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Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation

We double-tagged Xist (inactivated X chromosome-specific transcript), a prototype long non-coding RNA pivotal for X chromosome inactivation (XCI), using the programmable RNA sequence binding domain of Pumilio protein, one tag for live-cell imaging and the other replacing A-repeat (a critical domain...

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Autores principales: Ha, Norbert, Lai, Lan-Tian, Chelliah, Rosi, Zhen, Yashu, Yi Vanessa, Seet Pei, Lai, Soak-Kuan, Li, Hoi-Yeung, Ludwig, Alexander, Sandin, Sara, Chen, Lingyi, Zhang, Li-Feng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159346/
https://www.ncbi.nlm.nih.gov/pubmed/30266032
http://dx.doi.org/10.1016/j.isci.2018.09.007
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author Ha, Norbert
Lai, Lan-Tian
Chelliah, Rosi
Zhen, Yashu
Yi Vanessa, Seet Pei
Lai, Soak-Kuan
Li, Hoi-Yeung
Ludwig, Alexander
Sandin, Sara
Chen, Lingyi
Zhang, Li-Feng
author_facet Ha, Norbert
Lai, Lan-Tian
Chelliah, Rosi
Zhen, Yashu
Yi Vanessa, Seet Pei
Lai, Soak-Kuan
Li, Hoi-Yeung
Ludwig, Alexander
Sandin, Sara
Chen, Lingyi
Zhang, Li-Feng
author_sort Ha, Norbert
collection PubMed
description We double-tagged Xist (inactivated X chromosome-specific transcript), a prototype long non-coding RNA pivotal for X chromosome inactivation (XCI), using the programmable RNA sequence binding domain of Pumilio protein, one tag for live-cell imaging and the other replacing A-repeat (a critical domain of Xist) to generate “ΔA mutant” and to tether effector proteins for dissecting Xist functionality. Based on the observation in live cells that the induced XCI in undifferentiated embryonic stem (ES) cells is counteracted by the intrinsic X chromosome reactivation (XCR), we identified Kat8 and Msl2, homologs of Drosophila dosage compensation proteins, as players involved in mammalian XCR. Furthermore, live-cell imaging revealed the obviously undersized ΔA Xist cloud signals, clarifying an issue regarding the previous RNA fluorescence in situ hybridization results. Tethering candidate proteins onto the ΔA mutant reveals the significant roles of Ythdc1, Ezh2, and SPOC (Spen) in Xist-mediated gene silencing and the significant role of Ezh2 in Xist RNA spreading.
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spelling pubmed-61593462018-10-01 Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation Ha, Norbert Lai, Lan-Tian Chelliah, Rosi Zhen, Yashu Yi Vanessa, Seet Pei Lai, Soak-Kuan Li, Hoi-Yeung Ludwig, Alexander Sandin, Sara Chen, Lingyi Zhang, Li-Feng iScience Article We double-tagged Xist (inactivated X chromosome-specific transcript), a prototype long non-coding RNA pivotal for X chromosome inactivation (XCI), using the programmable RNA sequence binding domain of Pumilio protein, one tag for live-cell imaging and the other replacing A-repeat (a critical domain of Xist) to generate “ΔA mutant” and to tether effector proteins for dissecting Xist functionality. Based on the observation in live cells that the induced XCI in undifferentiated embryonic stem (ES) cells is counteracted by the intrinsic X chromosome reactivation (XCR), we identified Kat8 and Msl2, homologs of Drosophila dosage compensation proteins, as players involved in mammalian XCR. Furthermore, live-cell imaging revealed the obviously undersized ΔA Xist cloud signals, clarifying an issue regarding the previous RNA fluorescence in situ hybridization results. Tethering candidate proteins onto the ΔA mutant reveals the significant roles of Ythdc1, Ezh2, and SPOC (Spen) in Xist-mediated gene silencing and the significant role of Ezh2 in Xist RNA spreading. Elsevier 2018-09-13 /pmc/articles/PMC6159346/ /pubmed/30266032 http://dx.doi.org/10.1016/j.isci.2018.09.007 Text en © 2018 The Author(s) http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Ha, Norbert
Lai, Lan-Tian
Chelliah, Rosi
Zhen, Yashu
Yi Vanessa, Seet Pei
Lai, Soak-Kuan
Li, Hoi-Yeung
Ludwig, Alexander
Sandin, Sara
Chen, Lingyi
Zhang, Li-Feng
Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation
title Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation
title_full Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation
title_fullStr Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation
title_full_unstemmed Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation
title_short Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation
title_sort live-cell imaging and functional dissection of xist rna reveal mechanisms of x chromosome inactivation and reactivation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159346/
https://www.ncbi.nlm.nih.gov/pubmed/30266032
http://dx.doi.org/10.1016/j.isci.2018.09.007
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