Engreitz 1..8

RESEARCH ARTICLE hybridize to a target RNA to purify the endogenous RNA and its associated genomic DNA from cross-linked cell lysate (Fig. 1A) (35). We The Xist lncRNA Exploits Three- designed RAP to enable specific purification of chromatin associated with a target lncRNA, Dimensional Genome Architecture to achieve high-resolution mapping of the associated DNA target sites upon sequencing of the captured DNA, and robustly capture any lncRNA Spread Across the X Chromosome with minimal optimization. To achieve high spec- ificity, RAP uses 120-nucleotide antisense RNA 1,2 3 1 1 Jesse M. Engreitz, Amy Pandya-Jones, Patrick McDonel, Alexander Shishkin, probes to form extremely strong hybrids with 1 1 1 1 1 Klara Sirokman, Christine Surka, Sabah Kadri, Jeffrey Xing, Alon Goren, the target RNA, thereby enabling purification 1,4,5 3 1 Eric S. Lander, * Kathrin Plath, * Mitchell Guttman *† using denaturing conditions that disrupt nonspecific RNA-protein interactions and nonspecific hy- Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they bridization with RNAs or genomic DNA. RAP localize to genomic targets remain unexplored. We investigated the localization mechanisms of the uses deoxyribonuclease I (DNase I) to digest ge- Xist lncRNA during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin nomic DNA to ~150–base pair (bp) fragments, regulation. During the maintenance of XCI, Xist binds broadly across the X chromosome. which provides high-resolution mapping of bind- During initiation of XCI, Xist initially transfers to distal regions across the X chromosome that ing sites. To robustly capture a lncRNA, RAP are not defined by specific sequences. Instead, Xist identifies these regions by exploiting the uses a pool of overlapping probes tiled across the Downloaded from three-dimensional conformation of the X chromosome. Xist requires its silencing domain to entire length of the target RNA to ensure capture spread across actively transcribed regions and thereby access the entire chromosome. These even in the case of extensive protein-RNA inter- findings suggest a model in which Xist coats the X chromosome by searching in three dimensions, actions, RNA secondary structure, or partial RNA modifying chromosome structure, and spreading to newly accessible locations. degradation (supplementary text). To test our method, we used RAP to purify ammalian genomes encode thousands tional silencing requires the A-repeat domain the Xist RNA and associated DNA from female of large noncoding RNAs (lncRNAs) (26), which interacts with the PRC2 chromatin mouse lung fibroblasts (MLFs), a differentiated http://science.sciencemag.org/ M(1–5), many of which play key func- regulatory complex (14), whereas localization to cell line in which Xist is expressed from and coats tional roles in the cell (6–9). One emerging par- chromatin requires several distinct domains the inactive X chromosome. We designed anti- adigm is that many lncRNAs can regulate gene (26–28) and interactions with proteins associ- sense probes tiled every 15 nucleotides across the expression (6, 8–11)byinteractingwithchroma- ated with the nuclear matrix (29–31). Despite 17-kb Xist transcript, excluding those that showed tin regulatory complexes (6, 12–14) and localizing these advances in our understanding of Xist, we any complementarity to other RNAs or genomic these complexes to genomic target sites (15–17). still do not understand the process by which Xist DNA regions (35). This yielded a pool of 1054 Despite the central role of RNA-chromatin inter- localizes to chromatin and spreads across the X unique probes. We performed RAP and observed actions, the mechanisms by which lncRNAs iden- chromosome. an enrichment of the Xist RNA by more than a tify their genomic targets remain unexplored. We present a biochemical method that en- factor of 100 relative to either input or a control The Xist ncRNA provides a model to inves- ables high-resolution mapping of lncRNA lo- purification using “sense” probes from the same on August 6, 2021 tigate the mechanisms of lncRNA localization calization. Using this method, we explored Xist strand as Xist itself (Fig. 1B). When we se- (11, 18, 19). Xist initiates X-chromosome inacti- localization during initiation and maintenance quenced all RNAs in the purified fraction, we vation (XCI) by spreading in cis across the future of XCI. During maintenance, Xist localized broad- found that the Xist RNA constituted ~70% of inactive X chromosome (20, 21), recruiting poly- ly across the entire X chromosome, lacking focal alignable reads despite representing <0.1% of comb repressive complex 2 (PRC2) (14, 22, 23), binding sites. During initiation of XCI, Xist trans- the polyadenylated input RNA. The remaining and forming a