sign in sign up
<<
Home , Chromosome 17, Neil Brockdorff, PRC2, SUZ12

Spatial separation of RNA and polycomb revealed by superresolution microscopy

Andrea Cerasea, Daniel Smeetsa,1, Y. Amy Tangb,2, Michal Gdulac, Felix Krausd, Mikhail Spivakovb,3, Benoit Moindrota, Marion Leleub,4, Anna Tattermuscha, Justin Demmerlea, Tatyana B. Nesterovaa, Catherine Greenc, Arie P. Ottee, Lothar Schermelleha, and Neil Brockdorffa,5

aDepartment of , , Oxford OX1 3QU, United Kingdom; bUK MRC Clinical Sciences Centre, Faculty of Medicine ICSTM, Hammersmith Hospital, London W12 ONX, United Kingdom; cWellcome Trust Centre for Genetics, Oxford OX3 7BN, United Kingdom; dDepartment Biology II, Ludwig Maximilians University Munich, 82152 Martinsried, Germany; and eSwammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM, Amsterdam, The Netherlands

Edited by Joan A. Steitz, Howard Hughes Medical Institute, New Haven, CT, and approved December 23, 2013 (received for review July 9, 2013)

In female mammals, one of the two X is transcrip- biological work demonstrating enrichment of PRC2 proteins tionally silenced to equalize X-linked dosage relative to XY on Xi and a close overlap with sites of Xist RNA localization, males, a process termed X inactivation. Mechanisti- both at metaphase and interphase (9–11). PRC2 recruitment cally, this is thought to occur via directed recruitment of was seen to occur coincidently with the onset of XCI (10, 11) and modifying factors by the master regulator, X-inactive specific to be strictly dependent on continuous Xist RNA transcription transcript (Xist) RNA, which localizes in cis along the entire length (11, 12). Collectively, these observations point to PRC2 being of the chromosome. A well-studied example is the recruitment of recruited directly via interaction with Xist RNA, and this idea polycomb repressive complex 2 (PRC2), for which there is evidence has been reinforced by biochemical evidence supporting direct of a direct interaction involving the PRC2 proteins Enhancer of zeste interaction between the conserved A-repeat domain of Xist RNA and the PRC2 proteins Enhancer of zeste 2 (Ezh2) and/or 2 (Ezh2) and Supressor of zeste 12 (Suz12) and the A-repeat region – located at the 5′ end of Xist RNA. In this study, we have analyzed Supressor of zeste 12 (Suz12) (7, 13 15). There are nevertheless Xist-mediated recruitment of PRC2 using two approaches, microarray- some observations that are difficult to reconcile with this view. Notably, Xist expression in early mouse embryos precedes PRC2 based epigenomic mapping and superresolution 3D structured recruitment to Xi (16, 17), and expression of Xist RNA trans- illumination microscopy. Making use of an ES cell line carrying lacking the A-repeat nevertheless recruits PRC2, albeit an inducible Xist transgene located on mouse , less efficiently (12). we show that 24 h after synchronous induction of Xist expres- A second major polycomb group (PcG) complex, PRC1, that sion, acquired PRC2 binding sites map predominantly to gene- mediates monoubiquitylation of H2A lysine 119 is also rich regions, notably within gene bodies. Paradoxically, these new sites of PRC2 deposition do not correlate with Xist-mediated Significance . The 3D structured illumination microscopy was performed to assess the relative localization of PRC2 proteins and Xist RNA. Unexpectedly, we observed significant spatial sep- Polycomb repressor proteins are recruited to the inactive X aration and absence of colocalization both in the inducible Xist chromosome in mammals, and this has been attributed to a – transgene ES cell line and in normal XX somatic cells. Our obser- biochemical interaction between the non -coding RNA vations argue against direct interaction between Xist RNA and X-inactive specific transcript (Xist), which initiates the X inacti- PRC2 proteins and, as such, prompt a reappraisal of the mechanism vation process, and core polycomb subunits. We have studied for PRC2 recruitment in inactivation. this using a combination of mapping analysis and 3D

