title: Live cell imaging of the nascent inactive X chromosome during the early differentiation process of naive ES cells towards epiblast stem cells.
abstract: Random X-chromosome inactivation ensures dosage compensation in mammals through the transcriptional silencing of one of the two X chromosomes present in each female cell. Silencing is initiated in the differentiating epiblast of the mouse female embryos through coating of the nascent inactive X chromosome by the non-coding RNA Xist, which subsequently recruits the Polycomb Complex PRC2 leading to histone H3-K27 methylation. Here we examined in mouse ES cells the early steps of the transition from naive ES cells towards epiblast stem cells as a model for inducing X chromosome inactivation in vitro. We show that these conditions efficiently induce random XCI. Importantly, in a transient phase of this differentiation pathway, both X chromosomes are coated with Xist RNA in up to 15% of the XX cells. In an attempt to determine the dynamics of this process, we designed a strategy aimed at visualizing the nascent inactive X-chromosome in live cells. We generated transgenic female XX ES cells expressing the PRC2 component Ezh2 fused to the fluorescent protein Venus. The fluorescent fusion protein was expressed at sub-physiological levels and located in nuclei of ES cells. Upon differentiation of ES cell towards epiblast stem cell fate, Venus-fluorescent territories appearing in interphase nuclei were identified as nascent inactive X chromosomes by their association with Xist RNA. Imaging of Ezh2-Venus for up to 24 hours during the differentiation process showed survival of some cells with two fluorescent domains and a surprising dynamics of the fluorescent territories across cell division and in the course of the differentiation process. Our data reveal a strategy for visualizing the nascent inactive X chromosome and suggests the possibility for a large plasticity of the nascent inactive X chromosome.
authors.affiliation: Génétique Moléculaire Murine, CNRS URA2578, Institut Pasteur, Paris, France; Université de Technologie de Compiègne, Compiègne, France.
authors.forename: Aurélia
authors.initials: A
authors.lastname: Guyochin
authors.affiliation: Génétique Moléculaire Murine, CNRS URA2578, Institut Pasteur, Paris, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Maenner
authors.affiliation: Génétique Moléculaire Murine, CNRS URA2578, Institut Pasteur, Paris, France.
authors.forename: Erin Tsi-Jia
authors.initials: ET
authors.lastname: Chu
authors.affiliation: Génétique Moléculaire Murine, CNRS URA2578, Institut Pasteur, Paris, France; Université Blaise Pascal, Clermont-Ferrand, France.
authors.forename: Asma
authors.initials: A
authors.lastname: Hentati
authors.affiliation: Génétique Moléculaire Murine, CNRS URA2578, Institut Pasteur, Paris, France.
authors.forename: Mikael
authors.initials: M
authors.lastname: Attia
authors.affiliation: Génétique Moléculaire Murine, CNRS URA2578, Institut Pasteur, Paris, France; EMBL Monterotondo, Adriano Buzzati-Traverso Campus, Monterotondo, Italy.
authors.forename: Philip
authors.initials: P
authors.lastname: Avner
authors.affiliation: Génétique Moléculaire Murine, CNRS URA2578, Institut Pasteur, Paris, France; Epigénétique des Cellules Souches, Department of Developmental Biology, CNRS URA2578, Institut Pasteur, Paris, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Clerc
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D053595:Embryonic Stem Cells / Q000166:cytology*; D000071221:Enhancer of Zeste Homolog 2 Protein; D005260:Female; D005855:Germ Layers / Q000166:cytology*; D021621:Imaging, Three-Dimensional; D007399:Interphase; D051379:Mice; D008822:Mice, Transgenic; D008938:Mitosis; D063151:Polycomb Repressive Complex 2 / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism; D014960:X Chromosome / Q000235:genetics*; D049951:X Chromosome Inactivation / Q000235:genetics*
chemical_list: D062085:RNA, Long Noncoding; C077885:XIST non-coding RNA; D000071221:Enhancer of Zeste Homolog 2 Protein; C498410:Ezh2 protein, mouse; D063151:Polycomb Repressive Complex 2