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A protein assembly mediates Xist localization and gene silencing

Pandya-Jones, Amy and Markaki, Yolanda and Serizay, Jacques and Chitiashvili, Tsotne and Mancia-Leon, Walter R. and Damianov, Andrey and Chronis, Konstantinos and Papp, Bernadett and Chen, Chun-Kan and McKee, Robin and Wang, Xiao-Jun and Chau, Anthony and Sabri, Shan and Leonhardt, Heinrich and Zheng, Sika and Guttman, Mitchell and Black, Douglas L. and Plath, Kathrin (2020) A protein assembly mediates Xist localization and gene silencing. Nature, 587 (7832). pp. 145-151. ISSN 0028-0836.

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[img] Image (JPEG) (Extended Data Fig. 1: Depletion of PTBP1, MATR3, CELF1 and TDP-43 does not strongly affect gene silencing during the Xist-dependent stage of XCI initiation) - Supplemental Material
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[img] Image (JPEG) (Extended Data Fig. 2: Depletion of PTBP1, MATR3, CELF1 and TDP-43 affects Xist localization during XCI initiation without strongly altering Xist processing) - Supplemental Material
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[img] Image (JPEG) (Extended Data Fig. 3: PTBP1, MATR3, CELF1 and TDP-43 directly bind the Xist E-repeat, comprising a tandem array of 20–25nt C/U/G-rich elements) - Supplemental Material
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[img] Image (JPEG) (Extended Data Fig. 4: CELF1 and PTBP1 localize within the Xist-coated territory) - Supplemental Material
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[img] Image (JPEG) (Extended Data Fig. 5: ΔE ES cells undergo differentiation similar to wild-type ES cells and splicing of Xist-intron 6 proceeds in the absence of the E-repeat) - Supplemental Material
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[img] Image (JPEG) (Extended Data Fig. 6: Loss of the E-repeat does not affect Xist abundance, splicing or stability) - Supplemental Material
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[img] Image (JPEG) (Extended Data Fig. 7: The XistΔE-coated X chromosome displays decreased DAPI staining and less compact H3K27me3 accumulation at differentiation day 7) - Supplemental Material
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[img] Image (JPEG) (Extended Data Fig. 8: Loss of the E-repeat prevents continued gene silencing in differentiating ES cells) - Supplemental Material
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[img] Image (JPEG) (Extended Data Fig. 9: A site-specific recombination-based approach to rescue phenotypes associated with loss of the E-repeat) - Supplemental Material
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[img] Image (JPEG) (Extended Data Fig. 10: Expression of MCP–CIZ1 or MCP–GFP–MCP does not rescue phenotypes due to loss of the E-repeat) - Supplemental Material
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[img] Image (JPEG) (Extended Data Fig. 11: CELF1 enhances droplet formation of PTBP1 with the E-repeat in vitro and mutations in MATR3 and TDP-43 that abrogate their self-association do not rescue ΔE phenotypes) - Supplemental Material
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Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus and binding diverse proteins to achieve X-chromosome inactivation (XCI). The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off. Here we show that the Xist RNA-binding proteins PTBP1, MATR3, TDP-43 and CELF1 assemble on the multivalent E-repeat element of Xist and, via self-aggregation and heterotypic protein–protein interactions, form a condensate in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.

