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MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression*

  • Wen Hao Neo
    Affiliations
    Division of Molecular Genetics and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
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  • Karen Yap
    Affiliations
    Division of Molecular Genetics and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
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  • Suet Hoay Lee
    Affiliations
    Division of Molecular Genetics and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
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  • Liang Sheng Looi
    Affiliations
    Division of Molecular Genetics and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
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  • Piyush Khandelia
    Affiliations
    Division of Molecular Genetics and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
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  • Sheng Xiong Neo
    Affiliations
    Division of Molecular Genetics and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
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  • Eugene V. Makeyev
    Affiliations
    Division of Molecular Genetics and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore

    Medical Research Council Centre for Developmental Neurobiology, King's College London, New Hunt's House, Guy's Hospital Campus, London SE1 1UL, United Kingdom
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  • I-hsin Su
    Correspondence
    To whom correspondence should be addressed. Tel.: 65-65138687; Fax: 65-67913858
    Affiliations
    Division of Molecular Genetics and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
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  • Author Footnotes
    * This work was supported by research funding from National Medical Research Council Grants NMRC/IRG/1269/2010 (to W. H. N. and I. S.) and NMRC/CBRG/0028/2013 (to E. V. M.) and National Research Foundation Singapore Grant NRF-RF2008-06 (to E. V. M.).
Open AccessPublished:July 25, 2014DOI:https://doi.org/10.1074/jbc.M113.525493
      Polycomb group protein Ezh2 is a histone H3 Lys-27 histone methyltransferase orchestrating an extensive epigenetic regulatory program. Several nervous system-specific genes are known to be repressed by Ezh2 in stem cells and derepressed during neuronal differentiation. However, the molecular mechanisms underlying this regulation remain poorly understood. Here we show that Ezh2 levels are dampened during neuronal differentiation by brain-enriched microRNA miR-124. Expression of miR-124 in a neuroblastoma cells line was sufficient to up-regulate a significant fraction of nervous system-specific Ezh2 target genes. On the other hand, naturally elevated expression of miR-124 in embryonic carcinoma cells undergoing neuronal differentiation correlated with down-regulation of Ezh2 levels. Importantly, overexpression of Ezh2 mRNA with a 3′-untranslated region (3′-UTR) lacking a functional miR-124 binding site, but not with the wild-type Ezh2 3′-UTR, hampered neuronal and promoted astrocyte-specific differentiation in P19 and embryonic mouse neural stem cells. Overall, our results uncover a molecular mechanism that allows miR-124 to balance the choice between alternative differentiation possibilities through fine-tuning the expression of a critical epigenetic regulator.

      Introduction

      Since their discovery in 1993, microRNAs (miRNAs),
      The abbreviations used are: miRNA
      microRNA
      NS
      nervous system
      H3K27
      histone H3 Lys-27
      3meH3K27
      H3K27 trimethylation
      N2a
      Neuro2a
      NSC
      neural stem cell
      RT-qPCR
      quantitative RT-PCR
      qPCR
      quantitative PCR
      RA
      retinoic acid
      EGFP
      enhanced GFP.
      19–25-nucleotide-long non-coding RNA molecules, have emerged as versatile regulators of developmental and physiological processes in a large fraction of eukaryotic organisms (
      • Lee R.C.
      • Feinbaum R.L.
      • Ambros V.
      The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.
      ,
      • Ivey K.N.
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      MicroRNAs as regulators of differentiation and cell fate decisions.
      ). There are estimated to be over 1000 miRNAs in the human genome, and more than 50% of human genes are predicted to be miRNA targets (
      • Lewis B.P.
      • Burge C.B.
      • Bartel D.P.
      Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.
      ,
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      The microRNA sequence database.
      ). Mature miRNAs regulate their cognate mRNAs as a part of an miRNA-induced silencing complex containing an Argonaute protein subunit (
      • Kim V.N.
      MicroRNA biogenesis: coordinated cropping and dicing.
      ). In plants, miRNAs often bind to fully complementary target sites typically located in the mRNA 3′-UTRs, which leads to gene repression through Argonaute-dependent mRNA “slicing” (
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      • Meister G.
