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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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The state of β-catenin during self-renewal and differentiation in mESCs. (A) E14Tg2A cells were grown in self-renewing (serum+LIF) or differentiating conditions (SRA) for 4 days. Cell lysates were fractionated with concanavalin A to separate membrane-associated proteins (pellet) from the soluble proteins (soluble). Expression of proteins was assessed by western blot and a quantitative fluorescent system. Analysis of total β-catenin in western blots from whole extracts of mESCs under self-renewal conditions shows two pools: one specific - β-catenin; and a second, slower running, one, which has an unspecified component (Fig. 4A; supplementary material Fig. S4B). Quantification can be found in supplementary material Fig. S4C. (B) E14Tg2A cells grown in serum+LIF were stained for the different isoforms of β-catenin with specific antibodies (see text for details). (C) Mouse ESCs grown and processed as described in A, with the additional treatment consisting of exposure to the indicated conditions 4 hours prior to lysis. Quantifications of western blots are in supplementary material Fig. S6. (D) Confocal images of TLG cells grown in serum+LIF. Twenty-four hours prior to fixation, cells were exposed to L cell-conditioned medium to allow differentiation. Cells were stained for activated β-catenin (ABC) and Nanog. Cells that lack Nanog expression (arrows) elevate expression of the reporter and of ABC, which now can be observed in the nucleus. Scale bar: 50 μm.
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Figure 4: The state of β-catenin during self-renewal and differentiation in mESCs. (A) E14Tg2A cells were grown in self-renewing (serum+LIF) or differentiating conditions (SRA) for 4 days. Cell lysates were fractionated with concanavalin A to separate membrane-associated proteins (pellet) from the soluble proteins (soluble). Expression of proteins was assessed by western blot and a quantitative fluorescent system. Analysis of total β-catenin in western blots from whole extracts of mESCs under self-renewal conditions shows two pools: one specific - β-catenin; and a second, slower running, one, which has an unspecified component (Fig. 4A; supplementary material Fig. S4B). Quantification can be found in supplementary material Fig. S4C. (B) E14Tg2A cells grown in serum+LIF were stained for the different isoforms of β-catenin with specific antibodies (see text for details). (C) Mouse ESCs grown and processed as described in A, with the additional treatment consisting of exposure to the indicated conditions 4 hours prior to lysis. Quantifications of western blots are in supplementary material Fig. S6. (D) Confocal images of TLG cells grown in serum+LIF. Twenty-four hours prior to fixation, cells were exposed to L cell-conditioned medium to allow differentiation. Cells were stained for activated β-catenin (ABC) and Nanog. Cells that lack Nanog expression (arrows) elevate expression of the reporter and of ABC, which now can be observed in the nucleus. Scale bar: 50 μm.

Mentions: Crude fractionation with concanavalin A (ConA) of the extracts reveals that, under self-renewal conditions, the cytosolic fraction (soluble) contains little β-catenin and barely detectable amounts of the PS45 and ABC isoforms (Fig. 4A). However, the membrane-associated ConA-bound fraction (pellet) contains all forms of β-catenin, including ABC, and this is supported by the localization of β-catenin in immunostaining experiments (Fig. 4B). An association of ABC and PS45 with membranes has been described before in other cell lines (Maher et al., 2009; Maher et al., 2010) and might reflect the existence of a specific hub for their regulation. In these experiments, we find some Nanog and Oct4, but not Tcf3, in membrane-containing fractions (Fig. 4A). This localization of Oct4 is corroborated with a different and more-stringent membrane fractionation protocol (supplementary material Fig. S5). Activation of Wnt signaling leads to an increase in total β-catenin and ABC in the soluble fraction within 4 hours of the stimulation, an effect that is more prominent with chiron (Fig. 4C). During RA-induced differentiation, we observe a similar increase in cytosolic total β-catenin and ABC (Fig. 4A,C; supplementary material Fig. S4C). We also notice that the increases of β-catenin in the soluble pool induced by Wnt3A or chiron in differentiating cells are larger than those induced during self-renewal (Fig. 4C), reflecting the increased sensitivity of the reporters in differentiating conditions (see Fig. 3A).


