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The Xist lncRNA interacts with SHARP to silence transcription through HDAC3

McHugh, Colleen A. and Chen, Chun-Kan and Chow, Amy and Surka, Christine F. and Tran, Christina and McDonel, Patrick and Pandya-Jones, Amy and Blanco, Mario and Burghard, Christina and Moradian, Annie and Sweredoski, Michael J. and Shishkin, Alexander A. and Su, Julia and Lander, Eric S. and Hess, Sonja and Plath, Kathrin and Guttman, Mitchell (2015) The Xist lncRNA interacts with SHARP to silence transcription through HDAC3. Nature, 521 (7551). pp. 232-236. ISSN 0028-0836. PMCID PMC4516396. doi:10.1038/nature14443.

[img] PDF - Accepted Version
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[img] PDF (Supplementary Notes 1-5 and Supplementary Tables 1-2) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 1: RAP-MS recovers and enriches the majority of Xist RNA from mouse ES cells, and these cells can be efficiently labelled with SILAC) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 2: RAP-MS identifies proteins that are known to directly interact with specific ncRNAs, and separates specific RNA interacting proteins from background proteins) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 3: Immunoprecipitation of the identified Xist-interacting proteins confirms Xist RNA interaction) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 4: Previously identified proteins associated with XCI are not required for Xist-mediated transcriptional silencing) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 5: SHARP, LBR, SAF-A, SMRT, and HDAC3 are required for Xist-mediated transcriptional silencing) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 6: SHARP is required for silencing many genes across the X chromosome) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 7: Multiple independent siRNAs targeting SHARP, LBR, SAF-A, HDAC3, or SMRT demonstrate the same silencing defect) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 8: SHARP, LBR, SAF-A, SMRT, and HDAC3 are required for transcriptional silencing in differentiating female ES cells) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 9: SHARP is required for exclusion of RNA polymerase II from the Xist-coated territory in differentiating female ES cells) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 10: SHARP is required for PRC2 recruitment across the Xist-coated territory in differentiating female ES cells) - Supplemental Material
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Many long non-coding RNAs (lncRNAs) affect gene expression, but the mechanisms by which they act are still largely unknown. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell. Here we develop a method to purify a lncRNA from cells and identify proteins interacting with it directly using quantitative mass spectrometry. We identify ten proteins that specifically associate with Xist, three of these proteins—SHARP, SAF-A and LBR—are required for Xist-mediated transcriptional silencing. We show that SHARP, which interacts with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of RNA polymerase II (Pol II) from the inactive X. Both SMRT and HDAC3 are also required for silencing and Pol II exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for Xist-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X chromosome. Our results suggest that Xist silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude Pol II across the X chromosome.

Item Type:Article
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URLURL TypeDescription ReadCube access Information CentralArticle
Moradian, Annie0000-0002-0407-2031
Sweredoski, Michael J.0000-0003-0878-3831
Lander, Eric S.0000-0003-2662-4631
Hess, Sonja0000-0002-5904-9816
Guttman, Mitchell0000-0003-4748-9352
Additional Information:© 2015 Macmillan Publishers Limited. Received 25 November 2014; Accepted 02 April 2015; Published online 27 April 2015. We thank J. Engreitz for extensive discussions, help in adapting the RAP method, and critical comments on the manuscript; A. Gnirke, S. Carr, J. Jaffe and M. Schenone for initial discussions about the RAP-MS method; A. Collazo, E. Lubek, and L. Cai for microscopy help; A. Wutz for providing transgenic cell lines; R. Eggleston-Rangel for assistance with mass spectrometry; S. Grossman, I. Amit, M. Garber and J. Rinn for comments on the manuscript and helpful suggestions; and S. Knemeyer for illustrations. C.A.M. is supported by a post-doctoral fellowship from Caltech. C.-K.C. is supported by an NIH NRSA training grant (T32GM07616). Imaging was performed in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. This work was funded by the Gordon and Betty Moore Foundation (GBMF775), the Beckman Institute, and NIH (1S10RR029591-01A1 to S.H.), an NIH Director’s Early Independence Award (DP5OD012190), the Rose Hills Foundation, Edward Mallinckrodt Foundation, Sontag Foundation, Searle Scholars Program, and funds from the California Institute of Technology. Contributions: C.A.M. developed the RAP-MS method, designed, performed, and analysed RAP-MS experiments and data, C.-K.C. designed, performed, and analysed Xist functional experiments, A.C. designed, performed, and oversaw experiments, C.F.S. helped develop RAP-MS and performed experiments, C.T., P.M., A.P.-J., A.M., A.A.S., J.S. performed experiments, M.J.S., M.B., C.B. analysed data, E.S.L. helped develop initial ideas for adapting RAP for protein detection, S.H. oversaw mass spectrometry development and data analysis, K.P. helped design Xist RAP-MS and functional experiments and analysed data, M.G. conceived, designed and oversaw the entire project and integrated the data, C.A.M., C.-K.C. and M.G. wrote the manuscript with input from all authors. The authors declare no competing financial interests.
Funding AgencyGrant Number
Caltech Postdoctoral FellowshipUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Arnold and Mabel Beckman FoundationUNSPECIFIED
Gordon and Betty Moore FoundationGBMF775
NIH Director’s Early Independence AwardDP5OD012190
Rose Hills FoundationUNSPECIFIED
Edward Mallinckrodt FoundationUNSPECIFIED
Sontag FoundationUNSPECIFIED
Searle Scholars ProgramUNSPECIFIED
Issue or Number:7551
PubMed Central ID:PMC4516396
Record Number:CaltechAUTHORS:20150319-091012142
Persistent URL:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:55922
Deposited By: Katherine Johnson
Deposited On:28 Apr 2015 01:59
Last Modified:10 Nov 2021 20:52

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