      Argonaute proteins at a glance.
      ). On the other hand, animal miRNAs tend to be partially complementary to their target sequences, which affords regulation of target mRNAs through translational inhibition and slicer-independent destabilization (
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      MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship.
      ,
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      Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?.
      ).
      miRNAs are known to be crucial for neuronal differentiation, because conditional ablation of the endoribonuclease Dicer, an essential component of the microRNA maturation pathway, in neural stem cells or progenitors leads to dramatic defects in survival and differentiation of newborn neurons (
      • De Pietri Tonelli D.
      • Pulvers J.N.
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      miRNAs are essential for survival and differentiation of newborn neurons but not for expansion of neural progenitors during early neurogenesis in the mouse embryonic neocortex.
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      Different timings of Dicer deletion affect neurogenesis and gliogenesis in the developing mouse central nervous system.
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      The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing.
      ). One of the most abundant and perhaps best studied miRNAs in the brain is miR-124 (
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      MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-α-PU. 1 pathway.
      ). miR-124 is derived from three independent genes (miR-124-1, miR-124-2, and miR-124-3) contributing to the increased mature miR-124 levels during neuronal differentiation (
      • Griffiths-Jones S.
      The microRNA sequence database.
      ,
      • Deo M.
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      Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides.
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      Reciprocal actions of REST and a microRNA promote neuronal identity.
      ). Interestingly, however, knock-out of just the miR-124-1 gene in the mouse resulted in visible reduction of mature miR-124 levels, defective neuronal survival, and axonal outgrowth as well as smaller brain size (
      • Sanuki R.
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      • Irie S.
      • Uneo S.
      • Koyasu T.
      • Matsui R.
      miR-124a is required for hippocampal axogenesis and retinal cone survival through Lhx2 suppression.
      ).
      miR-124 may regulate hundreds and possibly thousands of distinct target genes (
      • Conaco C.
      • Otto S.
      • Han J.-J.
      • Mandel G.
      Reciprocal actions of REST and a microRNA promote neuronal identity.
      ,
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      • Zang J.B.
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      Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.
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      Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.
      ,
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      Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.
      ,
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      • Maniatis T.
      Multilevel regulation of gene expression by microRNAs.
      ). Important examples include genes encoding the SCP1 subunit of the global repressor of NS-specific genes REST, transcription factors Sox9 and cAMP-response element-binding protein, Notch ligand Jagged1, and the BAF53a subunit of a chromatin remodeling complex (
      • Cheng L.C.
      • Pastrana E.
      • Tavazoie M.
      • Doetsch F.
      miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche.
      ,
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      • Kandel E.
      Characterization of small RNAs in aplysia reveals a role for miR-124 in constraining synaptic plasticity through CREB.
      ,
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      • Lee B.
      • Lee J.W.
      • Lee S.K.
      The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development.
      ,
      • Yoo A.S.
      • Staahl B.T.
      • Chen L.
      • Crabtree G.R.
      MicroRNA-mediated switching of chromatin-remodelling complexes in neural development.
      ). We have previously shown that miR-124 also targets mRNA of Ptbp1 (polypyrimidine tract-binding protein), a global regulator of pre-mRNA splicing (
      • Makeyev E.V.
      • Zhang J.
      • Carrasco M.A.
      • Maniatis T.
      The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing.
      ). Ptbp1 is expressed at high levels in non-neuronal cells and neuronal precursors, where it suppresses the utilization of neuron-specific alternative exons. During neuronal differentiation, Ptbp1 expression is reduced by miR-124, which triggers a switch in alternative splicing patterns among a wide range of transcripts. Ptbp1 additionally controls the abundance of several neuron-specific mRNAs through nuclear and cytoplasmic RNA quality control mechanisms (
      • Makeyev E.V.
      • Zhang J.
      • Carrasco M.A.
      • Maniatis T.
      The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing.
      ,
      • Makeyev E.V.
      • Maniatis T.
      Multilevel regulation of gene expression by microRNAs.
      ,
      • Yap K.
