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A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells.

Faunes F, Hayward P, Descalzo SM, Chatterjee SS, Balayo T, Trott J, Christoforou A, Ferrer-Vaquer A, Hadjantonakis AK, Dasgupta R, Arias AM - Development (2013)

Bottom Line: Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency.A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain.Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.

ABSTRACT
The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

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Wnt reporter activity during self-renewal and differentiation of mESCs. (A) TopFlash assay assessing Wnt/β-catenin transcriptional activity in mESCs (E14Tg2A) under self-renewing (serum+LIF, blue bars) and differentiating conditions (SRA, red bars), or HEK293T cells (green bars). Results are representative of two experiments and the average of three replicates (B,D) Confocal images of Wnt reporter lines grown in serum+LIF and exposed to indicated conditions 24 hours prior to fixation. TK215 (RFP fluorescent, B) and TLG2 (eGFP fluorescence, D) were stained with DAPI to show nuclei (B) or for β-catenin to visualize cell outlines (blue channels, D). (C) Flow cytometry profiles of Wnt reporter lines TK215, TLC2 and TLG2 grown in serum+LIF (blue population) or SRA (red population) for the indicated number of days. (E) Characterization of the Wnt transcriptional inhibitor ICRT3 in TK215 and TLC2 cells. The response of the reporter to the indicated conditions was analyzed by flow cytometry after 72 hours. The increase in fluorescence of cells grown in serum+LIF+C3 is due to an increase in autofluorescence (see supplementary material Fig. S3C).
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Figure 3: Wnt reporter activity during self-renewal and differentiation of mESCs. (A) TopFlash assay assessing Wnt/β-catenin transcriptional activity in mESCs (E14Tg2A) under self-renewing (serum+LIF, blue bars) and differentiating conditions (SRA, red bars), or HEK293T cells (green bars). Results are representative of two experiments and the average of three replicates (B,D) Confocal images of Wnt reporter lines grown in serum+LIF and exposed to indicated conditions 24 hours prior to fixation. TK215 (RFP fluorescent, B) and TLG2 (eGFP fluorescence, D) were stained with DAPI to show nuclei (B) or for β-catenin to visualize cell outlines (blue channels, D). (C) Flow cytometry profiles of Wnt reporter lines TK215, TLC2 and TLG2 grown in serum+LIF (blue population) or SRA (red population) for the indicated number of days. (E) Characterization of the Wnt transcriptional inhibitor ICRT3 in TK215 and TLC2 cells. The response of the reporter to the indicated conditions was analyzed by flow cytometry after 72 hours. The increase in fluorescence of cells grown in serum+LIF+C3 is due to an increase in autofluorescence (see supplementary material Fig. S3C).

Mentions: In a first set of experiments, we used Luciferase-based β-catenin/Tcf (TOP-FLASH) reporters, which have been shown to be activated by β-catenin and repressed by Tcf proteins (Merrill et al., 2001) (Fig. 3A). In ESCs, these reporters display the same low levels of activity without stimulation of Wnt signaling as they do in HEK293T cells. This was confirmed using a fluorescent reporter, TK215, which is inactive under self-renewal conditions (Fig. 3B). Stimulation of Wnt signaling, through Wnt3A or chiron, induces activity of both reporters in a concentration-dependent manner (Fig. 3A,B; supplementary material Fig. S3A). Under self-renewal conditions, it is not easy to activate the reporter; even with 6 μM chiron it is not possible to obtain levels of activation observed by simply transferring the cells into differentiation medium.


A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells.

Faunes F, Hayward P, Descalzo SM, Chatterjee SS, Balayo T, Trott J, Christoforou A, Ferrer-Vaquer A, Hadjantonakis AK, Dasgupta R, Arias AM - Development (2013)

Wnt reporter activity during self-renewal and differentiation of mESCs. (A) TopFlash assay assessing Wnt/β-catenin transcriptional activity in mESCs (E14Tg2A) under self-renewing (serum+LIF, blue bars) and differentiating conditions (SRA, red bars), or HEK293T cells (green bars). Results are representative of two experiments and the average of three replicates (B,D) Confocal images of Wnt reporter lines grown in serum+LIF and exposed to indicated conditions 24 hours prior to fixation. TK215 (RFP fluorescent, B) and TLG2 (eGFP fluorescence, D) were stained with DAPI to show nuclei (B) or for β-catenin to visualize cell outlines (blue channels, D). (C) Flow cytometry profiles of Wnt reporter lines TK215, TLC2 and TLG2 grown in serum+LIF (blue population) or SRA (red population) for the indicated number of days. (E) Characterization of the Wnt transcriptional inhibitor ICRT3 in TK215 and TLC2 cells. The response of the reporter to the indicated conditions was analyzed by flow cytometry after 72 hours. The increase in fluorescence of cells grown in serum+LIF+C3 is due to an increase in autofluorescence (see supplementary material Fig. S3C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585656&req=5

Figure 3: Wnt reporter activity during self-renewal and differentiation of mESCs. (A) TopFlash assay assessing Wnt/β-catenin transcriptional activity in mESCs (E14Tg2A) under self-renewing (serum+LIF, blue bars) and differentiating conditions (SRA, red bars), or HEK293T cells (green bars). Results are representative of two experiments and the average of three replicates (B,D) Confocal images of Wnt reporter lines grown in serum+LIF and exposed to indicated conditions 24 hours prior to fixation. TK215 (RFP fluorescent, B) and TLG2 (eGFP fluorescence, D) were stained with DAPI to show nuclei (B) or for β-catenin to visualize cell outlines (blue channels, D). (C) Flow cytometry profiles of Wnt reporter lines TK215, TLC2 and TLG2 grown in serum+LIF (blue population) or SRA (red population) for the indicated number of days. (E) Characterization of the Wnt transcriptional inhibitor ICRT3 in TK215 and TLC2 cells. The response of the reporter to the indicated conditions was analyzed by flow cytometry after 72 hours. The increase in fluorescence of cells grown in serum+LIF+C3 is due to an increase in autofluorescence (see supplementary material Fig. S3C).
Mentions: In a first set of experiments, we used Luciferase-based β-catenin/Tcf (TOP-FLASH) reporters, which have been shown to be activated by β-catenin and repressed by Tcf proteins (Merrill et al., 2001) (Fig. 3A). In ESCs, these reporters display the same low levels of activity without stimulation of Wnt signaling as they do in HEK293T cells. This was confirmed using a fluorescent reporter, TK215, which is inactive under self-renewal conditions (Fig. 3B). Stimulation of Wnt signaling, through Wnt3A or chiron, induces activity of both reporters in a concentration-dependent manner (Fig. 3A,B; supplementary material Fig. S3A). Under self-renewal conditions, it is not easy to activate the reporter; even with 6 μM chiron it is not possible to obtain levels of activation observed by simply transferring the cells into differentiation medium.

Bottom Line: Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency.A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain.Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.

ABSTRACT
The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

Show MeSH
Related in: MedlinePlus