structured illumination microscopy (3D-SIM) that allows 3D im- GENETICS aging with eightfold volumetric resolution improvement com- chromosome inactivation (XCI) is the dosage compensation pared with previous state-of-the-art confocal microscopy. Our Xmechanism used by mammals to equalize levels of X-linked findings reveal that Xist-mediated recruitment of polycomb re- genes in females relative to males. The process is triggered in in cis pressors does not correlate well with gene silencing and, more- early development by expression and localization of the over, that using 3D-SIM, polycomb proteins and Xist RNA show noncoding RNA (ncRNA), X-inactive specific transcript (Xist). significant spatial separation. These observations challenge Chromosome coating by Xist RNA sets in motion a cascade of prevailing models and prompt a reappraisal of the role of Xist chromatin modifications culminating in mitotically stable silencing RNA in polycomb recruitment. of X-linked genes. Chromatin modifications on the inactive X chromosome (Xi) include gain or loss of specific histone tail Author contributions: L.S. and N.B. designed research; A.C., D.S., Y.A.T., A.T., J.D., and T.B.N. posttranslational modifications, histone variants, proteins that performed research; A.P.O. contributed new reagents/analytic tools; A.C., Y.A.T., M.G., F.K., modulate higher order chromosome structure, and gain of DNA M.S., B.M., M.L., and C.G. analyzed data; and L.S. and N.B. wrote the paper. methylation at promoter-associated CpG islands (CGIs) (1). Xist The authors declare no conflict of interest. transgenes located on function similarly, triggering This article is a PNAS Direct Submission. chromatin modification and gene silencing in cis on the transgene- Freely available online through the PNAS open access option. bearing chromosome (2, 3). 1Present address: Department Biology II, Ludwig Maximilians University Munich, 82152 The molecular mechanisms linking Xist RNA to the silencing Martinsried, Germany. machinery remain incompletely understood (4). Nevertheless, 2Present address: EMBL-European Bioinformatics Institute (EBI), Wellcome Trust Genome several factors have been identified as having a role in Xist- Campus, Hinxton, Cambridge CB10 1 SD, United Kingdom. mediated chromosome silencing. Notably, the transcription fac- 3Present address: The Babraham Institute, Babraham Research Campus, Cambridge CB22 tor YY1 (5); the nuclear matrix protein SAF-A/hnRNPU (6); 3AT, United Kingdom. and the polycomb repressive complex 2 (PRC2), which catalyzes 4Present address: Bioinformatics and Biostatistics Core Facility, EPFL/SIB, 1015 Lausanne, trimethylation of at lysine 27 () (7), have Switzerland. been suggested to interact directly with Xist RNA. 5To whom correspondence should be addressed. E-mail: [email protected]. Recruitment of PRC2 in XCI has been studied extensively. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Initial evidence came from a genetic study (8), with subsequent 1073/pnas.1312951111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1312951111 PNAS | February 11, 2014 | vol. 111 | no. 6 | 2235–2240 Downloaded by guest on September 29, 2021 recruited in XCI (18). Until recently, this finding was attrib- Results uted to binding of PRC1 to PRC2-mediated H3K27me3 via the PRC2 Is Recruited to Chromosome 17 Gene-Rich Domains in Response chromodomain of the core PRC1 subunit, Cbx2/4/6/7/8. However, to Xist RNA Induction. We set out to map Xist-mediated PRC2 recent studies have revealed that noncanonical PRC1 complexes binding sites at an early time point following the onset of Xist in which Cbx family proteins are not present are also recruited to RNA expression. This required a system in which Xist RNA ex- Xi, via an H3K27me3-independent pathway (19, 20). It is not pression could be induced synchronously and in a high proportion known, however, if this involves direct interaction of noncanonical of cells. We therefore made use of a previously characterized PRC1 with Xist RNA. Studies on the XCI system have prompted mouse ES cell line, 3E, that carries a doxycycline-inducible A suggestions of a wider role for ncRNA in PRC2 and PRC1 re- Xist transgene integrated on chromosome 17 (30) (Fig. 1 ). cruitment (recently reviewed in ref. 21). Notable examples are the We established 24 h postinduction as the earliest time point at which robust Xist RNA domains could be observed in a high Kcnqlot1 ncRNA that functions as a master regulator in parental proportion of interphase nuclei in undifferentiated 3E ES imprinting (22); the ncRNA HOTAIR implicated in recruitment cells (Fig. 1 B and C). Similar kinetics were observed for Hox in trans of PRC2 to gene loci (23); the COLDAIR ncRNA PRC2-mediated H3K27me3 (Fig. 1 B and C), as seen pre- proposed to play a role in PRC2 recruitment to the FLC in viously (12). Arabidopsis thaliana (24); and ANRIL ncRNA, which is implicated To map sites of PRC2-mediated H3K27me3, we developed a in recruitment of PRC1 complexes to the INK4a locus (25). high-density oligonucleotide-tiling array encompassing a 65.4-Mb An important question regarding the mechanism of XCI is the span of mouse chromosome 17 [Mb 20–86, Mus musculus 8 (mm8) identity of chromosomal sites to which Xist RNA binds. Cyto- assembly]. Control regions included known ES cell PRC2 target genetic analyses indicate that these sites are located discontin- genes, non-PRC2 target genes, and around 800 kb of randomly uously along the length of the chromosome, being concentrated selected regions on chromosomes 12 and 18 (Table S1). To de- in gene-rich domains (26). Xist RNA-mediated recruitment of termine sites of PRC2 activity, we carried out ChIP using an PRC2 has been analyzed by ChIP sequencing both in differen- antibody specific to H3K27me3. Immunoprecipitated material from multiple experiments was pooled and hybridized to tiling tiating XX ES cells (27, 28) and in trophoblast stem (TS) cells arrays to determine relative signal in doxycycline-induced and (29). These studies determined that sites of PRC2 enrichment on matched untreated control 3E ES cells. Data were obtained from at the Xi map broadly within gene-rich domains, consistent with least three biological replicates. Strong enrichment of H3K27me3 cytogenetic analysis, and further suggested primary localization was seen at positive control loci, for example, the HoxC cluster to preexisting sites of PRC2 occupancy and spreading to non- (Fig. S1A), and not at negative controls, for example, the Sox2 canonical genic and intergenic sites, short interspersed nuclear gene promoter (Fig. S1B). elements (SINEs), and simple repeats (27–29). In this study, we We defined H3K27me3 peaks using Tilemap software (http:// used a similar approach to map PRC2 binding sites at an early jilab.biostat.jhsph.edu/software/tilemap/index.htm)(Materials and time point following synchronous induction of a Xist transgene Methods) and determined peaks that are common to uninduced located on an , mouse chromosome 17. Using this and doxycycline-induced samples, as well as new peaks present system, we show that newly acquired sites of PRC2 activity map only in induced samples (H3K27me3 new peaks). As anticipated, predominantly to gene-rich regions but that Xist-linked PRC2 chromosome 17 peaks present in both induced and uninduced 3E cells correspond to known ES cell PRC2 target genes (31) recruitment does not correlate with gene silencing. Importantly, (Fig. S1C). An example, the Ptchd4 locus, is depicted in Fig. using superresolution microscopy, we find that Xist RNA and S1D. Peaks present only in induced 3E ES cells showed a highly PRC2 proteins do not colocalize and, in fact, exhibit significant significant association with chromosome 17 relative to regions on spatial separation. Our observations argue against direct inter- other chromosomes (P < 0.0005, χ2 test). Selected new peaks and action between the bulk of Xist RNA and PRC2 proteins and, as nonpeak regions were verified at 24 h by ChIP-quantitative PCR such, prompt a reexamination of the mechanism of PcG protein (qPCR) assay for H3K27me3 and also for the core PRC2 sub- recruitment in X inactivation. unit, Suz12 (Fig. S2 A and B). We also performed ChIP followed