      • Lim Z.Q.
      • Khandelia P.
      • Friedman B.
      • Makeyev E.V.
      Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention.
      ). Collectively, these studies demonstrate that miR-124 regulates several molecular pathways critical for proper progression of neuronal differentiation.
      Neuron-specific genes are frequently modified by Ezh2-mediated H3K27 trimethylation (3meH3K27) in stem cells, whereas both the Ezh2 levels and the density of 3meH3K27 marks are down-regulated upon neuronal differentiation (
      • Lee T.I.
      • Jenner R.G.
      • Boyer L.A.
      • Guenther M.G.
      • Levine S.S.
      • Kumar R.M.
      • Chevalier B.
      • Johnstone S.E.
      • Cole M.F.
      • Isono K.
      Control of developmental regulators by Polycomb in human embryonic stem cells.
      ,
      • Bracken A.P.
      • Dietrich N.
      • Pasini D.
      • Hansen K.H.
      • Helin K.
      Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions.
      ,
      • Boyer L.A.
      • Plath K.
      • Zeitlinger J.
      • Brambrink T.
      • Medeiros L.A.
      • Lee T.I.
      • Levine S.S.
      • Wernig M.
      • Tajonar A.
      • Ray M.K.
      Polycomb complexes repress developmental regulators in murine embryonic stem cells.
      ). Interestingly, overexpression of miR-124 in hepatocellular carcinoma cells, where it is normally present at negligibly low levels, has been shown to reduce Ezh2 expression (
      • Zheng F.
      • Liao Y.J.
      • Cai M.Y.
      • Liu Y.H.
      • Liu T.H.
      • Chen S.P.
      • Bian X.W.
      • Guan X.Y.
      • Lin M.C.
      • Zeng Y.X.
      The putative tumour suppressor microRNA-124 modulates hepatocellular carcinoma cell aggressiveness by repressing ROCK2 and EZH2.
      ). However, whether miR-124 contributes to down-regulation of Ezh2 expression during neurogenesis has not been investigated.
      To this end, we first expressed miR-124 in mouse neuroblastoma Neuro2a (N2a) cells and showed that this treatment was sufficient to up-regulate a significant fraction of neuron-specific Ezh2 target genes. We further found that in P19 cells undergoing neuronal differentiation, the Ezh2 protein level was significantly reduced in an inverse correlation with increasing expression of mature miR-124. Importantly, miR-124-specific antisense inhibitor restored Ezh2 expression in differentiating P19 cells, whereas disruption of the putative miR-124 target site in exogenously expressed Ezh2 3′-UTR abolished the miR-124-mediated down-regulation and led to reduced neuronal differentiation. A similar effect of miR-124-regulated Ezh2 expression on neurogenesis was also observed in differentiating embryonic mouse neural stem cells. Thus, our results implicate Ezh2 as an important miR-124 target in the context of neuronal differentiation.

      DISCUSSION

      Neuron-enriched miRNA miR-124 provides a compelling example of a non-coding RNA modulating cellular gene expression at multiple levels (
      • Makeyev E.V.
      • Maniatis T.
      Multilevel regulation of gene expression by microRNAs.
      ). Importantly, this miRNA targets several master regulators of elaborated transcriptional and post-transcriptional programs, including transcription factor Sox9 (
      • Cheng L.C.
      • Pastrana E.
      • Tavazoie M.
      • Doetsch F.
      miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche.
      ), transcriptional co-repressor SCP1 (
      • Visvanathan J.
      • Lee S.
      • Lee B.
      • Lee J.W.
      • Lee S.K.
      The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development.
      ), chromatin remodeling component BAF53A (
      • Yoo A.S.
      • Staahl B.T.
      • Chen L.
      • Crabtree G.R.
      MicroRNA-mediated switching of chromatin-remodelling complexes in neural development.
      ), and RNA-binding protein Ptbp1 (
      • Makeyev E.V.
      • Zhang J.
      • Carrasco M.A.
      • Maniatis T.
      The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing.
