer et al., 2003). Treatment of ESCs with NucView 488 enabled live visualization of active Casp3. In undifferentiated ESCs, Casp3 activation was rare in both WT and KO cells, but the proportion of cells with active Casp3 increased visibly after LIF withdrawal as a function of time (Figure 1D). Notably, a far greater proportion of Yap1 KO cells than WT cells possessed active Casp3 by 60 hr. Then, we performed flow cytometry to quantify active Casp3 as well as externalized phosphatidylserine. Both the relative proportion of Casp3 positive cells and the fluorescent intensity of the Casp3 substrate fluorescent probe were higher in Yap1 KO differentiating ESCs (dESCs), and this was correlated with an increased proportion of annexin V positive cells (Figure 1E and F). Immunoblot analysis confirmed faster and more intense cleavage of Casp9, Casp3, and Parp1 in Yap1 KO cells during differentiation (Figure 1G). To determine whether Yap1 KO dESCs were more sensitive to exogenous apoptosis-inducing stimuli, we treated dESCs with staurosporine (STS), a high-affinity, non-specific kinase inhibitor that has long been used to dissect the induction of intrinsic apoptosis in a myriad of cellular contexts (Belmokhtar et al., 2001; Preta and Fadeel, 2012; Xu et al., 2015). This treatment induced faster and more drastic activation of Casp3 and Parp1 in Yap1 KO than in WT dESCs as quickly as two hours after addition, reflecting a vastly heightened sensitivity to apoptosis-inducing stress (Figure 1—figure supplement 1G).

Next, we quantified caspase activity using a luminogenic substrate. By 60 hr after LIF removal, all caspases tested were approximately two-fold more active in Yap1 KO cells than in WT (Figure 1H). These observations demonstrate that lack of Yap1 accelerates and intensifies caspase activation during differentiation. We decided to dissect which part of the apoptotic pathway is affected first by loss of Yap1. Though Casp8 activity is elevated in Yap1 KO cells, we did not detect substantial differences in cell death after Casp8 KD (data not shown), so we decided to target Casp9 with two different shRNAs (Figure 1—figure supplement 1H). As expected, KD of Casp9 reduced cell death during differentiation, and this was particularly stark for Yap1 KO cells, where cell death was reduced to WT levels without Casp9 KD (Figure 1I). This implied that the abnormally high rates of apoptosis in Yap1 KO cells are sustained by heightened Casp9 activation. We observed that mRNA expression of caspases was relatively equal between Yap1 KO cells and WT cells during differentiation (Figure 1—figure supplement 1I). Additionally, protein levels of Casp3 showed similar fluctuations in dESCs for both WT and KO cells; although Casp8 and Casp9 were elevated in Yap1 KO cells (Figure 1G). However, since caspase activity is strongly activated by cleavage (Hu et al., 2013), we speculated that Yap1 may regulate other factors that indirectly affect the rate of caspase cleavage.

Yap1 protects against apoptosis regardless of differentiation method and acts directly after the exit from self-renewal

To determine whether the roles of Yap1 are either specific to LIF withdrawal or broadly applicable to the exit from self-renewal in different conditions, we utilized alternate differentiation methods (Figure 2A). Utilizing N2B27 medium (neural ectoderm fate) or low serum DMEM supplemented with IDE1 (definitive endoderm fate) (Borowiak et al., 2009), we again observed that dESCs without Yap1 experienced much higher rates of cell death compared to WT cells, which could be rescued by zVAD (Figure 2B and C). We verified by RT-qPCR that N2B27 medium indeed induced neural ectoderm marker expression (Figure 2—figure supplement 1A) whereas IDE1 treatment induced endoderm marker expression (Figure 2—figure supplement 1B), as well as repression of Nanog, an ESC self-renewal marker.

Figure 2 with 1 supplement see all
Loss of Yap1 augments apoptosis in several differentiation conditions, but its role is largely restricted to the exit from self-renewal.

(A) Schematic of 3 differentiation protocols (ectoderm, endoderm, and epiblast) used in Figures 2, 3 and 5. (B) LDH assay of WT and Yap1 KO ESCs in N2B27 with or without 2i and Z-VAD. (C) LDH assay of WT and Yap1 KO ESCs in low serum DMEM supplemented with IDE1 ±Z VAD (48 hr). (D) LDH assay of ESC towards EpiLC conversion in WT and Yap1 KO ESCs (72 hr). (E) Schematic of verteporfin (vert) treatment timings during late and early differentiation in WT ESCs in -LIF. (F) Timecourse LDH assay of verteporfin-treated dESCs at the indicated timepoints along with positive controls (treatment with verteporfin just after -LIF as well as untreated Yap1 KO ESCs, the latter of which are n = 8). All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.004

We also induced differentiation towards epiblast-like cells (EpiLCs) to mimic early embryo development in vitro; whereas mESCs are equivalent to the inner cell mass of the blastocyst at E3.5–4.5, EpiLCs represent the next developmental stage, the E5.5–6.0 epiblast (Hayashi et al., 2011). We confirmed repression of Nanog and upregulation of EpiLC-specific markers (Figure 2—figure supplement 1C). As expected, EpiLCs lacking Yap1 underwent substantially higher cell death than WT by d3, and zVAD reduced cell death in both genotypes to the low, basal rates experienced in 2i media (Figure 2D). Finally, we used a well-characterized inhibitor of Yap1, verteporfin (Brodowska et al., 2014), to investigate Yap1’s role during late -LIF differentiation (Figure 2E). While treatment with as low as 1 μM verteporfin before the exit from self-renewal phenocopied Yap1 KO, treatment during late differentiation had more modest effects on cell death, and treated cells had death rates nearly identical to untreated by d7 (Figure 2F). Together, these data suggest that loss of Yap1 increases rates of apoptosis in ESCs directly after the exit from self-renewal, regardless of the ultimate lineage those ESCs are destined for.

Yap1 modulates the expression of apoptosis-related genes during differentiation

Following our deduction that Casp9 hyperactivation distinguishes Yap1 KO dESCs from WT dESCs and that lack of Yap1 enhances apoptosis in several differentiation conditions, we examined the expression of anti- and pro-apoptotic genes that affect Casp9 activation. After 72 hr of LIF withdrawal, we detected a deficiency in three key anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1) in Yap1 KO cells by immunoblot (Figure 3A). Immunocytochemistry confirmed reduced expression of Bcl-2 and Mcl-1 in Yap1 KO dESCs compared to WT, as well as lower mitochondrial content as measured by MitoTracker dye (Figure 3—figure supplement 1A and B); as expected, Bcl-2 and Mcl-1 strongly colocalized with the mitochondria (weighted colocalization coefficient for all samples ~ 0.7–0.9). We then investigated the significance of this expression defect in the context of what normally happens during differentiation. In WT ESCs, we found that Bcl2 was strongly upregulated in all differentiation conditions tested; pro-apoptotic genes such as Puma (Bbc3) and Noxa (Pmaip1) were also activated, whereas Bcl2l1 and Mcl1 either stayed constant or were weakly upregulated (Figure 3B). Comparing Yap1 KO cells to WT ESCs, by d2, we found a general trend for decreased anti-apoptotic gene expression (most consistently Bcl2) and increased pro-apoptotic gene expression (Figure 3C). This defect worsened over time in -LIF (Figure 3D) and was particularly stark for Bcl2, which was upregulated as much as 80 to 100-fold by 96 hr in WT ESCs upon differentiation (Figure 3E).

Figure 3 with 1 supplement see all
Loss of Yap1 leads to abnormal expression of apoptosis-related genes.

(A) Immunoblot of Bcl-2, Bcl-xL, and Mcl-1 in WT and Yap1 KO cells in -LIF after 72 hr of differentiation. (B) RT-qPCR measuring the expression of anti-apoptotic (blue) and pro-apoptotic (red) genes in WT ESCs cultured in the indicated differentiation conditions (all at 48 hr) normalized to their respective self-renewal conditions. (C) RT-qPCR measuring the expression of anti- and pro-apoptotic genes in Yap1 KO vs. WT cells (log2) in various differentiation conditions (all at 48 hr). (D) RT-qPCR measuring the expression of Bcl2, Bcl2l1, and Mcl1 in Yap1 KO cells vs. WT cells during differentiation (timecourse). (E) RT-qPCR measuring the expression of Bcl2 in WT and Yap1 KO cells during differentiation (timecourse) relative to +LIF. All data are expressed as mean ±standard deviation (n = 3 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.006

To reinforce this observation, we examined the expression of apoptosis-related genes after 2d of transient OE of Yap1 after 3d of differentiation total, and found a modest induction in Bcl2, Bcl2l1, and Mcl1, as well as a modest repression of Bbc3 and Bmf (Figure 3—figure supplement 1C). Using RNA-seq data from a previous study (Chung et al., 2016), we found that differentiation induces the expression of a group of anti-apoptotic genes in WT cells, but this induction is debilitated after Yap1 KD (Figure 3—figure supplement 1D). Meanwhile, constitutive Yap1 OE during +LIF conditions appeared to slightly induce anti-apoptosis genes on average, though not significantly (Figure 3—figure supplement 1E). Collectively, these data show that Yap1 may function as a master regulator in proper maintenance or induction of anti-apoptotic genes (particularly Bcl2) during differentiation, and it may also dampen the upregulation of pro-apoptotic genes.

Yap1 directly regulates apoptosis-related genes via transcription

We performed ChIP-seq of Yap1 using ESCs overexpressing FLAG-Bio-Yap1 (FB-Yap1) under differentiation (-LIF, 72 hr) and self-renewal (+LIF) conditions, detecting 8453 peaks significantly enriched over the BirA control above threshold between duplicates during differentiation and only 699 peaks in +LIF, reflecting its known cytoplasmic localization during self-renewal. Many of the differentiation-related peaks were intergenic as well as in promoters (Figure 4—figure supplement 1A). Yap1 occupancy was positively correlated with degree of gene downregulation upon Yap1 KD, although some upregulated genes upon KD were associated with unusually low Yap1 occupancy (Figure 4A). By integrating data from a previous study investigating enhancer patterns at different stages of pluripotency (Buecker et al., 2014), we found that Yap1 peaks were strongly correlated with increased Ep300 (p300) occupancy during differentiation (Figure 4B). Although EpiLC differentiation induces a different cell fate than -LIF due to supplementation with activin A and bFGF, we reasoned that apoptosis-related regulation would be shared between the two conditions. We confirmed a physical interaction between Yap1 and p300 as well as one of its known cofactors, Tead4 (Chen et al., 2010), during ESC differentiation using co-immunoprecipitation (Figure 4—figure supplement 1B), consistent with known Yap1 nuclear localization in dESCs (Chung et al., 2016). Indeed, motif analysis revealed a significant enrichment of the Tead factor motif in addition to Zic3 and AP-1 complex (JunB and Fra1 (Fosl1)) motifs in the center of Yap1 peaks, whereas Esrrb (a negative control) was not found (Figure 4—figure supplement 1C and D). Finally, since p300 possesses histone acetyltransferase activity, we confirmed an increase in H3K27ac, an activating histone mark, in Yap1 peaks during differentiation (Figure 4—figure supplement 1E). Gene ontology (GO) analysis of genes bound by Yap1 and downregulated by Yap1 KD mainly yielded terms related to cell migration and motility, and regulation of cell death was also statistically significant (Figure 4—figure supplement 1F).

Figure 4 with 1 supplement see all
Yap1 directly regulates target apoptotic genes during differentiation.

(A) RNA-seq heatmap (Yap1 KD/empty vector KD, in both undifferentiated and differentiating ESCs) and line graph depicting Yap1 peak score, normalized to BirA, calculated using a moving window average (window = 150). Color bar indicates extent of upregulation (red) or downregulation (green) upon Yap1 KD. (B) ChIP-seq peak heatmaps using coordinates centered on the top Yap1 peaks (p-value cutoff, 1e-5) in dESCs (-LIF), which are shown in the second heatmap from the left. The other heatmaps represent occupancy of Yap1 in ESCs (first) or p300 in ESCs (third) or dESCs (fourth) corresponding to Yap1 dESC peak centers ± 3 kb (bin size = 100). (C) Signal tracks of Yap1 (red) and p300 (blue) occupancy on apoptosis-related genes in dESCs and EpiLCs, respectively. (E and F) Dual luciferase assay of Yap1-occupied cis-regulatory elements from anti- and pro-apoptotic genes in (E) Yap1 KO and WT cells ± LIF (48 hr) or (F) WT cells with Yap1 or empty OE (in -LIF, 48 hr), relative to pGL3-promoter, 24 hr after transfection. (G) Dual luciferase assay of Bcl-2 and Mcl-1 regulatory elements in Yap1 KO cells after transfection of empty vector or vectors containing FLAG-Bio Yap1 with or without a Ser79Ala mutation. (H) Dual luciferase assay of Mcl-1 with a deletion of its Tead binding motif (GGAAT on the reverse strand) in WT ESCs ± Yap1 OE. All data are expressed as mean ±standard deviation (n = 3 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.009

In addition to its co-activating properties, Yap1 also acts as a co-repressor in other contexts (Kim et al., 2015), and we observed Yap1 occupancy on both anti-apoptotic and pro-apoptotic genes (Figure 4C). To characterize Yap1 target putative cis-regulatory elements, we performed the dual luciferase assay in Yap1 KO cells, WT cells, and cells transfected with a Yap1 OE vector using the pGL3 promoter vector (Figure 4—figure supplement 1G). In Yap1 KO cells, luciferase constructs with regulatory elements associated with Mcl1, Bcl2, or Bcl2l1 have lower luciferase activity relative to WT cells, while regulatory elements associated with Bmf, Pmaip1, and Bbc3 led to higher luciferase activity (Figure 4D). Meanwhile, transient OE of Yap1 led to higher luciferase activity with anti-apoptotic gene regulatory elements and lower luciferase activity with pro-apoptotic gene regulatory elements (Figure 4E). Though the initial Bcl2 intronic regulatory element was unresponsive to Yap1 OE, combining it with another element in the same intron (Figure 4—figure supplement 1H) caused its activity to increase 2x during OE (Figure 4E).