Fig. 1. Analysis of Xist expression and H3K27me3 localization in undifferentiated 3E ES cells. (A) Schematic representation of chromosome 17 (Chr. 17) and magnification of the cytological region encompassing the inducible Xist transgene (Xist-tg) as determined by DNA-FISH mapping of flanking loci Txndc2 and Kntc2 (30). Genomic coordinates are from mm8 assembly. (B) Representative images of Xist RNA-FISH and H3K37me3 IF stainings on 3E cells 24 h after Xist induction with doxycycline (dox). No Xist up-regulation is detected in noninduced cells. (C) Quantification of cells with Xist RNA and H3K27me3 foci (n ≥ 200). Error bars represent SD in three independent experiments. Two asterisks (**) indicate high statistical significance (P ≤ 0.001, t test).

2236 | www.pnas.org/cgi/doi/10.1073/pnas.1312951111 Cerase et al. Downloaded by guest on September 29, 2021 Fig. 2. Summary of epigenomic and transcriptional profiling of chromosome 17 in 3E ES cells fol- lowing Xist-transgene induction. (A) Schematic showing tiled chro- mosome 17 region corresponding to Mb 20–85.4 (mm8). (B) Chromo- some 17 features are as follows: gene density on + and − strands (blue), CGI density (green), L1 re- peat density (%, orange), Xist- dependent new H3K27me3 peaks acquired 24 h after transgene in- duction (violet), Xist-mediated gene silencing 72 h after trans- gene induction indicating strongly (red) and weakly (green) down- regulated genes, strongly (dark blue) and weakly (light blue) down-regulated genes, and density of tiled probes (olive). 0/+1 indicates the absence or presence of a given feature (CGI, new H3K27me3 peaks, etc.).

