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    • Introduction MicroRNAs miRNAs are small non coding RNAs that

    2018-10-24

    Introduction MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate the stability and translation of protein-coding transcripts through partial complementarity with their 3′ UTRs (Jaskiewicz and Filipowicz, 2008). There have been reports that miRNAs affect histone modifications or DNA methylation in mammalian gssg (Kanellopoulou et al., 2005; Sinkkonen et al., 2008; Benetti et al., 2008; Nesterova et al., 2008; Han et al., 2007), although the mechanisms are often indirect. For example, miRNAs post-transcriptionally control negative regulators of DNA methyltransferases in mouse ESCs (Sinkkonen et al., 2008; Benetti et al., 2008; Nesterova et al., 2008). In mouse ESCs a set of developmental regulator genes is characterized by the simultaneous presence of activating and repressive histone modifications (Azuara et al., 2006; Bernstein et al., 2006). This bivalent state is resolved during ESC differentiation (Mikkelsen et al., 2007), suggesting that bivalency keeps genes in a poised state to enable rapid activation or stable silencing upon differentiation (Pietersen and van Lohuizen, 2008; Voigt et al., 2013). Bivalent genes are prematurely expressed in ESCs that lack the PRC2 components EED (Azuara et al., 2006; Boyer et al., 2006) or SUZ12 (Pasini et al., 2004). PRC2-deficient ESCs remain viable and can self-renew (Pasini et al., 2007; Chamberlain et al., 2008; Shen et al., 2008) but show reduced developmental potential (Pasini et al., 2007; Chamberlain et al., 2008; Shen et al., 2008), and PRC2 mutations are lethal during post-implantation in vivo (Faust et al., 1995; O\'Carroll et al., 2001; Pasini et al., 2004). Hence, polycomb group (PcG) repression of bivalent genes may safeguard the pluripotent state and the ability of ESCs to differentiate. miRNAs affect key features of ESCs, including their characteristic cell-cycle behavior (Wang et al., 2008). miRNAs are critical for ESC pluripotency and differentiation (Miyoshi et al., 2011; Anokye-Danko et al., 2011), and somatic miRNAs facilitate differentiation by terminating the expression of pluripotency factors such as Nanog (Melton et al., 2010). Although much is known about ESC miRNAs, their impact on the regulation of bivalent genes has not been systematically investigated. To address this point we have analyzed the distribution of PRC2 core components and the regulation of bivalent genes in ESCs deficient in the RNase III enzyme DICER, which is required for the biogenesis of most miRNAs (Jaskiewicz and Filipowicz, 2008). We find that miRNAs are required for the binding of EZH2 and SUZ12 at many bivalent promoters, and therefore for the maintenance of the bivalent state. These data define an unexpected role for miRNAs in maintaining the bivalent state of developmental regulator genes in mouse ESCs.
    Results and Discussion Previously characterized Dicer-deficient ESCs (Nesterova et al., 2008) were adapted to growth in feeder-free culture with inhibitors of MEK and GSK3β (see Experimental Procedures). We mapped the distribution of EZH2 by chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq). Control ESCs retained EZH2 promoter binding at 2,110 bivalent genes (Ku et al., 2008) in control ESCs under 2i/LIF culture conditions. Loss of DICER resulted in reduced EZH2 occupancy of bivalent gene promoters (Figure 1A, left). The number of bivalent promoters was reduced to 1,033, reflecting the loss of EZH2 binding to 1,077 (or 51%) of formerly bivalent genes (Figure 1A, right) as illustrated for the Pax6 promoter (Figure 1B). Loss of EZH2 was confirmed by ChIP-PCR (Figure 1C, top). At the sites we tested, the H3K27me3 chromatin mark deposited by PRC2 was reduced (Figure 1C, middle). As expected (Marks et al., 2012), the promoter mark H3K4me3 was unaffected (Figure 1C, bottom). To explore the kinetics of EZH2 loss from bivalent promoters, we induced the deletion of Dicer by treatment of ERt2Cre Dicerlox/lox ESCs with 4-hydroxytamoxifen (4-OHT). Dicer mRNA expression declined within 4 days of ERt2Cre induction, followed by the reduced expression of the miRNAs miR-291a-3p and miR-92 (Figure 1D). EZH2 occupancy of formerly bivalent promoters was reduced within 8 days (Figure 1E), indicating that EZH2 binding closely follows the reduced expression of miRNAs.