To determine the importance of the known Yap1-Tead interaction for the function of these regulatory elements, we chose the strongest enhancers (Mcl1 distal and Bcl2 intronic tandem) for further testing. Transient OE of Yap1 in Yap1 KO cells rescued enhancer function to levels comparable to Yap1 OE in WT cells, whereas OE of Yap1 S79A, a mutant less capable of binding to Tead factors (Schlegelmilch et al., 2011), only mildly rescued Mcl1’s enhancer’s activity and failed to rescue Bcl2’s enhancer’s activity at all (Figure 4F). Furthermore, ablation of the Tead binding sequence (ΔTBS) from the Mcl1 enhancer not only eliminated its Yap1 responsiveness, but also nearly abolished its enhancer activity (Figure 4G). Intriguingly, Yap1 occupancy on apoptosis-related genes seems to be relatively conserved (r =~0.5) among different human cancer cell types (Figure 4—figure supplement 1I and J). Thus, Yap1 may regulate apoptosis-related genes through conserved binding locations in both the human and mouse genome. Additionally, Yap1 peaks in mouse dESCs also correlated with Tead1 and Tead4 peaks in other mouse cell types, and signal tracks show similar occupancy patterns particularly for Bcl2 (Figure 4—figure supplement 1K and L). Taken together, our data as well as data reanalyzed from other labs suggest that Yap1 directly regulates apoptosis-related genes.

Loss of Yap1 contributes to heightened mitochondrial priming and dependence on anti-apoptotic proteins

Mitochondrial priming describes how close a cell is to the threshold of apoptosis and is a function of the balance between anti-apoptotic and pro-apoptotic proteins (Czabotar et al., 2013; Deng, 2017; Sarosiek et al., 2013). Since Yap1 KO cells already show higher expression of pro-apoptotic genes and lower expression of anti-apoptotic genes upon differentiation (Figure 3C), we surmised that loss of Yap1 would increase mitochondrial priming and thereby sensitize dESCs to activation of the apoptotic cascade.

Using the JC-10 assay, we measured differences in mitochondrial priming between Yap1 KO and WT ESCs during differentiation, initially. Whereas mitochondria were equally primed during self-renewal, all four differentiation conditions (-LIF, neural, endoderm, EpiLC) resulted in a greater loss of mitochondrial membrane potential (Δψ) in Yap1 KO cells normalized to WT ESCs (Figure 5A). Next, we treated ESCs with a small panel of BH3 mimetics capable of inhibiting Bcl-2, Bcl-xL, Mcl-1, and/or Bcl-w to measure addiction to anti-apoptotic proteins. As expected, inhibition of anti-apoptotic proteins increased Δψ in dESCs more than in self-renewing ESCs (Figure 5B). Furthermore, deletion of Yap1 significantly sensitized dESCs, but not undifferentiated ESCs, to Δψ loss post BH3 mimetic treatment. We then investigated whether the higher loss of Δψ in Yap1 KO cells correlated with greater rates of cell death. Our results showed that mere inhibition of anti-apoptotic proteins was sufficient to cause cell death, particularly in dESCs, even before apoptosis normally occurs during differentiation (Figure 5C). Strikingly, loss of Yap1 significantly amplified cell death in response to BH3 mimetics at almost all concentrations tested, but only during differentiation (Figure 5C). Ablation of Yap1 also enhanced addiction to Mcl-1 and Bcl-xL; inhibition of either protein resulted in 2-3x greater cell death in Yap1 KO than WT (Figure 5C). Thus, loss of Yap1 leads to increased mitochondrial priming during differentiation, which subsequently sensitizes Yap1 KO to excessive activation of the apoptotic cascade. Importantly, despite how critical mitochondrial priming is to biomedical applications such as successful chemotherapy, almost no genes that regulate mitochondrial priming upstream of apoptosis-related proteins have been shown in any context.

Figure 5 with 1 supplement see all
Yap1 regulates mitochondrial priming and addiction to anti-apoptotic proteins.

(A) JC-10 mitochondrial membrane potential assay in WT and Yap1 KO cells during various forms of differentiation (72 hr for Pan and EpiLC, 48 hr for Neural and Endo) and self-renewal (maintained for an equal amount of time). Values (525/570 nm ratio, n = 6) corresponding to loss in ∆ψ (mitochondrial membrane potential) in Yap1 KO cells were normalized to WT cells. (A) JC-10 assay in WT and Yap1 KO cells in ±LIF after 12 hr of treatment with BH3 mimetics ABT-737, Venetoclax, A-1210477, and A1155463 (total differentiation time: 36 hr). Values (525/570 nm ratio) corresponding to loss in ∆ψ were normalized to DMSO as a control. (C) LDH assays of BH3 mimetic dose response curves after 24 hr of treatment in WT and Yap1 KO cells in ±LIF (48 hr differentiation). (D) LDH assay of WT and Yap1 KO cells after KD of Bmf or Puma in -LIF conditions (72 hr). (E) LDH assay of inducible Bmf and Puma OE (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells in ±LIF (48 hr differentiation). (F) Immunoblot of cleaved Casp3, cleaved Parp1, and Mcl-1 in WT and Yap1 KO dESCs (28 hr) after 4 hr of treatment with BH3 mimetics A-1210477 (Mcl-1 inhibitor) and ABT-737 (inhibitor of Bcl-2, Bcl-xL, and Bcl-w). β-actin was used as a loading control. All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.012

Manipulation of the levels of BH3-only proteins Bmf or Puma mildly affect rates of ESC death in the absence of Yap1 during differentiation

BH3 mimetics promote cell death by mimicking pro-apoptotic BH3-only proteins (Dai et al., 2016). In addition to lower expression of anti-apoptotic proteins, we observed higher expression of BH3-only genes in Yap1 KO cells relative to WT cells (Figure 3C). Though it is known that KD of Puma in self-renewing ESCs reduces sensitivity to cytotoxic agents (Huskey et al., 2015), roles of Bmf and Puma during differentiation are relatively unknown. Therefore, we performed KD of Bmf and Puma (Figure 5—figure supplement 1A and B) and this mildly reduced cell death in Yap1 KO cells during differentiation but not in WT ESCs (Figure 5D). Inducible OE of either factor (Figure 5—figure supplement 1C and D) accelerated apoptosis during differentiation, particularly in Yap1 KO cells, and Puma promoted cell death more strongly than Bmf (Figure 5E). This difference may be because Puma promiscuously binds to all known anti-apoptotic Bcl-2 family proteins, whereas Bmf binds only weakly to Mcl-1, preferring Bcl-2, Bcl-xL, and Bcl-w (Chen et al., 2005). Thus, although Yap1’s pro-survival function is primarily via activation of anti-apoptotic proteins, heightened expression of individual pro-apoptotic BH3-only proteins seems to contribute to enhanced cell death during differentiation of Yap1 KO cells. Finally, we used BH3 mimetics to probe differential roles of anti-apoptotic proteins during early differentiation. Mcl-1 expression was already reduced in Yap1 KO cells 28 hr after LIF withdrawal compared to WT ESCs. Yap1 KO cells were acutely sensitive to inhibition (4 hr) of either Mcl-1 or Bcl-2/Bcl-xL/Bcl-w, indicating increased mitochondrial priming even at such an early timepoint (Figure 5F). Since Mcl-1 is much more highly expressed than both Bcl-2 and Bcl-xL, we surmise that deficiency in its expression helps explain heightened apoptotic activation in Yap1 KO cells even before differences in Bcl-2 expression become apparent (Figure 3E).

Modulation of anti-apoptotic proteins controls cell death during differentiation

Having shown that Yap1 directly regulates apoptosis-related genes and thus reduces mitochondrial priming during differentiation, we sought to characterize whether modulating individual Yap1 targets could control cell death during differentiation. We stably overexpressed Yap1 (as a positive control to complement the KO) and Bcl-xL (Figure 6—figure supplement 1A) and inducibly overexpressed Bcl-2 (Figure 6—figure supplement 1B, C and D) in Yap1 KO cells, which reduced cell death in Yap1 KO to levels comparable to WT (Figure 6A and B). Intriguingly, inducible OE of Taz, a Yap1 paralog also possessing a Tead-binding domain, reduced cell death in both Yap1 KO cells and WT cells to levels just below uninduced WT cells, perhaps via upregulation of Bcl-xL (Figure 6C and D), and Figure 6—figure supplement 1F). Conversely, KD of Bcl-xL, Mcl-1 (Figure 6—figure supplement 1F), or Bcl-2 (Figure 6—figure supplement 1G) in WT ESCs individually increased cell death during differentiation 1.5 to 2-fold compared to controls (Figure 6E and F, Figure 6—figure supplement 1H). Since Yap1 is crucial for ES differentiation, we questioned whether the apoptosis-related genes regulated by Yap1 might have some effect on differentiation efficiency. Surprisingly, we found that OE of Bcl-2 led to increased induction of trophectoderm (Cdx2 and Gata3) and mesoderm markers (Gsc and T), while KD of Bcl-2 tended to reduce induction of lineage markers. However, KD of Bcl-xL or Mcl-1 had no effect on lineage marker induction (Figure 6G and Figure 6—figure supplement 1I and J). Taken together, these data clearly demonstrate that anti-apoptotic factors transcriptionally regulated by Yap1 are critical for dESC survival, and that OE or KD of each apoptotic factor can significantly shift the balance between survival and death. The results additionally suggest the previously unknown roles of Bcl-2 in regulation of ESC lineage specification, as only its roles in self-renewal have been deeply probed (Yamane et al., 2005). Our combined model of Yap1’s role in ESC differentiation is provided in Figure 6H.

Figure 6 with 1 supplement see all
Overexpression of Taz or individual anti-apoptotic proteins fully rescues the survival defect in the absence of Yap1.

(A) LDH assay of WT, Yap1 KO, and Yap1 KO constitutively overexpressing Bcl-xL or Yap1 in -LIF (72 hr). (B) LDH assay of inducible Bcl-2 (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells -LIF (72 hr). (C) LDH assay of inducible Taz (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells ± LIF (72 hr differentiation). (D) Immunoblot of cleaved Parp1, cleaved Casp3, Bcl-xL, and Mcl-1 in Yap1 KO cells inducibly overexpressing Taz (±Dox, 48 hr, 500 ng/mL) in -LIF (72 hr). (E) LDH assay of WT ESCs during differentiation (72 hr) after 48 hr KD of Bcl-xL or Mcl-1. (F) LDH assay of WT ESCs ± LIF (72 hr)±KD of Bcl-2. (G) RT-qPCR measuring the expression of lineage markers (trophectoderm: Cdx2 and Gata3, ectoderm: Nes and Otx2, endoderm: Gata4, mesoderm: Gsc and T) in WT and Yap1 KO cells in -LIF (72 hr, n = 3). Expression is indicated as a fold change in +Dox samples relative to -Dox. (H) Model proposing roles for Yap1 specific to the exit from self-renewal. In complex with Tead factors like Tead4, Yap1 co-activates anti-apoptotic genes and mildly co-represses pro-apoptotic genes to dampen mitochondrial priming, which thus prevents hyperactivation of the apoptotic cascade through Casp9. All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.014

Discussion

Though ESCs experience 30% or more cell death during differentiation, regulators of this process remain largely unknown. In this study, we have shown that in the absence of Yap1, this proportion of cell death increases to 70–80%. Accordingly, we have demonstrated that Yap1 directly and strongly activates anti-apoptotic genes, in addition to mildly repressing pro-apoptotic genes, to promote survival during the stressful process of differentiation. Yap1 therefore attenuates the increase in mitochondrial priming during differentiation that threatens mitochondrial integrity and leads to Casp9 activation. OE of Yap1, its paralog Taz, or its anti-apoptotic targets in Yap1 KO cells reduces cell death to WT levels or even lower. Our proposed role for Yap1 as a pro-survival factor in ESCs is consistent with other studies done in cancer or epithelial contexts (Lin et al., 2015; Rosenbluh et al., 2012; Song et al., 2015; Zhao et al., 2016), but our work is the first study to show such a contextual, differentiation-specific role for Yap1 in ESCs.

Intriguingly, our Casp3 live imaging assay revealed that activation of Casp3 was extremely heterogeneous, with many cells changing their morphology during differentiation without detectable caspase activation. Future studies focusing on how individual cells make the molecular decision of differentiation vs. apoptosis will be desired. We hypothesize that relative changes in the expression of key pro- and anti-apoptotic genes at the single cell level, as well as lineage markers, shortly after LIF withdrawal could successfully predict whether an individual cell will differentiate or perish.