by qPCR at the same genomic locations at 72 h after Xist induction map to gene-rich chromosomal domains, with a significant (Fig. S2C). Data show correlated enrichment of H3K27me3 and proportion lying within gene bodies. However, there was no Suz12 at new peaks at both time points. The location of new peaks direct link between these sites and Xist-mediated silencing of relative to gene density, CGIs, and LINE1 (L1) repeat density is associated genes. summarized in Fig. 2. New peaks are located across chromosome 17 with no discernible clustering around the transgene location. Spatial Separation of PRC2 Proteins and Xist RNA Revealed by There is a strong correlation with the location of genes (P = Superresolution Microscopy. To explore the localization of PRC2 − 1.97 * 10 62, χ2 test) and a reciprocal relationship relative to L1 relative to Xist RNA further, we used immunofluorescence (IF) − repeats (P = 1.72 * 10 58, χ2 test). These observations are in detection in combination with RNA FISH (immuno-RNA- keeping with cytogenetic analyses (26) and previous ChIP map- FISH) and applied superresolution 3D structured illumination ping data on the X chromosome (27–29). microscopy (3D-SIM) (32, 33). In comparison to conventional fluorescence light microscopy, 3D-SIM increases the optical Xist-Mediated H3K27me3 Deposition Occurs at Genic Sites but Is Not resolution in both lateral and axial directions by a factor of 2 Linked to Transcriptional Silencing. We went on to test the re- below the diffraction limit, allowing multicolor 3D optical sec- lationship of new (Xist-induced) H3K27me3 peaks and underlying tioning of whole cells with an eightfold increase in volumetric chromosome features (Fig. 3A). We found a strong correlation resolution (32). We applied 3D-SIM to compare localization with gene bodies for both highly expressed and poorly expressed patterns of Xist RNA and core PRC2 proteins directly in the genes, as defined in an analysis of uninduced 3E ES cells (30). inducible Xist transgene ES cell line 3E. To assess spatial There was a slight preference for active vs. inactive genes. In- terestingly, new H3K27me3 peaks did not correlate with gene promoters and anticorrelated with CGIs, contrasting with canon- ical PRC2 target sites. Some new peaks occurred at preexisting

PRC2 target genes (31), most likely reflecting an expansion of GENETICS existing peaks in response to Xist-mediated silencing. There was also a small positive correlation with SINE repeats that are concentrated in gene-rich chromosomal domains, and an anti- correlation with L1 repeats, consistent with the chromosomal distribution of new H3K27me3 peaks (Fig. 3A). We anticipated that new genic peaks would indicate loci that are silenced by Xist RNA at an early time point. To investigate this, we assayed Xist-mediated gene silencing in undifferentiated 3E ES cells using expression microarrays. Down-regulated genes mapped predominantly to chromosome 17, as expected (Fig. S3A). Of a total of 119 significantly down-regulated genes, 85 were strongly repressed (21–45%), with the remainder showing relatively weak repression (5–20%) (Fig. S3B). For selected examples, changes in expression level were verified by quanti- tative RT-PCR assay (Fig. S3C). The total number of down- regulated genes was less than the 231 observed in a previous study, in which we analyzed differentiated 3E ES cells (30), in- dicating that Xist-mediated silencing is less efficient in un- Fig. 3. Summary of associations (preference factor) for chromosome 17 differentiated ES cells. We next assessed the correlation of Xist- Xist-dependent H3K27me3 new peaks. (A) Preference summary of genomic features associated with 24-h Xist-induced H3K27me3 new peaks. Preference dependent H3K27me3 sites with down-regulated genes. As B was expressed as the log ratio of H3K27me3 density at the genomic features shown in Fig. 3 , there is a negative correlation for strongly and of interest (gene bodies, gene 3′ regions, etc.) compared with H3K27me3 weakly down-regulated genes. This finding was unexpected, given density outside of these features (both computed for the area covered by that we anticipated that genes in the vicinity of Xist RNA binding our tiling microarray). (B) Preference summary for Xist-induced peaks vs. sites would be inactivated relatively efficiently. In summary, 72-h down-regulated genes. A single asterisk (*) and two asterisks (**) in- results from epigenomic mapping of H3K27me3 following Xist dicate statistical (F ≤ 0.05) and highly statistical (F ≤ 0.001) significance, re- induction in 3E ES cells confirm that early sites of PRC2 activity spectively (Fisher test).

