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Temporal perturbation of the Wnt signaling pathway in the control of cell reprogramming is modulated by TCF1.

ABSTRACT: Cyclic activation of the Wnt/?-catenin signaling pathway controls cell fusion-mediated somatic cell reprogramming. TCFs belong to a family of transcription factors that, in complex with ?-catenin, bind and transcriptionally regulate Wnt target genes. Here, we show that Wnt/?-catenin signaling needs to be off during the early reprogramming phases of mouse embryonic fibroblasts (MEFs) into iPSCs. In MEFs undergoing reprogramming, senescence genes are repressed and mesenchymal-to-epithelial transition is favored. This is correlated with a repressive activity of TCF1, which contributes to the silencing of Wnt/?-catenin signaling at the onset of reprogramming. In contrast, the Wnt pathway needs to be active in the late reprogramming phases to achieve successful reprogramming. In conclusion, continued activation or inhibition of the Wnt/?-catenin signaling pathway is detrimental to the reprogramming of MEFs; instead, temporal perturbation of the pathway is essential for efficient reprogramming, and the "Wnt-off" state can be considered an early reprogramming marker.

SUBMITTER: Aulicino F

PROVIDER: S-EPMC4050487 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Temporal perturbation of the Wnt signaling pathway in the control of cell reprogramming is modulated by TCF1.

Aulicino Francesco F Theka Ilda I Ombrato Luigi L Lluis Frederic F Cosma Maria Pia MP

Stem cell reports 20140508 5

Cyclic activation of the Wnt/β-catenin signaling pathway controls cell fusion-mediated somatic cell reprogramming. TCFs belong to a family of transcription factors that, in complex with β-catenin, bind and transcriptionally regulate Wnt target genes. Here, we show that Wnt/β-catenin signaling needs to be off during the early reprogramming phases of mouse embryonic fibroblasts (MEFs) into iPSCs. In MEFs undergoing reprogramming, senescence genes are repressed and mesenchymal-to-epithelial transit ...[more]

PMID: 24936456

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Project description:BACKGROUND:Clusterin (CLU) is an enigmatic molecule associated with various physiological processes and disease states. Different modes of cellular stress lead to increased CLU levels, and additionally numerous growth factors and cytokines affect the expression of the CLU gene. APC and c-MYC, both intimately linked to the Wnt signaling pathway have previously been shown to influence CLU levels, and we therefore investigated if changes in Wnt signaling activity in vitro could regulate the expression of one, or more, of several CLU mRNA and protein variants. RESULTS:Over-expression of the cytoplasmic domain of E-cadherin tagged with GFP was used to abrogate Wnt signaling activity in LS174T and HCT116 colon carcinoma cells. This fusion construct sequestered signaling competent beta-catenin whereby Wnt signaling was abrogated, and consequently cytoplasmic CLU protein levels increased as demonstrated by immunofluorescence. To determine which branch of the Wnt pathway was mediating the CLU response, we over-expressed dominant negative (dn) TCF1 and TCF4 transcription factors in stably transfected LS174T cells. We observed both intra- and extracellular levels of CLU protein to be induced by dnTCF1 but not dnTCF4. Subsequent analysis of the expression levels of three CLU mRNA variants by real time RT-PCR revealed only one CLU mRNA variant to be responsive to dnTCF1 over-expression. 5'-end RACE indicated that this CLU mRNA variant was shorter at the 5'-end than previously reported, and accordingly the translated protein was predicted to be shorter at the N-terminus and destined to the secretory pathway which fit our observations. Examination of the immediate expression kinetics of CLU after dnTCF1 over-expression using real time RT-PCR indicated that CLU might be a secondary Wnt target. CONCLUSION:In conclusion, we have demonstrated that the Wnt signaling pathway specifically regulates one out of three CLU mRNA variants via TCF1. This CLU transcript is shorter at the 5' end than reported by the RefSeq database, and produces the intracellular 60 kDa CLU protein isoform which is secreted as a ~80 kDa protein after post-translational processing.

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Temporal perturbation of the Wnt signaling pathway in the control of cell reprogramming is modulated by TCF1.

Publications

Temporal perturbation of the Wnt signaling pathway in the control of cell reprogramming is modulated by TCF1.

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