Spreading of X chromosome inactivation via a hierarchy of defined Polycomb stations.

X chromosome inactivation (XCI) provides an excellent model by which to study Polycomb regulation and the role of long noncoding RNAs (lncRNAs) in inducing facultative heterochromatin (Lyon 1999; Wutz and Gribnau 2007; Payer and Lee 2008; Lee 2011). XCI is controlled by the X-inactivation center (Xic), an X-linked region that controls the counting of X chromosomes, the mutually exclusive choice of Xa and Xi, and the recruitment and propagation of silencing complexes. The 17-kb Xist RNA initiates the silencing step as it accumulates on the X (Brockdorff et al. 1992; Brown et al. 1992; Clemson et al. 1996). Although recent studies have shown that Xist RNA directly recruits Polycomb repressive complex 2 (PRC2) to the Xi (Zhao et al. 2008) and that loading of the Xist-PRC2 complex occurs first at a YY1-bound nucleation center located within the Xic (Jeon and Lee 2011), how the silencing complexes spread throughout the X after this obligatory nucleation step remains a major unsolved problem. Because autosomes with ectopic Xic sequences are subject to long-range silencing (Wutz and Gribnau 2007; Payer and Lee 2008), it is thought that spreading elements cannot be unique to the X. One hypothesis suggests that repetitive elements of the LINE1 class facilitate spreading (Lyon 2000). However, this hypothesis has been difficult to test, as linking repeats to locus-specific function has been complicated by their repetitive nature. Some studies have provided correlative evidence (Bailey et al. 2000; Wang et al. 2006; Chow et al. 2010), whereas others find that species lacking active LINE1s nonetheless possess XCI (Cantrell et al. 2009). Other classes of repeats may be more enriched on the X (Chow et al. 2005). Matrix-associated proteins, such as HNRNPU (also known as SAF-A), have also been proposed to facilitate spreading (Helbig and Fackelmayer 2003; Hasegawa et al. 2010; Pullirsch et al. 2010), but a direct link has also not been demonstrated. In general, the identification of spreading elements has been thwarted by the lack of high-throughput approaches that distinguish Xi and Xa at sufficient resolution. Epigenomic studies have primarily focused on male cells (Bernstein et al. 2006; Boyer et al. 2006; Barski et al. 2007; Mikkelsen et al. 2007; Ku et al. 2008), though one recent ChIP-seq analysis with partial allele-specific coverage used female mouse embryonic stem (ES) cells but without addressing PRC2 binding. The reported 1.2-fold enrichment of H3K27me3 on Xi (Marks et al. 2009) is unexpectedly low and at odds with intense cytological H3K27me3 immunostaining (Plath et al. 2003; Silva et al. 2003)—likely caused by low-density polymorphisms between Xi and Xa. As a result, the quest for an Xi chromatin state map and spreading elements has remained unrealized. In principle, silencing complexes could initially load at the Xic and spread serially from nucleosome to nucleosome. Alternatively, they could spread outwardly via “way stations” located at defined sites along the X that would anchor and relay silencing complexes (Gartler and Riggs 1983). To test these models, we herein devise an allele-specific ChIP-seq strategy that enables the generation of chromosome-wide developmental profiles at unprecedented allelic resolution. We report a high-density Xi chromatin state map and identification of discrete Polycomb stations.