Cerase et al. PNAS | February 11, 2014 | vol. 111 | no. 6 | 2237 Downloaded by guest on September 29, 2021 relationships of the detected signals quantitatively, we used three correlation of the two signals (Figs. 5B and 4 A and B). These different complementary analysis methods: nearest neighbor results closely match expectations taking into account distances centroid distance measurements of segmented spot signals (SI between core PRC2 subunits (34) and measurements done on Materials and Methods and Fig. S4), scatter plots, and calculation the cells labeled with a single antibody against H3K27me3 but of pixel intensity-based colocalization coefficients using both with two different secondary antibodies (∼100 nm, arbitrary coloc- Pearson’sandManders’ correlations (additional details are alization baseline; Fig. S5). Comparable results were obtained provided in Materials and Methods). To determine system- analyzing colocalization of the PRC2 subunits Ezh2 and Eed related inherent colocalization error between the respective (Fig. 4 A and B and Fig. S6C). color channels, we imaged 0.2-μm diameter TetraSpeck micro- In light of recent evidence demonstrating that noncanonical spheres (Invitrogen) stained throughout with multiple fluorescent PRC1 complexes are recruited to the Xi via a PRC2-independent dyes and applied cross-correlation channel alignment. From this, pathway (19, 20), we extended our 3D-SIM analyses to determine we estimated the error distance to be about 25 nm (Fig. 4 A and B relative localization of Xist RNA and the core PRC1 protein and Fig. S5A). Additionally, signal separation attributable to bi- Ring1b, common to both canonical and noncanonical PRC1 com- ologically detected particles (antibody detection) was estimated plexes. Quantitative colocalization analysis and distance measure- by imaging SNL mouse fibroblast cells stained with H3K27me3 ments revealed spatial separation similar to that observed with primary antibody and then simultaneously labeled with Alexa PRC2 proteins (Fig. 4 A and B and Fig. S7B). We further analyzed B 488- and Alexa 594-conjugated secondary antibodies (Fig. S5 ). the localization of PRC1 relative to PRC2 within the inactive X Quantitative colocalization analysis determined the two signals domain, labeling the catalytic components Ezh2 and Ring1b. In line to be highly correlated and the nearest neighbor distances to ∼ A B B with expectations, the determined intermediate average distance be 100 nm (Fig. 4 and and Fig. S5 ). These values rep- and correlation coefficient values were between the colocalization resent the collective inaccuracy associated with dual-antibody/ controls (Eed-Ezh2/Eed-Suz12) and Xist-PRC2/1 (Fig. 4 A and B fluorophore detection. These include labeling specific variations and Fig. S7C). In summary, our 3D-SIM analyses indicate that there (e.g., by antibody competition, accessibility, epitope density, is little or no direct interaction between Xist RNA and core PcG quenching, epitope-to-fluorophore distance) but may also in- proteins. This is at apparent odds with the prevailing view that PcG volve intrinsic errors in the spot segmentation and nearest proteins interact directly with Xist RNA and suggests a need neighbor centroid determination. In addition, imaging-specific to consider and test alternative mechanisms for PcG protein error sources, such as wavelength-dependent variation in optical recruitment in XCI. resolution and channel alignment mismatch along the optical axis by chromatic aberrations, may contribute to some extent. We Discussion therefore assume that spatially associated biological structures/ proteins display the observed range of minimal distances (∼100 nm). In this study, we analyzed Xist-mediated deposition of H3K27me3 Previous studies have proposed that Xist RNA interacts di- in an ES cell line with an inducible Xist transgene located on rectly with the PRC2 subunit(s) Ezh2 and/or Suz12 (7, 13–15), mouse chromosome 17. This system offered the advantages of and we therefore assessed their relative spatial distribution by allowing assessment of H3K27me3 deposition at an early time 3D-SIM. We found that the two signals show significant sepa- point following synchronous induction of Xist RNA and a means to ration. The median minimal green-red distance of segmented correlate our observations with Xist-mediated gene silencing in an spots was ∼160 nm, and colocalization analyses indicated no cor- autosomal context. At the chromosomal level, we found that Xist- relation (exemplary cells are shown in Fig. 5A and Fig. S6A, and mediated H3K27me3 deposition occurs preferentially within gene- average values for 10 analyzed cells are shown in Fig. 4 A and B). rich domains, correlating well with prior studies using either cyto- We further analyzed colocalization of Xist RNA and the core genetic (26) or high-throughput epigenomic mapping (27–29). A PRC2 subunit Eed; again, we observed clear spatial separation of more detailed analysis of our data revealed that sites of H3K27me3 the signals (Fig. S6B). The estimated intersignal distances were deposition occur predominantly within the body of genes rather in the range of 150–180 nm, and colocalization analyses in- than at CGI promoters, as in the case of other PRC2 target loci dicated absence of correlation (Fig. 4 A and B and Fig. S6B). (31).WealsoobservedsomeincreaseinH3K27me3atpreexisting Similar results were obtained in an analysis of colocalization of PRC2 target gene promoters and additionally at SINEs. Xist RNA and Eed in a female somatic cell line, C2C12 (Fig. 4 A There are both similarities and differences compared with and B and Fig. S7A). By way of comparison and to validate our previous epigenomic mapping studies that analyzed PRC2 lo- approach, we imaged 3E cells with antibodies to two different calization on Xi. In differentiating XX ES cells and XX TS cells, PRC2 subunits, Suz12 and Eed. We observed a significant overlap increased PRC2 levels on Xi were seen at both genes and at with mean nearest neighbor distances of ∼110 nm and strong transcription start sites (27–29). Moreover, a recent analysis of

Fig. 4. Summary of 3D-SIM experiments. (A) Graphs illustrate the averages of Pearson’s correlation co- efficient (Upper) and Manders’ correlation coef- ficients 1 and 2 (Lower, hatched bars), respectively, for all 3D datasets analyzed for each indicated labeling pair. Error bars indicate SD. (B) Green-red distances shown as box plots (Upper, ±1.5 IQR) andmeandistances(Lower, SEM) of all analyzed datasets. For each indicated labeling pair, quan- tifications were performed for at least 10 datasets comprising ∼15 z-sections per cell or a comparably sized region of beads. Red bars indicate PRC2- PRC2 pairs, and blue bars indicate Xist-PRC2 pairs. A PRC1-PRC2 pair is shown in green, and controls with H3K27me3 dual labeling (H3K27me3 2s) and TetraSpeck beads are shown in gray. **, difference with high statistical significance P > 0.001, t test.