One unexpected finding from our study is that OE of Bcl-2 improved induction of essential trophectoderm and mesoderm markers, and KD of Bcl-2 (but not Mcl-1 or Bcl-xL) conversely hampered such induction. Elucidating the mechanism by which Bcl-2 accelerates induction of lineage markers is beyond the scope of this work but would enhance understanding of how apoptosis-related factors influence non-apoptotic processes such as differentiation. Additionally, we noted that Yap1 binding peaks on apoptosis-related genes are conserved across several human cancer cell types, which corroborates previous findings (Rosenbluh et al., 2012). Since addiction to anti-apoptotic factors is a defining characteristic of cancer cells, the regulation mechanisms we have elucidated may be broadly applicable to how Yap1 promotes tumorigenesis.

In sum, our study has clearly demonstrated that Yap1 robustly promotes survival of ESCs during differentiation by direct transcriptional regulation of apoptotic genes. Nearly all cells in the body originate from various progenitor cells, and since the process of differentiation is often fraught with error and stress, our research may spur advances in the regulation of the survival or death decision during cell fate changes in a broad variety of contexts.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
AntibodyMouse anti
-β-actin
AbgentCat#AM1829b
RRID:AB_10664137
1:20,000
in 5% BSA
AntibodyMouse
anti-Yap1
Santa Cruz
Biotechnology
Cat#sc-101199
RRID:AB_1131430
1:1000
in 5% milk
AntibodyRabbit anti-
cleaved
caspase-3
Cell Signaling
Technology
Cat#9661S
RRID:AB_2341188
1:1000
in 5% BSA
AntibodyMouse anti-
cleaved Parp1
Cell Signaling
Technology
Cat#9548S
RRID:AB_2160592
1:1000
in 5% BSA
AntibodyMouse anti-
caspase-9
Cell Signaling
Technology
Cat#9508S
RRID:AB_10695598
1:1000
in 5% BSA
AntibodyRabbit anti-
Bcl-2
Cell Signaling
Technology
Cat#3498S
RRID:AB_1903907
1:1000
in 5% BSA
(WB), 1:200 in
4% BSA,
1% NGS (ICC)
AntibodyRabbit anti-Bcl-xLCell Signaling
Technology
Cat#2764S
RRID:AB_10695729
1:1000 in 5% BSA
AntibodyRabbit anti-Mcl-1Cell Signaling
Technology
Cat#94296S
RRID:AB_2722740
1:1000 in 5% BSA,
1:800 in 4% BSA,
1% NGS (ICC)
AntibodyMouse anti-PumaSanta Cruz
Biotechnology
Cat#sc-374223
RRID:AB_10987708
1:500 in 5% BSA
AntibodyRabbit anti-BmfBiossCat#bs-7587R RRID:
AB_2722741
1:1000 in 5% BSA
AntibodyHorse anti-mouse
secondary, HRP-
conjugated
Cell Signaling T
echnology
Cat#7076P2 RRID:
AB_330924
1:10,000 in TBST
AntibodyGoat anti-rabbit
secondary, HRP-
conjugated
Cell Signaling
Technology
Cat#7074S
RRID:AB_2099233
1:10,000 in TBST
AntibodyGoat anti-rabbit
IgG Alexa Fluor 594
Thermo
Scientific
Cat#R37117
RRID:AB_2556545
1:1000 in 4% BSA,
1% NGS (ICC)
AntibodyDynabeads MyOne Streptavidin
T1
Thermo
Scientific
Cat#656011:2000 in 5% BSA
AntibodyRabbit anti-
Taz (Wwtr1)
Sigma AldrichHPA007415
RRID:AB_1080602
1:500 in 5% milk
Chemical
compound,
drug
Z-VAD-FMKApexBioCat#A1902
Chemical
compound,
drug
Necrostatin-1Selleck
Chemicals
Cat#S8037
Chemical
compound,
drug
CHIR99021Selleck
Chemicals
Cat#S2924
Chemical
compound,
drug
PD184352Selleck
Chemicals
Cat#S1020
Chemical
compound,
drug
IDE1Cayman
Chemical
Company
Cat#13816
Chemical
compound,
drug
StaurosporineCell Signaling
Technology
Cat#9953S
Chemical
compound,
drug
PolybreneMilliporeCat#TR‐
1003‐G
Chemical
compound,
drug
PuromycinThermo
Scientific
Cat#A111
3803-02
Chemical
compound,
drug
Geneticin
/G418
Thermo
Scientific
Cat#10131027
Chemical
compound,
drug
ABT-737SelleckchemCat#S1002
Chemical
compound,
drug
Venetoclax
/ABT-199
SelleckchemCat#S8048
Chemical
compound,
drug
A-1210477SelleckchemCat#S7790
Chemical
compound,
drug
A-1155463SelleckchemCat#S7800
Chemical
compound,
drug
Lipofectamine
3000
Life TechnologiesCat#L3000008
Chemical
compound,
drug
INTERFERinPolyplus
Transfection
Cat#409–10
Chemical
compound,
drug
VerteporfinSelleck
Chemicals
S1786
Chemical
compound,
drug
Recombinant
Human
/Mouse/Rat
Activin A Protein
R and D Systems338-AC-010
Chemical
compound,
drug
Gibco FGF Basic
Recombinant
Mouse
Protein
Thermo Fisher
Scientific
PMG0034
Chemical
compound,
drug
KnockOut
Serum
Replacement
Thermo Fisher
Scientific
10828028
Commercial
assay or kit
Pierce LDH
Cytotoxicity Assay
Kit
Thermo
Scientific
Cat#88954
Commercial
assay or kit
RNeasy Plus
Mini Kit
QiagenCat#74136
Commercial
assay or kit
qScript cDNA
SuperMix
QuantaBio
/VWR
Cat#101414–108
Commercial
assay or kit
PerfeCTa SYBR
Green FastMix
VWRCat#95072–012
Commercial
assay or kit
Caspase-Glo 3/7PromegaCat#G8090
Commercial
assay or kit
Caspase-Glo 8PromegaCat#G8200
Commercial
assay or kit
Caspase-Glo 9PromegaCat#G8210
Commercial
assay or kit
Cell Meter JC-10
Mitochondrion
Membrane
Potential Assay Kit
AAT BioquestCat#22800
Commercial
assay or kit
NEBNext Ultra II
DNA Library Prep Kit
for Illumina
New England
Biolabs
Cat#E7645S
Commercial
assay or kit
Dual-Glo
Luciferase
Assay System
PromegaCat#E2920
Recombinant
DNA reagent
pLKO-puroMillipore
Sigma
See table S3
Recombinant
DNA reagent
pLVX-TRE3G
-ZsGreen1
ClontechCat#631164
Recombinant
DNA reagent
pCMV-Tet3GClontechCat#631164
Recombinant
DNA reagent
pCMV3-
Bcl2l1/Bcl-xL
Sino BiologicalCat#MG50012-UT
Recombinant
DNA reagent
pGL3-promoterPromegaCat#E1761

Recombinant
DNA reagent
pRL-TKPromegaCat#E2231
Recombinant
DNA reagent
3149 pSFFV-neo
Bcl-2 cDNA
AddGeneCat#8750
Recombinant
DNA reagent
Mus musculus
BCL2
binding
component 3
(Bbc3), mRNA.
NM_
133234.2
GenScriptCat#OMu19350D
Recombinant
DNA reagent
pcDNA3.1/
HisC-mTAZ
AddGeneCat#31793
Cell line
(Mus musculus,
male)
J1 Embryonic
Stem Cells
ATCCATCC SCRC
-1010
Cell line
(M. musculus,
male)
CJ7 Embryonic
Stem Cells
ENCODERRID:CVCL_C316
Cell line
(M. musculus,
male)
ES-E14TG2a
Embryonic
Stem Cells
ATCCATCC CRL-1821
Cell line
(Homo sapiens)
HEK293T cellsATCCATCC CRL-3216
Software,
algorithm
FlowJoTreestar
Software,
algorithm
BoxPlotRhttp://shiny.chemgrid.org/boxplotr/
Software,
algorithm
Java TreeViewhttp://jtreeview.sourceforge.net/
Software,
algorithm
AmiGO 2http://amigo.geneontology.org
Software,
algorithm
Primer3http://primer3.ut.ee/
Software,
algorithm
HOMERhttp://homer.ucsd.edu/homer/
Software,
algorithm
GOrillahttp://cbl-gorilla.cs.technion.ac.il/
Software,
algorithm
Cistromehttp://cistrome.org/db/#/
Software,
algorithm
Galaxyhttp://cistrome.org/ap/
Software,
algorithm
SRA Toolkithttps://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=toolkit_doc
Software,
algorithm
Bowtie 2http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
Software,
algorithm
MACS2https://github.com/taoliu/MACS
Software,
algorithm
Vassar Statshttp://vassarstats.net/matrix2.html
Software,
algorithm
Integrated
Genome Viewer
http://software.broadinstitute.org/software/igv/
Software,
algorithm
ZEN Microscope
Software
https://www.zeiss.com/microscopy/int/downloads/zen.html
Software,
algorithm
ImageJhttps://imagej.nih.gov/ij/index.html
Software,
algorithm
STARhttps://github.com/alexdobin/STAR

Cell culture

J1, CJ7, and E14TG2a (E14) male mouse ESCs were cultured on 0.1% gelatin-coated plates in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 18% fetal bovine serum (BioWest), MEM nonessential amino acids (Gibco), EmbryoMax nucleosides (Millipore), 50 U/mL penicillin/streptomycin/L-glutamine (PSG, Gibco), 100 µM β-mercaptoethanol, and 1000 U/mL recombinant mouse leukemia inhibitory factor (LIF, Millipore) in a 37°C with 5% CO2. Media was changed daily, and cells were passaged every 2 days. J1, CJ7, E14TG2a mouse ES cells and HEK293T cells were all obtained from ATCC (except CJ7 line was obtained from Dr. Stuart Orkin), confirmed by partial genomic DNA sequencing. Mycoplasma contamination was not detected by PCR based methods.

Cell death assay

ESCs were seeded at a density of 2 × 105 cells/mL in the indicated media type in a clear 96-well plate. Inhibitors were administered 24 hr after seeding at a concentration of 50 μM (Z-VAD-FMK, ApexBio; Necrostatin-1, Selleckchem). At the indicated timepoints, lactate dehydrogenase (LDH) activity was quantified in the supernatant using the Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. A680nm values were first subtracted as background noise. Then, absorbance from an average of 3 media-only wells (reflecting background LDH activity) was subtracted from every sample’s A490nm value. Data were normalized to wells that had been lysed completely using the provided lysis buffer to establish a benchmark for 100% cell death.

Cell differentiation

For nonspecific differentiation, ESCs were washed in -LIF medium and then seeded at the cell densities specified below in each assay, as well as passaged on day one after seeding. Cells were assayed at 24, 48, 60, and/or 72 hr according to the experiment. For neural differentiation, ESC were first maintained in NDiff 227 medium (N2B27, Clontech) supplemented with 3 μM CHIR99021 (Selleck Chemicals) and 1 μM PD184352 (Selleck Chemicals), defined as 2i, to promote self-renewal in the absence of serum and LIF. Differentiation occurred in N2B27 in the absence of 2i and assayed at 24, 48, and 72 hr. For definitive endoderm differentiation, ESCs were grown in DMEM supplemented with 1% FBS, PSG, and 5 μM IDE1 (Cayman Chemical Company) and assayed at 48 hr. For EpiLC differentiation, ESCs were grown in N2B27 supplemented with 20 ng/mL activin A (R and D Systems), 12 ng/mL bFGF (Thermo Fisher), and 1% KOSR (Thermo Fisher) as previously shown (Hayashi et al., 2011), and assayed at 72 hr. For verteporfin-related experiments, verteporfin was diluted in DMSO to a concentration of 1 mM and then further diluted in fresh media during media changes, and cells were protected from light with aluminum foil. HEK293T cells (ATCC CRL-3216) were cultured in DMEM supplemented with 10% FBS and PSG. All cells were grown at 37°C in the presence of 5% CO2.

Flow cytometry

For analysis of externalized phosphatidylserine and active caspase-3, ESCs were seeded at a density of 4 × 105 cells per well on a six well plate in medium with or without LIF. After incubating cells for the indicated number of hours, cells were gently detached and dissociated into a single cell suspension using Accutase (Biolegend). Cells were then resuspended in 200 μL 1X Annexin V binding buffer plus 5 μL of 0.2 mM NucView 488 caspase-3 substrate solution and 5 μL CF 594 annexin V solution (Biotium). Cells were incubated in the dark at room temperature for 30 mins, then centrifuged (1000 rpm, five mins) at 4°C and washed. Then, stained cells were filtered using a 70 μm cell strainer to remove clumps (Celltreat). Flow cytometry was performed on a BD LSRFortessa SORP Flow Cytometer (BD Biosciences) and analysis was carried out with FlowJo (Treestar).