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)

The state of β-catenin during self-renewal and differentiation in mESCs. (A) E14Tg2A cells were grown in self-renewing (serum+LIF) or differentiating conditions (SRA) for 4 days. Cell lysates were fractionated with concanavalin A to separate membrane-associated proteins (pellet) from the soluble proteins (soluble). Expression of proteins was assessed by western blot and a quantitative fluorescent system. Analysis of total β-catenin in western blots from whole extracts of mESCs under self-renewal conditions shows two pools: one specific - β-catenin; and a second, slower running, one, which has an unspecified component (Fig. 4A; supplementary material Fig. S4B). Quantification can be found in supplementary material Fig. S4C. (B) E14Tg2A cells grown in serum+LIF were stained for the different isoforms of β-catenin with specific antibodies (see text for details). (C) Mouse ESCs grown and processed as described in A, with the additional treatment consisting of exposure to the indicated conditions 4 hours prior to lysis. Quantifications of western blots are in supplementary material Fig. S6. (D) Confocal images of TLG cells grown in serum+LIF. Twenty-four hours prior to fixation, cells were exposed to L cell-conditioned medium to allow differentiation. Cells were stained for activated β-catenin (ABC) and Nanog. Cells that lack Nanog expression (arrows) elevate expression of the reporter and of ABC, which now can be observed in the nucleus. Scale bar: 50 μm.
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Related In: Results  -  Collection

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Figure 4: The state of β-catenin during self-renewal and differentiation in mESCs. (A) E14Tg2A cells were grown in self-renewing (serum+LIF) or differentiating conditions (SRA) for 4 days. Cell lysates were fractionated with concanavalin A to separate membrane-associated proteins (pellet) from the soluble proteins (soluble). Expression of proteins was assessed by western blot and a quantitative fluorescent system. Analysis of total β-catenin in western blots from whole extracts of mESCs under self-renewal conditions shows two pools: one specific - β-catenin; and a second, slower running, one, which has an unspecified component (Fig. 4A; supplementary material Fig. S4B). Quantification can be found in supplementary material Fig. S4C. (B) E14Tg2A cells grown in serum+LIF were stained for the different isoforms of β-catenin with specific antibodies (see text for details). (C) Mouse ESCs grown and processed as described in A, with the additional treatment consisting of exposure to the indicated conditions 4 hours prior to lysis. Quantifications of western blots are in supplementary material Fig. S6. (D) Confocal images of TLG cells grown in serum+LIF. Twenty-four hours prior to fixation, cells were exposed to L cell-conditioned medium to allow differentiation. Cells were stained for activated β-catenin (ABC) and Nanog. Cells that lack Nanog expression (arrows) elevate expression of the reporter and of ABC, which now can be observed in the nucleus. Scale bar: 50 μm.
Mentions: Crude fractionation with concanavalin A (ConA) of the extracts reveals that, under self-renewal conditions, the cytosolic fraction (soluble) contains little β-catenin and barely detectable amounts of the PS45 and ABC isoforms (Fig. 4A). However, the membrane-associated ConA-bound fraction (pellet) contains all forms of β-catenin, including ABC, and this is supported by the localization of β-catenin in immunostaining experiments (Fig. 4B). An association of ABC and PS45 with membranes has been described before in other cell lines (Maher et al., 2009; Maher et al., 2010) and might reflect the existence of a specific hub for their regulation. In these experiments, we find some Nanog and Oct4, but not Tcf3, in membrane-containing fractions (Fig. 4A). This localization of Oct4 is corroborated with a different and more-stringent membrane fractionation protocol (supplementary material Fig. S5). Activation of Wnt signaling leads to an increase in total β-catenin and ABC in the soluble fraction within 4 hours of the stimulation, an effect that is more prominent with chiron (Fig. 4C). During RA-induced differentiation, we observe a similar increase in cytosolic total β-catenin and ABC (Fig. 4A,C; supplementary material Fig. S4C). We also notice that the increases of β-catenin in the soluble pool induced by Wnt3A or chiron in differentiating cells are larger than those induced during self-renewal (Fig. 4C), reflecting the increased sensitivity of the reporters in differentiating conditions (see Fig. 3A).

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