1. Volume 16, Issue 6, Pages 639-652 (June 2015)
β-Catenin Regulates Primitive Streak Induction through Collaborative Interactions with SMAD2/SMAD3 and OCT4 
Nina S. Funa, Karen A. Schachter, Mads Lerdrup, Jenny Ekberg, Katja Hess, Nikolaj Dietrich, Christian Honoré, Klaus Hansen, Henrik Semb 
Cell Stem Cell 
Volume 16, Issue 6, Pages (June 2015)
DOI: /j.stem
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2. Cell Stem Cell 2015 16, 639-652DOI: (10.1016/j.stem.2015.03.008)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 WNT and NODAL Signaling Regulate PS Formation
(A) Diagram describing the differentiation protocol from hESC to DE.
(B) Cells transduced with a FopFlash- or TopFlash-luciferase reporter were treated as indicated. Experiments were performed in triplicate, and average values are presented.
(C) Expression of β-catenin targets AXIN2 and LEF1 was analyzed at different time points after CHIR treatment and normalized to undifferentiated cells (hES).
(D and E) Cells treated for 24 hr as indicated. Gene expression for NODAL, LEFTY (D), T, MIXL1, and EOMES (E) was analyzed at different time points by qPCR and normalized to undifferentiated cells (hES).
(F and G) Cells were treated for 24 hr as indicated and immunostained for T (F) and SMAD2 (G). Scale bar, 25 μm.
(H) Cells were treated as indicated, and lysates were analyzed by immunoblot. Data are representative of three independent experiments.
(I) Cells transfected with siRNA pools for β-catenin, SMAD2, or a scrambled control (scrb) were treated with CHIR for 24 hr. Gene expression was analyzed by qPCR. Data were normalized to CHIR-treated scrb-transfected cells.
(J) Cells transfected with siRNA pools for β-catenin or a scrambled control (scrb) were treated with RPMI or 100 ng/ml AA for 3 days and analyzed as described above.
Data in this figure are presented as mean ± SEM of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p < See also Figures S1 and S2.
Cell Stem Cell  , DOI: ( /j.stem )
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4. Figure 2
β-Catenin Binds Directly to Regulatory Regions in the PS Genes
Cells treated with CHIR or CHIR+SB or mock treated (RPMI) for 6 hr were subjected to ChIP-seq analysis for β-catenin.
(A) Genomic distribution of binding sites as a percentage of the total peaks obtained in each condition.
(B) University of California, Santa Cruz (UCSC) tracks were generated for EOMES, MIXL1, and NODAL from the β-catenin ChIP-seq.
(C) Gene Ontology analysis for processes in which β-catenin-bound genes are enriched in cells treated with CHIR and CHIR+SB.
(D) Cells treated as indicated for 19 hr were subjected to ChIP with a β-catenin and isotype IgG control antibody. Enrichment at the indicated β-catenin binding sites in NODAL, EOMES, and MIXL1 was analyzed by qPCR and normalized to input. Data are presented as fold enrichment relative to an unbound control region.
Cell Stem Cell  , DOI: ( /j.stem )
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5. Figure 3
β-Catenin-Driven Expression of PS Genes Is Dependent on SMAD2/SMAD3
(A and B) Cells treated as indicated for 19 hr were subjected to ChIP with a SMAD2/SMAD3 and isotype IgG control antibody. Cells transfected with siRNA pools for β-catenin were treated with CHIR 48 hr after transfection and processed as the rest. Enrichment at the indicated β-catenin binding sites in NODAL, EOMES, and MIXL1 (A) and at the indicated SBE sites (B) was analyzed by qPCR and normalized to input. Data are presented as fold enrichment relative to an unbound control region.
(C) Cells were treated as indicated. Cellular lysates were immunoprecipitated with a SMAD2 or IgG control antibody. The presence of β-catenin, pSMAD2, and total SMAD2 in the precipitates was analyzed by western blot. Input levels are shown as controls on the right side. The data are representative of three independent experiments.
