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A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells

In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formati...

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Autores principales: Ng, Karen, Daigle, Nathalie, Bancaud, Aurélien, Ohhata, Tatsuya, Humphreys, Peter, Walker, Rachael, Ellenberg, Jan, Wutz, Anton
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135487/
https://www.ncbi.nlm.nih.gov/pubmed/21613549
http://dx.doi.org/10.1091/mbc.E11-02-0146
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author Ng, Karen
Daigle, Nathalie
Bancaud, Aurélien
Ohhata, Tatsuya
Humphreys, Peter
Walker, Rachael
Ellenberg, Jan
Wutz, Anton
author_facet Ng, Karen
Daigle, Nathalie
Bancaud, Aurélien
Ohhata, Tatsuya
Humphreys, Peter
Walker, Rachael
Ellenberg, Jan
Wutz, Anton
author_sort Ng, Karen
collection PubMed
description In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2‑tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome‑bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement.
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spelling pubmed-31354872011-09-30 A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells Ng, Karen Daigle, Nathalie Bancaud, Aurélien Ohhata, Tatsuya Humphreys, Peter Walker, Rachael Ellenberg, Jan Wutz, Anton Mol Biol Cell Articles In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2‑tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome‑bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement. The American Society for Cell Biology 2011-07-15 /pmc/articles/PMC3135487/ /pubmed/21613549 http://dx.doi.org/10.1091/mbc.E11-02-0146 Text en © 2011 Ng et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.
spellingShingle Articles
Ng, Karen
Daigle, Nathalie
Bancaud, Aurélien
Ohhata, Tatsuya
Humphreys, Peter
Walker, Rachael
Ellenberg, Jan
Wutz, Anton
A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells
title A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells
title_full A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells
title_fullStr A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells
title_full_unstemmed A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells
title_short A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells
title_sort system for imaging the regulatory noncoding xist rna in living mouse embryonic stem cells
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3135487/
https://www.ncbi.nlm.nih.gov/pubmed/21613549
http://dx.doi.org/10.1091/mbc.E11-02-0146
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