      ). Here we expand this list by showing that in cells undergoing neural differentiation (P19 as well as embryonic mouse neural stem cells), miR-124 represses the expression of a critical epigenetic factor, lysine methyltransferase Ezh2. We provide evidence that Ezh2 down-regulation by miR-124 in this context promotes neuronal and counters astrocyte-specific differentiation route.
      What could be a molecular mechanism underlying this effect? Ezh2 is known to limit neurogenic competence of neural progenitor cells and repress expression of several neurogenesis-promoting genes (
      • Bracken A.P.
      • Dietrich N.
      • Pasini D.
      • Hansen K.H.
      • Helin K.
      Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions.
      ,
      • Boyer L.A.
      • Plath K.
      • Zeitlinger J.
      • Brambrink T.
      • Medeiros L.A.
      • Lee T.I.
      • Levine S.S.
      • Wernig M.
      • Tajonar A.
      • Ray M.K.
      Polycomb complexes repress developmental regulators in murine embryonic stem cells.
      ,
      • Hirabayashi Y.
      • Suzki N.
      • Tsuboi M.
      • Endo T.A.
      • Toyoda T.
      • Shinga J.
      • Koseki H.
      • Vidal M.
      • Gotoh Y.
      Polycomb limits the neurogenic competence of neural precursor cells to promote astrogenic fate transition.
      ,
      • Marson A.
      • Levine S.S.
      • Cole M.F.
      • Frampton G.M.
      • Brambrink T.
      • Johnstone S.
      • Guenther M.G.
      • Johnston W.K.
      • Wernig M.
      • Newman J.
      Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells.
      ), including master regulator for neuronal lineage commitment and differentiation Ascl1/Mash1 (
      • Parras C.M.
      • Schuurmans C.
      • Scardigli R.
      • Kim J.
      • Anderson D.J.
      • Guillemot F.
      Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity.
      ,
      • Fode C.
      • Ma Q.
      • Casarosa S.
      • Ang S.-L.
      • Anderson D.J.
      • Guillemot F.
      A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons.
      ). Up-regulation of this gene is sufficient to promote neuronal differentiation (
      • Berninger B.
      • Guillemot F.
      • Götz M.
      Directing neurotransmitter identity of neurones derived from expanded adult neural stem cells.
      ,
      • Geoffroy C.G.
      • Critchley J.A.
      • Castro D.S.
      • Ramelli S.
      • Barraclough C.
      • Descombes P.
      • Guillemot F.
      • Raineteau O.
      Engineering of dominant active basic helix-loop-helix proteins that are resistant to negative regulation by postnatal central nervous system antineurogenic cues.
      ). Notably, Ascl1 is one of the genes consistently derepressed by miR-124 in N2a neuroblastoma cells (Table 1), and our future studies will determine the functional significance of this effect.
      TABLE 1miR-124-up-regulated Ezh2 target genes
      Gene symbolGene nameFCCNS-specific
      Morc4Microrchidia 42.932No
      Tpm1Tropomyosin 1, α2.705Yes
      Col8a2Collagen, type VIII, α22.494No
      Dusp8Dual specificity phosphatase 82.371Yes
      Atf3Activating transcription factor 32.244Yes
      PrlrProlactin receptor2.128Yes
      Mt1Metallothionein 12.102Yes
      SmoxSpermine oxidase2.096Yes
      Klhl22Kelch-like 22 (Drosophila)2.070Yes
      Plxna2Plexin A22.054Yes
      Fbn2Fibrillin 21.990Yes
      Sema7aSema domain, immunoglobulin domain (Ig), and GPI membrane anchor, (semaphorin) 7A1.989Yes
      Igfbp6Insulin-like growth factor-binding protein 61.946Yes
      Igfbp5Insulin-like growth factor binding protein 51.925Yes
      Atp1a3ATPase, Na+/K+ transporting, α3 polypeptide1.892Yes
      Chrm3Cholinergic receptor, muscarinic 3, cardiac1.862No
      Tgfb3Transforming growth factor, β31.852Yes
      Adora2bAdenosine A2b receptor1.841Yes
