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A protein assembly mediates Xist localization and gene silencing

A Publisher Correction to this article was published on 02 October 2020

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Abstract

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear1. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus2 and binding diverse proteins3,4,5 to achieve X-chromosome inactivation (XCI)6,7. The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off8. Here we show that the Xist RNA-binding proteins PTBP19, MATR310, TDP-4311 and CELF112 assemble on the multivalent E-repeat element of Xist7 and, via self-aggregation and heterotypic protein–protein interactions, form a condensate1 in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase8, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.

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Fig. 1: The E-repeat mediates Xist sequestration and controls the number of Xist foci.
Fig. 2: The E-repeat establishes heritable gene silencing.
Fig. 3: PTBP1, MATR3, TDP-43 and CELF1 confer gene silencing and Xist sequestration functions on the E-repeat.
Fig. 4: Self-association of E-repeat-binding RBPs is critical for formation of the Xi compartment.

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Data availability

All genomic data for Xist interactions and chromatin association have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE137305. Reagents are available upon request.

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Acknowledgements

We thank members of the Plath and Black laboratories for discussions and reading of the manuscript. A.P.-J. was supported by postdoctoral fellowships from the Helen Hay Whitney Foundation and NIH (F32 GM103139); K.P. by Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research (BSCRC) at UCLA, the David Geffen School of Medicine at UCLA, and the Jonnson Comprehensive Cancer Center at UCLA, the NIH (R01 GM115233), and a Faculty Scholar grant from the Howard Hughes Medical Institute; D.L.B. by the NIH (R01 GM049662 and R01 MH109166 (to K.P. and D.L.B.)); M.G. was funded by the New York Stem Cell Foundation, Searle Scholars Program and the Pew-Steward Scholars Program. M.G. is a NYSCF-Robertson Investigator. Y.M., B.P. and S.Z. were supported by the NIH (NICHD 5R03HD095086 to Y.M., R03HD088380 to B.P., R01NS104041 and R01MH116220 to S.Z.). Y.M. and H.L. were supported by the Deutsche Forschungsgemeinschaft (SFB1064/A17 and LE721/18-1). T.C., A.C. and S.S. are supported by graduate fellowships from the Boehringer Ingelheim Foundation (to T.C.); the UCLA Whitcome Fellowship (to A.C.); and the UCLA Broad Stem Cell Research Center – Rose Hills Foundation training award and the UCLA Dissertation Year Fellowship (to S.S.).

Author information

Authors and Affiliations

Authors

Contributions

K.P., A.P.-J., Y.M. and D.L.B. conceptualized the project and A.P.-J. performed the experiments unless stated otherwise. Y.M. and T.C. performed experiments for 3D-SIM imaging and acquired and analysed 3D-SIM data, overseen by H.L. Y.M. acquired high-resolution images and performed image analysis on immunostained cells. J.S. performed all aggregation measurements, helped with EMSAs and analysed RAP–seq data. R.M., W.M. and A.C. helped to create ES cell deletion lines. S.Z. performed the initial PTBP1/2 iCLIP–seq experiments, A.D. helped A.P.-J. with iCLIP–seq experiments, S.S. and J.S. analysed CLIP–seq data, B.P. and C.C. performed and analysed CHIP–seq experiments, X.-J.W. purified rPTBP1 and rCELF1, and C.-K.C. performed RAP–seq experiments. A.P.-J., J.S., Y.M., T.C. and K.P. analysed data, A.P.-J., Y.M., J.S., M.G. and K.P. interpreted the data and contributed towards methodology and model creation, K.P., D.L.B, M.G. and H.L. acquired funding to support the project, A.P.-J. and K.P. administered the project and A.P.-J. and K.P. wrote the manuscript, including edits from all authors.

Corresponding authors

Correspondence to Douglas. L. Black or Kathrin Plath.

