Live cell imaging of the nascent inactive X chromosome during the early differentiation process of naive ES cells towards epiblast stem cells

PLoS One. 2014 Dec 29;9(12):e116109. doi: 10.1371/journal.pone.0116109. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation / genetics*
  • Cell Nucleus / metabolism
  • Cell Survival
  • Embryonic Stem Cells / cytology*
  • Enhancer of Zeste Homolog 2 Protein
  • Female
  • Germ Layers / cytology*
  • Imaging, Three-Dimensional*
  • Interphase
  • Mice
  • Mice, Transgenic
  • Mitosis
  • Polycomb Repressive Complex 2 / metabolism
  • RNA, Long Noncoding / genetics
  • RNA, Long Noncoding / metabolism
  • X Chromosome / genetics*
  • X Chromosome Inactivation / genetics*

Substances

  • RNA, Long Noncoding
  • XIST non-coding RNA
  • Enhancer of Zeste Homolog 2 Protein
  • Ezh2 protein, mouse
  • Polycomb Repressive Complex 2

Grants and funding

AG, SM, ETC, MA, PA, and PC were supported by Institut Pasteur (www.pasteur.fr), Centre National pour la Recherche Scientifique (www.cnrs.fr), Agence Nationale pour la Recherche (www.agence-nationale-recherche.fr), Epigenome Network of Excellence, REVIVE Labex, (www.pasteur.fr/ip/easysite/pasteur/en/research/labex/revive), and Foundation Louis D. Institut de France (http://www.institut-de-france.fr/institutions/prix-amp-fondations/fondations/fondation-louis-d-florence-de-c). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.