      Ap3m2Adaptor-related protein complex 3, μ2 subunit1.839Yes
      Shroom3Shroom family member 31.830Yes
      OxtOxytocin1.816Yes
      Gnao1Guanine nucleotide-binding protein, αO1.780Yes
      Kcnk9Potassium channel, subfamily K, member 91.777No
      CrhbpCorticotropin-releasing hormone-binding protein1.768Yes
      Slc35f1Solute carrier family 35, member F11.749Yes
      FaahFatty acid amide hydrolase1.746Yes
      Nphs2Nephrosis 2 homolog, podocin (human)1.743No
      Pax7Paired box gene 71.729No
      Sypl2Synaptophysin-like 21.718Yes
      Gpc5Glypican 51.714Yes
      Kcne3Potassium voltage-gated channel, Isk-related subfamily, gene 31.707No
      Sp7Sp7 transcription factor 71.705Yes
      Epha5Eph receptor A51.703Yes
      Gpc4Glypican 41.701Yes
      ItpkaInositol 1,4,5-trisphosphate 3-kinase A1.691Yes
      Gprc5cG protein-coupled receptor, family C, group 5, member C1.682No
      Irx6Iroquois-related homeobox 6 (Drosophila)1.680No
      HhatHedgehog acyltransferase1.678No
      Gpr45G protein-coupled receptor 451.678Yes
      Celsr2Cadherin, EGF LAG seven-pass G-type receptor 2 (flamingo homolog, Drosophila)1.664Yes
      Kirrel3Kin of IRRE like 3 (Drosophila)1.662Yes
      Cyp46a1Cytochrome P450, family 46, subfamily a, polypeptide 11.661Yes
      Hoxc12Homeobox C121.656No
      Tcfap2bTranscription factor AP-2β1.640No
      Nkx1-2NK1 transcription factor-related, locus 2 (Drosophila)1.638No
      Nol3Nucleolar protein 3 (apoptosis repressor with CARD domain)1.632Yes
      Rab15RAB15, member RAS oncogene family1.630Yes
      Nuak2NUAK family, SNF1-like kinase, 21.629No
      Tmem28Transmembrane protein 281.628Yes
      Hmx1H6 homeobox 11.625No
      Spryd3SPRY domain-containing 31.624Yes
      Btbd11BTB (POZ) domain containing 111.613Yes
      Adamtsl5ADAMTS-like 51.610No
      C1qtnf4C1q and tumor necrosis factor related protein 41.607Yes
      Cacna2d2Calcium channel, voltage-dependent, α2/δ subunit 21.603Yes
      Dpp10Dipeptidylpeptidase 101.594Yes
      Zmiz1Zinc finger, MIZ-type-containing 11.591Yes
      Hoxa9Homeobox A91.589No
      Calb1Calbindin 11.585Yes
      Pstpip2Proline-serine-threonine phosphatase-interacting protein 21.583Yes
      Plagl1Pleiomorphic adenoma gene-like 11.581Yes
      Nkx2-6NK2 transcription factor related, locus 6 (Drosophila)1.579No
      Trim54Tripartite motif-containing 541.562No
      Gpr6G protein-coupled receptor 61.552Yes
      AplnApelin1.547Yes
      Snx22Sorting nexin 221.534Yes
      Alx4Aristaless-like homeobox 41.534No
      Nxph4Neurexophilin 41.526No
      En2Engrailed 21.522Yes
      Tmem25Transmembrane protein 251.520Yes
      Tubb2bTubulin, β2B1.513Yes
      Ybx2Y box protein 21.506No
      Il12rb1Interleukin 12 receptor, β11.505Yes
      Ascl1Achaete-scute complex homolog 1 (Drosophila)1.502Yes
      Our analysis also revealed that more than 1800 Ezh2 target genes are not up-regulated in miR-124-overexpressing cells. Non-CNS-specific genes (634 genes) are probably associated with silent chromatin in neuronal progenitors and therefore could not be up-regulated simply by miR-124-mediated Ezh2 down-regulation. A small fraction of Ezh2 target genes with predicted miR-124 target sites could potentially be down-regulated by miR-124 (81 genes; 7 non-CNS and 74 CNS-specific), but only 12 CNS-specific genes were down-regulated in miR-124-overexpressing cells, which is not statistically enriched. The remaining 1242 CNS-specific genes may require additional CNS-specific activators that are not expressed just 24 h after miR-124 transfection. Although our current analysis already revealed a significant overlap between Ezh2 target genes and the miR-124 up-regulated gene list, more Ezh2 target genes could be up-regulated by persistent miR-124 overexpression in differentiating neurons.
