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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 OPEN ACCESS
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https://hal.univ-lorraine.fr/hal-01715229
Contributor : Sylvain Maenner <>
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Aurélia Guyochin, Sylvain Maenner, Erin Tsi-Jia Chu, Asma Hentati, Mikael Attia, et al.. Live Cell Imaging of the Nascent Inactive X Chromosome during the Early Differentiation Process of Naive ES Cells towards Epiblast Stem Cells. PLoS ONE, Public Library of Science, 2014, 9 (12), pp.116109 - 116109. ⟨10.1371/journal.pone.0116109⟩. ⟨hal-01715229⟩

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