To equalize X-chromosome dosages between the sexes, the female mammal inactivates one of her two X-chromosomes. X-chromosome inactivation (XCI) is initiated by expression of Xist, a 17-kb noncoding RNA that accumulates on the X in cis. Because interacting factors have not been isolated, the mechanism by which Xist induces silencing remains unknown. Here, we discover a 1.6 kb ncRNA (RepA) within Xist and identify the polycomb complex, PRC2, as its direct target. PRC2 is initially recruited to the X by RepA RNA, with Ezh2 serving as the RNA-binding subunit. The antisense Tsix RNA inhibits this interaction. RepA depletion abolishes full-length Xist induction and H3-K27 trimethylation of the X. Likewise, PRC2 deficiency compromises Xist upregulation. Therefore, RepA/PRC2 is required for the initiation and spread of XCI. We conclude that a ncRNA cofactor recruits polycomb complexes to their target loci.The mouse X-inactivation center harbors several noncoding genes, including Xist (1,2) and its antisense repressor, Tsix (3). On the future Xa (active X), Tsix blocks Xist upregulation and prevents the recruitment of silencing factors in cis. On the future Xi (inactive X), Tsix is downregulated, enabling Xist transactivation and spread of Xist RNA along the chromosome (4). The accumulation of Xist transcripts correlates with a cascade of chromatin changes (5), but how Xist directs these changes is unknown. In principle, the act of transcribing Xist could induce structural changes which could alter chromosome-wide function (1). Alternatively, Xist could work as a transcript (1,2) by recruiting chromatin modifiers or by targeting the X to a specialized compartment (6). Though universally attractive, RNA-based models have remained hypothetical, as Xist-interacting proteins have yet to be identified.To circumvent conventional difficulties with purifying Xist-interacting proteins, we carried out RNA immunoprecipitations (RIP) and asked if Xist RNA can be found in a specific protein complex. We isolated nuclear RNAs and their binding proteins in the native state to avoid fixation artifacts and tested two cell types --mouse embryonic stem (ES) cells, which exist in the pre-XCI state but recapitulate XCI when induced to differentiate; and mouse embryonic fibroblasts (MEFs) which faithfully maintains Xi. Because H3-K27 trimethylation (H3-K27me3) closely follows Xist up-and down-regulation (6-9), we asked if Xist RNA binds the H3-K27 methylase, PRC2, the polycomb complex that includes Eed, Suzl2, RbAp48, and the catalytic subunit, Ezh2 (10). Indeed, α-Ezh2 and α-Suz12 antibodies co-immunoprecipitated Xist RNA (Fig. 1A-D). By contrast, Xist sequences were not detected in α-H3-K27me3, α-H4Ac, and no-antibody controls. Pre-treatment with RNases that digest single-stranded (RNase

Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully recapitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes. In addition, we observe a continuum of activation states, revealing a pseudotemporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs, allowing for novel insights into neuronal activation patterns in vivo.

The presence of two active X chromosomes (XaXa) is a hallmark of the ground state of pluripotency specific to murine embryonic stem cells (ESCs). Human ESCs (hESCs) invariably exhibit signs of X chromosome inactivation (XCI) and are considered developmentally more advanced than their murine counterparts. We describe the establishment of XaXa hESCs derived under physiological oxygen concentrations. Using these cell lines, we demonstrate that (1) differentiation of hESCs induces random XCI in a manner similar to murine ESCs, (2) chronic exposure to atmospheric oxygen is sufficient to induce irreversible XCI with minor changes of the transcriptome, (3) the Xa exhibits heavy methylation of the XIST promoter region, and (4) XCI is associated with demethylation and transcriptional activation of XIST along with H3K27-me3 deposition across the Xi. These findings indicate that the human blastocyst contains pre-X-inactivation cells and that this state is preserved in vitro through culture under physiological oxygen.