Immunoblotting

For generating lysates suitable for Western blot, ESCs were cultivated under various conditions (e.g., differentiation) in 6-well, 12-well, or 24-well gelatin-coated plates. After various conditions were met cells were quantified using 0.2% trypan blue to distinguish viable from nonviable cells. Then, up to 4 × 106 cells were directly lysed via addition of 2x Laemmli Sample Buffer (Bio-Rad) supplemented with 5% β-mercaptoethanol (Millipore Sigma). Lysates were heated at 95°C for five mins, then cooled to room temperature and routinely stored at −20°C. Lysates were loaded into gels such that either the absolute number of viable cells (quantified by trypan blue) or amount of protein (quantified by the Pierce BCA Assay, Thermo Scientific) loaded in each well was the same. Up to 15 μL of lysate was run on a 4–20% Mini-PROTEAN TGX Stain-Free protein gel (Bio-Rad) or a 10% TGX FastCast gel (Bio-Rad) in denaturing conditions at 130V for 50–70 mins followed by semi-dry transfer using the Trans-Blot Turbo Transfer System (Bio-Rad) onto 0.2 µm nitrocellulose or methanol-activated PVDF membranes (Bio-Rad). Successful protein transfer was verified with Ponceau S staining (Amresco) or stain-free fluorescent crosslinking.

After blocking with 5% bovine serum albumin (BSA) or 5% skim milk (for Yap1 and β-actin only) in Tris-buffered saline containing 0.1% Tween 20 (TBST), membranes were incubated overnight with primary antibodies, diluted in 5% BSA as described in the paragraph below. The following day, membranes were washed, incubated with secondary antibodies, washed again, incubated with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) and visualized on a ChemiDoc XRS+ (Bio-Rad). β-actin was used as a loading control.

Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000). HRP-conjugated secondary antibodies (purchased from Cell Signaling Technology), used at a dilution of 1:10,000 in TBST, were horse anti-mouse (#7076P2) and goat anti-rabbit (#7074S).

Lentiviral production and infection and transposon-mediated gene integration

Lentiviruses were used to transduce shRNA and overexpression constructs. Bacterial glycerol stocks containing the appropriate shRNA were purchased from Millipore Sigma. A complete list of shRNA can be found in supplemental table S3. HEK293T cells were seeded at a density of 1.2 × 106 cells per well on a six well plate. After reaching a confluency of 50–60%, cells were transfected with 1.2 μg of an shRNA-containing pLKO-puro vector (Millipore Sigma) as well as 800 ng pCMV‐Δ8.9 and 400 ng VSVG packaging plasmids with Fugene 6 (Promega) using the manufacturer’s protocol. For inducible overexpression (OE), a pLVX-IRES-ZsGreen1 vector (Clontech) containing the gene of interest and pLVX-TRE3G vector (Clontech) were transfected separately with packaging plasmids. After 18 hr of overnight incubation, HEK293T medium was replaced with ES medium. Then, two days after transfection, medium was supplemented with HEPES to a final concentration of 15 mM to act as an additional buffer, and the supernatant (which contains lentiviral particles) was filtered through a 0.45‐μm Supor membrane (PALL). ESCs were infected at a density of 2 × 105 cells/mL in medium supplemented with 10 μg/mL polybrene (Millipore). 48 hr post-infection, ESCs were selected with puromycin (Thermo Scientific) or geneticin/G418 (Thermo Scientific). Given that cells were passaged every two days, relevant experiments were performed within five passages of the initial infection.

As an alternative method for inducible OE, for the Wwtr1 gene (Taz), a pSBtet-GP vector (AddGene) with luciferase replaced by a multiple cloning site and cloned with the gene of interest. The resultant construct was transfected along with a transposase-containing pCMV(CAT)T7-SB100 vector (AddGene) into ESCs at a density of 6 × 105 cells/mL. Selection with puromycin occurred 24 hr later. All relevant experiments were performed within five passages of the initial transfection. Doxycycline (Fisher Scientific) was used at a concentration of 500 ng/mL for all inducible OE experiments. cDNAs for all OE experiments were obtained from either vectors (Bcl-xL - Sino Biological, Bcl-2–3149 pSFFV-neo Bcl-2 cDNA from AddGene, Puma - GenScript, Taz - pcDNA3.1/HisC-mTAZ from AddGene) or full-length mouse ESC cDNA reverse transcribed using the ProtoScript II First Strand cDNA Synthesis Kit from New England Biolabs (Bmf). All inserts were confirmed by Sanger sequencing.

Caspase activity assay

For determination of caspase activity, the Caspase-Glo 3/7, 8, and 9 Assay Systems (Promega) was used. ESCs were seeded at a density of 1 × 105 cells/mL in a white-walled 96-well plate (Millipore Sigma). At the indicated timepoints, cells were assayed using the respective kits according to manufacturer’s instructions. After subtracting the noise from blank wells (containing media but no cells), luminescent signals in each well were normalized to the cell number.

Gene expression analysis

RNA-seq data was downloaded from Gene Expression Omnibus. Yap1 KD data belonged to the series GSE69669. Samples corresponding to accession numbers GSM1706496, GSM1706495, GSM1706489, and GSM1706488 were used for analysis. Boxplots were generated using BoxPlotR (http://shiny.chemgrid.org/boxplotr/) where whiskers extend to the 5th and 95th percentiles. Gene lists were taken from AmiGO 2 (http://amigo.geneontology.org), specifically positive (GO:2001244) and negative (GO:2001243) regulation of intrinsic apoptotic signaling pathway. Lists were double-checked for any genes known to behave differently than annotated in the ES cell context. Genes that were not expressed were removed from the analysis to reduce noise. Gene ontology analysis was carried out using GOrilla (http://cbl-gorilla.cs.technion.ac.il/) using the ‘two unranked lists of genes’ option, where the background list was populated by all genes listed in the RNA-seq output.

For RT-qPCR, total RNA was extracted from cells with the RNeasy Plus Mini Kit (Qiagen). Then, 600 ng of RNA was reverse transcribed into cDNA using the qScript cDNA SuperMix from QuantaBio (VWR). Next, qPCR was performed in 20 μL reactions using the PerfeCTa SYBR Green FastMix (VWR) plus 6 ng of cDNA and 250 nM forward and reverse primers. Primers were designed using Primer3 (http://primer3.ut.ee/) such that each primer amplified the junction between two or more exons, and their specificity as well as lack of primer dimer formation was verified with melt curve analysis showing one peak. Relative expression was normalized to Gapdh using the 2-ΔΔCT method. All reactions were performed at least in triplicate on a StepOnePlus Real-Time PCR System (Applied Biosystems). All primer sequences are listed in Supplemental Table S1.

Immunofluorescence

ESCs were seeded (6 × 105 cells/mL) on a gelatin-coated µ-Slide VI 0.4 (Ibidi). For cells growing in -LIF conditions, cells were differentiated on a 10 cm plate for one day before seeding the µ-Slide and seeded at 2 × 105 cells/mL. After an additional 2 days, cells were thoroughly washed with Dulbecco’s phosphate-buffered saline (DPBS) and fixed using 4% paraformaldehyde (freshly cracked with 70 mM NaOH at 70°C) for 15 mins. For mitochondrial staining, after washing but before fixation, cells were incubated in 300 nM MitoTracker Deep Red FM in OptiMem for 30 mins. Cells were washed again with DPBS and then permeabilized with 0.3% Triton X-100 in PBS for five mins. After washing once more, samples were blocked using IF blocking solution (4% BSA and 1% normal goat serum diluted in DPBS) for one hour, then incubated overnight with Bcl-2 (1:200) or Mcl1 (1:800) primary antibodies diluted in IF blocking solution. Then, samples were washed thoroughly followed by incubation with fluorescent secondary antibody (goat anti-rabbit IgG Alexa Fluor 594 from Thermo Scientific) diluted 1:1000 for one hour. After further washing, ProLong Glass Antifade Mountant with NucBlue (Thermo Scientific) was added to the samples, which were allowed to cure for 18–24 hr at room temperature. Slides were then imaged using a Zeiss LSM 710 Confocal Microscope using the Plan-Apo 63X (oil) objective and images were processed using ZEN microscope software. Colocalization was quantified using Zen software by setting the crosshairs such that noise was restricted to the lower left quadrant, and the same crosshair coordinates were used for all samples. Intensity was quantified using ImageJ and normalized to the number of discrete nuclei (stained by NucBlue) that could reasonably be assigned to separate cells.

Chromatin immunoprecipitation followed by NextGen sequencing (ChIP-seq)

After reaching ~80% confluency in a 15 cm plate, BirA ESCs with or without FLAG-Bio-Yap1 were crosslinked with 1% formaldehyde for 7 min at room temperature and constant shaking. Formaldehyde was quenched with addition of glycine to a final concentration of 125 mM along with shaking for 5 min. Cells were then sonicated using a Bioruptor (Diagenode), and sheared chromatin including DNA fragments ~ 300 bp in length were used for immunoprecipitation with Dynabeads MyOne Streptavidin T1 (Thermo Scientific). Sequencing libraries were prepared with the enriched ChIP sample using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) and sequenced using the Illumina HiSeq 4000 at the UT Austin Genomic Sequencing and Analysis Facility (GSAF).

ChIP-seq data analysis

Public ChIP-seq data sets were downloaded from Cistrome (http://cistrome.org/db/#/). When possible, only data sets that passed all of Cistrome’s quality control conditions were used. To determine pairwise correlations between ChIP-seq data sets, human YAP1 ChIP-seq peaks.bed files were sent directly to Galaxy (http://cistrome.org/ap/) and peaks were assigned to genes using the BETA-minus functionality (assembly hg19). For mouse Tead factor ChIP-seq datasets and our own Yap1 ChIP-seq data, fastq files were directly processed using the SRA Toolkit, and 75 bp reads were mapped onto the mouse genome (assembly mm9) using Bowtie 2. Peaks were then called using model-based analysis of ChIP-seq (MACS2). For comparison with p300 ChIP-seq data, Bowtie two output was used to compare target overlap within a window of 6 kb of the Yap1 peak center (in dESCs) using a bin size of 100 bp. The apoptosis gene list was retrieved from AmiGO 2 (GO:0006915). Binding scores for all genes were then used for pairwise correlations using Vassar Stats (http://vassarstats.net/matrix2.html) and correlations were visualized using Java TreeView (http://jtreeview.sourceforge.net/). Signal tracks were visualized using Integrated Genome Viewer (IGV, http://software.broadinstitute.org/software/igv/).

For our own Yap1 ChIP-seq data, motif analysis was performed using HOMER (http://homer.ucsd.edu/homer/motif/fasta.html). Peak to gene features were assigned using in-house Perl code. Binding sites were assigned to genomic features according to the following hierarchy: promoter (±2 kb of the TSS)>upstream (2–20 kb upstream of the TSS)>intron > exon>intergenic (all other binding sites that did not fit the other categories). Gene ontology (GO) analysis was performed using GOrilla (http://cbl-gorilla.cs.technion.ac.il/) using the two unranked lists of genes (target and background lists) setting. For the target list, all the genes with a peak score (normalized to BirA) greater than two were included. These Yap1 target genes were further sorted into either upregulated upon Yap1 KD (log2(KD/control)≥0.5) or downregulated ((log2(KD/control)≤−0.5). For the background list, all the genes from the bed file (20,422) were included. The top five GO terms (relative to -log10(p-value)), plus the top apoptosis-related GO term, were then graphed.

Dual luciferase reporter assay

ESCs were seeded (6 × 105 cells/mL when comparing Yap1 OE to empty, 4 × 105 cells/mL when comparing Yap1 KO to WT) in a white-walled 96-well plate. Cells were transfected with 40 ng pGL3-promoter (Promega) containing firefly luciferase downstream of the SV40 promoter plus putative Yap1-responsive regulatory elements cloned from genomic mouse DNA. Simultaneously, as an internal control, cells were co-transfected with 40 ng pRL-TK containing Renilla luciferase downstream of the HSV-thymidine kinase promoter. During Yap1 OE experiments, half of the wells were transfected with 40 ng of a FLAG-Bio vector containing either Yap1, mutant Yap1 (Ser79Ala) generated by site-directed mutagenesis via the NEBuilder HiFi DNA Assembly Kit (New England Biolabs), or no insert downstream of the EF-1α promoter. All transfections related to luciferase were performed with Lipofectamine 3000 (Life Technologies). Cells were incubated for 16 hr before changing the media, and luciferase activity was measured by the Dual-Glo Luciferase Assay a total of 24 hr after transfection System (Promega). Firefly luciferase signal was normalized to Renilla luciferase signal, and then the signal of each regulatory element-containing construct was normalized to pGL3-promoter. All regulatory element sequences tested are listed in Supplemental Table S2. The NEBuilder HiFi DNA Assembly Kit was used to assemble the Bcl-2 tandem enhancer as well as the Mcl-1 distal enhancer with Tead site deletion (using overlapping homology excluding the Tead binding motif).

