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Published May 14, 2015 | Accepted Version + Supplemental Material
Journal Article Open

The Xist lncRNA interacts with SHARP to silence transcription through HDAC3


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.

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.

Attached Files

Accepted Version - nihms677968.pdf

Supplemental Material - nature14443-s1.pdf

Supplemental Material - nature14443-sf1.jpg

Supplemental Material - nature14443-sf10.jpg

Supplemental Material - nature14443-sf2.jpg

Supplemental Material - nature14443-sf3.jpg

Supplemental Material - nature14443-sf4.jpg

Supplemental Material - nature14443-sf5.jpg

Supplemental Material - nature14443-sf6.jpg

Supplemental Material - nature14443-sf7.jpg

Supplemental Material - nature14443-sf8.jpg

Supplemental Material - nature14443-sf9.jpg


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August 20, 2023
October 20, 2023