SMADs and YAP Compete to Control Elongation of β-Catenin:LEF-1-Recruited RNAPII during hESC Differentiation  Conchi Estarás, Chris Benner, Katherine A. Jones  Molecular Cell  Volume 58, Issue 5, Pages 780-793 (June 2015) DOI: 10.1016/j.molcel.2015.04.001 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 58, 780-793DOI: (10.1016/j.molcel.2015.04.001) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Analysis of β-Catenin Binding to LEF-1 Enhancers in Wnt3a-Signaling hESCs (A) Venn diagrams showing the number of β-catenin and LEF-1 overlapping peaks in untreated and Wnt3a-treated (200 mg/ml, 4 hr) H1 hESCs. (B) Genomic distribution of LEF-1 peaks in hESCs. (C) Heatmaps show the density of ChIP-seq reads for LEF-1, β-catenin, total RNAPII, Ser5P-, or Ser7P-RNAPII in untreated and Wnt3a-treated H1 hESCs, across a 2 Kb region centered on LEF-1 peaks. (D) Histogram showing the effect of Wnt3a treatment on H3K27ac and H3K27me3 levels around the activated LEF-1 enhancers (≥3-fold). (E) Genome browser captures show examples of the ChIP-seq binding profiles at specific ME genes, identified at the top, covering at least one putative LEF-1 enhancer nearest to the gene. Gene diagrams and scale bars are depicted above and below the captures, respectively. Immunoprecipitated proteins and cell treatments are shown on the left. Dashed boxes highlight the LEF-1 enhancer peaks. See also Figure S1. Molecular Cell 2015 58, 780-793DOI: (10.1016/j.molcel.2015.04.001) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 RNAPII Complexes at LEF-1 Sites and hESC Enhancers Are Enriched in CTD-Ser5P, but Not -Ser7P (A) Metaprofile of Ser5P- and Ser7P-RNAPII distribution on LEF-1 enhancers, other hESC (H3K4me1+/H3K4me3−) enhancers, and genes. (B) Genome browser capture shows examples of the ChIP-seq binding profiles of the indicated proteins (at left) on the pluripotency gene PRDM14. Scale bar and gene diagram are depicted above and below the capture, respectively. The dashed boxes highlight active enhancer and promoter regions, as indicated at the top. (C) Heatmap ordered by the ratio of Ser5P:Ser7P-RNAPII. The distribution of RNAPII forms and chromatin marks are shown and indicated above each map. Right, GRO-seq heatmap shows transcription from positive (sense strand; red) and negative (antisense strand; blue) strands. Left, Ser5P+,Ser7P−-RNAPII complexes (eRNAPII) are located predominantly in intergenic regions and overlap with sequence motifs shown at the left. (D) Scatterplot showing inverse correlation between Ser5P:Ser7P RNAPII ratio and nascent transcription genome-wide, measured by GRO-seq reads. Pearson correlation coefficient and p value are shown in the graph. Scheme below shows that eRNAPII associates with high Ser5P:Ser7P ratio and low transcription. See also Figure S2. Molecular Cell 2015 58, 780-793DOI: (10.1016/j.molcel.2015.04.001) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 GRO-seq Analysis of Wnt3a-Induced Genes in hESCs (A) Cumulative distribution frequency of LEF-1 activated peaks in Wnt3a regulated genes and all the other genes in hESCs. (B) Examples of GRO-seq profiles in untreated and Wnt3a-treated H1 hESCs (200 ng/ml, 6 hr). Sense (+) and antisense (−) DNA strands are depicted. Scale bars are shown above the GRO-seq profiles. The β-catenin peaks mark potential nearby gene enhancers. (C) Metaprofile of nascent transcription from LEF-1 activated enhancers (eRNAs) in untreated and Wnt3a stimulated hESCs. (D) The activity of the EOMES, MIXL1, NODAL, and KLF5 promoter region was assessed alone (P, see plasmid graph), or together with a LEF-1 enhancer (∗; PE) in transfection assays with a luciferase reporter. The graph plots luciferase activity normalized to Renilla (from three independent replicates in untreated (−) or Wnt3a (+)-treated hESCs (200 ng/ml, 9 hr). (E) Immunoblot showing LEF-1 and β-catenin protein levels in hESCs, mesendodermal (ME) cells, and ectodermal (EC) precursor cells. Specific markers for hESCs (OCT4), ME cells (GATA6), and EC cells (SOX1) are shown. The DDX39 blot was used as a loading control. A diagram of the protocol used for EM and EC differentiation (days: 2 days, 3 days) is shown at the top. The graph depicts normalized luciferase activity from three independent replicates of the PE constructs (from part D) in the ME and EC precursor cells. See also Figure S3. Molecular Cell 2015 58, 780-793DOI: (10.1016/j.molcel.2015.04.001) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 β-Catenin Recruits Cohesin and Induces long-Range Enhancer-Promoter Looping in Wnt3a-Signaling hESCs (A) Heatmaps showing ChIP-seq read density of NIPBL and β-catenin at LEF-1 peaks in response to Wnt3a (200 ng/ml; 4 hr) in H1 hESCs. (B) Coimmunoprecipitation of endogenous β-catenin and NIPBL proteins in untreated and Wnt3a-signaling hESCs. (C) Genome browser image of the NIPBL and β-catenin binding profiles at the MIXL1 and EOMES loci in untreated and Wnt3a-treated hESCs. Below, 3C experiment