Mitochondrial priming and loss of membrane potential

Mitochondrial membrane potential loss (∆ψ) was measured as a change in the 525/570 nm ratio relative to the DMSO-treated control using the Cell Meter JC-10 Mitochondrion Membrane Potential Assay Kit (AAT Bioquest) according to the manufacturer’s instructions after 12 hr of incubation with either BH3 mimetic or various timepoints of differentiation (72 hr for -LIF and EpiLC, 48 hr for neural ectoderm and endoderm). BH3 mimetics ABT-737 (Oltersdorf et al., 2005), Venetoclax/ABT-199 (Souers et al., 2013), A-1210477 (Leverson et al., 2015), and A-1155463 (Tao et al., 2014) were applied to ESCs or dESCs (after 24 hr of differentiation) at the concentrations indicated in the figure. Cell death was measured using the LDH assay as described above after 24 hr of incubation with the BH3 mimetic (48 hr total after LIF withdrawal).

siRNA knockdown

MISSION siRNA was purchased from Millipore Sigma. Duplexes targeting Mcl1 (NM_008562: SASI_Mm01_00048593, SASI_Mm02_00314161, SASI_Mm01_00048594) as well as Bcl2l1 (NM_009743: SASI_Mm02_00316924, SASI_Mm02_00316925, SASI_Mm02_00316926) were ordered and resuspended at a concentration of 25 μM in 5X siRNA buffer (Dharmacon) diluted to 1X RNase-free water (Thermo Scientific). siRNA was reverse transfected into ESCs (6 × 105 cells/mL) at a final concentration of 75 nM using INTERFERin according to the manufacturer’s protocol (Polyplus Transfection). MISSION siRNA Fluorescent Universal Negative Control #1 conjugated to 6-FAM was used as both a transfection control and as a non-targeting siRNA control. After verifying KD at the protein level by Western blot, the best two siRNAs were chosen for further experiments. All shRNA and siRNA TRC/ID numbers, and shRNA sequences (or the target position where siRNA is predicted to bind) are listed in supplemental table S3.

Data, software, and code availability

Yap1 ChIP-seq data generated in this study has been uploaded to Gene Expression Omnibus under accession number GSE112606. Code used to analyze raw sequencing files using the programs STAR, Bowtie2, MACS, and Homer is available in the code file included with this manuscript (Source code file 1).

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Decision letter

  1. Marianne E Bronner
    Senior and Reviewing Editor; California Institute of Technology, United States
  2. Jacob H Hanna
    Reviewer; Weizmann Institute of Science, Israel

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for sending your article entitled "Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation" for peer review at eLife. Your article is being evaluated by three peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor.

As you will see, the reviewers think the paper is potentially very interesting and well-executed but also feel like some major revisions would be required. There was concern by some reviewers that in vivo validation would take too long and may not be necessary, so we would appreciate hearing your opinion in that regard. We hope you find the reviews helpful.

Reviewer #1:

In this manuscript the authors analyzed the role of YAP1 during differentiation of stem cells in culture. For this purpose, endogenous YAP1 was deleted using Crispr/Cas technology. The authors demonstrate that while 30% of the control WT cells die after exiting self-renewal, up to 70% of YAP1 KO cells die via apoptotic cell death. YAP1 was necessary for prominent upregulation of anti-apoptotic genes and attenuation of pro-apoptotic genes during stem cell differentiation and this plays an important role for protection of differentiating stem cells from apoptotic cell death. Authors also demonstrate that forced overexpression of anti-apoptotic BCl-xl or Bcl2 in YAP1 KO cells rescue cell death upon differentiation. The authors propose that YAP1 directly regulates pro- and anti-apoptotic genes to protect stem cells from apoptotic cell death during differentiation.

Extensive literature implicates YAP1 in protection of cells from apoptotic cell death. This manuscript extends this knowledge and shows that YAP1 is necessary to protect cultured stem cells from apoptotic cell death during differentiation. While this finding is well documented, the molecular mechanisms responsible for this YAP1 function are not completely clear. The physiological relevance of this mechanism in a live organism is also not obvious.

Specific points:

1) Physiological relevance. Differentiation induced by growth factor withdrawal in culture is a somewhat artificial process. It is not clear if such an extensive activation of apoptosis happens during differentiation of stem cells in vivo and whether YAP1 protects from it. To add physiological relevance, authors should have analyzed YAP1-/- embryos.

2) Mechanism. It still not clear if YAP1 is playing an instructive or permissive role in protection from apoptosis. The authors show tremendous upregulation of Bcl2, BCl-xl and MCl-1 during differentiation of control wildtype stem cells and the lack of this upregulation in YAP1 KO cells (Figure 2A). Overexpression of Bcl2 or BCl-xl rescues YAP1 KO cells (Figure 4DE). YAP1 is binding to Bcl2 and other anti-apoptotic genes and the YAP1-binding elements from these genes better activate Luciferase in wild-type than in YAP1 KO cells. It appears that YAP1 directly activates anti-apoptotic genes which protect cells from apoptotic death. On the other hand, the authors show very little upregulation of Bcl2 and BCl-xl in cells overexpressing YAP1 (Figure 3E); however, the apoptosis phenotype is rescued completely by exogenous YAP1(Figure 4D). If YAP1 activation directly upregulates Bcl2 and BCl-xl to protect cells from apoptosis, why there is a complete rescue without significant changes in Bcl2 and BCl-xl expression?

3) Figure 2. Are all these changes in YAP1 KO cells the drivers or the consequences of apoptotic process? What happens is apoptosis is chemically induced in normal WT ES cells and then the ratios of anti- and pro-apoptotic mRNAs is compared between apoptotic and non-apoptotic cells? If the same changes are observed in that experiment, then these are likely the consequences of apoptosis, not the drivers.

4) Subsection “Yap1 modulates the expression of apoptosis-related genes during differentiation”; "Meanwhile, constitutive Yap1 OE slightly induced anti-apoptosis genes on average (Figure 2—figure supplement 2C). These data suggest that Yap1 may be a master regulator of anti-apoptotic genes during ESC differentiation." Is the difference significant in Figure 2—figure supplement 2C? If it is not significant, then the statement is not completely correct.

5) In the same section; "Intriguingly, analyzing publicly available RNA-seq data from four human cell types using gene set expression analysis (GSEA) showed dysregulation of genes involved in apoptotic signaling after YAP1 KD relative to control KD, showing that Yap1 may transcriptionally regulate apoptosis in both mouse and human cells (Figure 2—figure supplement 2D). Is the GSEA overlaps significant in Figure 2—figure supplement 2D? What are the p and q values? If it is not significant, then the statement is not completely correct.

6) Subsection “Yap1 directly regulates apoptosis-related genes via transcription”: "Meanwhile, transient OE of Yap1 led to higher luciferase activity with anti-apoptotic gene regulatory elements and lower luciferase activity with pro-apoptotic gene regulatory elements (Figure 3E)." What happens to endogenous Bcl2 and BCl-xl genes in YAP1 overexpressing cells? They should be prominently upregulated, if the authors model is correct?

7) Figure 3D. Is TEAD-YAP1 -binding necessary for upregulation of Luciferase expression in Figure 3D? This can be analyzed by mutating TEAD-YAP-binding consensus sequences in these constructs. If YAP1 role is direct, TEAD-YAP binding to the promoter should be critical.

8) Figure 2 is not well organized. What happens to pro-apoptotic transcripts during differentiation in WT ES cells? Are they up or downregulated in comparison to non-differentiated cells? It appears that the direction of the changes in gene expression are similar in WT and YAP KO cells, but only the magnitude is different. This is not obvious the way the data are presented.

9) Figure 3C. Is there any change in YAP1 binding to pro- and anti-apoptotic genes during differentiation? This static data are not very informative. Especially considering that some YAP1 binding genes are upregulated while others are downregulated.

10) Figure 4C should have data for Bmf and Puma knockdowns in WT cells. If the difference between WT and YAP1 KO cells significantly reduced upon Bmf and Puma knockdowns in both cell types, then Bmf and Puma genes play a specific role. If the difference between WT and KO cells is not significantly reduced upon Bmf and Puma knockdowns in both cell types, then Bmf and Puma genes do not play a specific role.

11) Figure 1—figure supplement 1J. Knockdown of what gene?

Reviewer #2:

This is an interesting study that tries to address the poorly explored phenomena that about 30% of ESCs undergo apoptosis upon differentiation initiation. The authors show convincingly that YAP1 keeps these levels low relatively, and KO of Yap1 increases apoptosis through increase caspase mediate cell death and mitochondrial priming. They show that YAP1 directly regulates transcription of key anti apoptotic genes.

Overall this is an interesting study, however some important issues need to be examined and clarified before publication:

1) Please show rescue of phenotype in YAP1 KO ESCs upon ectopic expression of Yap1 in order to exclude off target phenotypes.

2) Does overexpression of YAP1 in WT ESCs further decreases apoptosis upon LIF removal ?

3) What about the other hippo pathway effector – can TAZ overexpression rescue the phenotype of YAP1 KO? Does TAZ overexpression in WT ESCS reduce apoptosis upon LIF withdrawal?

4) The authors indicate "Thus, Yap1 is key for survival during ESC differentiation, and ablation of Yap1 exacerbates apoptosis specifically rather than triggering an alternate cell death pathway." However, they do not show survival rate of yap KO in a few passages in differentiated state. How do they know lethality is during the differentiated state transition, and not just more lethal in the differentiated state itself?

5) The authors indicate "This implied that the abnormally high rates of apoptosis in Yap1 KO cells are sustained by heightened Casp9 activation. However, since mRNA expression of caspases was relatively equal between Yap1 KO cells and WT cells during differentiation, we speculated that Yap1 may regulate other factors that indirectly affect the rate of caspase cleavage". This is over speculation. If mRNA levels are the same, protein levels should be tested.

6) The authors indicate "Immunocytochemistry shows that Yap1 nuclear localization tends to be associated with higher expression of BCl-2 in -LIF, but not in +LIF conditions, where undifferentiated ESCs show cytoplasmic Yap1 and weaker BCl-2 staining" (Figure 2—figure supplement 2A)". The ICC looks of poor quality and looks like a multicellular effect and not a single cell phenotype. Can this be explained or improved?

7) The authors indicate "Collectively, these data show that Yap1 is critical for proper BCl-2 induction during differentiation". However Yet in Figure 3E they show no change in BCL2 luci with OE of YAP? Can this be better explained and addressed.

8) The authors indicated "Using RNA-seq data from a previous study (Chung et al., 2016), we found that differentiation induces the expression of a group of anti-apoptotic genes in WT cells, but this induction is debilitated after Yap1 KD (Figure 2—figure supplement 2B)." No t-test performed for any of these boxplots in Figure 2—figure supplement 2. This should be added and verified.

9) The authors indicate "Additionally, we confirmed a physical interaction between Yap1 and Tead4 during ESC differentiation using co-immunoprecipitation (Figure 3—figure supplement 3D)." Did the authors conduct an IP for p300 as well, if they show a gene correlation in ChIP? This is also what they present in the end Figure (5), but they don't show any IP for it to substantiate this claim?

10) The authors indicate "Furthermore, deletion of Yap1 significantly sensitized ESCs, but not undifferentiated ESCs, to loss of Δѱ in response to BH3 mimetic treatment. We then investigated whether the higher loss of Δѱ in Yap1 KO cells correlated with greater rates of cell death". I wonder why did the authors use only response to BH3 treatment? Why not +/- LIF like before? Could this be also shown and explained.

Reviewer #3:

Yap1 plays numerous roles in development and cancer but its roles during ESC differentiation and early development remains unclear. Previous studies showed the death of 30% or more of ESCs upon differentiation and proposed that apoptosis during ESC differentiation is to cull cells that fail to exit self-renewal in order to promote efficient differentiation. This function of apoptosis is not limited to ESC differentiation, as it also happens in other biological processes. However, mechanisms that regulate the balance between survival and death during ESC differentiation remain unclear. In this manuscript, by using the Yap1 KO ESCs established in their previous studies, LeBlanc et al. show that Yap1 KO ESCs experience massive cell death upon the exit from self-renewal and Yap1 contextually protects differentiating, but not self-renewing ESC from hyperactivation of the apoptotic cascade. Yap1 attenuates mitochondrial apoptosis during ESC differentiation by upregulating anti-apoptotic factors through transcriptional regulation and Yap1 KO cells develop a high degree of mitochondria priming that precedes elevated rates of apoptosis.

This study is well executed and timely, and the message is clear and significant in contributing to our understanding of the functions of Yap1 during ESC differentiation and early development and of the balance control between survival and death during cell fate changes. While I am strongly supportive for its publication in eLife, I do have some major and minor points listed below that could further enhance the clarity and improve the manuscript. And the authors should pay more attention to the labeling in the figures and provide a few minimal additional experiments and more explanations to the experiment design to make the overall story more straightforward and clearer.

Major points:

1) At beginning the authors tested several mESC differentiation models (LIF withdraw, N2B27, and IDE1). However, it will be nicer if they also test cell apoptosis of WT/Yap1KO ESCs in EpiLC differentiation, because 1) EpiLC differentiation mimics the in vivo epiblast maturation at pre-to-post implantation stage, and 2) it is more related to the context of Yap1 and p300 ChIP-seq analysis as performed in Figure 3.

2) Figure 1 shows the upregulation of Casp and apoptosis in Yap1 KO starts early (24-48 hrs) after LIF withdrawal, but Figure 2A and 2B show the upregulation of anti-apoptotic factors (BCl-2, BCl-xL and MCl-1) in WT ES starts late (72 hrs) after LIF withdrawal. BCl-2 is the regulator upstream of Casp. The authors should provide some explanations about these results.

3) In the Figure 1E, 1F and 2D, please indicate the time after LIF withdrawal (or the time of induced differentiation) in hours to better compare them with other results (like Figure 1D, 2A, 2B). I didn't find their time point information in Figure/Figure legends/Text/Experimental procedures.

4) About Figure 3D and 3E: (1) the authors should at least provide one representation of the reporter construct; (2) the authors should indicate the regulatory elements (e.g., by using bars) used in the luciferase constructs in the Yap1 and p300 ChIP-seq tracks; (3) I am curious to see the expression level of Yap1 in Yap1 OE: was it rescued to a WT level or an overexpression?