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Peer review information Nature thanks Jernej Ule and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Depletion of PTBP1, MATR3, CELF1 and TDP-43 does not strongly affect gene silencing during the Xist-dependent stage of XCI initiation.

a, Experimental schematic. b (i), Immunoblot confirming the siRNA-mediated knockdown of PTBP1, MATR3, and CELF1, normalized to GAPDH. Asterisks indicate non-specific bands. (ii), As in (i), except for TDP-43. Error represents the s.e.m. from three independent experiments. For source data see Supplementary Fig. 1. c (i), Graph showing nascent transcription patterns of the X-linked gene Gpc4 after 3 days of differentiation and knockdown of the indicated factor (spot refers to nascent transcription event on one chromosome) (n = 50 from 1 experiment). (ii), Same as (i) but for Chic1. (iii), Same as (i) but for Atrx. d, Representative images of siRNA-treated differentiating cells immunostained for indicated proteins (red), probed for Xist (green) and DAPI stained (blue). e (i), Schematic for aggregation score calculation. (ii), Box plots showing Xist aggregation scores upon depletion of indicated proteins. Independent siGFP controls were used for CELF1 and TDP-43 experiments. (iii), Box plots showing the Xist mask values used to calculate the aggregation scores in (ii). (iv), Box plots showing the bounding circle area values encompassing the Xist mask used to calculate the Xist aggregation scores in (ii). For box plots in (ii)–(iv): (n = 25): *P < 0.05, ***P < 0.0005, ****P < 0.00005; two-tailed Kolmogorov–Smirnov test from one replicate in b. Horizontal lines denote the median, whiskers indicate 1.5× the interquartile range, dots represent outliers.

Extended Data Fig. 2 Depletion of PTBP1, MATR3, CELF1 and TDP-43 affects Xist localization during XCI initiation without strongly altering Xist processing.

a, Proportion of Xist-positive cells with co-localizing exclusion of RNA Pol II or enrichment of H3K27me3 or the PRC2 components EZH2, EED, SUZ12 on the Xi, in female ES cells differentiated for 3 days and treated with siGFP or siPTBP1 (n = 50 from one experiment). b, Percentage of Xist-positive cells with H3K27me3 Xi-enrichment in day 3 differentiated female ES cells treated with siGFP, siPTBP1, siMATR3, siTDP-43 or siCELF1 (n = 100, from one experiment). The siPTBP1 sample is independent from that in a. c, Xist splicing events assessed below. d, Histogram showing Xist abundance (exon 1 PCR above) upon siRNA-mediated knockdown of indicated RBPs in female ES cells at differentiation day 3. e, As in d, except for the abundance of spliced Xist exon 1–2 and exon 6–7 amplicons upon knockdown. For d and e, samples were normalized against siGFP and Snrnp27 RNA and assessed in triplicate from three independent experiments. Error bars represent s.e.m; *P < 0.05, two-tailed Student’s t-test. f, Snapshot of expected spliced exon 6 (green) to exon 7 (red) sequence. Correct exon 6–7 ligation occurs after 72 h of siGFP or siPTBP1 treatment in differentiating female ES cells (black sequence) in two independent experiments. g (i), A tet-inducible full-length Xist cDNA transgene was inserted into the X-linked Hprt locus in male ES cells. (ii), Percentage of cells with an Xist cloud after 48 h of siPTBP1, and dox treatment starting at 24 h of siRNA treatment, in cells described in (i) (n = 80, from one experiment). (iii), Representative RNA FISH images of Xist, co-immunostained for PTBP1 and DAPI labelled, in cells described and treated as in (i), (ii). Note Xist dispersal upon PTBP1 knockdown, despite the absence of Xist  splicing.

