Many long non-coding RNAs (lncRNAs) affect gene expression1, but the mechanisms by which they act are still largely unknown2. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals3,4. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role3. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell5. 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-repressor6 that activates HDAC37, 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.
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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.
The authors declare no competing financial interests.
Extended data figures and tables
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.
a, RT-qPCR measuring the percentage of the total cellular Xist or 18S recovered after RAP-MS of Xist. Values are computed as the amount of each RNA in the elution divided by the amount of RNA in the starting (‘input’) lysate material. Error bars represent the standard error of the mean from 5 biological replicates. b, Enrichment of Xist after RAP-MS captures from pSM33 cells as measured by qPCR. Bars indicate RNA levels of Xist, 18S, and Oct4 after purification of Xist, normalized to RNA in input sample. Each bar represents the RNA levels of Xist, 18S, and Oct4 after purification of Xist, normalized to RNA in input sample, from 3 biological replicates. c, SILAC labelling efficiency of a representative culture of pSM33 mouse ES cells after 10 days of growth (3 cell passages) in SILAC medium. Peptides were analysed by mass spectrometry, and values indicate the fraction of identified peptides with heavy-label incorporation with different levels of peptide labelling (shown in bins).
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.
a, SILAC ratios of top proteins enriched in the RAP-MS U1 snRNA, 18S rRNA, and 45S pre-rRNA experiments. b, SILAC ratio plot of replicate captures of U1 snRNA versus 18S rRNA from one of two biologically independent label-swap experiments. Proteins associated with U1 are consistently found in U1 samples, both light and heavy labelled (top right quadrant), and proteins specifically associated with 18S are consistently identified in 18S, both light and heavy (lower left quadrant). Background contaminant proteins have low enrichments (centre of panel) or are consistently found in the light channel and do not replicate between experiments (that is, keratin, streptavidin). c, SILAC ratio plot of replicate captures of U1 snRNA versus 45S pre-rRNA from one label-swap experiment. Proteins that are known to associate with 45S pre-rRNA are consistently identified in 45S captures.
Extended Data Figure 3 Immunoprecipitation of the identified Xist-interacting proteins confirms Xist RNA interaction.
RNA immunoprecipitation experiments were performed for seven Xist-interacting proteins (black bars), two control RNA binding proteins that were not identified by RAP-MS and IgG (grey bars) in UV-crosslinked cell lysate after 6 h of Xist induction by doxycycline addition (Methods). The RNA associated with each protein was measured and enrichment levels were computed relative to the level of the RNA in total cellular input and normalized to the total efficiency of capture in each sample to allow for direct comparison across all immunoprecipitation experiments (Methods). a, Enrichment of the Xist lncRNA after immunoprecipitation from a sample of pSM33 male cells. b, Immunoprecipitation of SHARP was performed from a sample of UV-crosslinked females ES cells that were treated with retinoic acid for 24 h. The levels of recovered Xist lncRNA (black bars), Neat1 lncRNA (white bars), and 45S pre-ribosomal RNA (grey bars) were measured by RT-qPCR. Enrichment of each RNA after capture with anti-SHARP antibody was calculated relative to the level of RNA captured with IgG control antibody. c, The enrichment of various lncRNAs after immunoprecipitation in pSM33 male cells—including Neat1, Malat1, Firre, and Tug1—are shown. d, The enrichment of various mRNA controls after immunoprecipitation in pSM33 male cells—including Oct4, Nanog, Stat3, and Suz12—are shown.
Extended Data Figure 4 Previously identified proteins associated with XCI are not required for Xist-mediated transcriptional silencing.
a, To confirm the specificity of our assay, we tested the function of several proteins that were previously identified to associate with Xist, but not to silence transcription, for their role in transcriptional silencing in our inducible male ES cells before Xist induction (−Dox; left) or after Xist induction for 16 h (+Dox; middle and right). Representative images are shown after knockdown of each protein. DAPI (blue), Xist (red), and Gpc4 (green). b, Quantification of the copy number of Gpc4 before and after Xist induction upon treatment with different siRNAs. Error bars represent the standard error of the mean across 50 individual cells from one experiment. ****P value < 0.001 between +Dox and –Dox cells based on an unpaired two-sample t-test. Scale bars on the images represent 5 μm. Importantly, while these proteins do not have a role in the initiation of transcriptional silencing, we do not mean to imply that they do not have other roles in XCI.