transcriptionally silent nuclear ferred directly from its transcription locus to distal reads were broadly distributed across ~7500 ex- compartment (24, 25) enriched for repressive sites across the X chromosome that are defined pressed transcripts, with no single transcript ex- chromatin modifications including trimethylation not by specific sequences but by their spatial ceeding 2% of the total purified RNA (Fig. 1C). of histone 3 lysine 27 (H3K27me3) (22, 23). These proximity in the nucleus to the Xist transcription We sequenced the genomic DNA that copurified functions of Xist—localization to chromatin locus. Furthermore, we show that Xist initially with the Xist RNA and observed a strong enrich- and silencing of gene expression—are medi- localized to the periphery of actively transcribed ment, with >70% of the DNA sequencing reads ated by distinct RNA domains (26): Transcrip- regions but gradually spread across them through from the Xist purification originating from the a mechanism dependent on the A-repeat domain. X chromosome, versus ~5% from the input DNA Together, these results suggest that Xist initially samples (Fig. 1D). 1Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. 2Division of Health Sciences and Technology, Massachu- localizes to distal sites across the chromosome by To ensure that the DNA purified by Xist RAP setts Institute of Technology, Cambridge, MA 02139, USA. exploiting chromosome conformation, and may reflected the endogenous localization of Xist, 3Department of Biological Chemistry, Jonsson Comprehensive spread to new sites through its ability to modify we performed three control experiments (fig. S1) Cancer Center, Molecular Biology Institute, and Eli and Edythe chromatin structure. (35): (i) To confirm that captured chromatin re- Broad Center of Regenerative Medicine and Stem Cell Research, flected preexisting interactions occurring in vivo, David Geffen School of Medicine, University of California, Los Results and Discussion – Angeles, CA 90095, USA. 4Department of Biology, Massachusetts we purified Xist from non cross-linked cellular Institute of Technology, Cambridge,MA02139,USA.5Depart- extracts. In this condition, we did not obtain any ment of Systems Biology, Harvard Medical School, Boston, MA RNAAntisensePurification(RAP):AMethodto detectable DNA signal by quantitative polymerase 02114, USA. Map lncRNA Interactions with Chromatin chain reaction (qPCR) despite obtaining com- *Corresponding author. E-mail: [email protected] (M.G.); To determine the genomic localization of lncRNAs, parable enrichments of the Xist RNA (35). (ii) [email protected] (K.P.); [email protected] (E.S.L.) we developed a method termed RAP, which is To rule out the possibility that RAP captured ge- †Present address: Division of Biology and Biological Engineering, conceptually similar to previous methods (32–34) nomic DNA through nonspecific hybridization California Institute of Technology, Pasadena, CA 91125, USA. in that it uses biotinylated antisense probes that with the probes or the target RNA, we tested www.sciencemag.org SCIENCE VOL 341 16 AUGUST 2013 1237973-1 RESEARCH ARTICLE X chromosome (Fig. 2C) (35). The most enriched regions showed higher H3K27me3 occupancy in MLFs (factor of 1.7) and higher gene density (factor of 3) relative to the chromosome average, consistent with the chromosome-wide correlations. The least enriched regions contained genes known to escape XCI (36). Consistent with their preferential positioning outside of the Xist domain (37, 38), escape genes displayed a ~50% reduction in Xist occupancy relative to silenced X-chromosome genes, with the level of Xist enrichment roughly reflecting the previously reported ratio of expression from the inactive versus active X chromosome (Pearson correlation = –0.66; Fig. 2D) (35, 39). Closer examination of Xist localization at some escape genes revealed sharp boundaries separating escaped and non-escaped domains (Fig. 2E). One of the least enriched regions resided immediately distal to the Xist locus and included the lncRNA genes Jpx and Ftx, both of which have been previously reported Downloaded from Fig. 1. RNA antisense purification (RAP) accurately purifies lncRNA-chromatin interactions. (A) to escape XCI and act as positive regulators of Schematic diagram of RAP. Biotinylated probes (blue) are hybridized to the target RNA (red) cross-linked Xist (40, 41) (Fig. 2F). to proteins and DNA. Gray inset: 120-nucleotide antisense capture probes are designed to tile across the Together, the least enriched regions con- entire target RNA. (B) Reverse transcription qPCR of the RNA captured using antisense probes to Xist tained 53 genes with more than 40% depletion (Xist RAP, black)

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