RESEARCH ARTICLE3806

Development 139, 3806-3816 (2012) doi:10.1242/dev.082198© 2012. Published by The Company of Biologists Ltd

INTRODUCTIONDuring cortical development, neural progenitor cells (NPCs) giverise to various types of neurons – initially deep layer neurons andthen upper layer neurons – and finally differentiate into astrocytesvia changes in differentiation competency (Alvarez-Buylla et al.,2001; Fishell and Kriegstein, 2003; Fujita, 2003; Götz and Huttner,2005; Miller and Gauthier, 2007; Kageyama et al., 2007). Thesechanges in competency are highly ordered and unidirectional(Frantz and McConnell, 1996), suggesting that a ‘clock’ isintegrated into the differentiation system of NPCs to ensure that thecell composition and final size of the brain are characteristicallysimilar among members of a given species. However, the precisemechanism by which the differentiation competency of NPCsswitches at the proper time remains largely unknown.

It has been shown that NPCs switch differentiation competencyby changing gene expression profiles that are regulated bytranscription factors. For example, the zinc-finger transcriptionfactors Fezf2 and Ctip2 (Bcl11b – Mouse Genome Informatics) andthe basic helix-loop-helix (bHLH) transcription factors neurogenin1 (Ngn1, Neurog1) and Ngn2 regulate the generation of deep layerneurons (Arlotta et al., 2005; Molyneaux et al., 2005; Chen, B. etal., 2005; Chen, J. G. et al., 2005; Chen et al., 2008), whereas thehomeodomain transcription factor Pax6 is involved in thedifferentiation of upper layer neurons (Fode et al., 2000;Schuurmans et al., 2004). By contrast, the orphan nuclear receptorsCOUP-TFI and COUP-TFII (Nr2f1 and Nr2f2 – Mouse Genome

Informatics) induce astrocytic differentiation (Naka et al., 2008).Recent reports suggest that chromatin remodeling and epigeneticregulation also play important roles in controlling celldifferentiation (Seuntjens et al., 2009; Strobl-Mazulla et al., 2010).For example, neuron-specific chromatin remodeling complexessuch as BAF53 regulate dendrite development (Lessard et al.,2007; Yoo et al., 2009), while polycomb complexes such asPolycomb repressive complex (PRC) 1 and 2, which regulatehistone modification, facilitate the switching from neurogenesis toastrogenesis by repressing the proneural gene Ngn1 (Hirabayashiet al., 2009). Histone modifications provide fast and reliableregulation of gene expression, but their significance in neuraldevelopment has not been fully investigated.

The histone H3 lysine 9 (H3K9) methyltransferase ESET (alsoknown as Setdb1 or KMT1E) represses gene expression ineuchromatin by interacting with the co-repressor KAP1 (Trim28)(Schultz et al., 2002). Loss of ESET accelerates differentiation ofembryonic stem (ES) cells toward the trophectoderm lineage byderepressing genes encoding developmental regulators such asCdx2 and Hand1 (Bilodeau et al., 2009; Yuan et al., 2009; Yeap etal., 2009). Interestingly, ESET, as well as KAP1, also plays a majorrole in repressing the emergence of proviral particles in ES cells(Matsui et al., 2010; Rowe et al., 2010; Karimi et al., 2011). Thereare ~1000 copies of the endogenous retroviral-like sequencesknown as the intracisternal A-particle (IAP) and their expression ispartly repressed by ESET in ES cells (Matsui et al., 2010; Karimiet al., 2011). This regulatory mechanism is of particular importancebecause upregulation of IAP expression has been associated withretrotransposition and increased susceptibility to cancer (Heidmannand Heidmann, 1991; Howard et al., 2008). Because ESET ablationleads to embryonic lethality before implantation (Dodge et al.,2004), the functions of ESET in the development ofpostimplantation embryos, such as neural development, have notbeen examined. To circumvent this problem, we investigated therole of ESET in brain development using ESET conditionalknockout (cKO) mice.

1Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. 2KyotoUniversity Graduate School of Medicine, Kyoto 606-8501, Japan. 3Kyoto UniversityGraduate School of Biostudies, Kyoto 606-8502, Japan. 4Research Center forAdvanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan.5Japan Science and Technology Agency, CREST, Shogoin-Kawahara, Sakyo-ku, Kyoto606-8507, Japan.

*Author for correspondence (rkageyam@virus.kyoto-u.ac.jp)

Accepted 30 July 2012

SUMMARYIn the developing brain, neural progenitor cells switch differentiation competency by changing gene expression profiles that aregoverned partly by epigenetic control, such as histone modification, although the precise mechanism is unknown. Here we foundthat ESET (Setdb1), a histone H3 Lys9 (H3K9) methyltransferase, is highly expressed at early stages of mouse brain development butdownregulated over time, and that ablation of ESET leads to decreased H3K9 trimethylation and the misregulation of genes,resulting in severe brain defects and early lethality. In the mutant brain, endogenous retrotransposons were derepressed and non-neural gene expression was activated. Furthermore, early neurogenesis was severely impaired, whereas astrocyte formation wasenhanced. We conclude that there is an epigenetic role of ESET in the temporal and tissue-specific gene expression that results inproper control of brain development.