2238 | www.pnas.org/cgi/doi/10.1073/pnas.1312951111 Cerase et al. Downloaded by guest on September 29, 2021 Fig. 5. Spatial separation of Xist RNA and Ezh2 determined by 3D-SIM. (A) Example of immuno-RNA-FISH for Xist (red) and Ezh2 (green) illustrating a maximum intensity z-projection of DAPI-stained cell nuclei (Left and Center), a magnified region surrounding the Xist RNA-enriched domain in one of the cells (Center), and four further magnified serial z-sections (125-nm z-distance, Right) centered on the “Barr body.” Graphs illustrate distance and colocalization analysis from the entire Barr body region (∼15 z-sections). (Upper) Nearest neighbor distances for segmented green and red signals (Distance g-r) are displayed as box plots (median, Q1, Q3), with whiskers indicating the 1.5× interquartile range (1.5 IQR), and as averages plus SD. (Lower) Colocalization analysis based on pixel intensities displayed as a green-red scatter plot with Pearson’s (Ps), Manders’ 1 (M1), and Manders’ 2 (M2) correlation coefficients is indicated. Averaged values from multiple cells are shown in Fig. 4 A and B.(B) IF detection of PRC2 proteins Suz12 (red) and Eed (green) with panels arranged as in A.

differentiating XX ES cells reported moderate PRC2 enrich- in 3D-SIM experiments might represent more than a single ment sites broadly distributed within genic and intergenic inter- molecule/complex, although this does not have a bearing on our vals and at SINE elements, also noting an approximate 40:1 ratio finding that Xist RNA and PcG proteins are spatially separated. of noncanonical vs. canonical PRC2 sites in XCI (28). We con- One potential caveat to our conclusions is that the sample pro- sider that the absence of PRC2 enrichment at gene promoters in cessing for immuno-RNA-FISH and IF detection alone is not our study likely reflects the relatively inefficient gene silencing of identical. More generally, we cannot entirely rule out that cell chromosome 17 that we observe in response to Xist expression in fixation contributes to the observed spatial separation of Xist undifferentiated 3E ES cells, contrasting with the complete and RNA and PRC2 proteins. efficient silencing on Xi that occurs in differentiated XX ES cells Although our 3D-SIM analyses argue against a direct in- and in TS cells (27–29). Extrapolating from this, our data in- teraction between Xist RNA and PcG complexes, this is in GENETICS dicate that H3K27me3 deposition in gene bodies and at SINE contrast to biochemical evidence (7, 13–15), as well as recent elements likely occurs early in response to Xist RNA expression, molecular mapping of Xist RNA binding sites (35, 36). At with subsequent expansion or spreading of H3K27me3 to pro- present, we cannot account for this apparent discrepancy, but moters and other sites being linked to progressive gene silencing. it is possible that the biochemical assays, being more sensitive, Our initial assumption was that early PRC2 deposition sites detect a low level of Xist RNA/PRC2 binding. Such an in- would indicate primary Xist RNA binding sites, and that genes teraction may be physiologically relevant or, alternatively, may linked to these sites would therefore be inactivated relatively reflect nonspecific binding of PRC2 proteins to RNA. Our findings quickly. However, analysis of gene silencing in 3E ES cells indicate that it will be important to reappraise the proposed direct revealed a negative correlation between Xist-mediated gene si- interactions of PRC2 with other ncRNAs, given that the Xist- lencing and new sites of H3K27me3 deposition. This observation PRC2 model has served as a paradigm in many cases. In some argues against a role for PRC2 in establishment of gene silencing instances, it may be possible to apply 3D-SIM or other high- in XCI, a conclusion that is consistent with the relatively mild resolution approaches to address this issue further. In light of the XCI phenotypes observed in PRC2-null embryos (8, 10), and evidence presented here indicating that the bulk of PRC2 and also with the observation that Xist-mediated PRC2 recruitment Xist RNA do not interact directly, it is interesting to consider and gene silencing are separable (12). That early H3K27me3 alternative hypotheses to explain PcG recruitment in XCI. One deposition occurs in gene bodies rather than promoters is likely possibility is that there is an RNA binding adaptor protein that a factor in the lack of correlation with gene silencing. forms a bridge between PRC2 and Xist RNA. We consider this to Our epigenomic mapping studies prompted us to apply super- be unlikely, given the estimated additional 50–100 nm of spatial resolution microscopy to reexamine the proposed direct re- separation (beyond the ∼100-nm measured inter-PRC2 subunit lationship between Xist RNA and PRC2 recruitment. Using distances), and also because no such candidates have been 3D-SIM, we found a clear spatial separation of Xist RNA and identified in biochemical purifications of PRC2 complexes. A PRC2 proteins, estimated to be between 50 nm and 100 nm. This second possibility, suggested recently (21), is that PRC2 recruitment conclusion is reinforced by the observation that different PRC2 occurs as an indirect consequence of changes in underlying subunits show significant colocalization relative to one another, chromatin configuration associated with Xist-RNA–mediated si- consistent with the size of the PRC2 complex as determined in lencing. To investigate this idea, it will be important to define the a recent EM study (34). It should be noted that foci observed underlying chromatin configuration at sites of Xist-dependent

