title: Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells.
abstract: In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell-fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA-sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast-like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial-to-mesenchymal transition (EMT), and DNA-(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1-binding sites correlate with loss-of-methylation in neural-stimulating conditions, however, after the cells initially acquired the correct DNA-methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re-adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA-methylation with irreversible commitment to differentiation. Stem Cells 2017;35:611-625.
authors.affiliation: Department of Development and Regeneration, KU Leuven, Leuven, 3000, Belgium.
authors.forename: Agata
authors.initials: A
authors.lastname: Stryjewska
authors.affiliation: Department of Development and Regeneration, KU Leuven, Leuven, 3000, Belgium.|Department of Cell Biology, Erasmus University Medical Center, Rotterdam, 3015 CN, The Netherlands.
authors.forename: Ruben
authors.initials: R
authors.lastname: Dries
authors.affiliation: VIB Inflammation Research Center (IRC), Unit Vascular Cell Biology.|Department of Biomedical Molecular Biology.|VIB-IRC, Unit Molecular and Cellular Oncology, Ghent University, Ghent, 9052, Belgium.|Center for Medical Genetics, Ghent University Hospital, Ghent, 9000, Belgium.
authors.forename: Tim
authors.initials: T
authors.lastname: Pieters
authors.affiliation: Department of Development and Regeneration, KU Leuven, Leuven, 3000, Belgium.
authors.forename: Griet
authors.initials: G
authors.lastname: Verstappen
authors.affiliation: Department of Cell Biology, Erasmus University Medical Center, Rotterdam, 3015 CN, The Netherlands.
authors.forename: Andrea
authors.initials: A
authors.lastname: Conidi
authors.affiliation: Department of Development and Regeneration, KU Leuven, Leuven, 3000, Belgium.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Coddens
authors.affiliation: Department of Development and Regeneration, KU Leuven, Leuven, 3000, Belgium.
authors.forename: Annick
authors.initials: A
authors.lastname: Francis
authors.affiliation: Department of Development and Regeneration, KU Leuven, Leuven, 3000, Belgium.
authors.forename: Lieve
authors.initials: L
authors.lastname: Umans
authors.affiliation: Department of Cell Biology, Erasmus University Medical Center, Rotterdam, 3015 CN, The Netherlands.|Center for Biomics, Erasmus University Medical Center, Rotterdam, 3015 CN, The Netherlands.
authors.forename: Wilfred F J
authors.initials: WF
authors.lastname: van IJcken
authors.affiliation: Department of Biomedical Molecular Biology.|VIB-IRC, Unit Molecular and Cellular Oncology, Ghent University, Ghent, 9052, Belgium.
authors.forename: Geert
authors.initials: G
authors.lastname: Berx
authors.affiliation: Department of Cell Biology, Liver Cell Biology Lab, Vrije Universiteit Brussel, Jette, 1090, Belgium.
authors.forename: Leo A
authors.initials: LA
authors.lastname: van Grunsven
authors.affiliation: Department of Cell Biology, Erasmus University Medical Center, Rotterdam, 3015 CN, The Netherlands.
authors.forename: Frank G
authors.initials: FG
authors.lastname: Grosveld
authors.affiliation: VIB Inflammation Research Center (IRC), Unit Vascular Cell Biology.|Department of Biomedical Molecular Biology.|VIB-IRC, Unit Molecular and Cellular Oncology, Ghent University, Ghent, 9052, Belgium.|ACBD - Blood Cancers and Stem Cells, Group Mammalian Functional Genetics, Monash University, Melbourne, VIC, 3004, Australia.
authors.forename: Steven
authors.initials: S
authors.lastname: Goossens
authors.affiliation: VIB Inflammation Research Center (IRC), Unit Vascular Cell Biology.|Department of Biomedical Molecular Biology.|ACBD - Blood Cancers and Stem Cells, Group Mammalian Functional Genetics, Monash University, Melbourne, VIC, 3004, Australia.
authors.forename: Jody J
authors.initials: JJ
authors.lastname: Haigh
authors.affiliation: Department of Development and Regeneration, KU Leuven, Leuven, 3000, Belgium.|Department of Cell Biology, Erasmus University Medical Center, Rotterdam, 3015 CN, The Netherlands.
authors.forename: Danny
authors.initials: D
authors.lastname: Huylebroeck
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D019175:DNA Methylation / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D058732:Embryoid Bodies / Q000166:cytology / Q000378:metabolism; D005855:Germ Layers / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D009474:Neurons / Q000166:cytology; D010641:Phenotype; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D025341:Principal Component Analysis; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic; D000074165:Zinc Finger E-box Binding Homeobox 2 / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; C540839:TET1 protein, mouse; C120602:ZEB2 protein, mouse; D000074165:Zinc Finger E-box Binding Homeobox 2
keywords: Cell differentiation; DNA-methylation; Embryonic stem cells; Pluripotent stem cells; RNA-sequencing; Repressors; Transcription factors; Transcriptom