(D) Cells were treated for 19 hr as indicated. β-catenin ChIP was performed followed by ChIP with SMAD2/SMAD3 or isotype IgG control antibody. Enrichment at the MIXL1 −13 kb locus was determined by qPCR and normalized to input. Data are presented as fold enrichment relative to an unbound control region.
Data in this figure are presented as mean ± SEM of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p <
Cell Stem Cell  , DOI: ( /j.stem )
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6. Figure 4 Suppression of NODAL Signaling Allows WNT-Induced NCp Fate
(A) UCSC tracks were generated for GBX2, TFAP2A, and MSX1 from the β-catenin ChIP-seq.
(B) Cells were treated as indicated, and gene expression was analyzed at different time points by qPCR. Data were normalized to undifferentiated cells (hES).
(C and D) Cells transfected with siRNA pools for β-catenin, SMAD2, NODAL, or a scrambled control (scrb) were treated with CHIR for 24 hr. Gene expression was analyzed by qPCR. Data were normalized to CHIR-treated scrb-transfected cells.
(E) Cells were treated as indicated. Gene expression was analyzed at different time points by qPCR. Data were normalized to undifferentiated cells (hES).
(F) Cells were treated as indicated for 24 hr and immunostained for T and TFAP2A. Scale bar, 50 μm.
Cell Stem Cell  , DOI: ( /j.stem )
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7. Figure 5 OCT4 Promotes PS Gene Induction
(A) Putative TCF/LEF and OCT4 binding sequences identified by motif enrichment analysis in the β-catenin peaks.
(B) Histograms showing the distribution of distances from each β-catenin peak (left histogram) or randomized control region (right histogram) to the nearest peak in previously published OCT4 ChIP-seq data (GSM539547).
(C) Cells were treated with CHIR for 19 hr and subjected to ChIP with an OCT4 or isotype IgG control antibody. Enrichment at the indicated β-catenin binding sites in NODAL, EOMES, and MIXL1 was analyzed by qPCR and normalized to input. Data are presented as fold enrichment relative to an unbound control region.
(D) Cells were treated for 19 hr as indicated. β-catenin ChIP was performed followed by ChIP with OCT4 or isotype IgG control antibody. Enrichment at the indicated loci was determined by qPCR and normalized to input. Data are presented as fold enrichment relative to an unbound control region.
(E) Cells were transfected with siRNA pools for OCT4 or a scrambled control (scrb). Twelve hours after transfection, cells were treated with RPMI or CHIR for 24 hr, and lysates were analyzed by immunoblot.
(F) Cells were transfected as in (E) and treated for 24 hr as indicated. Expression of markers for PS was analyzed by qPCR and normalized to scrb-transfected control cells.
Data in this figure are presented as mean ± SEM of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p <
Cell Stem Cell  , DOI: ( /j.stem )
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8. Figure 6 Graded NODAL Signaling Modulates Fate of PS-like Cells
(A–C) Cells were treated with CHIR for 24 hr, followed by various concentrations of AA or SB for an additional 48 hr as indicated. Gene expression for markers of DE (A), PS (B), and NCp (C) was analyzed by qPCR and normalized to D1 CHIR-treated cells.
(D and E) Cells were treated as indicated and co-immunostained for T/SOX17 (D) and T/TFAP2A (E). Scale bars, 100 μm (left) and 50 μm (right).
Data in this figure are presented as mean ± SEM of at least three independent experiments.∗∗p < and ∗∗∗p < See also Figure S3.
Cell Stem Cell  , DOI: ( /j.stem )
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9. Figure 7
Mechanism for the Regulation of Wnt-Induced Early Lineage Choices
Under pluripotency conditions, OCT4 and SMAD2 co-occupy upstream regulatory regions on PS genes. CHIR treatment promotes stabilization β-catenin, which is recruited to regulatory regions in PS and NCp genes. Initially, activation of Wnt signaling induces expression of NCp. Binding of β-catenin, SMAD2/SMAD3, and OCT4 to chromatin promotes a sustained increase in NODAL levels and SMAD2/SMAD3 activity, resulting in downregulation of NCp gene expression and induction of PS genes. See text for more details.
Cell Stem Cell  , DOI: ( /j.stem )
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