Cerase et al. PNAS | February 11, 2014 | vol. 111 | no. 6 | 2239 Downloaded by guest on September 29, 2021 H3K27me3 deposition identified in this and other epigenomic IF and DNA/RNA FISH Analyses. Preparation of cells for conventional IF de- mapping studies. tection, RNA-FISH, and immuno-RNA-FISH was as previously described (18, 38). IF staining and immuno-RNA-FISH for superresolution microscopy were Materials and Methods performed using 3D-preserving conditions described elsewhere (32) and in SI Materials and Methods. Cell Culture. Growth, maintenance, and doxycycline induction of the Xist- inducible ES cell line 3E were as previously described (30). C2C12 cells were Image Acquisition and Analysis. Conventional IF imaging was performed as grown in DMEM supplemented with 10% (vol/vol) FBS, 2 mM glutamine, and previously described (30). Superresolution imaging is described in detail in 1% penicillin/streptomycin. All cells were incubated at 37 °C in a humidified SI Materials and Methods. Colocalization was assessed as in the study by 5% (vol/vol) CO incubator. 2 Ronnemberg et al. (39). Full details are provided in SI Materials and Methods.

ChIP and DNA Microarrays. H3K27me3 ChIP and hybridization to custom ACKNOWLEDGMENTS. We thank Haruhiko Koseki for Ring 1b antibody; microarrays were performed essentially as described previously (37). Full Marion Cremer, Sarah Cooper, and Anca M. Farcas for critical reading of the details are provided in SI Materials and Methods. manuscript; Nathan Rose and Alfredo Castello for RNA binding analysis PRC2 components; and Rob Klose and all members of N.B. and Klose Analysis. Gene expression change after doxycycline treat- laboratory for helpful comments and stimulating discussions. This study was funded by Wellcome Trust Grant 081385 (to N.B.) and Wellcome Trust ment was monitored by Affymetrix microarray as previously described (30). Strategic Award 091911, supporting advanced microscopy at Micron Oxford Genes were classified as strongly and weakly down-regulated, correspond- (http://micronoxford.com). D.S. was supported by a fellowship of the German ing to 21–45% and 5–20% down-regulation, respectively. Maximum allelic Academy Exchange Service, and F.K. was supported by an Erasmus Fellowship down-regulation is 50%. of the European Union.

