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Wnt3a-Activin A Synergy Induces eRNAPII Pause-Release and Counteracts a Yap1 Elongation Block during hESC Differentiation

ABSTRACT: The Wnt3a/β-catenin and Activin/Smad2,3 signaling pathways synergize to induce endodermal differentiation of human embryonic stem cells, however the mechanism is not well-understood. Using ChIP-seq and GRO-seq analyses, we report here that hESC enhancers, including Wnt3a/LEF-1 sites, hold enhancer RNAPII complexes (eRNAPII) containing high levels of Ser5P and low Ser7P. In Wnt3a signaling, β-catenin recruits cohesin to the LEF-1:eRNAPII sites to induce enhancer-promoter looping and activate transcription of mesoendodermal (ME) genes. However, paused Ser5P-RNAPII complexes accumulate at these genes, indicating that elongation remains limiting. Subsequent Activin/Smad2,3 signaling increases P-TEFb occupancy, CTD-Ser7P, and productive elongation at ME genes. Additionally, ME genes, including EOMES and MIXL1, are repressed by the Hippo regulator, Yap1, an essential pluripotency factor. GRO-seq experiments indicate that Yap1 blocks nascent transcription and controls NELF occupancy on ME genes. Thus, Wnt3a/β-catenin and Activin/Smad2,3 pathways up-regulate transcription initiation and elongation, respectively, to overcome Yap1 repression during early hESC differentiation

ORGANISM(S): Homo sapiens

PROVIDER: GSE64758 | GEO | 2015/05/04

SECONDARY ACCESSION(S): PRJNA271823

REPOSITORIES: GEO

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Wnt3a-Activin A Synergy Induces eRNAPII Pause-Release and Counteracts a Yap1 Elongation Block during hESC Differentiation

Project description:The Wnt3a/?-catenin and Activin/Smad2,3 signaling pathways synergize to induce endodermal differentiation of human embryonic stem cells, however the mechanism is not well-understood. Using ChIP-seq and GRO-seq analyses, we report here that hESC enhancers, including Wnt3a/LEF-1 sites, hold enhancer RNAPII complexes (eRNAPII) containing high levels of Ser5P and low Ser7P. In Wnt3a signaling, ?-catenin recruits cohesin to the LEF-1:eRNAPII sites to induce enhancer-promoter looping and activate transcription of mesoendodermal (ME) genes. However, paused Ser5P-RNAPII complexes accumulate at these genes, indicating that elongation remains limiting. Subsequent Activin/Smad2,3 signaling increases P-TEFb occupancy, CTD-Ser7P, and productive elongation at ME genes. Additionally, ME genes, including EOMES and MIXL1, are repressed by the Hippo regulator, Yap1, an essential pluripotency factor. GRO-seq experiments indicate that Yap1 blocks nascent transcription and controls NELF occupancy on ME genes. Thus, Wnt3a/?-catenin and Activin/Smad2,3 pathways up-regulate transcription initiation and elongation, respectively, to overcome Yap1 repression during early hESC differentiation ChIP-seq and GROseq experiments in H1 hESCs. Cells were treated with Wnt3a (200ng/ml), Activin A (100ng/ml) or Wnt3a+Activin A (W200ng/ml+A100ng/ml) for 4h (ChIP-seq) or 6h (GRO-seq). GRO-seq in YAP depleted cells were carried out following transfection with control or YAP siRNAs . After 48h transfection, cells were left untreated or treated with Wnt3a+Activin (W200ng/ml+A100ng/ml) for additional 6h.

2015-05-04 | E-GEOD-64758 | biostudies-arrayexpress

Homo sapiens

Project description:Wnt3a-Activin A Synergy Induces eRNAPII Pause-Release and Counteracts a Yap1 Elongation Block during hESC Differentiation

| PRJNA271823 | ENA

TCF/LEF dependent and independent regulation of Wnt/beta-catenin transcription

Project description:During canonical Wnt signalling the activity of nuclear beta-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones simultaneously carrying loss-of-function alleles of all four TCF/LEF genes. Exploiting unbiased whole transcriptome sequencing studies, we found that a subset of beta-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that beta-catenin occupied specific genomic regions in the absence of TCF/LEF. Finally, we revealed the existence of a transcriptional activity of beta-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as beta-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of beta-catenin that bypasses the TCF/LEF transcription factors.

2018-11-22 | E-MTAB-7028 | biostudies-arrayexpress

TCF/LEF dependent and independent regulation of Wnt/beta-catenin transcription

2018-11-02 | E-MTAB-7029 | biostudies-arrayexpress

YAP Repression of the WNT3 Gene Controls hESC Differentiation Along the Cardiac Mesoderm Lineage

Project description:In hESCs, Wnt3/β-catenin activity is low and Activin/SMAD signaling ensures NANOG expression to sustain pluripotency. In response to exogenous Wnt3 effectors, Activin/SMADs switch to cooperate with β-catenin and induce mesendodermal differentiation genes. We show here that the HIPPO effector YAP binds to the WNT3 gene enhancer and prevents the gene from being induced by Activin in proliferating hESCs. In the absence of YAP, Activin signaling is sufficient to induce expression of the endogenous Wnt3 cytokine, which stabilizes β-catenin and selectively activates genes required for cardiac mesoderm (ME) formation. Interestingly, Activin-stimulated YAP-knockout hESCs strongly express β-catenin-dependent cardiac mesoderm markers (BAF60c and HAND1), but unlike WT hESCs, fail to express cardiac inhibitor genes (CDX2, MSX1). Accordingly, YAP-/- cells treated with Activin alone can differentiate efficiently to beating cardiomyocytes in culture, bypassing the need for sequential treatment with exogenous Wnt ligand and Wnt inhibitors. Similarly, Activin in combination with small-molecule YAP inhibitors generates beating cardiomyocytes from wild-type hESCs following a one- step protocol. Our findings highlight an unanticipated role of YAP as an upstream regulator of WNT3 to maintain hESC pluripotency in the presence of Activin, and uncover a direct route for the development of human embryonic cardiac mesoderm.

