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Published November 5, 2020 | Submitted + Supplemental Material + Accepted Version
Journal Article Open

A protein assembly mediates Xist localization and gene silencing


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.

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.


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.

Attached Files

Accepted Version - nihms-1605140.pdf

Submitted - 2020.03.09.979369v2.full.pdf

Supplemental Material - 41586_2020_2703_Fig10_ESM.webp

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Supplemental Material - 41586_2020_2703_MOESM1_ESM.pdf

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