Investigating the role of Wnt/β-catenin pathway in pluripotency and somatic cell reprogramming

The adaptive response of cells to external stimuli is an intriguing mechanism at the basis of the existence of life itself. For this purpose, signalling pathways and gene regulatory networks elegantly evolved translating extracellular signals into finely tuned cellular responses. Among them, the Wnt/ß-catenin signalling pathway converges on the regulation of ß-catenin protein, which, in turn regulates target gene expression. In particular the Wnt/ß-catenin pathway plays a pivotal role in sustaining pluripotency and somatic cell reprogramming. Here we identified a temporal of Wnt/ß-catenin activity during somatic cell reprogramming, controlling the expression levels of mesenchymal-to-epithelial transition and senescence-associated genes through TCF1. We demonstrated that the “Wnt-OFF” state is an early reprogramming marker and that dynamic modulation can be effectively used to increase the reprogramming efficiency. Furthermore the Wnt/ß-catenin pathway is a key regulator of pluripotency and self-renewal of mouse embryonic stem cells (mESCs) and a small-molecule activator of the Wnt pathway is widely used to maintain embryonic stem cells in a ground state of pluripotency. The role of ß-catenin in mESCs is however still controversial. We noticed available ß-catenin knock-out models are flawed by the production of N-terminally truncated proteins with unknown functions. We therefore generated a novel ß-catenin knock-out using CRISPR/Cas9 technology, hoping to have clearer insight of ß-catenin functions in mESCs. We have also found that ground state pluripotency promoted by sustained Wnt pathway activation cannot be maintained indefinitely, resulting in a “lapsed” ground state possibly due, among other factors, to regulatory negative feedback loops that impair Wnt/ß-catenin activity.La respuesta adaptativa de las células a estímulos externos es un mecanismo fundamental de la existencia de la vida en sí misma. Para este fin, rutas de señalización y redes de regulación génica evolucionaron elegantemente, traduciendo señales extracelulares en respuestas celulares calibradas con precisión. Entre ellas, la ruta de señalización de Wnt/ß-catenin converge en la regulación de la proteína ß-catenin, que a su vez regula la expresión de genes diana. En particular, la ruta de Wnt/ß-catenin tiene un rol fundamental en el mantenimiento de la pluripotencia y la reprogramación de células somáticas. En esta tesis hemos identificado un papel temporal de actividad de Wnt/ß-catenin durante la reprogramación de células somáticas, lo que controla los niveles de expresión de genes asociados a transición mesenquima-epitelial y senescencia a través de TCF1. Además, la ruta de Wnt/ß-catenin es un regulador clave de la pluripotencia y la auto-renovación de células madre embrionarias de ratón (mESCs). Una pequeña molécula, activadora de la ruta Wnt es usada comúnmente para mantener las células madre embrionarias en “ground state” de pluripotencia. Sin embargo, el rol de la ß-catenin en las mESCs es aún controvertido. Observamos que los modelos disponibles de Knock-Out de ß-catenin producen proteínas truncadas en N-terminal con funciones desconocidas. Por ello, generamos un nuevo Knock-Out usando CRISPR/Cas9, al fin de clarificar funciones de ß-catenina en mESCs. Hemos encontrado también que el “ground state” de pluripotencia promovido por la activación sostenida de Wnt no puede ser mantenido indefinidamente, resultando esto en un“ lapsed ground state”; debido posiblemente, entre otros factores, a feedback-loop negativos que afectan negativamente la actividad de Wnt/ß-catenin

Reduced expression of Paternally Expressed Gene-3 enhances somatic cell reprogramming through mitochondrial activity perturbation

