Supplementary Components2: Desk S1. Xist growing. Therefore, Polycomb and Xist complexes need one another to propagate along the Xi, recommending a feedforward mechanism between RNA protein and initiator effectors. Perturbing Xist/Polycomb growing causes failing of Xi silencing, with compensatory downregulation from the energetic X, and disrupts topological Xi reconfiguration also. Thus, Do it again B can be a multifunctional element that integrates codependent Xist/Polycomb spreading, silencing, and chromosome architecture. along the future inactive X (Xi) and induces conversion to a heterochromatic state (Brown et al., 1992; Clemson et al., 1996; Marahrens et al., 1997). The functions of Xist are manifold. On one hand, Xist acts as a modular RNA scaffold in the assembly of repressive protein factors. Two well-known factors, Polycomb repressive complexes 1 and 2 (PRC1/PRC2), are responsible for monoubiquitylating histone H2A at lysine 119 (H2AK119ub) and trimethylating histone H3 at lysine 27 (H3K27me3), respectively (Schoeftner et al., 2006; Zhao et al., 2008; Chu et al., 2015; McHugh et al., 2015; Minajigi et al., 2015; Moindrot et al., 2015; Isomangiferin Monfort et al., 2015). On the other hand, Xist forms a repressive compartment by repelling transcriptional and architectural factors to establish Isomangiferin a Isomangiferin unique Xi chromosome conformation (Nora et al., 2012; Rao et al., 2014; Deng et al., 2015; Minajigi et al., 2015; Giorgetti et al., 2016). Although broad functions have been associated with Xist, specific mechanisms have not been clarifiedin particular, how Xist RNA spreads transgenes and ectopic insertions, often in male cells (Lee et al., MUC1 1999; Wutz et al., 2002; Jeon and Lee, 2011; Pintacuda et al., 2017), with the caveat that these non-physiological perturbations might not inform Xist function in the endogenous context. Here, we carry out a systematic deletional analysis of endogenous Xist and identify a specific RNA motifRepeat Bfor RNA spreading and Polycomb targeting. In doing so, we reveal the surprising discovery that Xist and Polycomb complexes depend on each other to spread across the Xi. RESULTS Comprehensive deletional analysis of native Xist in female cells To performed a systematic CRISPR/Cas9 deletion screen, guide RNA (gRNA) pairs were designed Isomangiferin to remove consecutive 1-2 kb regions across the locus in female mouse embryonic fibroblasts (MEFs), where XCI has already been established (Fig. 1A). The transformed MEFs were tetraploid (with genome duplication after XCI) carrying two Xis and two Xas within the same nucleus (Yildirim et al., 2011), thus enabled isolation of Xi+/? clones (deletion on only one Xi) and Xi?/? clones (deletion on both Xis) (Fig. 1B). Xi+/? cells provided an internal control Xist cloud within the same nucleus for comparative microscopy, while Xi?/? cells provided a homogeneous system for genomic experiments. We screened for mutant clones by two-color RNA FISHcyan probes external and red probes Isomangiferin internal to each deletionand selected clones exhibiting cyan with no overlapping red signal (Fig. 1B) and validated them by Sanger sequencing (File S1). Open in a separate window Figure 1. CRISPR/Cas9 deletion screen identifies Xist functional domains.See also Figures S1-S3. (A) Diagram of Xist locus, repeat elements, gRNA target sites, and qPCR amplicons. (B) Schematic of screening method using tandem two-color RNA FISH. (C) Xist RNA FISH for deletions showing altered Xist cloud morphology in Xi+/? MEFs. Arrowhead indicates WT and arrow indicates mutant Xist cloud. Right panels show 3x zoom-in of each cloud. (D) RT-qPCR showing effect of deletions on Xist RNA levels in Xi?/? MEFs. Error bars show standard deviation for 3 biological replicates. (E) 3D STORM imaging and size measurements of Xist clouds in RepB and RepE Xi+/? MEFs. Epifluorescent images of same cells shown to the right, with arrowhead indicating WT and arrow indicating mutant Xist cloud. p-values by two-tailed t-test. (F) H3K27me3 and H2AK119ub IF for deletions showing phenotype in Xi+/? MEFs. Arrowhead signifies WT and arrow signifies mutant Xist cloud. We started with a visible inspection of Xist cloud morphology by RNA Seafood. From the 13 deletions, 7 exhibited some phenotype. While Do it again A is well known for its function in gene silencing (Wutz et al., 2002; Zhao et al., 2008), its deletion continues to be reported to trigger decreased deposition and/or lack of Xist appearance in both individual and mouse cells (Chow et al., 2007; Zhao et al., 2008; Hoki et al., 2009). A minor Do it again A deletion allowed us to derive clones with an unchanged Xist cloud and general RNA.