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Xist spatially amplifies SHARP/SPEN recruitment to balance chromosome-wide silencing and specificity to the X chromosome

Jachowicz, Joanna W. and Strehle, Mackenzie and Banerjee, Abhik K. and Blanco, Mario R. and Thai, Jasmine and Guttman, Mitchell (2022) Xist spatially amplifies SHARP/SPEN recruitment to balance chromosome-wide silencing and specificity to the X chromosome. Nature Structural and Molecular Biology, 29 (3). pp. 239-249. ISSN 1545-9993. doi:10.1038/s41594-022-00739-1. https://resolver.caltech.edu/CaltechAUTHORS:20211102-173017796

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Abstract

Although thousands of long non-coding RNAs (lncRNAs) are encoded in mammalian genomes, their mechanisms of action are poorly understood, in part because they are often expressed at lower levels than their proposed targets. One such lncRNA is Xist, which mediates chromosome-wide gene silencing on one of the two X chromosomes (X) to achieve gene expression balance between males and females. How a limited number of Xist molecules can mediate robust silencing of a much larger number of target genes while maintaining specificity exclusively to genes on the X within each cell is not well understood. Here, we show that Xist drives non-stoichiometric recruitment of the essential silencing protein SHARP (also known as SPEN) to amplify its abundance across the inactive X, including at regions not directly occupied by Xist. This amplification is achieved through concentration-dependent homotypic assemblies of SHARP on the X and is required for chromosome-wide silencing. Expression of Xist at higher levels leads to increased localization at autosomal regions, demonstrating that low levels of Xist are critical for ensuring its specificity to the X. We show that Xist (through SHARP) acts to suppress production of its own RNA which may act to constrain overall RNA levels and restrict its ability to spread beyond the X. Together, our results demonstrate a spatial amplification mechanism that allows Xist to achieve two essential but countervailing regulatory objectives: chromosome-wide gene silencing and specificity to the X. This suggests a more general mechanism by which other low-abundance lncRNAs could balance specificity to, and robust control of, their regulatory targets.


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https://doi.org/10.1038/s41594-022-00739-1DOIArticle
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https://doi.org/10.1101/2021.10.27.466149DOIDiscussion Paper
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ORCID:
AuthorORCID
Jachowicz, Joanna W.0000-0002-1599-682X
Strehle, Mackenzie0000-0003-1410-8701
Banerjee, Abhik K.0000-0001-9127-5461
Blanco, Mario R.0000-0002-9852-2231
Guttman, Mitchell0000-0003-4748-9352
Alternate Title:Xist spatially amplifies SHARP/SPEN recruitment to balance chromosome-wide silencing and specificity to the X chromosome, Xist spatially amplifies SHARP recruitment to balance chromosome-wide silencing and specificity to the X chromosome
Additional Information:© 2022 Nature Publishing Group. Received 15 January 2021; Accepted 28 January 2022; Published 17 March 2022. We thank M. Elowitz, S. Chong, I. Goronzy and D. Honson (Caltech) for their critical comments on the manuscript, A. Pandya-Jones, Y. Markaki and K. Plath (UCLA) for initial discussions and guidance on SHARP visualization, and F. Dossin, A. Loda and E. Heard (EMBL) for sharing their cell lines and cell culture protocols. We would like to thank A. Chow (Caltech) for helpful comments and support with the cell culture work done at the Guttman laboratory, the Biological Imaging Facility at Caltech for their help with microscopy, the Flow Cytometry Facility at Caltech for their help with cell sorting, and the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech for their help with sequencing. We also thank S. Hiley for contributions to writing and editing this manuscript, G. Riddihough and M. Bao (Life Science Editors) for editorial assistance, and I.M. Strazhnik (Caltech) for helping with illustrations. This work was supported by the National Institutes of Health (NIH) 4DN program (U01 DA040612 and U01 HL130007), NIH Directors’ Transformative Research Award (R01 DA053178), the New York Stem Cell Foundation and funds from the California Institute of Technology. M.G. is a NYSCF-Robertson Investigator. J.W.J. was supported by a Biology and Biological Engineering postdoctoral fellowship from Caltech. A.K.B. was funded by National Heart, Lung, and Blood Institute F30-HL136080 and the University of Southern California MD/PhD Program. Data availability: Previously published RAP-DNA sequencing data used in Extended Data Fig. 5b,f is available at GEO accession GSE46918. RNA-DNA SPRITE data used in Fig. 5f,g and Extended Data Fig. 5 is available at GEO accession GSE151515. CLAP sequencing data generated in this study and used in Fig. 3c,d and RAP-DNA sequencing data generated in this study and used in Fig. 5d–f is available at GEO accession GSE192574. Additional source data files are available for Figs. 1c,d,e,g, 2e,g, 3b, 4c,e,g and 5c,i,j. Source data are provided with this paper. Contributions: These authors contributed equally: J. W. Jachowicz, M. Strehle. J.W.J. conceived of this project with M.G. J.W.J. and M.S. performed experiments, analyzed and interpreted data, generated figures and wrote the paper. A.K.B. performed all CLAP sequencing experiments and provided comments and edits for the manuscript. J.T. created the SHARP rescue constructs with A.K.B. and assisted with cell culture. M.R.B. worked with A.K.B. on CLAP sequencing experiments, worked with J.W.J. on RAP sequencing experiments, analyzed sequencing data and provided comments and edits for the manuscript. M.G. oversaw all experiments and analysis, performed analyses and generated figures, and wrote the paper with J.W.J. and M.S. The authors declare no competing interests. Peer review information: Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Anke Sparmann, Beth Moorefield and Carolina Perdigoto were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Funders:
Funding AgencyGrant Number
NIHU01 DA040612
NIHU01 HL130007
NIHR01 DA053178
New York Stem Cell FoundationUNSPECIFIED
Caltech Division of Biology and Biological EngineeringUNSPECIFIED
NIH Postdoctoral FellowshipF30-HL136080
University of Southern CaliforniaUNSPECIFIED
Subject Keywords:Dosage compensation; Gene silencing; Long non-coding RNAs; Non-coding RNAs; Transcription
Issue or Number:3
DOI:10.1038/s41594-022-00739-1
Record Number:CaltechAUTHORS:20211102-173017796
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20211102-173017796
Official Citation:Jachowicz, J.W., Strehle, M., Banerjee, A.K. et al. Xist spatially amplifies SHARP/SPEN recruitment to balance chromosome-wide silencing and specificity to the X chromosome. Nat Struct Mol Biol 29, 239–249 (2022). https://doi.org/10.1038/s41594-022-00739-1
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:111705
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:02 Nov 2021 21:34
Last Modified:17 Mar 2022 23:08

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