Gene expression can be regulated at multiple levels, but it is not known if and how there is broad coordination between regulation at the transcriptional and post-transcriptional levels. Transcription factors and chromatin regulate gene expression transcriptionally, whereas microRNAs (miRNAs) are small regulatory RNAs that function post-transcriptionally. Systematically identifying the post-transcriptional targets of miRNAs and the mechanism of transcriptional regulation of the same targets can shed light on regulatory networks connecting transcriptional and post-transcriptional control. We used individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) for the RNA-induced silencing complex (RISC) component AGO2 and global miRNA depletion to identify genes directly targeted by miRNAs. We found that Polycomb repressive complex 2 (PRC2) and its associated histone mark, H3K27me3, is enriched at hundreds of miRNArepressed genes. We show that these genes are directly repressed by PRC2 and constitute a significant proportion of direct PRC2 targets. For just over half of the genes corepressed by PRC2 and miRNAs, PRC2 promotes their miRNA-mediated repression by increasing expression of the miRNAs that are likely to target them. miRNAs also repress the remainder of the PRC2 target genes, but independently of PRC2. Thus, miRNAs post-transcriptionally reinforce silencing of PRC2-repressed genes that are inefficiently repressed at the level of chromatin, by either forming a feed-forward regulatory network with PRC2 or repressing them independently of PRC2.

Gene expression can be regulated at multiple levels, but it is not known if and how there is broad coordination between regulation at the transcriptional and posttranscriptional levels. Transcription factors and chromatin regulate gene expression transcriptionally, while microRNAs (miRNAs) are small regulatory RNAs that function post-transcriptionally. Systematically identifying the posttranscriptional targets of miRNAs and the mechanism of transcriptional regulation of the same targets can shed light on regulatory networks connecting transcriptional and post-transcriptional control. We used iCLIP (individual crosslinking and immunoprecipitation) for the RISC (RNA-induced silencing complex) component AGO2 and global miRNA depletion to identify genes directly targeted by miRNAs. We found that PRC2 (Polycomb repressive complex 2) and its associated histone mark, H3K27me3, is enriched at hundreds of miRNArepressed genes. We show that these genes are directly repressed by PRC2 and constitute a significant proportion of direct PRC2 targets. For just over half of the genes co-repressed by PRC2 and miRNAs, PRC2 promotes their miRNAmediated repression by increasing expression of the miRNAs that are likely to target them. miRNAs also repress the remainder of the PRC2 target genes, but independently of PRC2. Thus, miRNAs post-transcriptionally reinforce silencing of PRC2-repressed genes that are inefficiently repressed at the level of chromatin, by either forming a feed-forward regulatory network with PRC2 or repressing them independently of PRC2.

Polycomb repressive complex 2 (PRC2) is a chromatin binding complex that represses gene expression by methylating histone H3 at K27 to establish repressed chromatin domains. PRC2 can either regulate genes directly through the methyltransferase activity of its component EZH2 or indirectly by regulating other gene regulators. Gene expression analysis of glioblastoma (GBM) cells lacking EZH2 showed that PRC2 regulates hundreds of interferon-stimulated genes (ISGs). We found that PRC2 directly represses several ISGs and also indirectly activates a distinct set of ISGs. Assessment of EZH2 binding proximal to miRNAs showed that PRC2 directly represses miRNAs encoded in the chromosome 14 imprinted DLK1-DIO3 locus. We found that repression of this locus by PRC2 occurs in immortalized GBM-derived cell lines as well as in primary bulk tumors from GBM and anaplastic astrocytoma patients. Through repression of these miRNAs and several other miRNAs, PRC2 activates a set of ISGs that are targeted by these miRNAs. This PRC2-miRNA-ISG network is likely to be important in regulating gene expression programs in GBM.

Polycomb repressive complex 2 (PRC2) is a chromatin binding complex that represses gene expression by methylating histone H3 at K27 to establish repressed chromatin domains. PRC2 can either regulate genes directly through the methyltransferase activity of its component EZH2 or indirectly by regulating other gene regulators. Gene expression analysis of glioblastoma (GBM) cells lacking EZH2 showed that PRC2 regulates hundreds of interferon-stimulated genes (ISGs). We found that PRC2 directly represses several ISGs and also indirectly activates a distinct set of ISGs. Assessment of EZH2 binding proximal to miRNAs showed that PRC2 directly represses miRNAs encoded in the chromosome 14 imprinted DLK1-DIO3 locus. We found that repression of this locus by PRC2 occurs in immortalized GBM-derived cell lines as well as in primary bulk tumors from GBM and anaplastic astrocytoma patients. Through repression of these miRNAs and several other miRNAs, PRC2 activates a set of ISGs that are targeted by these miRNAs. This PRC2-miRNA-ISG network is likely to be important in regulating gene expression programs in GBM.