a sleek w “exagees | + More an ROA Sey LETTER 2c" High -resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation Matthew D. Simon!2*, Stefan F. Pinter!*, Rui Fang”, Kavitha Sarma!?, Michael Rutenberg-Schoenberg”, Sarah K. Bowman’, Barry A. Kesner"™, Verena K, Maier", Robert E. Kingston’ & Jeannie T. Lee “The Xist long noncoding RNA (ncRNA) is essential for X-chromosome inactivation (XCD, the process by which mammals compensate for ‘unequal numbers of sex chromosomes". During XCI, Xist coats the future inactive X chromosome (Xi and recruits Polycom repre ‘ve complex 2 (PRC2) to the X-inacivation centre (Xi0’. How Xist ‘Spreads silencing on a 150-megabases scale is unclear. Here we gen: Crate high-resolution maps of Xist binding on the X chromosome cross a developmental time course using CHART-q. In female ‘els undergoing XCI de nove, Xist follows a two-step mechanism, initially targeting gene-rich slands before spreading to intervening igene-poor domains. Xist is depleted from genes that escape XCI but may concentrate near escapee boundaries. Xist binding is ine arly proportional to PRC2 density and H3 lysine 27 trimethyation (H3K27me3), indicating co-migration of Xist and PRC2, Interes- tingly, when Xist fs acutely stripped off from the Xi in post-XCI cells, Xist recovers quickly within both gene-rich and gene-poor ‘domains on a timescale of hours instead of days, indicating a prev ously primed Xi chromatin state. We conclude that Xist spreading takes distinct stage-specific forms. During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist ‘spreads rapidly to both gene-rich and gene-poor regions. Kist RNA isa protorypelncRNA with global epigenetic function’ “The initiation of XCI depends on Xis’ and loading ofthe Xist-PRC2 ‘complex at a nucleation site within the Xic, Thereafter, Xist RNA forms a “elold™ over the X-chromosome, signalling the initiation of chromosome-wide slencing’ Concurrently, PRC2 accumulates broadly slong the X-chromosome’, Although Xist RNA coats the Xiat eyolo- tical resolution, whether and where Xist binds at molecular resolution femains unknown, In one model, Xist targets PRC2 to the Xi, but ‘outward spreading of PRC2 doesnot involve Xist. Alternatively both hhuceation and spread involve Xstin which case Xist and PRC2 would Co-migrae ata molecular sale. ‘We mapped genome-wide binding locations of Xist RNA by per forming CHART-seq (capture hybridization analysis of RNA targets with deep sequencing) technique to localize IncRNAS on chromatin ‘sing complementary oligonucleotides to enrich for DNA targets (Extended Data Fig 1a). We designed a cocktail of 11 complementary, ‘oligonucleotides for Xist CHART based on conserved or functional, ist domains” and RNase H mapping for accessibility (Extended Data Fig 1b,cand Extended Data Table 1)- Allele pectic CHART-seq ‘was performed at four developmental stages (Extended Data Fig. 1a) ‘before XCTin undifferentiated female mouse embryonic stem (ES) cells (G0, <19 of nuclei XCI postive, showing an Xist loud or H3K27me3 focus) early-XCI (43; <10% postive), mid-XCI(d7; 40-50% postive), and post-XCI (mouse embryonic fibroblast (MEF) clone, >95% posi tive), About 600,000 sequence polymorphisms between the Mus musculus (mus) and Mus castaneu (cas) X-chromosomes enabled ~35% allele specifie mapping to Xi and Xa (active X chromosome), respectively”. Disabling the mus Tsiallele in the female ES cells ensured that the mus X willbe Xi", We validated results by comparing two independent cap ture oligonucleotide sub-mixtures and an alternative 40-ligonucleotide Cocktail targeting across the length of Xist (Extended Data Fig. 22-e dnd Extended Data Table 1). Regions with significant Xist enrichment Tocalized almost exclusively to Xi (>99% X-linked, P< 0.001; >90% iskewed, P< 005, Extended Data Fig 2, i)-On autosomes, bind ing was minimal and of questionable significance. Enriched segments ‘were not complementary to capture oligonucleotides and showed min. {mal enrichment on Xa do, 3, d7 and MEF cells. Enrichment was not ‘observed using sense contol oligonucleotides (Extended Data Fig. 28) "These experiments excluded artefactual enrichment, validating Xist, CCHART-seq specific. "The dominant CHART peak was in Xist exon 1 and was specific to Xi Fig La). A developmental ime course