Extended Data Fig. 3 PTBP1, MATR3, CELF1 and TDP-43 directly bind the Xist E-repeat, comprising a tandem array of 20–25nt C/U/G-rich elements.

a (i), Top, diagram of the Xist genomic locus. The IVT E-repeat RNA used in d is indicated. Bottom, PTBP1, MATR3 and CELF1 i/eCLIP–seq profiles across the Xist locus in male tetO-Xist ES cells after 6 h of dox induction. CELF1 input profile is shown, read counts indicated on left. (ii), PTBP1 ChIP–seq profiles across the Xist locus before or after 20 h of dox treatment in male tetO-Xist ES cells. (iii), PTBP1, PTBP2 and TDP-43 iCLIP–seq profiles across the Xist locus in the female mouse brain. b, Table of mapping statistics for PTBP1, MATR3 and CELF1 i/eCLIP–seq data in a. Note that Xist is overexpressed in this experiment, which influences the number of reads mapping to the locus. c (i), The first 1,500 nt of exon 7 of Xist are shown, capturing the E-repeat. The sequence remaining after splicing of the XistΔE transcript is underlined and italicized. The C/U/G tandem repeats within the 5′ half of the E-repeat are indicated (pink-full and blue-truncated repeats) as are the CU-tracts (green) in the 3′ half. Potential TDP-43 sites are indicated in orange. (ii), Alignment of the 25 full C/U/G-tandem repeats (pink) from (i). Brown tracts encode putative PTBP1/MATR3 binding sites, red tracts putative CELF1/TDP-43 binding sites. (iii), Alignment of the nine truncated C/U/G-tandem repeats (blue) from (i). Orange coloured nucleotides are variable within each truncated repeat unit. d, Left: EMSA of IVT E-repeat RNA (see a) and either none, or increasing amounts of rPTBP1 (0, 1.95 nM, 3.9 nM, 7.8 nM, 15.6 nM, 31.3 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1 μM and 2 μM). Right, quantification of the bound RNA fraction (dissociation constant, Kd ≈ 200 nM, from two independent experiments, with s.e.m shown). For source data see Supplementary Fig. 1.

Extended Data Fig. 4 CELF1 and PTBP1 localize within the Xist-coated territory.

a, Experimental schematic. b, Left, confocal-Airyscan sections of wild-type ES cells at differentiation day 3 and 7, immunostained for CELF1 and H3K27me3. Inset, enlargement of the Xi territory. Right, CELF1 staining in greyscale. c, Histogram showing the proportion of H3K27me3-marked Xi’s with a co-localizing CELF1 enrichment. Error bars indicate s.e.m. (n = 50 from 3 coverslips across 2 independent differentiations); *P < 0.05, two-tailed Student’s t-test. d (i), Intensity values for CELF1 fluorescence were recorded across a 2 μm line over the Xi (identified on the basis of the H3K27me3 Xi-staining) or within the nucleoplasm of the same nucleus in z-stack projections. (ii), Box plot showing the distribution of the ratio between the top 10% CELF1 Xi intensity values compared to the top 10% intensity values from the nucleoplasm (n = 12, from one experiment); *P < 0.05, two-sample Kolmogorov–Smirnov test. e (i), Left, As in b, but showing PTBP1 immunostaining at differentiation day 3. Right, PTBP1 staining in greyscale. (ii), As in (i), except at differentiation day 7. Note that these images highlight a mesh-like PTBP1 concentration within the Xi observed in a small fraction of cells, distinct from that observed in the nucleoplasm of these cells or from the pattern within the Xi at day 3. f (i), As in e (i), but showing MATR3 immunostaining and Xi-zoom ins. (ii), As in e (ii), except showing MATR3 immunostaining. g, As in f, except for TDP-43. h, As in d (ii), except showing data for PTBP1, MATR3 and TDP-43, (n = 5, from one experiment). Red dots, data points for the top 10% Xi/Nucleoplasmic intensity values from 5 cells. For box plots in d (ii) and h, horizontal lines denote the median, whiskers indicate 1.5× the interquartile range, dots represent outliers.