      Interestingly, examination of published genomic maps of the Ezh2-specific 3meH3K27 modifications suggests that promoter regions of all three mouse miR-124 genes are associated with this repressive mark as well as Suz12, a component of the PRC2 complex, in ES cells (Table 2) (
      • Marson A.
      • Levine S.S.
      • Cole M.F.
      • Frampton G.M.
      • Brambrink T.
      • Johnstone S.
      • Guenther M.G.
      • Johnston W.K.
      • Wernig M.
      • Newman J.
      Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells.
      ). It is therefore possible that Ezh2 controls miR-124 levels in stem cells, synergizing with the repressive effect of REST (
      • Singh S.K.
      • Kagalwala M.N.
      • Parker-Thornburg J.
      • Adams H.
      • Majumder S.
      REST maintains self-renewal and pluripotency of embryonic stem cells.
      ). During the neurogenic phase, the H3K27-specific demethylase Jmjd3 is up-regulated, leading to derepression of neuron-specific genes, possibly including miR-124 (
      • Jepsen K.
      • Solum D.
      • Zhou T.
      • McEvilly R.J.
      • Kim H.-J.
      • Glass C.K.
      • Hermanson O.
      • Rosenfeld M.G.
      SMRT-mediated repression of an H3K27 demethylase in progression from neural stem cell to neuron.
      ), that can now dampen Ezh2 expression. This hypothetical double-negative feedback between miR-124 and Ezh2/PRC2 would be similar to the previously reported relationship between miR-124 and SCP1/REST (
      • Visvanathan J.
      • Lee S.
      • Lee B.
      • Lee J.W.
      • Lee S.K.
      The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development.
      ).
      TABLE 2Enrichment of PRC2 at all three miR-124 loci
      LociPositions of miR-124 promoterPositions of Pre-miR-124Suz12 binding sitesOther transcription factors or histone modification associated with miR-124 loci
      mmu-mir-124-1chr14:63540450–63546275chr14:63544772–63544848 (+)chr14:63540776–63544525Oct4Nanog3meH3K27
      mmu-mir-124-2chr3:17986635–17986835chr3:17987829–17987903 (+)chr3:17985751–17988075Oct4Sox2NanogTcf33meH3K27
      mmu-mir-124-3chr2:180819000–180825000chr2:180823445–180823520 (+)chr2:180820551–180823275Oct43meH3K27
      Although further work will be needed to address the miR-124-Ezh2/PRC2 cross-regulation model, the results of our preliminary studies are consistent with this possibility. Indeed, induction of Ezh2 expression following RA treatment led to dramatic down-regulation of miR-124 expression in differentiating P19 cultures (data not shown). Thus, it is possible that the stimulatory effect of miR-124-resistant Ezh2 on astrocyte generation observed in our study might be caused by Ezh2-mediated down-regulation of miR-124 expression. Underscoring the biological relevance of these regulatory events, miR-124 is naturally expressed in neurons but not in astrocytes (
      • Smirnova L.
      • Gräfe A.
      • Seiler A.
      • Schumacher S.
      • Nitsch R.
      • Wulczyn F.G.
      Regulation of miRNA Expression during Neural Cell Specification.
      ), and it is predicted to directly down-regulate a number of astrocyte-enriched genes (
      • Lim L.P.
      • Lau N.C.
      • Garrett-Engele P.
      • Grimson A.
      • Schelter J.M.
      • Castle J.
      • Bartel D.P.
      • Linsley P.S.
      • Johnson J.M.
      Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.
      ,
      • Cahoy J.D.
      • Emery B.
      • Kaushal A.
      • Foo L.C.
      • Zamanian J.L.
