To address whether the bivalent state requires miRNAs or oth

To address whether the bivalent state requires miRNAs or other Dicer-dependent functions, we reconstituted Dicer-deficient ESCs with miR-294, a member of the miR-290–295 miRNA family. ChIP-PCR showed that EZH2 binding was restored 48 hr later at the Gata3, Pax6, and Nr2f2 promoters (Figure 4B). Thus, reconstitution of Dicer-deficient ESCs with a member of the 290–295 miRNA family was sufficient to rescue PRC2 occupancy of at least a subset of loci that lost the bivalent state in Dicer-deficient ESCs. The dihydrofolate reductase inhibitor antibacterial of bivalent genes is regulated at multiple levels (Brookes et al., 2012; Margueron and Reinberg, 2011; Simon and Kingston, 2013; Jia et al., 2012). We have shown that transcripts from bivalent genes are preferentially targeted by miRNAs because their 3′ UTRs contain significantly more miRNA binding sites. The pluripotency-associated transcription factors OCT4, SOX2, and NANOG control miRNA expression in ESCs (Marson et al., 2008), linking the regulation of bivalent genes by ESC miRNAs to the ESC core regulatory network (Figure S4). An unexpected finding of our study is that miRNAs are required for the recruitment of PRC2 components to bivalent genes, and thereby the state of bivalency itself.
Acknowledgments
Introduction In most cellular contexts Notch signaling acts as a gatekeeper to differentiation, promoting maintenance of stem or progenitor cell fates (Andersson et al., 2011; Guruharsha et al., 2012). Modulation of Notch signaling is used to control stem or progenitor cell differentiation in vitro, for example toward neural, intestinal, or hematopoietic lineages (Lowell et al., 2006; Schmitt et al., 2004; Yin et al., 2014). Deregulated Notch signaling is increasingly linked to cancer, and Notch receptor mutations are found in, for example, T cell leukemia, non-small cell lung cancer, and breast cancer as well as in several types of tumor cell lines (Mutvei et al., 2015; Robinson et al., 2011; Weng et al., 2004; Westhoff et al., 2009). Notch signaling is also frequently hyperactivated in a range of tumors, including breast cancer (for review see Andersson and Lendahl, 2014). Notch signaling ensues when transmembrane Notch ligands of the Jagged or Delta-like type interact with Notch receptors on a juxtaposed cell. This results in proteolytic cleavage and liberation of the intracellular domain of the Notch receptor (Notch ICD), which relocates to the cell nucleus and interacts with the DNA-binding protein CSL (also known as RBP-Jk or CBF1), thus making CSL the central node in the signaling cascade for all four Notch receptors (Notch 1–4) (Andersson et al., 2011). In the “Notch off” state, CSL acts as a repressor and binds a number of transcriptional co-repressors, such as SHARP/MINT, KDM5A, and KyoT2 (for review see Borggrefe and Oswald, 2014). In the “Notch on” state, i.e., upon binding to Notch ICD, CSL sheds the co-repressors and instead recruits co-activators, such as p300 and PCAF, converting it to an activator. The interaction between Notch ICD and CSL is stabilized by the MAML protein, and the ternary Notch ICD/MAML/CSL complex induces expression of Notch downstream genes (Nam et al., 2006; Wilson and Kovall, 2006). It has traditionally been assumed that CSL serves as a DNA-bound repressor in the absence of Notch, and in line with this, CSL can bind to DNA in the absence of Notch and remains bound to DNA even during mitosis (Lake et al., 2014). Recent studies, however, provide support for a more dynamic view whereby CSL is recruited to the DNA by Notch ICD (Castel et al., 2013; Krejcí and Bray, 2007). It is an open question whether CSL only transmits the signal from the Notch receptors or also plays a role in other, non-Notch-related signaling transductions. Gene-targeting experiments show that phenotypes resulting from targeting of Notch ligands or receptors in some situations are phenocopied by targeting of CSL, for example during somitogenesis (Conlon et al., 1995; Oka et al., 1995) or in memory T cells (Maekawa et al., 2015), which is in line with CSL functioning exclusively as the central hub in the Notch signaling cascade (Guruharsha et al., 2012). On the other hand, there are also an increasing number of proteins, such as CTCF, EBNA3c, interferon regulatory factor 4, and RITA (see Collins et al., 2014 and references therein), which are not part of the Notch signaling mechanism but interact with CSL, suggesting that CSL has a broader range of actions extending beyond only transmitting Notch signaling.