Imprinted genes control several cellular and metabolic processes in embryonic and adult tissues. In particular, paternally expressed gene-3 (Peg3) is active in the adult stem cell population and during muscle and neuronal lineage development. Here we have investigated the role of Peg3 in mouse embryonic stem cells (ESCs) and during the process of somatic cell reprogramming towards pluripotency. Our data show that Peg3 knockdown increases expression of pluripotency genes in ESCs and enhances reprogramming efficiency of both mouse embryonic fibroblasts and neural stem cells. Interestingly, we observed that altered activity of Peg3 correlates with major perturbations of mitochondrial gene expression and mitochondrial function, which drive metabolic changes during somatic cell reprogramming. Overall, our study shows that Peg3 is a regulator of pluripotent stem cells and somatic cell reprogramming.We thank Marie Victoire Neguembor, Martina Pesaresi and Sergi Angel Bonilla Pons for suggestions on the manuscript and Umberto Di Vicino for technical support. We thank Angelique di Domenico and the group of Antonella Consiglio for providing us the OPA1, DRP1 and VDAC1 antibodies. We thank Mari Carmen Ortells and the group of Bill Keyes for providing us the OXPHOS antibody. The pInducer10-mir-RUP-PheS plasmid was a gift from Stephen Elledge (Addgene plasmid # 44011). We are grateful for support from Ministerio de Economia y Competitividad and FEDER funds (SAF2011-28580, BFU2014-54717-P, and BFU2015-71984-ERC to M.P.C.), Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat de Catalunya (2014 SGR1137 to M.P.C.), the European Union's Horizon 2020 research and innovation programme under grant agreement CellViewer No 686637 (to M.P.C.), Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017 and the CERCA Programme/Generalitat de Catalunya (to M.P.C.), People Programme Marie Curie Actions of the European Union's Seventh Framework Programme (FP7/2007-2013/, n° 290123 to I.T.), La Caixa international PhD fellowship (to F.S.), Ministerio de Ciencia e Innovació FPI (to F.A.) and Ministerio de Ciencia e Innovación FPI-Severo Ochoa (to A.C.G)

(Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes

CRISPR/dCas9-based labeling has allowed direct visualization of genomic regions in living cells. However, poor labeling efficiency and signal-to-background ratio have limited its application to visualize genome organization using super-resolution microscopy. We developed (Po)STAC (Polycistronic SunTAg modified CRISPR) by combining CRISPR/dCas9 with SunTag labeling and polycistronic vectors. (Po)STAC enhances both labeling efficiency and fluorescence signal detected from labeled loci enabling live cell imaging as well as super-resolution fixed-cell imaging of multiple genes with high spatiotemporal resolution.European Union’s Horizon 2020 Research and Innovation Programme [CellViewer No 686637 to M.L., M.P.C.]; Ministerio de Economia y Competitividad [BFU2013–49867-EXP to M.L., M.P.C.]; Fundació Cellex Barcelona (to M.L); European Union Seventh Framework Programme under the European Research Council Grants [337191-MOTORS to M.L.]; ‘Severo Ochoa’ Programme for Centres of Excellence in R&D [SEV-2015- 0522 to M.L.]; Ministerio de Economia y Competitividad and FEDER Funds [BFU2014–54717-P, BFU2015–71984-ERC to M.P.C.]; AGAUR Grant [2014 SGR1137 to M.P.C.]; Spanish Ministry of Economy and Competitiveness (to M.P.C.); Centro de Excelencia Severo Ochoa [2013–2017 to M.P.C.]; CERCA Programme/Generalitat de Catalunya (to M.P.C); Ministerio de Ciencia e Innovacion FPI (to F.A.); People Program (Marie Curie Actions) FP7/2007–2013 under REA grant [608959 to M.V.N.]. Funding for open access charge: European Union’s Horizon 2020 Research and Innovation Programme [CellViewer No 686637]

A tunable dual-input system for on-demand dynamic gene expression regulation

Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells

Wnt/Tcf1 pathway restricts embryonic stem cell cycle through activation of the Ink4/Arf locus

Understanding the mechanisms regulating cell cycle, proliferation and potency of pluripotent stem cells guarantees their safe use in the clinic. Embryonic stem cells (ESCs) present a fast cell cycle with a short G1 phase. This is due to the lack of expression of cell cycle inhibitors, which ultimately determines naïve pluripotency by holding back differentiation. The canonical Wnt/β-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/β-catenin controls the cell cycle of mESCs remains unknown. Here we show that the Wnt-effector Tcf1 is recruited to and triggers transcription of the Ink4/Arf tumor suppressor locus. Thereby, the activation of the Wnt pathway, a known mitogenic pathway in somatic tissues, restores G1 phase and drastically reduces proliferation of mESCs without perturbing pluripotency. Tcf1, but not Tcf3, is recruited to a palindromic motif enriched in the promoter of cell cycle repressor genes, such as p15Ink4b, p16Ink4a and p19Arf, which mediate the Wnt-dependent anti-proliferative effect in mESCs. Consistently, ablation of β-catenin or Tcf1 expression impairs Wnt-dependent cell cycle regulation. All together, here we showed that Wnt signaling controls mESC pluripotency and proliferation through non-overlapping functions of distinct Tcf factors.We are grateful for the support from ERC grant (242630-RERE) (MPC), the Ministerio de Economia y Competitividad y FEDER (SAF2011-28580, and BFU2014-54717-P, BFU2015-71984-ERC to MPC), an AGAUR grant from Secretaria d´Universitats i Investigació del Departament d´Economia i Coneixement de la Generalitat de Catalunya (2014SGR1137 to MPC), Ministerio de Ciencia e Innovación FPI (to FA), the European Union's Horizon 2020 research and innovation programme under grant agreement CellViewer No 686637 (to MPC), the Spanish Ministry of Economy and Competitiveness, Centro de Excelencia Severo Ochoa 2013-2017, the CERCA Programme/Generalitat de Catalunya (to MPC); KU Leuven starting grant (STG) and KU Leuven C1 funds (C14/16/078) (to FL), AFR Postdoctoral Grant from the Luxembourg National Research Fund (FNR); ANEMO project N. 4001584/PDR 2012-1 and Dutch Province of Limburg (to GE), Short Term Mobility Award, CNR (to AC)