1. Morey C, Avner P (2011) The demoiselle of X-inactivation: 50 years old and as trendy 20. Tavares L, et al. (2012) RYBP-PRC1 complexes mediate H2A ubiquitylation at poly- and mesmerising as ever. PLoS Genet 7(7):e1002212. comb target sites independently of PRC2 and H3K27me3. Cell 148(4):664–678. 2. Lee JT, Jaenisch R (1997) Long-range cis effects of ectopic X-inactivation centres on 21. Brockdorff N (2013) Noncoding RNA and Polycomb recruitment. RNA 19(4):429–442. a mouse autosome. 386(6622):275–279. 22. Pandey RR, et al. (2008) Kcnq1ot1 antisense noncoding RNA mediates lineage-specific 3. Wutz A, Jaenisch R (2000) A shift from reversible to irreversible X inactivation is transcriptional silencing through chromatin-level regulation. Mol Cell 32(2):232–246. triggered during ES cell differentiation. Mol Cell 5(4):695–705. 23. Rinn JL, et al. (2007) Functional demarcation of active and silent chromatin domains 4. Brockdorff N (2011) Chromosome silencing mechanisms in X-chromosome in- in human HOX loci by noncoding . Cell 129(7):1311–1323. activation: Unknown unknowns. Development 138(23):5057–5065. 24. Heo JB, Sung S (2011) Vernalization-mediated epigenetic silencing by a long intronic 5. Jeon Y, Lee JT (2011) YY1 tethers Xist RNA to the inactive X nucleation center. Cell noncoding RNA. Science 331(6013):76–79. 146(1):119–133. 25. Yap KL, et al. (2010) Molecular interplay of the noncoding RNA ANRIL and methyl- 6. Hasegawa Y, et al. (2010) The matrix protein hnRNP U is required for chromosomal ated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Mol – localization of Xist RNA. Dev Cell 19(3):469–476. Cell 38(5):662 674. 7. Zhao J, Sun BK, Erwin JA, Song JJ, Lee JT (2008) Polycomb proteins targeted by a short 26. Duthie SM, et al. (1999) Xist RNA exhibits a banded localization on the inactive repeat RNA to the mouse X chromosome. Science 322(5902):750–756. X chromosome and is excluded from autosomal material in cis. Hum Mol Genet 8(2): – 8. Wang J, et al. (2001) Imprinted X inactivation maintained by a mouse Polycomb group 195 204. gene. Nat Genet 28(4):371–375. 27. Marks H, et al. (2009) High-resolution analysis of epigenetic changes associated with – 9. Mak W, et al. (2002) Mitotically stable association of polycomb group proteins eed X inactivation. Genome Res 19(8):1361 1373. 28. Pinter SF, et al. (2012) Spreading of X chromosome inactivation via a hierarchy of and enx1 with the inactive x chromosome in trophoblast stem cells. Curr Biol 12(12): defined Polycomb stations. Genome Res 22(10):1864–1876. 1016–1020. 29. Calabrese JM, et al. (2012) Site-specific silencing of regulatory elements as a mecha- 10. Silva J, et al. (2003) Establishment of histone h3 methylation on the inactive nism of X inactivation. Cell 151(5):951–963. X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes. 30. Tang YA, et al. (2010) Efficiency of Xist-mediated silencing on autosomes is linked to Dev Cell 4(4):481–495. chromosomal domain organisation. Chromatin 3(1):10. 11. Plath K, et al. (2003) Role of histone H3 lysine 27 methylation in X inactivation. Sci- 31. Mikkelsen TS, et al. (2007) Genome-wide maps of chromatin state in pluripotent and ence 300(5616):131–135. lineage-committed cells. Nature 448(7153):553–560. 12. Kohlmaier A, et al. (2004) A chromosomal memory triggered by Xist regulates histone 32. Schermelleh L, et al. (2008) Subdiffraction multicolor imaging of the nuclear pe- methylation in X inactivation. PLoS Biol 2(7):E171. riphery with 3D structured illumination microscopy. Science 320(5881):1332–1336. 13. Kaneko S, et al. (2010) Phosphorylation of the PRC2 component Ezh2 is cell cycle- 33. Clemson CM, McNeil JA, Willard HF, Lawrence JB (1996) XIST RNA paints the inactive – regulated and up-regulates its binding to ncRNA. Genes Dev 24(23):2615 2620. X chromosome at interphase: Evidence for a novel RNA involved in nuclear/chro- 14. Maenner S, et al. (2010) 2-D structure of the A region of Xist RNA and its implication mosome structure. J Cell Biol 132(3):259–275. for PRC2 association. PLoS Biol 8(1):e1000276. 34. Ciferri C, et al. (2012) Molecular architecture of human polycomb repressive complex 15. Kanhere A, et al. (2010) Short RNAs are transcribed from repressed polycomb target 2. Elife 1:e00005. – genes and interact with polycomb repressive complex-2. Mol Cell 38(5):675 688. 35. Simon MD, et al. (2013) High-resolution Xist binding maps reveal two-step spreading 16. Mak W, et al. (2004) Reactivation of the paternal X chromosome in early mouse during X-chromosome inactivation. Nature 504(7480):465–469. – embryos. Science 303(5658):666 669. 36. Engreitz JM, et al. (2013) The Xist lncRNA exploits three-dimensional genome archi- 17. Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E (2004) Epigenetic dynamics of tecture to spread across the X chromosome. Science 341(6147):1237973. imprinted X inactivation during early mouse development. Science 303(5658): 37. O’Geen H, Nicolet CM, Blahnik K, Green R, Farnham PJ (2006) Comparison of sample 644–649. preparation methods for ChIP-chip assays. Biotechniques 41(5):577–580. 18. de Napoles M, et al. (2004) Polycomb group proteins Ring1A/B link ubiquitylation of 38. Casanova M, et al. (2011) Polycomblike 2 facilitates the recruitment of PRC2 Polycomb to heritable gene silencing and X inactivation. Dev Cell 7(5):663–676. group complexes to the inactive X chromosome and to target loci in embryonic stem 19. Leeb M, Wutz A (2007) Ring1B is crucial for the regulation of developmental control cells. Development 138(8):1471–1482. genes and PRC1 proteins but not X inactivation in embryonic cells. J Cell Biol 178(2): 39. Ronneberger O, et al. (2008) Spatial quantitative analysis of fluorescently labeled 219–229. nuclear structures: Problems, methods, pitfalls. Chromosome Res 16(3):523–562.

2240 | www.pnas.org/cgi/doi/10.1073/pnas.1312951111 Cerase et al. Downloaded by guest on September 29, 2021