demonstrated a progression in Xist density, with enriched segments increasing from <0.1% cov- ‘rage ofthe Xin pre-XCIcellsto approximately 20% ineary-and mid. ‘XCL and approximately 4% in post-XCI cells (Fig. 1b, cand Extended Data 2h). Thus, Xist RNA not only forms a cytological loud but also binds broad swaths ofthe X at molecular resolution, Xist could either spread uniformly along the Xi or target specific regions. Intriguingly, {in eals undergoing XCI (43, d7), Xist preferentially targeted mult ‘megabase domains (Fig, 1c) In post-XCI MEFs, Xist spread into inter ‘ening gene-poor regions throughout the Xi. The d3 and d7 patterns ‘were more similar to eachother than to MEF patterns (Fig. 1d e and Extended Data Fig 32) Purthermore, comparative analysis identified MEF-specfle domains not found during XCI (Fig. 1e). Despite het erogeneity in the onset of XCT in th ex vivo ES differentiation system, thehighly similar d3 and d7 distributions show that Xist targets gene. rich domains first. Extension of ES differentiation to dl0 showed Statistically significant filing in of gene-poor domains (Extended Data Fig, 3b,c) although not tothe extent observed in somatic els (MEFS). ‘We infer that fall spreading across Xi may only be achieved later in evelopment, once differentiation into somatic lineages occurs. Ths, during de novo XCI in the embryo, Xist probably follows a two-step patter of spreading, frst targeting gene-rich clusters (hereafter, ‘carly omains) and eventually spreading to intervening gene-poor regions ‘late’ domains). Throughout the proces, gene bodies of escapees! ‘were depleted of Kist, but ocasionally demonstrated Xist enrichment in flanking regions (Fig, If and Extended Data Fig. 4), indicating boundaries that sequester Xist and prevent spreading into neighbour ing privileged escapee loc ‘We investigated what might target Xist to early domains by compar- isons with various chromatin features (see Methods)". Interestingly. ist s more likely to target genes in regions of active chromatin in ES, calls Allele-specific RNA sequencing analysis demonstrated the pref ‘rence of Xistforgenes that areacive (for example, onthe Xaandin dO ‘and 7 cell) and showed skewed expression in d7 ES cellsand in MEFs sas 014 EA opment acu BophaGt MEF nih ai , ae aa sasinade a i i ne dei, tome Ba xetesaa nate nna xetesar ails in » * eee 4 me ge a il = pees ie inh eee ‘ . Act econ abecaen ocr eae = : eo] de a ge a § as a i med "ear vases ery top) = : . oe img os wats sate Be ead a 8 cytological bance om "51 S47 normalized dons o9,) vara 68 [owe epee =P = - LINE [pia gO eg onsen prep eee eee rete Mabe oem eet thems ite Mt ‘SINES A Nak alt on ha ae, DNase-HS i Me alleen eae dl, Wee abate deeded werretuinseayong® ME ope eater ais ver onan GMS i ao ke lt TT a a wool sae act eon a san HASTA i pany ee et Figure 1| CHART seq reveae a two-step mechanism of Kis spreading daring de novo XCI. a Xist RNA irenriched on Xi Normale read dents CHART signals (40-4 bins) from LNA-4978 8h comreated with MEF (ei LNA4978 3h cotlated against LNA-4978 Bh (0), Regions showing ‘more than tenfold ference after normalization ae coloured 2s shown in dg Xist ecovery in indicated samples, wih 40-Kb binned Xist densities normallaed to motian levels feat domains ofeach sample to determine how ‘arly and late domains recover from kpockofcomparedto during de nove XC ‘Noralied median values for each sample indicated above box. *P< 0.05; ;P 0", Wilcoxon ts at in Extended Data Figs and 3h, Model Astin! mets of Xist spreading during establishment and maintenance.(r= -054) and tioning did not. ing onthe X chromosome (Fig 1h)" LINEIs have Been proposed as spreading elements”, but repetitive reads from Xit CHARTT-sq align- ing to LINE] were not enriched over input (Extended Data Fig. 5). The localization of Xist showed modest postive correlation with Xi loop- ing contacts inferred from HiC (high-throughput chromosome con- formation capture)" through an anchor within the Xstlocus (Fig. th and Extended Data Fig. 5b). Together, these data support a role for sweasked whether _Xist continues to associate with PRC2 during spreading. Comparison ‘of Xist, EZH2 and H3K27me3 eneichment revealed strikingly similar ‘chromosome profiles acros time (Fig, 2a and Extended Data Fig 62-0), By contrast, PRC2 