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    • qRT PCR showed that the expression of the PRC core

    2018-10-24

    qRT-PCR showed that the expression of the PRC2 core components Ezh2, Suz12, and Eed was not reduced upon Dicer deletion (Figure 2A). Similarly immunoblotting showed no reduction of EZH2, SUZ12, or JARID2 proteins or global H3K4me3 or H3K27me3 (Figure 2B). Cellular fractionation experiments confirmed that total and chromatin-associated EZH2 protein remained unchanged (Figure 2C), suggesting selectively reduced binding of EZH2 at the promoters of bivalent genes. Exclusion of PcG from CpG-methylated regions may focus PcG binding to sites of low CpG methylation, namely CpG islands and CG-rich promoter regions, to facilitate the formation of bivalent domains (Ku et al., 2008; Lynch et al., 2012; Brinkman et al., 2012). Global DNA methylation levels are reduced in most Dicer-deficient ESC lines (Sinkkonen et al., 2008; Benetti et al., 2008; Nesterova et al., 2008; Ip et al., 2012) and DNA hypomethylation is linked to PcG redistribution in DNA methyltransferase (Dnmt)-deficient ESCs (Lynch et al., 2012; Brinkman et al., 2012; Cooper et al., 2014). PRDM14 controls Dnmt3b expression (Ma et al., 2011) and is upregulated under 2i conditions (Ying et al., 2008), but Prdm14 mRNA did not further increase in Dicer-deficient ESCs (log2 fold change −0.36, adjusted p = 0.446). Nevertheless, there were differences in Dnmt mRNA (Figure S1A) and DNMT protein (Figure S1B) expression between control and Dicer-deficient ESC under 2i/LIF culture conditions (Figure S1A). We therefore used high-performance liquid chromatography to directly monitor the fraction of methylated dihydrofolate reductase inhibitor residues in response to Dicer deletion. Treatment of ERt2Cre Dicer ESCs with 4-OHT did not affect the fraction of methylated cytosines (Figure 2D) even though miRNAs were efficiently depleted (Figure 1D) and EZH2 occupancy was reduced at bivalent promoters (Figures 1A–1D). Hence, the observed loss of bivalency was not due to globally reduced DNA methylation. Consistent with these data, ChIP-seq and ChIP-PCR analysis showed no substantial redistribution of EZH2 to repeat regions or to other genomic regions that had been reported to gain PRC2 binding in DNMT-deficient ESCs (Figures S1C–S1F). We explored other potential mechanisms for the observed redistribution of PcG proteins in Dicer-deficient ESCs. First, although ERK2 is required for PRC2 occupancy and poised RNA polymerase at developmental genes (Tee et al., 2014), immunoblotting showed no significant differences in ERK1 and -2 protein expression between control and Dicer-deficient ESCs (Figure S1G). Second, NUP153 promotes PcG binding to developmentally regulated genes in ESCs (Jacinto et al., 2015), but our microarray and proteomics data show robust expression of Nup153 mRNA and NUP153 protein in both control and Dicer-deficient ESCs, without any indication of reduced expression in Dicer-deficient ESCs (GSE60161). Finally, PRC2 occupancy may reflect, rather than cause transcriptional repression (Riising et al., 2014). Eviction of PRC2 by transcription could account for the loss of EZH2 from some but not all of the formerly bivalent promoters in Dicer-deficient ESCs, since not all formerly bivalent genes showed increased expression of primary transcripts (see below). We profiled gene expression in control and Dicer-deficient ESCs and identified sequence motif enrichment in 3′ UTRs using miReduce. Highly enriched sequence motifs corresponded to binding sites for the ESC miRNAs miR-290–295 and miR-302, as well as miR-17-92 (Figure 3A). The miR-290–295 cluster is the most highly expressed miRNA population in ESCs (Zheng et al., 2011), and miR-290-3p, miR-291a-3p, miR-291b-3p, miR-292-3p, miR-294, and miR-295 contain the hexamer seed AAGUGC, which is shared by the miR-302 cluster. The miR-17-92 family contains the shifted seed AAAGUG and likely shares targets with miR-290–295 and miR-302 (Leung et al., 2011; Ciaudo et al., 2009; Babiarz et al., 2008). As expected, transcripts with 3′ UTR motifs for these abundant miRNAs were significantly upregulated in Dicer-deficient ESCs (Figure 3A). In addition, miReduce identified 3′ UTR motifs that matched binding preferences of RNA binding proteins (Figure S2A). We found the 3′ UTRs of bivalent genes enriched for miRNA motifs (p < 10−15, odds ratio = 2.91), including miR-290 (p = 1.17 × 10−6, odds ratio = 2.00) and miR-302 (p = 1.1 × 10−6, odds ratio = 1.95) (Figure S2A). Similar enrichment (p < 2.2 × 10−16, odds ratio = 2.22) was observed for an independently defined set of bivalent genes (Mikkelsen et al., 2007) and for the 2,110 bivalent genes that were bound by the EZH2 in our ESCs (p < 2.2 × 10−16, odds ratio = 3.18 for any miRNA motif; p < 2.2 × 10−16, odds ratio = 3.95 for miR-290; and p < 10−10, odds ratio = 4.32 for miR-302). The genomic basis for the increased occurrence of miRNA motifs was a greater than average 3′ UTR length, rather than a higher density of miRNA motifs in the 3′ UTRs of bivalent genes (Figure S2B).