Wnt/Tcf1 pathway restricts embryonic stem cell cycle through activation of the Ink4/Arf locus

Understanding the mechanisms regulating cell cycle, proliferation and potency of pluripotent stem cells guarantees their safe use in the clinic. Embryonic stem cells (ESCs) present a fast cell cycle with a short G1 phase. This is due to the lack of expression of cell cycle inhibitors, which ultimately determines naïve pluripotency by holding back differentiation. The canonical Wnt/β-catenin pathway controls mESC pluripotency via the Wnt-effector Tcf3. However, if the activity of the Wnt/β-catenin controls the cell cycle of mESCs remains unknown. Here we show that the Wnt-effector Tcf1 is recruited to and triggers transcription of the Ink4/Arf tumor suppressor locus. Thereby, the activation of the Wnt pathway, a known mitogenic pathway in somatic tissues, restores G1 phase and drastically reduces proliferation of mESCs without perturbing pluripotency. Tcf1, but not Tcf3, is recruited to a palindromic motif enriched in the promoter of cell cycle repressor genes, such as p15Ink4b, p16Ink4a and p19Arf, which mediate the Wnt-dependent anti-proliferative effect in mESCs. Consistently, ablation of β-catenin or Tcf1 expression impairs Wnt-dependent cell cycle regulation. All together, here we showed that Wnt signaling controls mESC pluripotency and proliferation through non-overlapping functions of distinct Tcf factors.We are grateful for the support from ERC grant (242630-RERE) (MPC), the Ministerio de Economia y Competitividad y FEDER (SAF2011-28580, and BFU2014-54717-P, BFU2015-71984-ERC to MPC), an AGAUR grant from Secretaria d´Universitats i Investigació del Departament d´Economia i Coneixement de la Generalitat de Catalunya (2014SGR1137 to MPC), Ministerio de Ciencia e Innovación FPI (to FA), the European Union's Horizon 2020 research and innovation programme under grant agreement CellViewer No 686637 (to MPC), the Spanish Ministry of Economy and Competitiveness, Centro de Excelencia Severo Ochoa 2013-2017, the CERCA Programme/Generalitat de Catalunya (to MPC); KU Leuven starting grant (STG) and KU Leuven C1 funds (C14/16/078) (to FL), AFR Postdoctoral Grant from the Luxembourg National Research Fund (FNR); ANEMO project N. 4001584/PDR 2012-1 and Dutch Province of Limburg (to GE), Short Term Mobility Award, CNR (to AC)

The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency

Supplemental material files: supplementary information: online appendix; replication fileEarly during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.We would like to thank Dr. Brad Merrill for providing the WT and Tcf7l1−/− mESCs cells and Samantha Zaunz for helping on FACS experiments. We are grateful to Susan Schlenner and the KU Leuven FACS core team for providing the facility and especially Reena Chinaraj who helped us during flow cytometry experiments. We also thank Lotte Schoeters and Alvaro Cortes Calabuig from the KULeuven Genomics Core (http://genomicscore.be) for RNA sequencing, data processing and analysis. The authors would like to extend their gratitude to the FWO Research Foundation – Flanders for the Ph.D. fellowships awarded to P.A. (11M7822N), B.V. (11E7920N), A.J. (1158318 N), postdoctoral funding to A.B. (1298722 N), KU Postdoctoral Mandate awarded to ADJS (PDM/18/212) and FWO-Vlaanderen Research Project Grants G097618N, G091521N (F.L.L.), G073622N (F.L.L., B.H.), G092518N, G0C6820N (K.P.K.), G0C9320N, G0B4420N (V.P.), EOS grant G0I7822N (V.P.), Ministerio de Ciencia e Innovación 008506-PID2020-114080GB-I00 (M.P.C.) and AGAUR grant 006712 2017-SGR 689 (M.P.C.) and C1 KU Leuven internal grants C14/21/115 (F.L.L.), C14/21/119 (V.P.)