Xist spatially amplifies SHARP recruitment to balance chromosome-wide silencing and specificity to the X chromosome [RAP-DNA]

Although thousands of lncRNAs are encoded in mammalian genomes, their mechanisms of action are largely uncharacterized because they are often expressed at significantly lower levels than their proposed targets. One such lncRNA is Xist, which mediates chromosome-wide gene silencing on one of the two X chromosomes to achieve gene expression balance between males and females. How a limited number of Xist molecules can mediate robust silencing of a significantly larger number of target genes (~1 Xist RNA: 10 gene targets) while maintaining specificity to genes on the X within each cell is unknown. Here, we show that Xist drives non-stoichiometric recruitment of the essential silencing protein SHARP to amplify its abundance across the inactive X, including at regions not directly occupied by Xist. This amplification is achieved through concentration-dependent homotypic assemblies of SHARP on the X and is required for chromosome-wide silencing. We find that expressing Xist at higher levels leads to increased localization at autosomal regions, demonstrating that low levels of Xist are critical for ensuring its specificity to the X chromosome. We show that Xist (through SHARP) acts to suppress production of its own RNA which may act to constrain overall RNA levels and restrict its ability to spread beyond the X. Together, our results demonstrate a spatial amplification mechanism that allows Xist to achieve two essential but countervailing regulatory objectives: specificity to the X and chromosome-wide gene silencing, and suggest a more general mechanism by which other low abundance lncRNAs can balance specificity to, and robust control of, their regulatory targets. Overall design: Mouse embryonic stem cells expressing Xist under a Dox inducible promoter were treated with increasing amounts of Dox to titrate expression of Xist. After Dox induction, cells were harvested and crosslinked using 3% Formaldehyde + DSG. Lysates prepared from cells were subsequently hybridized with biotinylated antisense probes to Xist and resulting complexes purified using streptavidin beads. DNA crosslinked to Xist was purified and subjected to next-generation sequencing technologies.