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Spatial Separation of Xist RNA and Polycomb Proteins Revealed by Superresolution Microscopy

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Spatial Separation of Xist RNA and Polycomb Proteins Revealed by Superresolution Microscopy Spatial separation of Xist RNA and polycomb proteins 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 Biochemistry, University of Oxford, 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 Human 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 chromosomes is transcrip- cell biological work demonstrating enrichment of PRC2 proteins tionally silenced to equalize X-linked gene dosage relative to XY on Xi and a close overlap with sites of Xist RNA localization, males, a process termed X chromosome inactivation. Mechanisti- both at metaphase and interphase (9–11). PRC2 recruitment cally, this is thought to occur via directed recruitment of chromatin 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 genes lacking the A-repeat nevertheless recruits PRC2, albeit an inducible Xist transgene located on mouse chromosome 17, 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 histone 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 gene silencing. 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 protein-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 X chromosome inactivation. this using a combination of genome 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 autosomes 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 histone H3 at lysine 27 (H3K27me3) (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 locus 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).
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