In addition, although the absence of -catenin severely impaired upregulation upon Chiron treatment, it did not completely abrogate it, suggesting that downstream targets of GSK3 contribute to partially regulate its expression (Figure?2J)

In addition, although the absence of -catenin severely impaired upregulation upon Chiron treatment, it did not completely abrogate it, suggesting that downstream targets of GSK3 contribute to partially regulate its expression (Figure?2J). -Catenin Ablation Promotes a Weak Upregulation of Primitive Endoderm Genes According to previously published works, the canonical Epacadostat (INCB024360) Wnt pathway is active in mESCs (Ten Berge et?al., 2011) and -catenin nuclear translocation reinforces the pluripotency network by upregulating pluripotency genes such as (Martello et?al., 2012; Pereira et?al., 2006; Qiu et?al., 2015) mainly by inhibiting TCF3 repressive activity on their promoters (Pereira et?al., 2006). this study, we generated a novel -catenin knockout model in mESCs to delete putatively functional N-terminally Epacadostat (INCB024360) truncated isoforms observed in previous knockout models. We showed that aberrant N-terminally truncated isoforms are not functional in mESCs. In the generated knockout line, we observed that canonical Wnt signaling is not active, as -catenin ablation does not alter mESC transcriptional profile in serum/LIF culture conditions. In addition, we observed that Wnt signaling activation represses mESC spontaneous differentiation in a -catenin-dependent manner. Finally, -catenin (C) isoforms can rescue -catenin knockout self-renewal defects in mESCs cultured in serum-free medium and, albeit transcriptionally silent, cooperate with TCF1 and LEF1 to inhibit mESC spontaneous differentiation in a GSK3-dependent manner. (Lyashenko et?al., 2011; Wray et?al., 2011). Surprisingly, LIF dependency can be rescued by transcriptionally defective -catenin isoforms (C mutants), challenging the hypothesis that -catenin transcriptional activity could be relevant for mESC pluripotency and self-renewal (Lyashenko et?al., 2011; Wray et?al., 2011). More recently, however, it has been demonstrated that the most widely used inducible -catenin knockout alleles lead to the production of uncharacterized N-terminally truncated -catenin isoforms (N -cat) during pre-implantation embryo development (Messerschmidt et?al., 2016). Here, we confirmed the production of N -cat isoforms in mESCs and generated a novel -catenin full knockout model in mESCs using CRISPR/Cas9. We found that complete -catenin deletion produces similar Epacadostat (INCB024360) phenotypes observed in the previously described knockout models retaining N -cat fragments, suggesting that N-terminally truncated isoforms are biologically inactive. We furthermore analyzed the impact of -catenin loss at transcriptomic level, in presence or absence of GSK3 chemical inhibition. Our results show that the Wnt/-catenin pathway is not transcriptionally active in mESCs cultured in serum/LIF. However, upon GSK3 inhibition, we observed -catenin-dependent inhibition of differentiation markers, while the expression of pluripotency genes remained unchanged. Finally, we showed that transcriptionally impaired C-terminally truncated -catenin rescue isoforms (C -cat) can inhibit mESC differentiation in the absence of LIF when GSK3 is inhibited, as their full-length counterpart. However, this phenotype is impaired upon silencing of TCF1/LEF1, suggesting that C -cat isoforms are not entirely transcriptionally silent and their nuclear function could depend on TCF/LEF factors. Results Inducible -Catenin Knockout Alleles Generate N-Terminally Truncated Isoforms in mESCs -Catenin (sites encompassing exons 3C6 (and alleles, respectively, and stably express the CRE-ERT2. Full-length -catenin was successfully excised upon 4-hydroxytamoxifen (4OHT) treatment in both cell lines, and N -cat isoforms with a molecular weight of approximately 48 and 52?kDa, respectively, were detected upon CRE recombination (Figures 1B and 1C). Immunofluorescence staining did not show any clear subcellular localization of N isoforms, which instead appeared distributed among cytoplasm and nuclei showing little or no membrane localization (Figures 1D and 1E). Open in a separate window Figure?1 Inducible -Catenin Knockout Alleles Produce N-Terminally Truncated Isoforms in mESCs (A) Schematic representation of murine (alleles used for -catenin studies in mESCs. Black boxes represent exons, yellow boxes coding exons, dashed red lines indicate sites, and white boxes represent exons excised upon CRE-mediated recombination of sites. (B and C) Western blot (B) and relative quantification (C) of NLC1 and SR18 cell lines upon 72-h 4-hydroxytamoxifen treatment (+4OHT) and respective untreated controls (CTRs). SR18 untreated cell line is heterozygous for full-length -catenin deletion. Western blot band intensities (C) are normalized on NLC1 full-length CTTNB1. (D) -Catenin immunofluorescence staining on fixed SR18 or NLC1 parental cell lines or upon 72-h?+4OHT treatment. A primary antibody raised against the C-terminal portion of -catenin was used. DAPI was used to counterstain nuclei. Scale bar represents 50?m. (E) Multichannel fluorescence intensity PP2Bgamma measurement of immunofluorescence images in (D). Image quantification has been performed across the dashed yellow line depicted in (D), merge panel. (F) Schematic representation of short-hairpin targeted regions (red triangles, 1, 2, and 3) and qRT-PCR amplicons (blue lines, #1 and #2) along the allele. (G) Western blot of -catenin of mESCs harboring the.