Item Type:Article
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URLURL TypeDescription ReadCube access Paper Correction Correction - Free ReadCube access
Pandya-Jones, Amy0000-0001-5519-4613
Markaki, Yolanda0000-0001-6013-2417
Serizay, Jacques0000-0002-4295-0624
Chitiashvili, Tsotne0000-0002-4887-1097
Mancia-Leon, Walter R.0000-0002-1920-6514
Chen, Chun-Kan0000-0002-1194-9137
Sabri, Shan0000-0002-0885-6159
Leonhardt, Heinrich0000-0002-5086-6449
Zheng, Sika0000-0002-0573-4981
Guttman, Mitchell0000-0003-4748-9352
Plath, Kathrin0000-0001-7796-3372
Alternate Title:An Xist-dependent protein assembly mediates Xist localization and gene silencing
Additional Information:© 2020 Nature Publishing Group. Received 10 August 2019; Accepted 17 June 2020; Published 09 September 2020. We thank members of the Plath and Black laboratories for discussions and reading of the manuscript. A.P.-J. was supported by postdoctoral fellowships from the Helen Hay Whitney Foundation and NIH (F32 GM103139); K.P. by Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research (BSCRC) at UCLA, the David Geffen School of Medicine at UCLA, and the Jonnson Comprehensive Cancer Center at UCLA, the NIH (R01 GM115233), and a Faculty Scholar grant from the Howard Hughes Medical Institute; D.L.B. by the NIH (R01 GM049662 and R01 MH109166 (to K.P. and D.L.B.)); M.G. was funded by the New York Stem Cell Foundation, Searle Scholars Program and the Pew-Steward Scholars Program. M.G. is a NYSCF-Robertson Investigator. Y.M., B.P. and S.Z. were supported by the NIH (NICHD 5R03HD095086 to Y.M., R03HD088380 to B.P., R01NS104041 and R01MH116220 to S.Z.). Y.M. and H.L. were supported by the Deutsche Forschungsgemeinschaft (SFB1064/A17 and LE721/18-1). T.C., A.C. and S.S. are supported by graduate fellowships from the Boehringer Ingelheim Foundation (to T.C.); the UCLA Whitcome Fellowship (to A.C.); and the UCLA Broad Stem Cell Research Center – Rose Hills Foundation training award and the UCLA Dissertation Year Fellowship (to S.S.). Data availability: All genomic data for Xist interactions and chromatin association have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE137305. Reagents are available upon request. Author Contributions: K.P., A.P.-J., Y.M. and D.L.B. conceptualized the project and A.P.-J. performed the experiments unless stated otherwise. Y.M. and T.C. performed experiments for 3D-SIM imaging and acquired and analysed 3D-SIM data, overseen by H.L. Y.M. acquired high-resolution images and performed image analysis on immunostained cells. J.S. performed all aggregation measurements, helped with EMSAs and analysed RAP–seq data. R.M., W.M. and A.C. helped to create ES cell deletion lines. S.Z. performed the initial PTBP1/2 iCLIP–seq experiments, A.D. helped A.P.-J. with iCLIP–seq experiments, S.S. and J.S. analysed CLIP–seq data, B.P. and C.C. performed and analysed CHIP–seq experiments, X.-J.W. purified rPTBP1 and rCELF1, and C.-K.C. performed RAP–seq experiments. A.P.-J., J.S., Y.M., T.C. and K.P. analysed data, A.P.-J., Y.M., J.S., M.G. and K.P. interpreted the data and contributed towards methodology and model creation, K.P., D.L.B, M.G. and H.L. acquired funding to support the project, A.P.-J. and K.P. administered the project and A.P.-J. and K.P. wrote the manuscript, including edits from all authors. The authors declare no competing interests. Peer review information: Nature thanks Jernej Ule and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Errata:In Fig. 1b of this Article, owing to an error in the production process, there are minus signs missing before the words ‘LIF’ and ‘feeders’ in the label ‘Plate ES cells in media (−LIF, −feeders)’. The Article has been corrected online.
Funding AgencyGrant Number
Helen Hay Whitney FoundationUNSPECIFIED
NIHF32 GM103139
NIHR01 GM115233
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NIHR01 GM049662
NIHR01 MH109166
New York Stem Cell FoundationUNSPECIFIED
Searle Scholars ProgramUNSPECIFIED
Pew-Steward Scholars ProgramUNSPECIFIED
Deutsche Forschungsgemeinschaft (DFG)SFB1064/A17
Deutsche Forschungsgemeinschaft (DFG)LE721/18-1
Boehringer Ingelheim FoundationUNSPECIFIED
Rose Hills FoundationUNSPECIFIED
Subject Keywords:Epigenetics; Long non-coding RNAs; Nucleus; RNA; RNA-binding proteins
Issue or Number:7832
Record Number:CaltechAUTHORS:20200310-130644860
Persistent URL:
Official Citation:Pandya-Jones, A., Markaki, Y., Serizay, J. et al. A protein assembly mediates Xist localization and gene silencing. Nature 587, 145–151 (2020).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:101826
Deposited By: Tony Diaz
Deposited On:10 Mar 2020 20:35
Last Modified:06 Nov 2020 18:01

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