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...
| Main Authors: | , , , , , , , , , , |
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| Format: | Journal Article |
| Language: | English |
| Published: |
2019
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| Online Access: | https://hdl.handle.net/10356/85701 http://hdl.handle.net/10220/49844 |
| _version_ | 1824454127856386048 |
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| author | Ha, Norbert How Ong Lai, Lan-Tian Chelliah, Rosi Zhen, Yashu Seet, Vanessa Pei Yi Lai, Soak-Kuan Li, Hoi-Yeung Ludwig, Alexander Sandin, Sara Chen, Lingyi Zhang, Li-Feng |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Ha, Norbert How Ong Lai, Lan-Tian Chelliah, Rosi Zhen, Yashu Seet, Vanessa Pei Yi Lai, Soak-Kuan Li, Hoi-Yeung Ludwig, Alexander Sandin, Sara Chen, Lingyi Zhang, Li-Feng |
| author_sort | Ha, Norbert How Ong |
| collection | NTU |
| 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. |
| first_indexed | 2025-02-19T03:17:23Z |
| format | Journal Article |
| id | ntu-10356/85701 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2025-02-19T03:17:23Z |
| publishDate | 2019 |
| record_format | dspace |
| spelling | ntu-10356/857012023-02-28T17:01:14Z Live-cell imaging and functional dissection of Xist RNA reveal mechanisms of X chromosome inactivation and reactivation Ha, Norbert How Ong Lai, Lan-Tian Chelliah, Rosi Zhen, Yashu Seet, Vanessa Pei Yi Lai, Soak-Kuan Li, Hoi-Yeung Ludwig, Alexander Sandin, Sara Chen, Lingyi Zhang, Li-Feng School of Biological Sciences Science::Biological sciences::Molecular biology Genetics Cell Biology 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. NMRC (Natl Medical Research Council, S’pore) NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Published version 2019-09-03T03:57:02Z 2019-12-06T16:08:35Z 2019-09-03T03:57:02Z 2019-12-06T16:08:35Z 2018 Journal Article Ha, N. H. O., Lai, L.-T., Chelliah, R., Zhen, Y., Seet, V. P. Y., Lai, S.-K., . . . Zhang, L.-F. (2018). Live-cell imaging and functional dissection of Xist RNA reveal mechanisms of X chromosome inactivation and reactivation. iScience, 8, 1-14. doi:10.1016/j.isci.2018.09.007 https://hdl.handle.net/10356/85701 http://hdl.handle.net/10220/49844 10.1016/j.isci.2018.09.007 en iScience © 2018 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 37 p. application/pdf |
| spellingShingle | Science::Biological sciences::Molecular biology Genetics Cell Biology Ha, Norbert How Ong Lai, Lan-Tian Chelliah, Rosi Zhen, Yashu Seet, Vanessa Pei Yi 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 | Science::Biological sciences::Molecular biology Genetics Cell Biology |
| url | https://hdl.handle.net/10356/85701 http://hdl.handle.net/10220/49844 |
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