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Canonical Wnt pathway controls mESCs self-renewal through inhibition of spontaneous differentiation via β-catenin/TCF/LEF functions

ABSTRACT: Canonical Wnt pathway controls mESCs self-renewal through inhibition of spontaneous differentiation via β-catenin/TCF/LEF functions

PROVIDER: PRJNA600166 | ENA |

REPOSITORIES: ENA

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ChIP-seq experiment of mouse embryonic stem cells (mESCs), assessing beta-catenin DNA binding in wild type mESCs and TCF/LEF quadruple knock-out mESCs.

Project description:Mouse embryonic stem cells (mESCs), derived from pre-implantation blastocyst cells, can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Many efforts have been devoted to identify genes that promote exit from the pluripotent state and the transition to a primed state of differentiation. One of the first identified players in this process was the Wnt/b-catenin effector TCF7L1 (previously referred to as TCF3), belonging to the family of four TCF/LEF transcription factors, which acts as pro-differentiation factor by repressing pluripotency genes. Of note, there is little evidence that the genetic abrogation of the mechanisms required for the exit from the pluripotent state is sufficient to enable self-renewal in the absence of 2iL. Here, we found that complete loss-of-function of Tcf7, Lef1, Tcf7l1 and Tcf7l2, the genes encoding for the four TCF/LEF transcription factors, (refered to as qKO) allows mESCs to become fully 2iL-independent and to propagate in basal N2B27. In addition to RNA-seq data deposited under accession number E-MTAB-10564, chromatin immunoprecipitation followed by deep DNA sequencing (ChIP-seq) has been performed in CHIR-stimulated mESCs. Here, the DNA binding landscape of β-catenin has been analyzed to assess its possible connection to TCF/LEF-mediated pluripotency.

2021-06-15 | E-MTAB-10575 | biostudies-arrayexpress

Canonical Wnt pathway controls mESCs self-renewal through inhibition of spontaneous differentiation via β-catenin/TCF/LEF functions

Project description:The Wnt/β-catenin signalling pathway is a key regulator of embryonic stem cell self-renewal and differentiation. Constitutive activation of this pathway has been shown to significantly increase mouse embryonic stem cell (mESC) self-renewal and pluripotency marker expression. In this study, we generated a novel β-catenin knock-out model in mESCs by using CRISPR/Cas9 technology to delete putatively functional N-terminally truncated isoforms observed in previous knock-out models. While we showed that aberrant N-terminally truncated isoforms are not functional in mESCS, we observed that canonical Wnt signalling is not active in mESCs, as β-catenin ablation does not alter mESC transcriptional profile in LIF-enriched culture conditions; on the other hand, Wnt signalling activation represses mESC spontaneous differentiation. We also showed that transcriptionally silent β-catenin (ΔC) isoforms can rescue β-catenin knock-out self-renewal defects in mESCs, cooperating with TCF1 and LEF1 in the inhibition of mESC spontaneous differentiation in a Gsk3 dependent manner.

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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.

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

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RNA-seq experiment of mouse embryonic stem cells (mESCs), comparing TCF/LEF quadruple knock-out cells with wild type cells

Project description:Mouse embryonic stem cells (mESCs), derived from pre-implantation blastocyst cells, can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Many efforts have been devoted to identify genes that promote exit from the pluripotent state and the transition to a primed state of differentiation. One of the first identified players in this process was the Wnt/b-catenin effector TCF7L1 (previously referred to as TCF3), belonging to the family of four TCF/LEF transcription factors, which acts as pro-differentiation factor by repressing pluripotency genes. Of note, there is little evidence that the genetic abrogation of the mechanisms required for the exit from the pluripotent state is sufficient to enable self-renewal in the absence of 2iL. Here, we found that complete loss-of-function of Tcf7, Lef1, Tcf7l1 and Tcf7l2, the genes encoding for the four TCF/LEF transcription factors, (refered to as qKO) allows mESCs to become fully 2iL-independent and to propagate in basal N2B27. To understand the genetic program that allows qKO cells to achieve 2iL-independent self-renewal, we performed RNA sequencing (RNA-seq) of qKO and wild type mESCs.

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ChIP-seq experiment of mouse embryonic stem cells (mESCs), assessing beta-catenin DNA binding in wild type mESCs and TCF/LEF quadruple knock-out mESCs.

Project description:ChIP-seq experiment of mouse embryonic stem cells (mESCs), assessing beta-catenin DNA binding in wild type mESCs and TCF/LEF quadruple knock-out mESCs.

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Genome-wide map of Tcf/Lef transcription factors, β-catenin, Six2 and chromatin marks in mouse nephron progenitor cells

Project description:During embryonic kidney development, nephron progenitor cells (NPCs) self-renew and differentiate into all cells in mature nephrons. The Wnt signaling components Wnt9b and β-catenin are required for both NPC self-renewal and differentiation. A low level of Wnt/β-catenin are associated with NPC self-renewal while a high level with differentiation. To investigate the transcriptional mechanisms behind Wnt/β-catenin-driven regulation of NPCs states, we modeled NPC self-renewal and differentiation in vitro with NPEM (Brown et al., 2015) supplemented with low (1.25 μM) or high (5 μM) concentration of CHIR22091 (CHIR), a small molecule GSK3β inhibitor that stabilizes β-catenin. We have found the Tcf/Lef family repressors Tcf7l1 and Tcf7l2 are enriched in low CHIR condition, while the activators Tcf7 and Lef1 in high CHIR condition, correlating with the NPC differentiation program transiting from being repressed to activated. To identify direct transcriptional targets of Tcf/Lef factors and β-catenin, here we generated ChIP-Seq data from primary NPC, as well as NPC cultured in low and high CHIR concentration.

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

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

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

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

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