Extended Data Fig. 5 ΔE ES cells undergo differentiation similar to wild-type ES cells and splicing of Xist-intron 6 proceeds in the absence of the E-repeat.

a, Homologous recombination strategy used to delete the Xist E-repeat in female ES cells. b, Southern blot strategy with a 5′ external probe for identification of deletion clones. c, Southern blot (described in b) on targeted ES cells with a loxP-flanked puromycin cassette in place of the E-repeat on one Xist allele. d, Sequencing analysis (black) of the wild-type Xist-PCR amplicon in ΔE cells (red line in b). 129-allele SNPs are shown in red and do not match those in PCR amplicon, confirming E-repeat deletion on the XistMS2(129) allele. e, Tsix RNA FISH on undifferentiated wild-type and ΔE ES cells confirms the presence of two Tsix nascent transcription units, used as a proxy to confirm targeted cells maintain two X chromosomes. f, Bright-field images of wild-type and ΔE cells at day 4 of differentiation, showing that differentiating cells are morphologically similar. g, Immunoblot of differentiation day 2 wild-type and ΔE cell lysate, showing equal loss of NANOG expression. h, Sequence of genomic and cDNA amplicons of the XistΔE allele after puromycin cassette removal, confirming correct targeting and the use of a cryptic splice site in ΔE cells. i, Exon 6–7 RT–PCR amplicons generated from RNA isolated from day 4 differentiated wild-type (primers APJ248/624) or ΔE (primers APJ248/631) cells. The ΔE PCR amplicon was shorter than expected. Sequencing revealed a cryptic 3′ splice site downstream of the loxP site that extended the E-repeat deletion within the Xist transcript (but not the Xist genomic DNA) by 42 nt (see (h)). j, PCR amplicons from wild-type or ΔE genomic DNA using the same primers as in i. The intron 6-containing products can be amplified, indicating non-detection of intron 6-containing Xist transcripts is not due to amplification problems. k, Schematic outlining primers used to assess Xist DNA and RNA in i and j. For c, g, i and j, see Supplementary Fig. 1 for source data.

Extended Data Fig. 6 Loss of the E-repeat does not affect Xist abundance, splicing or stability.

a, RT–qPCR quantification of the fold upregulation of XistMS2 RNA during differentiation of wild-type or ΔE cells normalized against undifferentiated samples and an internal control (Rrm2). b, RT–qPCR measurements of XistMS2 RNA half-life (upon actinomycin D treatment) at day 3 of differentiation in wild-type or ΔE cells, calculated as MS2 transcript copy number per μg of total RNA. For a and b, error bars represent the s.e.m. (n = 3, measured in triplicate). Differences were not significant by two-tailed Student’s t-test. c, Epifluorescence images of differentiation day 4 wild-type and ΔE cells probed for exonic regions of Xist (red) or Xist intron 1 (yellow), and DAPI stained, indicating that the XistΔE transcripts within the cloud are spliced. d, Same as Fig. 1h, except two additional XistΔE-expressing nuclei are shown. Scale bar, 5 μm. e, Same as Fig. 1h, except for the nuclei in d. Note aberrant localization of XistΔE at the nuclear lamina. f, 3D Amira reconstructions of the cells shown in Fig. 1h. g, Representative epifluorescence images of RNA FISH against Xist and MS2 with DAPI staining for comparison to super-resolution images in d, e and Fig. 1e, h. Inset, enhanced image of the marked area. h, Box plot showing the distribution of the area (in pixels) covered by the Xist RNA FISH signal, used to calculate the Xist aggregation score in Fig. 1f (n = 30, from one experiment). i, Same as h except showing distribution of the bounding circle area, (n = 30, from one experiment); ***P < 0.00005, two-sample Kolmogorov–Smirnov test. j, Box plot of the average distance between Xist foci within Xist-MS2 clouds in differentiation day 7 wild-type and ΔE ES cells, as measured by IMARIS. 50 measurements were made per cell, 5 cells per sample; ****P < 0.000005, two-sample Kolmogorov–Smirnov test. For hj, horizontal lines denote the median, whiskers indicate 1.5× the interquartile range, dots represent outliers.

