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Xist-seeded nucleation sites form local concentration gradients of silencing proteins to inactivate the X-chromosome

Markaki, Yolanda and Chong, Johnny Gan and Luong, Christy and Tan, Shawn Y. X. and Wang, Yuying and Jacobson, Elsie C. and Maestrini, Davide and Dror, Iris and Mistry, Bhaven A. and Schöneberg, Johannes and Banerjee, Abhik and Guttman, Mitchell and Chou, Tom and Plath, Kathrin (2020) Xist-seeded nucleation sites form local concentration gradients of silencing proteins to inactivate the X-chromosome. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20201123-130437407

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Abstract

The long non-coding RNA Xist exploits numerous effector proteins to progressively induce gene silencing across the X chromosome and form the inactive X (Xi)-compartment. The mechanism underlying formation of the chromosome-wide Xi-compartment is poorly understood. Here, we find that formation of the Xi-compartment is induced by ~50 locally confined granules, where two Xist RNA molecules nucleate supra-molecular complexes (SMCs) of interacting proteins. Xist-SMCs are transient structures that concentrate rapidly recycling proteins in the X by increasing protein binding affinity. We find that gene silencing originates at Xist-SMCs and propagates across the entire chromosome over time, achieved by Polycomb-mediated coalescence of chromatin regions and aggregation, via its intrinsically disordered domains, of the critical silencing factor SPEN. Our results suggest a new model for X chromosome inactivation, in which Xist RNA induces macromolecular crowding of heterochromatinizing proteins near distinct sites which ultimately increases their density throughout the chromosome. This mechanism enables deterministic gene silencing without the need for Xist ribonucleoprotein complex-chromatin interactions at each target gene.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2020.11.22.393546DOIDiscussion Paper
ORCID:
AuthorORCID
Markaki, Yolanda0000-0001-6013-2417
Guttman, Mitchell0000-0003-4748-9352
Plath, Kathrin0000-0001-7796-3372
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This version posted November 23, 2020. We thank David Baker (UW) for sharing the ct-60 gene fused to GFP, Joost Gribnau (Oncode Institute) for the MS2-targeting construct, Alexander Shishkin for the SPEN entry clone, Irina Solovei for DNA from flow sorted mouse X-chromosomes, and Yi-Yun Ho for help optimizing the chromosome barcoding experiments. We thank Amy Pandya-Jones for help with the establishment of the ΔB-Xist cell lines and Tsotne Chitiashvili for help with FISH and providing XIST-488 probes. We thank Edith Heard and Francois Dossin for sharing the gene expression profiles after Bgl-GFP-SPOC rescue in SPEN depleted cells. We thank Douglas Black and Emilie Marcus and all members of the Plath laboratory for critical reading of the manuscript and helpful discussions. We also thank the David Geffen School of Medicine (DGSOM) at UCLA, the Jules Stein Eye Institute, David Williams, the Department of Biological Chemistry for supporting the imaging approaches. Funding: The imaging core was supported by the NIH (R01GM115233). Y.M. was supported by the NIH (R03HD095086), K.P. was supported by an Innovation Award from and facilities of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, DGSOM, and the Jonsson Comprehensive Cancer Center at UCLA, the NIH (R01GM115233, 1R01MH109166, R21HD094172), the Keck Foundation, and a Faculty Scholar grant from the Howard Hughes Medical Institute. D.M. and T.C. were supported by the NSF (DMS-1814364) and NIH (R01HL146552), and A.B. was supported by the NIH (F30HL136080) and the USC MD/PhD Program. Author Contributions: Y.M. and K.P. conceived the project and Y.M. performed the experiments unless stated otherwise. S.T., C.L., Y.M. and J.C.G. created engineered cell lines and J.C.G., J.W. and Y.M. performed 3D-SIM imaging, data analyses and visualization. K.P. created the ΔB-Xist cell lines. C.L. and Y.M. performed and analyzed FRAP experiments. J.C.G and Y.M. synthesized probes and performed RNA/DNA FISH experiments. T.C. developed all modeling included in this study. E.J. analyzed gene silencing kinetics and correlation of RAP-seq data to Hi-C and expression data. D.M. performed the fitting of FRAP experiments and extracted parameters, and inferred the confining potential of Xist granules, under supervision of T.C. B.M. derived the X-chromosome configurations, which was also overseen by T.C. S.T. performed RAP-seq experiments and I.D. analyzed the data. A.B. cloned the WT- and ΔIDR-SPEN constructs. J.S. wrote the code for subtracting the developmental motion in live-cell 3D-SIM experiments. Y.M., K.P. and T.C. interpreted the data and contributed towards methodology and model creation. K.P. supervised all experimental work. K.P., Y.M., T.C. and M.G. acquired funding to support the project and Y.M., K.P. and T.C. wrote the manuscript with edits from all authors. The authors declare no competing interests. Data and materials availability: All data and material derived from this study are available to researchers upon request.
Funders:
Funding AgencyGrant Number
NIHR01GM115233
NIHR03HD095086
UCLAUNSPECIFIED
NIHR01GM115233
NIH1R01MH109166
NIHR21HD094172
W. M. Keck FoundationUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NSFDMS-1814364
NIHR01HL146552
NIH Postdoctoral FellowshipF30HL136080
University of Southern CaliforniaUNSPECIFIED
DOI:10.1101/2020.11.22.393546
Record Number:CaltechAUTHORS:20201123-130437407
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20201123-130437407
Official Citation:Xist-seeded nucleation sites form local concentration gradients of silencing proteins to inactivate the X-chromosome. Yolanda Markaki, Johnny Gan Chong, Christy Luong, Shawn Y.X. Tan, Yuying Wang, Elsie C. Jacobson, Davide Maestrini, Iris Dror, Bhaven A. Mistry, Johannes Schoeneberg, Abhik Banerjee, Mitchell Guttman, Tom Chou, Kathrin Plath. bioRxiv 2020.11.22.393546; doi: https://doi.org/10.1101/2020.11.22.393546
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106787
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:23 Nov 2020 22:31
Last Modified:16 Nov 2021 18:56

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