was done in untreated or Wnt3a-treated hESCs. The diagram below each graph indicates the position of the primers (arrows indicate orientation) and the probe (green star). Blue and yellow boxes highlight upstream enhancers and the promoter, respectively. Mean (SD; n = 2). (D) Graphs on the right show relative mRNA levels of several Wnt3a induced genes in NIPBL, SMC3 and MED26 siRNA transfected cells. The immunoblot on the left shows the knockdown efficiency for each siRNA. Mean (SD; n = 2–3). See also Figure S4. Molecular Cell 2015 58, 780-793DOI: (10.1016/j.molcel.2015.04.001) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Analysis of Wnt-Activin Synergy at ME Differentiation Genes (A) Scheme showing signaling pathways interplay during early endoderm differentiation. (B) Left, immunofluorescence experiment showing GATA6 and OCT4 levels in hESCs treated with a GSK3 inhibitor (GSK3i, 50 nM) and Activin A (50 ng/ml), alone or together for 48 hr. DAPI stain marks nuclear DNA. Right panel, mRNA levels at OCT4 and GATA6 genes in response to signaling, as indicated. (C) Analysis of MIXL1 and EOMES mRNA levels in response to Activin A, Wnt3a, or combination of both cytokines (6 hr). The maximum fold change for each treatment is indicated above the bars. Mean (SD; n = 2). (D) Top, ChIP-seq binding profile of the indicated proteins (at left). Below, GRO-seq profiles in hESCs that were untreated, or exposed to Wnt3a (200 ng/ml), Activin A (100 ng/ml), or Wnt3a plus Activin A (W200A100) for 6 hr. Gene diagrams are depicted at the bottom. Scale bars and SMAD:FOXH1 and β-catenin sites are indicated at the top. To simplify, only the coding strand (marked as + or −) is depicted. (E) Metaprofile showing normalized ChIP-seq read counts of NIPBL and Ser5P-RNAPII within 1 Kb from SMAD2,3 and β-catenin peaks in response to the indicated treatments. (F) Following siRNA transfection (48 hr), hESCs were treated with Wnt + Activin (W200A100, 4 hr). The immunoblot shows the effects of β-catenin- and SMAD2,3-siRNAs on endogenous protein levels. The top graph shows MIXL1 mRNA levels in the transfected cells, and the bottom graph shows ChIP-qPCR at the MIXL1 gene, using the indicated antibodies. The positions of the primers are shown in the bottom diagram. Mean (SD; n = 2). See also Figure S5 and Table S1. Molecular Cell 2015 58, 780-793DOI: (10.1016/j.molcel.2015.04.001) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 Activin/SMAD2,3 Signaling Stimulates P-TEFb Elongation at ME Genes (A) ChIP-seq binding profile of the indicated proteins (left) in untreated, Wnt3a (200 ng/ml)-, Activin A (100 ng/ml)-, or Wnt3a plus Activin A (W200A100)-treated hESCs (4 hr). Gene diagram and scale bar are depicted at the bottom. (B) Left, ChIP-seq metaprofile for Ser5P- and Ser7P-RNAPII within 1 Kb of the TSS for the 189 Wnt-Activin coregulated genes. Right, ChIP-qPCR analysis of P-TEFb/CDK9 levels at the MIXL1 and EOMES genes in response to signaling, as indicated. Mean (SD; n = 2–3). See also Figure S6. Molecular Cell 2015 58, 780-793DOI: (10.1016/j.molcel.2015.04.001) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 Wnt-Activin Synergy Overcomes YAP Repression at ME Genes (A) Left panels show immunoblot analysis of hESCs transfected with control (siC) or YAP-, TAZ-, or TEAD4-specific siRNAs for 48 hr. Right graphs show RT-qPCR analysis of EOMES, MIXL1, and T/Brachyury mRNA levels in untreated or Wnt-Activin (W200A100) cotreated hESCs exposed to the different siRNAs, as indicated. Mean (SD; n = 3). (B) GRO-seq analysis in YAP-depleted H1 hESCs. Following control or YAP siRNA transfection (48 hr), cells were either left untreated or stimulated with W200A100 for 6 hr. Captures show the levels of nascent transcription at four Wnt-Activin coregulated genes, one pluripotency gene (POU5F1), and canonical target genes (AXIN2, LEFTY1) that are not coregulated by Wnt3a+Activin A. Note that YAP depletion predominantly affects nascent transcription at the coregulated genes. Scale bars and gene diagrams are depicted at top and at the bottom, respectively. (C) RNAi-ChIP analysis in YAP-depleted hESCs. Left, diagram shows the SMAD2,3 and YAP sites at the MIXL1 and EOMES genes. Following siRNA transfection (48 hr), cells were treated with W200A100 (4 hr) prior to ChIP-qPCR analysis of the MIXL1 and EOMES genes, using the antibodies indicated above each graph. The positions of the primers are indicated below each graph. Mean (SD; n = 2). (D) Box diagram shows the Ser5P:Ser7P ratio at SMAD and LEF-1 sites in the absence (−) or presence (+) of TEAD4 cobinding peaks. (E) Metaprofile showing Ser5P-RNAPII levels in Activin A-treated hESCs within 1 Kb of SMAD2,3 peaks alone (blue) or at composite SMAD:TEAD peaks (orange). (F) Diagram showing that YAP counteracts SMAD2,3 activity at ME genes in self-renewing hESCs. See also Figure S7. Molecular Cell 2015 58, 780-793DOI: (10.1016/j.molcel.2015.04.001) Copyright © 2015 Elsevier Inc. Terms and Conditions