5) Figure 3 – data are clear but too shallow/superficial: According to the increased p300 (and H3K27ac) ChIP-seq intensities at Yap1 peaks during differentiation, it is implied that Yap1 may function as an activator of transcription for the anti-apoptotic genes BCl-2, BCl-xL, MCl-1. ChIP-qPCR are required to confirm the Yap1 binding at those loci (Figure 3C). Furthermore, authors should also compare the Yap1 and p300 ChIP intensity between ES and dES situations, to support their conclusion that Yap1 activates the anti-apoptotic genes during differentiation. Does Yap1 physically interact with p300? If so, the authors may want to show if Yap1 recruits p300 to chromatin.

6) Figure 4C: the label is misleading. In the figure, the authors should indicate the Bmf KD and Puma KD are performed in Yap1 KO.

https://doi.org/10.7554/eLife.40167.034

Author response

We appreciate all the reviewers for their insightful and constructive comments. We recognize that the top concern is a lack of clarity in elucidating Yap1’s molecular mechanisms in promoting survival via transcriptional regulation. We have attempted to address this concern and others by performing a considerable number of additional experiments to support our claims. The following is a summary of our responses to major or recurring comments:

To address whether Yap1 truly binds more to target apoptosis-related gene loci during differentiation rather than self-renewal, we use both ChIP-seq and ChIP-qPCR to show that Yap1 occupancy indeed increases on target loci (most notably MCl-1 and BCl-2) after the exit from self-renewal. Additionally, to further characterize enhancers linked to apoptosis-related genes, we generated a tandem BCl-2 enhancer that is more responsive to OE of Yap1 than our previous enhancer, deleted the Tead-binding consensus sequence from the MCl-1 enhancer, and demonstrated that OE of a mutant Yap1 that cannot bind to Tead factors is deficient in rescue of luciferase expression. Finally, in response to concerns that in vivovalidation was lacking, we performed several experiments using the EpiLC differentiation condition in vitro, which more closely mimics the transition from inner cell mass to epiblast that occurs in vivo, compared to mere LIF withdrawal.

Reviewer #1:

[…] Extensive literature implicates YAP1 in protection of cells from apoptotic cell death. This manuscript extends this knowledge and shows that YAP1 is necessary to protect cultured stem cells from apoptotic cell death during differentiation. While this finding is well documented, the molecular mechanisms responsible for this YAP1 function are not completely clear. The physiological relevance of this mechanism in a live organism is also not obvious.

We sincerely appreciate the reviewer’s constructive feedback for the improvement of our manuscript. We have performed new experiments that hopefully address the reviewer’s major concerns and illuminate the molecular mechanisms involved.

Specific points:

1) Physiological relevance. Differentiation induced by growth factor withdrawal in culture is a somewhat artificial process. It is not clear if such an extensive activation of apoptosis happens during differentiation of stem cells in vivo and whether YAP1 protects from it. To add physiological relevance, authors should have analyzed YAP1-/- embryos.

We thank you for this critical comment. Yap1 -/- embryos have been made previously and show morphological defects by E7.5 and developmental arrest by E8.5, culminating in embryonic lethality by E10.5 (Morin-Kensicki et al., 2006). Key defects included yolk sac vasculogenesis and failure of the allantois to attach to the chorion, which implicated Yap1 in regulating cell number, morphogenetic movements, or cell-cell interactions, but the authors could not pin down any concrete mechanism as to why Yap1 -/- led to such broad developmental defects. Thus, we agree that generating and analyzing our own Yap1 -/- embryos in greater mechanistic depth would have been a considerable contribution.

However, we bring up the comment from reviewer 3, who helpfully suggested epiblast-like cell (EpiLC) differentiation as a way to more closely reflect what occurs in vivo. The ESC to EpiLC cell fate conversion is reminiscent of the in vivo transition of the inner cell mass (ICM) to the epiblast, and in vitro differentiation techniques produce EpiLCs that are highly similar to epiblasts according to principal component analysis of RNA-seq data (Hayashi et al., 2011). We performed this differentiation method and analyzed cell death, apoptosis-related gene expression, and mitochondrial depolarization, finding them to be relatively consistent with differentiation experiments utilizing LIF withdrawal (new results presented in Figures 2D, 3B, and 5A).

Furthermore, stem cell therapy often requires culturing ESC or patient-specific iPS cells and differentiation of them in vitro, before transplantation to treat various diseases (Jiaojiao et al., 2018; Shi et al., 2016). Therefore, we believe thatin vitrostudies of ESC differentiation are still valuable because of their application to biotechnological and biomedical advances.

2) Mechanism. It still not clear if YAP1 is playing an instructive or permissive role in protection from apoptosis. The authors show tremendous upregulation of Bcl2, BCl-xl and MCl-1 during differentiation of control wildtype stem cells and the lack of this upregulation in YAP1 KO cells (Figure 2A). Overexpression of Bcl2 or BCl-xl rescues YAP1 KO cells (Figure 4DE). YAP1 is binding to Bcl2 and other anti-apoptotic genes and the YAP1-binding elements from these genes better activate Luciferase in wild-type than in YAP1 KO cells. It appears that YAP1 directly activates anti-apoptotic genes which protect cells from apoptotic death. On the other hand, the authors show very little upregulation of Bcl2 and BCl-xl in cells overexpressing YAP1 (Figure 3E); however, the apoptosis phenotype is rescued completely by exogenous YAP1(Figure 4D). If YAP1 activation directly upregulates Bcl2 and BCl-xl to protect cells from apoptosis, why there is a complete rescue without significant changes in Bcl2 and BCl-xl expression?

This comment makes an excellent point, as the aforementioned data do seem to be somewhat contradictory. First, we should clear up some misconceptions. We showed upregulation of only BCl-2 during differentiation; our new Figure 2B shows that BCl-xL and MCl-1 are either slightly upregulated or merely maintained during differentiation, at least by 48 hr. Yap1 KO cells fail to upregulate BCl-2, but they also seem to fail to maintain levels of BCl-xL or MCl-1.

The seeming lack of upregulation of BCl-2 and BCl-xL via luciferase expression after OE of Yap1 can be explained by looking at the asymmetric transcriptional effects of KO and OE. Complete lack of Yap1 seems to be more impactful on BCl-2 levels than transient overexpression, where a 16-fold induction of Yap1 only upregulated anti-apoptotic genes by approximately 2-fold (new Figure 3—figure supplement 1C). Therefore, there is indeed upregulation of these genes after OE, it was simply not detected by our previous luciferase assays.

Furthermore, enhancers rarely work alone in cells, and Yap1 showed multiple peaks on all target apoptosis-related genes discussed in this study. Thus, we surmised that creating a tandem enhancer consisting of two elements occupied by Yap1 according to ChIP-seq may more closely approximate what is occurring in the cell. Indeed, combining our old BCl-2 enhancer with an additional element in the same intron (new Figure 4—figure supplement 1H) granted upon it the ability to increase expression 2x upon Yap1 OE (modified Figure 4E), consistent with our RT-qPCR data without changing basal enhancer activity.

3) Figure 2. Are all these changes in YAP1 KO cells the drivers or the consequences of apoptotic process? What happens is apoptosis is chemically induced in normal WT ES cells and then the ratios of anti- and pro-apoptotic mRNAs is compared between apoptotic and non-apoptotic cells? If the same changes are observed in that experiment, then these are likely the consequences of apoptosis, not the drivers.

We appreciate this comment, as apoptosis-related genes not only contribute to cell death but are in turn regulated by cell death itself. Indeed, it is known that apoptosis initiates global mRNA decay, which contributes to amplifying cell death when anti-apoptotic transcripts are depleted, forming a feedforward loop (Thomas et al., 2015). Complicating this picture, anti-apoptotic proteins can function as transcriptional regulators in and of themselves (Wu et al., 2017). We believe that the best way to disentangle this highly interconnected phenomenon is by modulation of individual genes to genetically dissect requirements for apoptosis in the absence of Yap1.

However, we did perform the requested experiment in an attempt to determine whether changes in ratios of anti- and pro-apoptotic mRNAs are sufficient drive apoptosis. We have chemically induced apoptosis using STS and measured the expression of pro- and anti-apoptotic genes over a timecourse. Surprisingly, we found that genes involved in intrinsic apoptosis (both pro and anti) were strongly repressed over the course of STS-induced apoptosis, whereas Fas and Fasl which are involved in promoting extrinsic apoptosis were significantly increased (Author response image 1). However, this did not seem to support or refute any of our major claims in our study.

Thus, as an alternative approach to your comment, we proposed that individual repression or overexpression of apoptosis-related genes would determine whether such changes in expression could in and of themselves drive changes in rates of cell death. We had already knocked down or overexpressed most factors, and these single factor manipulations did rescue or exacerbate cell death (Figures 5D, 6A, 6B, 6E, and 6F); to strengthen this point, we inducibly overexpressed pro-apoptotic factors Bmf and Puma. OE accelerated, but did not cause, cell death in dESCs, particularly in Yap1 KO cells, and particularly with Puma, agreeing with our KD data (new Figure 5E, Figure 5—figure supplement 1C and D). We believe that these experiments provide sufficient evidence that at least individually, changes in the expression of these apoptosis-related genes can shift cell fate to either survival or death in dESCs.

Author response image 1
dESCs were treated with 1 μM STS for the indicated hours and expression of various anti- and pro-apoptotic genes was measured at each treatment timepoint (n = 2).

Expression values were normalized to Gapdh and to 0 hr (DMSO-treated) dESCs

4) Subsection “Yap1 modulates the expression of apoptosis-related genes during differentiation”; "Meanwhile, constitutive Yap1 OE slightly induced anti-apoptosis genes on average (Figure 2—figure supplement 2C). These data suggest that Yap1 may be a master regulator of anti-apoptotic genes during ESC differentiation." Is the difference significant in Figure 2—figure supplement 2C? If it is not significant, then the statement is not completely correct.

As requested, we have performed statistical testing on the box plots in question (two-tailed paired t-test), given that each gene (n=88) has two expression values corresponding to either control KD or Yap1 KD. Accordingly, we obtained a P-value of 0.0096, indicating that the difference in expression after differentiation in control KD vs. Yap1 KD is significant (modified Figure 3—figure supplement 1D). We also performed the same test on pro-apoptotic genes as a control and obtained a P-value of 0.9141, indicating lack of significance. Both have been marked in the panels as ** (since it falls between the intervals of 0.01 and 0.001) and NS, respectively.

5) In the same section; "Intriguingly, analyzing publicly available RNA-seq data from four human cell types using gene set expression analysis (GSEA) showed dysregulation of genes involved in apoptotic signaling after YAP1 KD relative to control KD, showing that Yap1 may transcriptionally regulate apoptosis in both mouse and human cells (Figure 2—figure supplement 2D). Is the GSEA overlaps significant in Figure 2—figure supplement 2D? What are the p and q values? If it is not significant, then the statement is not completely correct.

We have checked the p- and q-values. Unfortunately, we found that only the HCC364 (lung cancer) cell line showed significant dysregulation. Its p- and q-values were 0.029 and 0.0325, respectively, whereas the values were generally >0.49 or higher in the other cancer cell types. We thus contend that our initial claim is not justified by the data without performing a much more comprehensive analysis or cancer-related experiments, which would be outside the scope of this manuscript, and thus we propose removal of that panel (Figure 3—figure supplement 1F) in our revised manuscript.

6) Subsection “Yap1 directly regulates apoptosis-related genes via transcription”: "Meanwhile, transient OE of Yap1 led to higher luciferase activity with anti-apoptotic gene regulatory elements and lower luciferase activity with pro-apoptotic gene regulatory elements (Figure 3E)." What happens to endogenous Bcl2 and BCl-xl genes in YAP1 overexpressing cells? They should be prominently upregulated, if the authors model is correct?

Thank you for this comment. That is indeed what is expected. As was mentioned in response to comment 1-2, we performed transient OE of Yap1 and found a modest increase in expression of anti-apoptotic factors (new Figure 3—figure supplement 1C).

One may question why the effects of Yap1 OE were modest and generally did not exceed two-fold changes in expression. We surmise that this is because Yap1 is already relatively highly expressed in WT dESCs, and since Yap1 is merely a co-regulator that can only function in complex with several other factors that then mediate DNA binding and histone modification, we propose that limiting factors of this complex may restrict the ability for excess Yap1 to strongly upregulate its targets. In addition, apoptosis-related genes must be tightly regulated by the cell by a myriad of mechanisms to prevent tumorigenesis; just as an example, a miRNA known as miR-136-5p represses BCl-2 (Li et al., 2017) and miR-302b represses MCl-1 (Khodayari et al., 2016).

7) Figure 3D. Is TEAD-YAP1 -binding necessary for upregulation of Luciferase expression in Figure 3D? This can be analyzed by mutating TEAD-YAP-binding consensus sequences in these constructs. If YAP1 role is direct, TEAD-YAP binding to the promoter should be critical.

This is an excellent point. Indeed, a careful search of the apoptosis-related enhancers showed that almost all of them had at least one occurrence of the Tead binding consensus sequence, GG(A/T/C)AT, or its reverse strand counterpart. Initially, we attempted to delete this motif from several of our putative regulatory elements, but it was more difficult than expected, as most of the regulatory elements had multiple instances of this motif and their overall sequences were extremely repetitive, making site-directed mutagenesis more prone to error due to slippage.