and H3K27me3 densities on Xa didnot coreelate with Xist, nor did those on chromosome 13, representative autosome (Extended Data Fig. 6a). Consistent with the idea that Xist directs PRC2 localization onto Xi, Xist densities demonstrated an extensive ees raat Sosone ina tree lets Pe coats trap ome 2) Comeen ono ai section ee ee ee Sees ee ee Seco tinea iota one eee ee ee ‘We then asked if localization mechanisms were inherent to Xist RNA or chromatin context In perturbation experiments, we stripped st RN covery on the Xiof MEFsat Lb Shand irected against repeat C of Xi ore spreading” d that LNA-497 Xist displacement by 3h, wi reassociation at 8h (Fig. 3a) As reassocation requires newly s aed Xist rather , ‘ytologically by RNA FISH. At 1h, when Xist was sil visualized on Xi (Fig. 3a), CHART-seq demonstrated a relative loss in late domains (Fig, 36-d), indicating that Xist binds more weakly to gene-poor than togene-rich regions, and consistent with the banded patter of Xist on the metaphase Xi observed cytologically", At 3h, Xist was strongly Can sequence DNA to determine genomic loci ‘where RNA is bound. In this paper they used 11 different oligonucleotides based on conserved or functional xist domains. They performed this Chart-Seq on four different developmental stages of embryonic mouse cells, 1) before XCI in undifferentiated mouse embryonic stem cells (ES) DO 2) Imearly XCI ES D3 3) In mid XCIES D7 4), Post XCI mouse cells, which are referred to as mouse embryonic fibroblasts (MEFs) Figure 1. c) Shows the preferential targeting by Xist of “multi-megabase” domains and the preference for genes that are active Figure 1. d) This igure shows the ist CHART-seq regions from 47 correlate strongly with those at ¢3. And are stronger of course in D7. When this graph is compared against the plot showing D7 versus [MEF cell and their place/density of Xist you ean see that while d3 and d7 are very similar, d7 and MEF vary quite abit. This i atributed in the paper to the differential targeting of active gene domains early in XCI compared to the later targeting of gene poor regions late in XCI. Figure 4. g) This figure shows the preferential targeting by Xist of genes in active chromatin. (Active genes were identified by histone methylation patterns). This pattern was interpreted as indicative that Xist targets gene-rich domains and i a sense “guided” by open chromatin.Figure 1 f. This figure also.uses CHART-ieq to showuthe targeting by Xist RNA to,enriched segménts ‘ietneatchaomosome which willbe silenced, mus, and the lack of targeting in the non slenced X- ehromosome eas Again nti igure canbe seen tha 3 and d target enriched segmgnt ite heavily while MEF targets regions which are hét heavily enriched. Figure 2. a) It serves to show the correlation between Xist RNA location and the location of PRC2. EZH2 is a subunit of the complex and causes histone methylation. Again, the cas X chromosome is. Used as a control to show that only the X chromosome being inactivated is targeted by Xist and PRC2. Again, note the preference for enriched segments. ———Figie 3) The researchers used RNA FISH (RNA fluorescence in situ hybrization) to determine the effect that locked nucleic acid (LNA) directed against repeat C of xist RNA which prevents nucleation and spreading, This C region of Xist has been shown in other papers to lead toa loss of Xist from the XI when itis removed and thus itwas chosen asthe target forthe LNAs, This FISH analysis, followed bby more Chart-sequencing revealed two interesting patterns, 1) that the xist density was lowered ‘more quickly in late domains than in the earlier (gene-rich) domains - which indicates stronger xist binding to those regions _ ‘and 2) that the spreading of xist in the somatic maintenance phase (MEF) DID NOT follow the two step pattern as it did in de novo XCI. The respreading of xist after removal in somatic maintenance phase was not only more general, in other words, both domains were repopulated with xist at the sare rat, but also tis rate was much faster, curing in hours rather than in days as ld nde novoxc. > He BX bickey occured via nw Gt fom te xc jest mM drow lS We of He LWA Weelererce, ‘The research group also used another LNA and found the pattern was very similar to the one observed/shown with LNA-4978,