Extended Data Fig. 7 The XistΔE-coated X chromosome displays decreased DAPI staining and less compact H3K27me3 accumulation at differentiation day 7.

a, Epifluorescence images of cells immunostained for H3K27me3 and probed for MS2. b, Quantification of XistMS2 RNA FISH clouds with a co-localizing accumulation of H3K27me3 at day 3 or 7 of differentiation in wild-type or ΔE cells (n = 60/coverslip, 3 coverslips over 2 experiments); *P = 0.05, two-tailed Student’s t-test. c (i), Top left, 3D-SIM section of wild-type and ΔE cells at differentiation day 7 stained for H3K27me3 and DAPI and probed for MS2. Inset, DAPI staining of marked region. Right, magnification of inset area with (top) or without DAPI (bottom). Bottom left, Z-stack projection of inset without DAPI. (ii), 3D Amira reconstruction of images in (i). d, Graph showing the number of pixels with indicated DAPI fluorescence intensity from XistMS2-expressing X chromosome in wild-type and ΔE cells, masked by H3K27me3 enrichment (n = 10, from one experiment). e, Epifluorescence images of wild-type and ΔE cells probed for MS2. Arrowheads point to the Xist cloud and highlight the DAPI-bright staining for the X-territory. f (i), Epifluorescence images of wild-type cells stained for EZH2 and Xist, with (left) and without (right) EZH2 Xi-enrichment at differentiation day 7. (ii), Histogram of the percentage of Xist clouds with co-localized EZH2 enrichment (n = 60 per coverslip, 3 coverslips from 2 experiments), *P < 0.05, **P < 0.005, ***P < 0.0005, two-tailed Student’s t-test. g, 3D-SIM sections through day 3.5 differentiated wild-type or ΔE ES cells (EpiLC differentiation), immunostained for RNA Pol II and probed for Xist, showing exclusion of RNA Pol II from the X-territory. Inset, signals derived from marked area. Small images: top left, same as inset without DAPI; bottom left, same as inset with only DAPI; top right, Z-stack projection of the cell; bottom right: Z-stack projection of the Xist-coated X chromosome. Scale bar, 5 μm; inset, 1 μm. h, 3D Amira reconstruction of cells in Fig. 2e. Inset, enlargement of the XistMS2-expressing X. Right, same as left without DAPI. i, Quantification of RNA Pol II exclusion from XistMS2-coated territory (n = 50 per coverslip, 2 coverslips from 1 experiment), *P = 0.05, two-tailed Student’s t-test.

Extended Data Fig. 8 Loss of the E-repeat prevents continued gene silencing in differentiating ES cells.

a, Histograms of nascent transcription pattern of indicated X-linked genes (Rnf12 (Rlim), Atrx, Mecp2, Gpc4 and Chic1) in undifferentiated wild-type and ΔE ES cells, demonstrating that heterozygous deletion of the E-repeat does not interfere with X-linked gene expression in undifferentiated ES cells (n = 60, from one experiment). b, Representative epifluorescence images of cells counted in a. Tsix, the antisense transcript of Xist, was also detected here to identify both X chromosomes. Co-localized foci appear yellow. c, Histograms of nascent expression patterns of the X-linked genes Gpc4 and Atrx in wild-type and ΔE cells displaying an Xis tMS2-coated X chromosome (n = 50), across 5 days of differentiation. These data were derived from an independent differentiation from that shown in Fig. 2c. d, Histograms of nascent expression patterns of indicated X-linked genes in wild-type and ΔE cells displaying an Xis tMS2-coated X chromosome (n = 50), across 7 days of differentiation derived from an independent differentiation from that shown in c and Fig. 2c. e, Histogram of nascent expression patterns of the X-linked gene Tsix in wild-type and ΔE cells across 5 days of differentiation. Note that these data were not scored relative to XistMS2 expression (that is, the monoallelic Tsix signal can be derived from either the 129 or cas allele (n = 70, except for the ΔE cells at day 5 with only 47 cells counted).