Extended Data Figure 5 SHARP, LBR, SAF-A, SMRT, and HDAC3 are required for Xist-mediated transcriptional silencing.
a, Representative images showing staining of DAPI (blue), Xist (red), and Gpc4 (green) for different siRNA knockdown in male ES cells before Xist induction (−Dox; left) or after Xist induction for 16 h (+Dox; middle and right). b, Quantification of the copy number of Gpc4 in –Dox and +Dox cells after knockdown with siRNAs targeting different mRNAs. Error bars represent the standard error of the mean across 50 individual cells from one experiment. NS, not significantly different between +Dox and –Dox cells; ****P value < 0.001 between +Dox and –Dox cells based on an unpaired two-sample t-test. Scale bars on the images represent 5 μm. c, Knockdown of SHARP, LBR, or SAF-A abrogates Xist-mediated gene silencing without causing pluripotency defects. Representative images showing staining of Nanog (cyan), Xist (red), and Gpc4 (green) upon knockdown of SHARP, LBR or SAF-A after 16 h of Xist induction with doxycycline. Scale bars on the images represent 5 μm.
a, A diagram showing the locations of Xist (red), X-linked silenced genes (black), and X-linked escaped genes (green) along the X chromosome. b, Representative images showing staining of DAPI (blue), Xist (red), X-linked silenced genes (green), and X-linked escaped genes (yellow) upon knockdown of SHARP or control male ES cells before Xist induction (−Dox) or after Xist induction for 16 h (+Dox). Knock of SHARP abolishes the silencing of Atrx, Gpc4, Rbmx, Smc1a and Mecp2, which are normally silenced upon Xist expression, but has no effect on Mid1 and Pir, which normally escape Xist-mediated silencing. The bar graphs show the quantification of the copy number of the mRNA for each gene for –Dox and +Dox cells upon transfection with SHARP siRNA or control siRNA; error bars represent the standard error of the mean across 50 individual cells from one experiment. NS, not significantly different, ****P value < 0.001, and **P value < 0.01 between +Dox and –Dox cells based on an unpaired two-sample t-test. Scale bars on the images represent 5 μm.
Extended Data Figure 7 Multiple independent siRNAs targeting SHARP, LBR, SAF-A, HDAC3, or SMRT demonstrate the same silencing defect.
a, Representative images showing staining of DAPI (blue), Xist (red), and Gpc4 (green) after knockdown of proteins using independent, non-overlapping, siRNA pools, or individual siRNA deconvoluted from the pool before Xist induction (−Dox; left) or after Xist induction for 16 h (+Dox; middle and right). Cells were either transfected with the siRNA pool from Dharmacon (siRNA-D), Qiagen (siRNA-Q) or Ambion/Life Technologies (siRNA-A), or each individual siRNA deconvoluted from the pool from Dharmacon (siRNA-D1, 2, 3, 4) or Qiagen (siRNA-Q1, 2, 3, 4). b, Quantification of the copy number of Gpc4 in –Dox and +Dox cells after knockdown with siRNAs targeting different mRNAs. Error bars represent the standard error of the mean across 50 individual cells from one experiment. NS, not significantly different between +Dox and –Dox cells based on an unpaired two-sample t-test. Scale bars on the images represent 5 μm. We excluded all siRNAs that did not reduce the targeted mRNA level by >70% (Methods). The sequences of deconvoluted siRNAs are shown in Supplementary Table 2.
Extended Data Figure 8 SHARP, LBR, SAF-A, SMRT, and HDAC3 are required for transcriptional silencing in differentiating female ES cells.
a, Representative images showing staining of DAPI (blue), Xist (red), and Gpc4 (green) upon knockdown of specific proteins using different siRNAs in female ES cells before differentiation (−RA; left) or after differentiation for 24 h (+RA; middle and right). RA, retinoic acid. b, Quantification of the copy number of Gpc4 for –RA and +RA cells upon transfection with different siRNAs. Error bars represent the standard error of the mean across 50 individual cells from one experiment. NS, not significantly different between +RA and –RA cells; ****P value < 0.001, **P value < 0.01, and *P value < 0.05 between +RA and –RA cells based on an unpaired two-sample t-test. Scale bars on the images represent 5 μm.
Extended Data Figure 9 SHARP is required for exclusion of RNA polymerase II from the Xist-coated territory in differentiating female ES cells.
Images of individual cells that are labelled with Xist (red), RNA Polymerase II (green), and DAPI (blue) across different siRNA conditions (rows) in female ES cells after 24 h of retinoic acid treatment. The dashed white region represents the outlined Xist-coated territory.
Extended Data Figure 10 SHARP is required for PRC2 recruitment across the Xist-coated territory in differentiating female ES cells.
Images of individual cells that are labelled with Xist (red), Ezh2 (green) and DAPI (blue) across different siRNA conditions (rows) in female ES cells after 24 h of differentiation. The dashed white region represents the outlined Xist-coated territory.
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McHugh, C., Chen, CK., Chow, A. et al. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521, 232–236 (2015). https://doi.org/10.1038/nature14443
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