      • Christopherson K.S.
      • Xing Y.
      • Lubischer J.L.
      • Krieg P.A.
      • Krupenko S.A.
      • Thompson W.J.
      • Barres B.A.
      A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function.
      ).
      In addition to its role in balancing neurogenesis versus astrogenesis, the miR-124/Ezh2 circuitry may function in other biological scenarios. For example, it has been recently proposed to control aggressiveness of hepatocellular carcinoma (
      • Zheng F.
      • Liao Y.J.
      • Cai M.Y.
      • Liu Y.H.
      • Liu T.H.
      • Chen S.P.
      • Bian X.W.
      • Guan X.Y.
      • Lin M.C.
      • Zeng Y.X.
      The putative tumour suppressor microRNA-124 modulates hepatocellular carcinoma cell aggressiveness by repressing ROCK2 and EZH2.
      ). Another recent study showed that miR-124 may prevent the activation of microglia, immune cells residing in the central nervous system (
      • Ponomarev E.D.
      • Veremeyko T.
      • Barteneva N.
      • Krichevsky A.M.
      • Weiner H.L.
      MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-α-PU. 1 pathway.
      ). Interestingly, Ezh2 is known to be up-regulated in activated lymphocytes and play an essential role in this process (
      • Su I.-H.
      • Dobenecker M.-W.
      • Dickinson E.
      • Oser M.
      • Basavaraj A.
      • Marqueron R.
      • Viale A.
      • Reinberg D.
      • Wülfing C.
      • Tarakhovsky A.
      Polycomb group protein ezh2 controls actin polymerization and cell signaling.
      ,
      • Su I.-H.
      • Basavaraj A.
      • Krutchinsky A.N.
      • Hobert O.
      • Ullrich A.
      • Chait B.T.
      • Tarakhovsky A.
      Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement.
      ),
      W. H. Neo and I.-H. Su, unpublished data.
      and it would be interesting to examine the role of Ezh2 in the context of microglia activation, which contributes to pathogen clearance in health or the progression of neurodegenerative and neoplastic diseases (
      • Saijo K.
      • Glass C.K.
      Microglial cell origin and phenotypes in health and disease.
      ).
      Although we show here that miR-124 represents one of the most potent miRNA regulators of Ezh2 expression, our data are also consistent with the possibility of combinatorial regulation by miRNA. Other than miR-124, miR-26a, and miR-101, six additional miRNAs consistently down-regulated the expression of Ezh2 3′-UTR reporter genes (Fig. 2D). Of these, only miR-20a, miR-26a, and miR-124 are known to be up-regulated in differentiating P19 cells (
      • Huang B.
      • Li W.
      • Zhao B.
      • Xia C.
      • Liang R.
      • Ruan K.
      • Jing N.
      • Jin Y.
      MicroRNA expression profiling during neural differentiation of mouse embryonic carcinoma P19 cells.
      ), which predicts possible synergistic effects of these three miRNAs on Ezh2 abundance. However, miR-124 is likely to be the major regulator of Ezh2 expression in differentiating neurons, because it is the most abundant miRNA in the brain (
      • Lagos-Quintana M.
      • Rauhut R.
      • Yalcin A.
      • Meyer J.
      • Lendeckel W.
      • Tuschl T.
      Identification of tissue-specific microRNAs from mouse.
      ) and is also highly up-regulated in differentiating P19 cells (20 times for miR-124 versus 2 times for miR-20 and miR-26a) (
      • Huang B.
      • Li W.
      • Zhao B.
      • Xia C.
      • Liang R.
      • Ruan K.
      • Jing N.
      • Jin Y.
      MicroRNA expression profiling during neural differentiation of mouse embryonic carcinoma P19 cells.
      ). The binding sites of these miRNAs are not overlapping. Other miRNAs identified in our study do not appear to be relevant for differentiating neurons.
      In conclusion, our study suggests that miRNA control of an important epigenetic regulator can be used as a regulatory paradigm for modulating the choice between alternative differentiation scenarios.

      Acknowledgments

      We thank Weijun Dai for helpful discussions.

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