Extended Data Fig. 9 A site-specific recombination-based approach to rescue phenotypes associated with loss of the E-repeat.

a, Flp-In approach taken to constitutively express Flag-tagged MCP fusion proteins in ES cells (Methods). The Flag–MCP–GFP fusion protein was only expressed in wild-type ES cells. All other rescue constructs were expressed in ΔE ES cells. b, Flag–MCP–GFP fusion protein recruitment to XistMS2 in wild-type cells at differentiation day 7 shown with representative epifluorescence images. Arrows indicate MS2+Xist129 clouds with co-localizing Flag–MCP–GFP enrichment. c, Tsix expression was used as a proxy to confirm presence of two X chromosomes in rescue ES cell lines. d (i), PTBP1-probed immunoblot on lysates from undifferentiated ΔE ES cells expressing full-length MCP–PTBP1 or MCP-PTBP1 mutants. (ii), As in d (i) except for MATR3 immunoblot for various MATR3 rescue lines. (iii), TDP-43-probed immunoblot on lysates from undifferentiated ΔE ES cells expressing MCP–TDP-43. (iv), CELF1-probed immunoblot on lysates from undifferentiated ΔE ES cells expressing MCP–CELF1. e, Histogram of the percentage of XistMS2 clouds that also show enrichment of H3K27me3 in wild-type or ΔE cells, or ΔE cells expressing the indicated MCP-fusion protein at differentiation day 7 (n = 80, from one experiment). f, Representative epifluorescence images of RNA FISH against Xist (green) and MS2 (red) in day 7 differentiated ΔE cell lines expressing the indicated variants of MCP fusion proteins. Inset, enlargement of the marked area. Arrowheads indicate wild-type Xist clouds in ΔE cells, derived from the cas allele. g, Immunoprecipitation of PTBP1, MATR3, CELF1, TDP-43 and CIZ1 from ES cell nuclear extracts (RNase treated) and detection of co-precipitated proteins with the same antibodies by immunoblotting (to accompany Fig. 3f). For images in d and g, see Supplementary Fig. 1 for source data.

Extended Data Fig. 10 Expression of MCP–CIZ1 or MCP–GFP–MCP does not rescue phenotypes due to loss of the E-repeat.

a, RNA FISH images of Tsix transcripts for detection of two X chromosomes. Two ΔE MCP–CIZ1 ES cell clones (9 and 10) are shown. b, Immunoblot result for undifferentiated ΔE ES cell clones expressing MCP–CIZ1. c, Representative epifluorescence images of day 7 differentiated MCP–CIZ1-expressing ΔE clones, probed for Xist and MS2. d, Proportion of Xist clouds also displaying a co-localizing MS2 signal at differentiation day 7. The results for both CIZ1 rescue clones from one experiment were merged and the error bars represent s.e.m (n = 120), P: not significant, two-tailed Student’s t-test. e, Quantification of nascent Gpc4 or Atrx expression patterns in wild-type, ΔE, or ΔE cells expressing MCP–CIZ1 (clone 9) displaying XistMS2 expression, at differentiation day 7 (n = 50, from one experiment). See k for legend. f, Representative epifluorescence images of in wild-type, ΔE or indicated ΔE rescue cell lines at differentiation day 7 immunostained for CIZ1 and probed for MS2. Arrowheads indicate rescued cloud from the ΔE XistMS2 allele. Fraction of MS2+Xist clouds showing CIZ1 enrichment is given. g, RNA FISH images of Tsix transcripts in ΔE MCP–GFP–MCP ES cells to demonstrate the presence of two X chromosomes. h, Representative epifluorescence images of day 7 differentiated MCP–GFP–MCP-expressing ΔE ES cells probed for Xist and MS2, and illustration of Flag-tagged MCP–GFP–MCP fusion protein (see Fig. 3b for key). i, Immunoblot against the Flag-tag and GAPDH using lysates from undifferentiated MCP–GFP–MCP ΔE ES cells. j, Histogram showing the proportion of nuclei with Xist FISH signal that also displayed a co-localizing MS2 signal at differentiation day 7 for indicated cell lines (n = 100); P: not significant, two-tailed Student’s t-test, using 2 independent MCP–GFP–MCP expressing clones from one experiment. k, Quantification of nascent Gpc4 or Atrx expression patterns in cells displaying XistMS2 expression at differentiation day 7 (n = 50, from one experiment). For images in b and i, see Supplementary Fig. 1 for source data.

