Polycomb Proteins Targeted by a Short Repeat RNA to the Mouse X Chromosome

Abstract

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

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“…Researches demonstrate that lncRNA can combine with chromatin remodeling complexes resulting in a specific mode of histone modification, activating or inhibiting transcription; therefore, lncRNA play a key role in genomic imprinting and dosage compensation effect [18][19][20][21] . For example, XIST is a well-known lncRNA which plays an important role in the X chromosome inactivation in female mammals 22) . The 5´ end of XIST has a repeated motif known as "repeat A" which can directly bond to EZH2, the catalytic subunit of Polycomb repressive get MEF2C and MAML1, respectively, to regulate myoblast differentiation.…”

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