Int J Stem Cells.  2020 Jul;13(2):192-201. 10.15283/ijsc20044.

The Distinct Role of Tcfs and Lef1 in the Self-Renewal or Differentiation of Mouse Embryonic Stem Cells

• Kim S
• Kim H
• Tan A
• Song Y
• Lee H
• Ying QL
• Jho Eh

• KMID: 2504332
• DOI: http://doi.org/10.15283/ijsc20044

Abstract

Background and Objectives
Tcfs and Lef1 are DNA-binding transcriptional factors in the canonical Wnt signaling pathway. In the absence of β-catenin, Tcfs and Lef1 generally act as transcriptional repressors with co-repressor proteins such as Groucho, CtBP, and HIC-5. However, Tcfs and Lef1 turn into transcriptional activators during the interaction with β-catenin. Therefore, the activity of Tcfs and Lef1 is regulated by β-catenin. However, the intrinsic role of Tcfs and Lef1 has yet to be examined. The purpose of this study was to determine whether Tcfs and Lef1 play differential roles in the regulation of self-renewal and differentiation of mouse ES cells.
Methods and Results
Interestingly, the expression of Tcfs and Lef1 was dynamically altered under various differentiation conditions, such as removal of LIF, EB formation and neuronal differentiation in N2B27 media, suggesting that the function of each Tcf and Lef1 may vary in ES cells. Ectopic expression of Tcf1 or the dominant negative form of Lef1 (Lef1-DN) contributes to ES cells to self-renew in the absence of leukemia inhibitory factor (LIF), whereas ectopic expression of Tcf3, Lef1 or Tcf1-DN did not support ES cells to self-renew. Ectopic expression of either Lef1 or Lef1-DN blocked neuronal differentiation, suggesting that the transient induction of Lef1 was necessary for the initiation and progress of differentiation. ChIP analysis shows that Tcf1 bound to Nanog promoter and ectopic expression of Tcf1 enhanced the transcription of Nanog.
Conclusions
The overall data suggest that Tcf1 plays a critical role in the maintenance of stemness whereas Lef1 is involved in the initiation of differentiation.

Keyword

Tcf/Lef1; Wnt; ES cell; Self-renewal; Differentiation

Figure

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  Fig. 1 The pattern of Tcfs and Lef1 expression in ES cells. (A) A6P10 ES cells were differentiated in the absence of LIF (left), in N2B27 medium without LIF (middle), or via EB formation without LIF (right). The differentiated ES cells were harvested on the days indicated. The amount of Tcfs and Lef1 transcripts was measured by RT-PCR analysis. The bands for Tcfs and Lef1 represent wild-type transcripts including β-catenin-binding domain. Self-renewal marker (Nanog), neural precursor markers (Sox1 and Fgf5) for N2B27 differentiation and mesodermal markers (Brachyury, Nkx2.5, αMHC) for EB differentiation were used to confirm proper differentiation; 18s rRNA was used as a loading control. (B) The endogenous protein levels of Tcfs/Lef1 were detected by western blotting. β-actin was used as a loading control.

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  Fig. 2 The effect of ectopic expression of Tcfs and Lef1 after LIF depletion. (A) A schematic diagram of Tcfs and Lef1 constructs (FL, full length; and DN, dominant negative). (B, C) A6P10 ES cells were transfected with control vector, Tcf1 (FL or DN), Lef1 (FL or DN), Tcf3 (FL or DN), or Tcf4 (FL or DN) and then selected using Zeocin. Selected ES cells were stained with alkaline phosphatase (AP), a marker of self-renewal, on the days indicated after LIF removal.

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  Fig. 3 Ectopic expression of Tcf1 maintains self-renewal and delays differentiation of ES cells. (A) mRNA expressions for Oct4, Sox2 and Nanog at ES cells stage and Day 4 of differentiation upon TCF1 overexpression were determined by using qPCR. RNA was isolated from cells expressing empty vector (EV) or Tcf1-FL (TCF1) on specified stage. GAPDH was used for normalization. (B) The amount of Nanog transcript was measured by RT-PCR analysis. RNA was isolated from ES cells expressing control vector or Tcf1-FL on the days specified. 18s rRNA was used as a loading control. (C) ChIP assay was performed to examine the binding of Tcf1, Lef1, and Tcf3 on Nanog promoter. Samples for ChIP were isolated from ES cells grown with LIF and from ES cells expressing control vector or Tcf1-FL cultured for 2 days without LIF. For immunoprecipitiation, endogenous antibody against Tcf1, Lef1, or Tcf3 was used. (D) To differentiate into neural precursors, 46C ES cells expressing control vector, Tcf1-FL, or Tcf1-DN were plated for 6 days in N2B27 medium. GFP fluorescence showed that 46C ES cells expressing control vector or Tcf1-DN, but not Tcf1-FL effectively differentiated into neural precursor (upper panel). The amount of Nanog or Sox1 transcript on the days indicated was measured via RT-PCR analysis (lower panel). β-actin was used as a loading control. (E) RNA samples were isolated from A6P10 ES cells expressing control vector or Tcf1-FL on the days indicated after EB formation. Self-renewal (Nanog), mesoderm (Brachyury), and cardiomyocyte (Islet1, αMHC) markers were used for RT-PCR analysis.

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  Fig. 4 Transient expression of Lef1 is required for proper differentiation. (A) A6P10 ES cells were infected with retroviruses expressing control or Lef1 shRNA, followed by selection using puromycin. After 2 days without LIF, the selected ES cells were harvested and analyzed by western blotting with anti-Lef1 antibody. β-actin was used as a loading control. (B) 46C ES cells expressing control shRNA or Lef1 shRNA were plated for 7 days in N2B27 medium. GFP fluorescence indicating a neural precursor was not induced in shLef1-46C cells (left panel). The amount of Nanog or Sox1 transcript was measured by RT-PCR analysis (right panel). (C) 46C ES cells were transfected with control vector, Lef1-FL, or Lef1-DN and then selected using Zeocin. 46C ES cells expressing each vector were plated for 7 days in N2B27 medium and GFP fluorescence was captured. (D) RNA samples were isolated from A6P10 ES cells expressing shGFP or shLef1 on the days indicated post-EB formation. Cardiomyocyte (α-MHC) and endoderm (GATA4) markers and loading control (18s rRNA) were used for RT-PCR analysis.

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  Fig. 5 A model describing the role of Tcfs and Lef1s in ES cells.

Reference

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