Ultimately, we used two approaches. We performed site-directed mutagenesis on Yap1 itself, mutating Ser79 to Ala, which virtually abolishes the Yap1-Tead interaction (Schlegelmilch et al., 2011). We then performed transient OE of mutant and regular Yap1 in Yap1 KO cells in an attempt to rescue luciferase expression of our strongest two enhancers, MCl-1 distal and the newly created BCl-2 intronic tandem (mentioned in the earlier comment, 1-2). OE of unmutated Yap1 rescued luciferase expression in Yap1 KO cells to levels comparable to WT ESCs with Yap1 OE, whereas OE of Yap1 Ser79Ala was defective in rescue of either construct (new Figure 4F).

For our second approach, we decided to alter the sequence of our strongest enhancer, MCl-1 distal, and delete the Tead binding sequence (TBS) entirely, as there was only one occurrence of it (BCl-2 tandem had three). We deleted ATTCC (indicating the presence of GGAAT on the reverse strand) from the middle of the enhancer and found that it not only abrogated ability to respond to Yap1 OE in WT ESCs, but also broke enhancer function entirely (new Figure 4G). Although the positive control (MCl-1 distal, unmutated) showed an increase in luciferase expression that was just barely not statistically significant by our threshold (p = 0.0613), we argue that these experiments still collectively demonstrate that the Tead-Yap1 interaction is essential for the regulation of these cis-regulatory elements.

8) Figure 2 is not well organized. What happens to pro-apoptotic transcripts during differentiation in WT ES cells? Are they up or downregulated in comparison to non-differentiated cells? It appears that the direction of the changes in gene expression are similar in WT and YAP KO cells, but only the magnitude is different. This is not obvious the way the data are presented.

We agree with this feedback and have checked the expression of all apoptosis-related genes in WT ESCs in all 4 differentiation conditions (new Figure 3B). This reveals that overall, BCl-2 expression is upregulated, BCl-xL and MCl-1 expression is maintained, and pro-apoptotic factors are strongly upregulated, though the magnitude of upregulation depends on differentiation method. Thus, the direction is indeed mostly the same. This also gives more context to what appear to be only mild differences in pro-apoptotic gene expression between WT and Yap1 KO; though Yap1 KO cells only have ~2x more Puma than WT in -LIF conditions (modified Figure 3C), Puma is already being upregulated 4x during differentiation in WT ESCs to begin with (new Figure 3B).

9) Figure 3C. Is there any change in YAP1 binding to pro- and anti-apoptotic genes during differentiation? This static data are not very informative. Especially considering that some YAP1 binding genes are upregulated while others are downregulated.

Thank you for the comment. We agree that static data are generally not as informative. Before submission, we had attempted to perform ChIP-seq of Yap1 in +LIF conditions. However, we did not submit this to NCBI or present it in our manuscript, as relatively a small number of peaks were enriched over background. This was completely expected, as Yap1 is almost entirely cytoplasmic during self-renewal (Chung et al., 2016) and therefore should not be binding DNA. Due to your feedback, we have submitted this sequencing data to NCBI under the same GSE number and added the L+ ChIP-seq data to modified Figure 4B, which has also been slimmed down for clarity (removed the low Yap1 peaks, as showing the L+ data is a better control and matches the p300 data presentation better). We also reanalyzed the data overall and have updated the number of peaks in the main text.

As shown in Author response image 2, binding of Yap1 and p300 at loci MCl-1 and BCl-2 increases upon differentiation according to ChIP-seq (Author response image 2A). Pro-apoptotic factors Bmf and Puma (Bbc3) also gain both Yap1 and p300 occupancy although not very strong. This increase in p300 occupancy, even on pro-apoptotic genes, is consistent with our RT-qPCR data during WT ESC differentiation (new Figure 3B). Yap1’s co-repressor function has been characterized previously (Kim et al., 2015). Perhaps it recruits HDAC to fine-tune the expression of pro-apoptotic genes during differentiation (since p300 is also present), but exploring this further to determine how Yap1 knows whether to co-repress or co-activate targets on binding may be outside the scope of this manuscript.

To validate this ChIP-seq data, we decided to perform ChIP-qPCR for our two strongest enhancers (corresponding to the two genes that seem to have the strongest phenotypic effect), matching our additional luciferase assays in new Figures 4F and 4G. Indeed, ChIP-qPCR confirms a significant enrichment of Yap1 at BCl-2 and MCl-1’s regulatory elements upon differentiation (Author response image 2B). Collectively, these data plus our luciferase assays, RT-qPCR, and other experiments demonstrate Yap1’s context-dependent role in maintenance of MCl-1 expression and upregulation of BCl-2.

Author response image 2
(A) Signal tracks indicating Yap1 (red) and p300 (blue) ChIP-seq performed in ESCs (+LIF for Yap1, 2i for p300) and dESCs (-LIF for Yap1, EpiLC for p300).

Grey bars indicate regions during primer design for ChIP-qPCR. (B) ChIP-qPCR of regulatory elements associated with apoptosis-related genes in +LIF (black) and -LIF (grey) FLAG-Bio-Yap1 ESCs (n = 2). Stars indicate p-values of 0.0015 and 0.0066, respectively (two-tailed t-test).

10) Figure 4C should have data for Bmf and Puma knockdowns in WT cells. If the difference between WT and YAP1 KO cells significantly reduced upon Bmf and Puma knockdowns in both cell types, then Bmf and Puma genes play a specific role. If the difference between WT and KO cells is not significantly reduced upon Bmf and Puma knockdowns in both cell types, then Bmf and Puma genes do not play a specific role.

We thank the reviewer for this comment. Pro-apoptotic factors are somewhat redundant in their function. KD of Bmf or Puma reduced cell death in Yap1 KO cells because the abnormally high activation of intrinsic apoptosis in such cells is easily perturbed by reducing the pool of available Casp9 (old Figure 1I) which is downstream of such factors. Due to this comment, we performed KD of the same factors in WT, and we found that KD did not consistently reduce cell death in WT between constructs (modified Figure 5D). This is consistent with the rest of our data that rates of apoptosis in WT are typically quite robust and difficult to decrease. Thus, Bmf and Puma do play a specific role in Yap1 KO cells.

11) Figure 1—figure supplement 1J. Knockdown of what gene?

This panel was referring to knockdown of Casp9. To increase clarity, this has been indicated to the panel below the x-axis. Due to figure reorganization, this is now modified Figure 1—figure supplement 1H.

Reviewer #2:

[…] Overall this is an interesting study, however some important issues need to be examined and clarified before publication:

We thank the reviewer for critically evaluating our manuscript. We hope that our additional experiments and clarifications offer sufficient evidence to justify our claims.

1) Please show rescue of phenotype in YAP1 KO ESCs upon ectopic expression of Yap1 in order to exclude off target phenotypes.

We have already performed the rescue experiment of ectopic expression of Yap1 in Yap1 KO ESCs, as this was the positive control for our BCl-xL OE experiment in old Figure 6A. We had generated three stable Yap1 KO cell lines overexpressing Yap1 (old Figure 6—figure supplement 1A) and performed the cell death assay (old Figure 6A) showing reduction of cell death during differentiation to levels below WT ESCs just like BCl-xL OE.

2) Does overexpression of YAP1 in WT ESCs further decreases apoptosis upon LIF removal?

We thank the reviewer for this comment, as we did not sufficiently address whether excess Yap1 can augment survival. We generated three stable WT ES cell lines overexpressing Yap1 (new Figure 1—figure supplement 1F) and performed the cell death assay, showing reduction of cell death during differentiation to levels below WT ESCs (~10%) (new Figure 1C).

3) What about the other hippo pathway effector – can TAZ overexpression rescue the phenotype of YAP1 KO? Does TAZ overexpression in WT ESCS reduce apoptosis upon LIF withdrawal?

We greatly appreciate this comment, because the literature has shown both overlapping and distinct functions for Yap1 and its paralog, Taz (Hong et al., 2005; Matsushita et al., 2018; Plouffe et al., 2018). We inducibly overexpressed Taz and this not only rescued the phenotype of Yap1 KO cells, but also slightly reduced cell death in WT ESCs from ~30% to 25% (new Figure 6C and Figure 6—figure supplement 1E). Though this reduction in cell death was consistent enough compared to uninduced cells to be statistically significant, it is not a dramatic reduction, and OE of Yap1 in WT ESCs was more effective in reducing cell death (new Figure 1C). Taz OE increases BCl-xL protein levels but does not seem to affect MCl-1 (new Figure 6D). Thus, excess Taz phenotypically rescues complete lack of Yap1 during differentiation.

4) The authors indicate "Thus, Yap1 is key for survival during ESC differentiation, and ablation of Yap1 exacerbates apoptosis specifically rather than triggering an alternate cell death pathway." However, they do not show survival rate of yap KO in a few passages in differentiated state. How do they know lethality is during the differentiated state transition, and not just more lethal in the differentiated state itself?

This is an intriguing comment, as we did not consider whether Yap1 had any roles during later differentiation, as most Yap1 studies occur in cancer cells or at very specific developmental stages. We decided to split the question into two parts. First, do Yap1 KO cells survive if they are continuously cultured? Second, does depletion of Yap1 after the exit from self-renewal influence apoptosis?

We found that by d7 of differentiation, cell death rates in Yap1 KO remained high (new Figure 2F). Some cells remained in somewhat round, small colonies, consistent with the differentiation defect phenotype previously reported (Chung et al., 2016), whereas other cells attained a flattened, stretched morphology similar to WT dESCs at this stage (data not shown). We did not passage the cells further, as even WT dESCs became sensitive to dissociation and replating at this stage, which would have obfuscated any subsequent cell death assays.

Secondly, we used a well-characterized inhibitor of Yap1, verteporfin, at various stages of differentiation (new Figure 2E) and found that inhibition was most deleterious during the exit from self-renewal (new Figure 2F). By d7, even the highest dosage had no effect on cell survival, and WT dESCs were relatively healthy at this point. Collectively, we propose that with regards to apoptosis, Yap1’s importance is primarily during the transition period from self-renewal to differentiation, and a few Yap1 KO dESCs do manage to survive and continue growing.

5) The authors indicate "This implied that the abnormally high rates of apoptosis in Yap1 KO cells are sustained by heightened Casp9 activation. However, since mRNA expression of caspases was relatively equal between Yap1 KO cells and WT cells during differentiation, we speculated that Yap1 may regulate other factors that indirectly affect the rate of caspase cleavage". This is over speculation. If mRNA levels are the same, protein levels should be tested.

To address this comment, we have performed Western blots for uncleaved Casp3 as well as uncleaved Casp8 and its first processed fragment (p43), and we had already performed Casp9 Western blot (modified Figure 1G). For Casp3, levels fluctuate wildly during differentiation, but the pattern remains roughly the same. However, for both Casp8 and Casp9, total protein levels are higher in Yap1 KO cells. As this does not reflect mRNA expression, we speculate that there must be some post-translational mechanism involved, and we do not deny that this may contribute to the enhanced cell death observed in Yap1 KO dESCs.

However, we believe that cleavage of caspases is generally more critical for caspase activity than relative protein levels. For example, Casp9 cleavage relieves inhibition of its activity by XIAP, allowing it to be fully active in the apoptosome holoenzyme complex (Twiddy and Cain, 2007). Thus, we believe that our focus on upstream regulators of mitochondrial outer membrane integrity (which affect apoptosome formation and thus Casp9 activation) is reasonable.

6) The authors indicate "Immunocytochemistry shows that Yap1 nuclear localization tends to be associated with higher expression of BCl-2 in -LIF, but not in +LIF conditions, where undifferentiated ESCs show cytoplasmic Yap1 and weaker BCl-2 staining" (Figure 2—figure supplement 2A)". The ICC looks of poor quality and looks like a multicellular effect and not a single cell phenotype. Can this be explained or improved?

We agree that the ICC previously presented did not provide sufficient evidence for our claims. We redesigned the experiment to make it more rigorous; instead of looking at +LIF and -LIF conditions, we compared WT and Yap1 KO ESCs, both in -LIF. Our new ICC shows reduced BCl-2 and MCl-1 in Yap1 KO dESCs compared to WT as quantified by ImageJ (new Figure 3—figure supplement 1A and B). As a form of antibody validation, we also stained for the mitochondria using a MitoTracker dye, verifying that our anti-apoptotic proteins colocalized with the mitochondria, especially MCl-1 (weighted colocalization coefficient of BCl-2 or MCl-1 with the mitochondria, ~0.7-0.9). Additionally, we found reduced mitochondrial content in Yap1 KO dESCs. This is consistent with prior observations that ESCs increase mitochondrial biogenesis during differentiation (Wanet et al., 2015); thus, it is logical that defects in differentiation may hamper this process.

7) The authors indicate "Collectively, these data show that Yap1 is critical for proper BCl-2 induction during differentiation". However Yet in Figure 3E they show no change in BCL2 luci with OE of YAP? Can this be better explained and addressed.

We are thankful for this comment. Reviewer 1 brought up a similar point (comment 1-2). We believe that testing Yap1-bound putative regulatory elements does not always yield a functional enhancer that behaves as it should in cells. To address this contradiction, we created a tandem enhancer composed of our old BCl-2 intronic regulatory element and another Yap1-occupied sequence (which also possessed a Tead binding site), and the schematic of this is seen in new Figure 4—figure supplement 1H. This tandem enhancer had nearly identical basal activity to the old enhancer, but upon Yap1 OE, its activity increased 2x, consistent with our RT-qPCR data (modified Figure 4E).