2018-01-08 | GSE99202 | GEO

Cell-line-specific gene expression changes following inactivation of the CTNNB1 gene in human colorectal cancer cell lines

Project description:Unrestrained transcriptional activity of β-CATENIN and its binding partner TCF7L2 frequently underlies colorectal tumor initiation and is considered an obligatory oncogenic driver throughout intestinal carcinogenesis. Yet, the TCF7L2 gene carries inactivating mutations in about 10 % of colorectal tumors and is non-essential in colorectal cancer (CRC) cell lines. To determine whether CRC cells acquire TCF7L2-independence through cancer-specific compensation by other T-cell factor (TCF)/lymphoid enhancer‑binding factor (LEF) family members, or rather lose addiction to β-CATENIN/TCF7L2-driven gene expression altogether, we generated multiple CRC cell lines entirely negative for TCF/LEF or β-CATENIN expression. Viability of these cells demonstrates complete β‑CATENIN- and TCF/LEF-independence, albeit one β-CATENIN-deficient cell line eventually became senescent. Absence of TCF/LEF proteins and β-CATENIN consistently impaired CRC cell proliferation, reminiscent of mitogenic effects of WNT/β-CATENIN signaling in the healthy intestine. Despite this common phenotype, β-CATENIN-deficient cells exhibited highly cell-line-specific gene expression changes with little overlap between β-CATENIN- and TCF7L2-dependent transcriptomes. Apparently, β‑CATENIN and TCF7L2 control sizeable fractions of their target genes independently from each other. The observed divergence of β-CATENIN and TCF7L2 transcriptional programs, and the finding that neither β-CATENIN nor TCF/LEF activity is strictly required for CRC cell survival has important implications when evaluating these factors as potential drug targets.

2022-12-13 | GSE199835 | GEO

Specifying the Anterior Primitive Streak by Modulating YAP1 Levels in Human Pluripotent Stem Cells

Project description:Specifying the primitive streak (PS) guides stem cell differentiation in vitro, however much remains to be learned about the transcription networks that direct anterior and posterior PS cells (APS and PPS, respectively) to differentiate to distinct mesendodermal subpopulations. Here, we show that APS genes are predominantly induced in YAP1-/- hESCs in response to ACTIVIN. This finding establishes the Hippo effector YAP1 as a master regulator of PS specification, functioning to repress ACTIVIN-regulated APS genes in hESCs. Moreover, transient exposure of wild-type hESCs to dasatinib, a potent C-SRC/YAP1 inhibitor, enables differentiation to APS-derived endoderm and cardiac mesoderm in response to ACTIVIN. Importantly, these cells can differentiate efficiently to normal beating cardiomyocytes without the cytoskeletal defect seen in YAP1-/- hESC-derived cardiomyocytes. Overall, we uncovered an induction mechanism to generate APS cells using a cocktail of ACTIVIN and YAP1i molecules that holds practical implications for hESC and iPSC differentiation into distinct mesendodermal lineages.

2018-11-14 | GSE121877 | GEO

Wnt signaling involved in mouse myoblast differentiation

Project description:Wnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However, both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown. Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation. These results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.

2011-07-15 | GSE30077 | GEO

Multiple regulatory intrinsically disordered motifs control FOXO4 transcription factor binding and function

Project description:Transcription factors harbour defined intrinsically disordered regulatory regions, which raises the question of how they mediate binding to structured co-regulators and how this regulates activity. Here, we present a detailed molecular regulatory mechanism of Forkhead box O4 (FOXO4) by the structured transcriptional co-regulator β-catenin. We find that the largely disordered FOXO4 C-terminal region, which contains its transactivation domain binds β-catenin through two defined interaction sites, and this is regulated by combined PKB/AKT- and CK1-mediated phosphorylation. Binding of β-catenin competes with the auto-inhibitory interaction of the FOXO4 disordered region with its DNA-binding forkhead domain, and thereby enhances FOXO4 transcriptional activity. Furthermore, we show that binding of the β-catenin inhibitor protein ICAT is compatible with FOXO4 binding to β-catenin, suggesting that ICAT acts as a molecular switch between anti-proliferative FOXO and pro-proliferative Wnt/TCF/LEF signalling. Together these data illustrate how the interplay of intrinsically disordered regions, post-translational modifications and co-factor binding contribute to transcription factor function. Highlights • The interaction network between FOXO4 and β-catenin was deciphered • FOXO4 auto-inhibition interferes with DNA binding and is counter-acted by β-catenin • FOXO4 exists in multiple conformations regulated by phosphorylation and co-factors • ICAT switches between FOXO4 and TCF/LEF transcription factors

2022-04-15 | PXD026263 | Pride

Inhibition of canonical Wnt/β-catenin signaling is involved in leflunomide (LEF)-mediated cytotoxic effects on renal carcinoma cells

Project description:Inhibition of canonical Wnt/β-catenin signaling is involved in leflunomide (LEF)-mediated cytotoxic effects on renal carcinoma cells

2016-03-31 | GSE77433 | GEO

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