Extended Data Fig. 11 CELF1 enhances droplet formation of PTBP1 with the E-repeat in vitro and mutations in MATR3 and TDP-43 that abrogate their self-association do not rescue ΔE phenotypes.

a, Images showing lack of droplets with 60 μM rPTBP1, 3.2 μM E-repeat or control RNA at 40 min. b, Droplets formed from 60 μM rPTBP1 and 0.5 μM E-repeat RNA over time. c, Same as a except with 0.5 μM control RNA and different concentrations of rPTBP1 (40 min). d, Same as a except with 60 μM rPTBP1 and 20 μM rCELF1, or 38 μΜ rCELF1 with 0.5 μM E-Repeat RNA. Arrowheads indicate solution boundary with sample on left. e, Bright-field images showing aggregate-like formations of 20 μM rCELF1, 0.5 μM E-repeat RNA with varied concentrations of rPTBP1. f, RNA FISH images of Tsix transcripts in indicated ES cell lines to show presence of both X chromosomes. g (i), MATR3 immunoblot on extracts from ΔE ES cells expressing MCP–MATR3(S85C). (ii), TDP-43 immunoblot on ΔE ES cells expressing MCP–TDP-43(EGGG). h, Epifluorescence images of day 7 differentiated ΔE cells expressing MCP–MATR3(S85C) (i) or MCP–TDP-43(EGGG) (ii) probed for Xist and MS2. i, MEFs (Xist2lox/2lox, R26M2rtTA/tetO-Cre) probed for Xist, before or after dox treatment (96 h). Percentage of cells with displayed Xist pattern is given (n = 50, two biological replicates). j, Histogram showing percentage of MEFs with H3K27me3 or CELF1 Xi-enrichment under conditions described in i. Error bars represent s.e.m, (n = 50, from two biological replicates). k, Histogram showing relative Xist abundance over time of dox treatment for cells in i (see Fig. 4g.). l, Experimental schematic for knockdown experiment in mq. m, Immunoblot showing knockdown of indicated factors in the experiment described in l. n, Percentage of MEFs (no dox) with an Xist cloud for indicated knockdowns (n = 50, from one experiment). o, Percentage of MEFs (no dox) with an Xi-enrichment of H3K27me3 that show a co-localizing accumulation of CELF1 (n = 50, from one experiment). p, Same as n except with dox treatment. q, Percentage of MEFs with CELF1 enrichment (n = 50, from one experiment). For images in g and m, see Supplementary Fig. 1 for source data.

Supplementary information

Supplementary Figures

Supplementary Figure 1: PDF of original source images for EMSA and immunoblots shown in Fig 3 and Extended Data Figs. 1, 3, 5 and 9-11.

Reporting Summary

Supplementary Table

Supplementary Table 1: PDF with information on the primers used in this study.

Supplementary Table

Supplementary Table 2: PDF with information on the primary antibodies used in this study.

Supplementary Information

Supplementary File 1: Text file containing supplementary notes on experimental interpretation, separate to the methods used for data generation.

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Pandya-Jones, A., Markaki, Y., Serizay, J. et al. A protein assembly mediates Xist localization and gene silencing. Nature 587, 145–151 (2020). https://doi.org/10.1038/s41586-020-2703-0

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