8) The authors indicated "Using RNA-seq data from a previous study (Chung et al., 2016), we found that differentiation induces the expression of a group of anti-apoptotic genes in WT cells, but this induction is debilitated after Yap1 KD (Figure 2—figure supplement 2B)." No t-test performed for any of these boxplots in Figure 2—figure supplement 2. This should be added and verified.

We have performed t-tests for the boxplots in previous Figure 2—figure supplement 2 (now modified Figure 3—figure supplement 1D and E), and only found one comparison (Yap1 KD vs. control KD during differentiation) was significant. Thus, the data support our initial assertion.

9) The authors indicate "Additionally, we confirmed a physical interaction between Yap1 and Tead4 during ESC differentiation using co-immunoprecipitation (Figure 3—figure supplement 3D)." Did the authors conduct an IP for p300 as well, if they show a gene correlation in ChIP? This is also what they present in the end Figure (5), but they don't show any IP for it to substantiate this claim?

We thank the reviewer for this feedback. We have performed a Co-IP and confirmed a physical interaction between Yap1 and p300 (modified Figure 4—figure supplement 1B) in dESCs, which supports our contention of Yap1 and p300 co-occupancy according to our ChIP-seq data.

10) The authors indicate "Furthermore, deletion of Yap1 significantly sensitized ESCs, but not undifferentiated ESCs, to loss of Δѱ in response to BH3 mimetic treatment. We then investigated whether the higher loss of Δѱ in Yap1 KO cells correlated with greater rates of cell death". I wonder why did the authors use only response to BH3 treatment? Why not +/- LIF like before? Could this be also shown and explained.

We appreciate this comment; initially, we only used BH3 treatment because most assays measuring Δѱ in the literature used either BH3 mimetics or fragments of BH3-only proteins. We have performed the mitochondrial membrane potential assay in both WT ESCs and Yap1 KO ESCs during 4 different differentiation and 2 different self-renewal conditions (Figure 5A). Loss of Δѱ as measured by A525/A590 was 1.5 to 2-fold higher in Yap1 KO ESCs compared to WT ESCs in all differentiation conditions tested, but not in ES+ or 2i media. This supports our BH3 mimetic data, and collectively, the results show that mitochondria in Yap1 KO cells are more sensitive to depolarization.

Reviewer #3:

[…] This study is well executed and timely, and the message is clear and significant in contributing to our understanding of the functions of Yap1 during ESC differentiation and early development and of the balance control between survival and death during cell fate changes. While I am strongly supportive for its publication in eLife, I do have some major and minor points listed below that could further enhance the clarity and improve the manuscript. And the authors should pay more attention to the labeling in the figures and provide a few minimal additional experiments and more explanations to the experiment design to make the overall story more straightforward and clearer.

We are grateful for the reviewer’s comments and overall support of our manuscript. Following this feedback and that of other reviewers, we have performed additional experiments, reorganized several of our figures, and fixed the labeling to improve the flow and clarity of the story.

Major points:

1) At beginning the authors tested several mESC differentiation models (LIF withdraw, N2B27, and IDE1). However, it will be nicer if they also test cell apoptosis of WT/Yap1KO ESCs in EpiLC differentiation, because 1) EpiLC differentiation mimics the in vivo epiblast maturation at pre-to-post implantation stage, and 2) it is more related to the context of Yap1 and p300 ChIP-seq analysis as performed in Figure 3.

Thank you for this comment. We have performed not only the cell death assay during EpiLC differentiation (verification of markers shown in new Figure 2—figure supplement 1C), but we also repeated a couple of other experiments related to the various differentiation methods (RT-qPCR and JC-10 assay).

In EpiLC conditions, Yap1 KO dESCs experience significantly higher cell death, and this can be rescued almost completely by zVAD (new Figure 2D). They also have a similar defect in anti-apoptotic gene expression (modified Figure 3C) and increased mitochondrial depolarization during differentiation (new Figure 5A). Thus, even in EpiLC differentiation (which mimics epiblast formation), Yap1 is instrumental for cell survival.

2) Figure 1 shows the upregulation of Casp and apoptosis in Yap1 KO starts early (24-48 hrs) after LIF withdrawal, but Figure 2A and 2B show the upregulation of anti-apoptotic factors (BCl-2, BCl-xL and MCl-1) in WT ES starts late (72 hrs) after LIF withdrawal. BCl-2 is the regulator upstream of Casp. The authors should provide some explanations about these results.

We appreciate this feedback, as the issue of timing of expression differences vs. the activation of the apoptotic cascade does require more explanation. Although BCl-2 is critical for preventing caspase activation, we would like to emphasize that MCl-1 is also important. Even though Yap1 KO cells have only a mild (~50%) reduction in MCl-1 compared to WT ESCs, it is much more highly expressed than BCl-xL or BCl-2. Loss of MCl-1 leads to the death of undifferentiated ESCs (Huskey et al., 2015). Although the same authors contend that BCl-2 and BCl-xL gain in importance during later stages of differentiation, we reason that because apoptosis starts so early (24-48 hr after -LIF), MCl-1 is still essential for survival in early differentiation. This is supported by our BH3 mimetic data showing that ESCs and dESCs (at 48 hr) are exquisitely sensitive to MCl-1 inhibition (old data in current Figure 5C) and KD on d1 potentiates cell death in dESCs by d3 (old data in current Figure 6E).

To address whether MCl-1 plays a role in inhibiting caspase activation during early differentiation, after 1 day of -LIF, we acutely inhibited either MCl-1 using a selective inhibitor or BCl-2/BCl-xL/BCl-w using a different inhibitor in both WT and Yap1 KO cells. Treatment lasted for only 4 hours until harvest of protein lysates.

The results in new Figure 5F show that inhibition of MCl-1 strongly increases Casp3 cleavage in both WT and Yap1 KO ESCs. We confirmed Yap1 KO cells have less MCl-1 protein than WT cells do, even just 28 hr after -LIF, and they are much more sensitive to inhibition of BCl-2/BCl-xL/BCl-w than WT ESCs. This is logical, since all of these proteins collaborate at the mitochondrial outer membrane to prevent apoptosis, and they are known to compensate for one another in chemotherapy-related studies (Moujalled et al., 2018).

Thus, we contend that lower expression of the most abundant anti-apoptotic protein, MCl-1, in Yap1 KO cells contributes to an increase in caspase cleavage that is already apparent early in differentiation, even before the defect of BCl-2 expression is particularly noticeable.

3) In the Figure 1E, 1F and 2D, please indicate the time after LIF withdrawal (or the time of induced differentiation) in hours to better compare them with other results (like Figure 1D, 2A, 2B). I didn't find their time point information in Figure/Figure legends/Text/Experimental procedures.

We have added the appropriate time of differentiation for the aforementioned panels directly to the figures.

4) About Figure 3D and 3E: (1) the authors should at least provide one representation of the reporter construct;

As requested, we have provided a representation of the firefly reporter construct in new Figure 4—figure supplement 1G.

(2) the authors should indicate the regulatory elements (e.g., by using bars) used in the luciferase constructs in the Yap1 and p300 ChIP-seq tracks;

The grey bars currently on the ChIP-seq tracks do indicate the regulatory elements used in the luciferase constructs. However, since this is not indicated in the panel and the bars are extremely light, it is indeed confusing, and the width of the bars is much larger than the sequence actually used for cloning. Thus, we have slimmed down the grey bars, made them darker, and added a key to reduce any misinterpretation (modified Figure 4C).

(3) I am curious to see the expression level of Yap1 in Yap1 OE: was it rescued to a WT level or an overexpression?

We apologize for any confusion. We used WT ESCs in modified Figure 4F, so this would be an overexpression. Though we did not perform RT-qPCR to determine the fold change in Yap1 expression after 24 hr of transfection when the assay was performed, you may refer to new Figure 3—figure supplement 1C, where Yap1 OE was measured after 48 hr of transfection. There was a 16x increase in mRNA levels, which establishes an upper bound. We expect that the actual increase in mRNA levels during the luciferase assay would be quite a bit lower, as only 24 hr had elapsed after transfection, and the amount of DNA transfected was 1/3 of the normal amount, as 3 vectors were transfected simultaneously.

5) Figure 3data are clear but too shallow/superficial: According to the increased p300 (and H3K27ac) ChIP-seq intensities at Yap1 peaks during differentiation, it is implied that Yap1 may function as an activator of transcription for the anti-apoptotic genes BCl-2, BCl-xL, MCl-1. ChIP-qPCR are required to confirm the Yap1 binding at those loci (Figure 3C). Furthermore, authors should also compare the Yap1 and p300 ChIP intensity between ES and dES situations, to support their conclusion that Yap1 activates the anti-apoptotic genes during differentiation.

This is an excellent point that reviewer 1 also brought up. Please refer to Author response image 2 in comment 1-9. In sum, we in turn performed ChIP-seq and the data showed that both Yap1 and p300 occupancy increase on apoptosis-related genes during differentiation. ChIP-qPCR showed enrichment for BCl-2 and MCl-1. Collectively, our luciferase, RT-qPCR, immunoblot, ICC, and now ChIP-qPCR data all agree that Yap1 activates BCl-2 and MCl-1 upon differentiation, but we have less evidence for its direct regulation of the other apoptosis-related genes.

Does Yap1 physically interact with p300? If so, the authors may want to show if Yap1 recruits p300 to chromatin.

Immunoblot shows that pulldown of FLAGBio-Yap1 also brings down p300, showing that a physical interaction between the two factors does indeed exist during ESC differentiation (Figure 4—figure supplement 1B). Although we intended to do ChIP-qPCR of Yap1 KO cells during differentiation to test whether ablation of Yap1 reduced p300 recruitment to Yap1 peaks, it was difficult to obtain a sufficient quantity of cells during differentiation for ChIP, which requires a lot of starting material for the fixation step. We considered adding zVAD to boost survival and cell yield so that we could do ChIP on Yap1 KO dESCs, but we were concerned that this may have some unintended effects. Therefore, we did not test whether Yap1 actively recruits p300 to chromatin. However, it is known from previous literature investigating YAP1 occupancy in human SF268 glioblastoma cells that KD of Yap1 reduced p300 occupancy as well as H3K27ac at Yap1’s peaks (Stein et al., 2015). Our Co-IP is thus consistent with prior observations and it is likely that Yap1 recruits p300, although we did not explicitly test that hypothesis.

6) Figure 4C: the label is misleading. In the figure, the authors should indicate the Bmf KD and Puma KD are performed in Yap1 KO.

Thank you for pointing this out. We have additionally performed Bmf and Puma KD in WT ESCs (modified Figure 5D) in response to a comment brought up by reviewer 1 using the same shRNA constructs. We also have added the label to modified Figure 5—figure supplement 1B as suggested.

https://doi.org/10.7554/eLife.40167.035

Article and author information

Author details

  1. Lucy LeBlanc

    1. Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    2. Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, United States
    Contribution
    Conceptualization, Resources, Formal analysis, Funding acquisition, Validation, Investigation, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5945-3133
  2. Bum-Kyu Lee

    1. Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    2. Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, United States
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  3. Andy C Yu

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  4. Mijeong Kim

    1. Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    2. Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Aparna V Kambhampati

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  6. Shannon M Dupont

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  7. Davide Seruggia

    1. Division of Hematology/Oncology, Boston Children’s Hospital, Boston, United States
    2. Harvard Stem Cell Institute, Harvard Medical School, Boston, United States
    3. Department of Pediatric Oncology, Dana-Farber Cancer Institute (DFCI), Boston, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  8. Byoung U Ryu

    Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  9. Stuart H Orkin

    1. Division of Hematology/Oncology, Boston Children’s Hospital, Boston, United States
    2. Howard Hughes Medical Institute, Boston, United States
    3. Department of Pediatric Oncology, Dana-Farber Cancer Institute (DFCI), Boston, United States
    4. Harvard Stem Cell Institute, Harvard Medical School, Boston, United States
    Contribution
    Supervision, Investigation
    Competing interests
    No competing interests declared
  10. Jonghwan Kim

    1. Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    2. Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    jonghwankim@mail.utexas.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9919-9843

Funding

National Institute of General Medical Sciences (R01GM112722)

  • Jonghwan Kim

Burroughs Wellcome Fund

  • Jonghwan Kim

National Science Foundation (GRFP)

  • Lucy LeBlanc

Hamilton Seed Grant (Departmental Grant from Molecular Biosciences)

  • Lucy LeBlanc

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This study was supported by R01GM112722 (NIH) and the Preterm Birth Research Grant (Burroughs Welcome Fund) to JK, as well as the NSF GRFP and Hamilton Seed Grant to LL. We thank the Genome Sequencing and Analysis Facility (GSAF) and Texas Advanced Computing Center (TACC) at UT Austin for ChIP-seq analysis as well as the Center for Biomedical Research Support at UT Austin for flow cytometry and confocal microscopy.

Senior and Reviewing Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewer

  1. Jacob H Hanna, Weizmann Institute of Science, Israel

Publication history

  1. Received: July 17, 2018
  2. Accepted: December 17, 2018
  3. Accepted Manuscript published: December 18, 2018 (version 1)
  4. Version of Record published: December 27, 2018 (version 2)

Copyright

© 2018, LeBlanc et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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