KEY WORDS: ESET, Astrocyte, Epigenetic control, Histone methyltransferase, Neural progenitor

Essential roles of the histone methyltransferase ESET in theepigenetic control of neural progenitor cells duringdevelopmentSiok-Lay Tan1,2, Miyuki Nishi1,3, Toshiyuki Ohtsuka1, Toshiyuki Matsui1, Keiko Takemoto1, Asuka Kamio-Miura4, Hiroyuki Aburatani4, Yoichi Shinkai1,3 and Ryoichiro Kageyama1,5,*

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MATERIALS AND METHODSMouse linesESET flox (f) (Matsui et al., 2010), Emx2-Cre (Kimura et al., 2005) andNes-Cre (Isaka et al., 1999) mice were described previously. Emx2-Cre;ESET(f/+) or Nes-Cre;ESET(f/+) male mice were crossed withESET(f/f) female mice, and the day when a vaginal plug was observed wasscored as embryonic day (E) 0.5.

In situ hybridization, immunohistochemistry and antibodiesEmbryos were dissected, fixed in 4% paraformaldehyde (PFA)/PBSovernight and cryoprotected with 20% sucrose for at least 10 hours. On thefollowing day, embryos were embedded in OCT compound (Tissue-Tek)and frozen at –80°C. Fixed samples were sectioned at 16 m with acryostat and dried for 2 hours. In situ hybridization was then performed aspreviously described (Imayoshi et al., 2008). Primers used to produce insitu hybridization probes are listed in supplementary material Table S1. Forimmunohistochemistry, embryos at different ages were fixed in 4% PFAfor 2 hours, equilibrated and sectioned as described above. Sections wereblocked with 5% normal goat serum or donkey serum in 0.1% Triton X-100/PBS and incubated with primary antibodies at 4°C overnight. Thefollowing primary antibodies were used: BrdU (1:100, BD Biosciences),Brn1 (Pou3f3) (1:100, Santa Cruz), Brn2 (Pou3f2) (1:250, Santa Cruz),cleaved Casp3 (1:500, Cell Signaling), Ctip2 (1:500, Abcam), Cux1 (1:100,Santa Cruz), ESET (1:1000, Cell Applications), FoxP2 (1:100, Santa Cruz),Gfap (1:1000, Sigma), IAP (1:500, a gift from Dr Bryan R. Cullen, DukeUniversity, Durham, USA), Ki67 (1:100, BD Pharmingen), nestin (1:500,BD Pharmingen), Pax6 (1:500, Abcam), PH3 (1:500, Sigma), Satb2(1:100, Abcam), Sox9 (1:500, a gift from Dr Michael Wegner, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany), Tbr1 (1:500,Millipore), Tbr2 (Eomes) (1:500, Abcam) and Tuj1 (Tubb3) (1:500,Babco). The following secondary antibodies were used: Alexa 488-conjugated mouse, rabbit, rat or goat and Alexa 594-conjugated mouse,rabbit or rat (1:250, Molecular Probes). Immunofluorescence images wereobtained with a Zeiss Axiophot confocal microscope.

Acquisition and analysis of microarray dataThe dorsal telencephalon of E14.5 wild-type (WT) and Emx2-Cre;ESET(f/f)(ESET cKO) mouse embryos were subjected to RNA extraction andexpression profiling using Affymetrix GeneChip Mouse Genome Array(n3) as previously described (Shimojo et al., 2008). Analysis of microarraydata was performed using GeneSpring GX version 11. Microarray data areavailable at Gene Expression Omnibus under the following accessionnumbers: GSM990998, GSM990999, GSM991000, GSM991001,GSM991002 and GSM991003 (for telencephalon WT 1-3 and cKO 1-3,respectively).

RNA preparation and quantitative (q) RT-PCRRNA was extracted from the dorsal telencephalon at different stages.Tissues were dissociated with trypsin and lysed with Trizol (Invitrogen);RNA was then extracted with phenol-chloroform and precipitatedaccording to the manufacturer’s protocol. RNA samples were subjected toreverse transcription as previously described (Shimojo et al., 2008). qRT-PCR was performed with Thunderbird SYBR Green PCR Mix (TOYOBO)using the primers listed in supplementary material Table S1.

RNA deep sequencingTotal RNA was isolated from E14.5 WT and ESET cKO embryonictelencephalon (male), and an RNA integrity number (RIN) was confirmed tobe greater than 8.0 using a 2100 Bioanalyzer (Agilent Technologies). TotalRNA (1 g) was used for generation of poly(A)+ RNA Seq libraries. Thesequencing libraries were prepared using the Illumina TruSeq RNA SamplePreparation Kit following the manufacturer’s protocol with the slightmodification that fragmentation time was reduced to 2 minutes. Afteramplification for 15 cycles, PCR products were purified with AgencourtAMPure XP magnetic beads (Beckman Coulter), and their size distributionand concentration were evaluated using a DNA 1000 Series II Assay on the2100 Bioanalyzer. cDNA at 4.2 pM/lane was applied to the flow cell andpaired-end 75 nt reads were sequenced on an Illumina GAIIx following the

manufacturer’s instructions (Wang et al., 2011). RNA sequencing results werepresented using Integrative Genomics Viewer (Broad Institute) version 2.0.

Bromodeoxyuridine (BrdU) labelingBrdU (2.5 mg; 5 mg/ml) was given to each pregnant mouse byintraperitoneal injection every 4 hours for a total of 12 hours at theindicated embryonic stage. Brains were fixed with 4% PFA, and sectionswere incubated in 2 M HCl for 30 minutes at 37°C, followed byneutralization in 0.1 M sodium tetraborate buffer for 10 minutes. Sectionswere incubated with mouse anti-BrdU antibody overnight at 4°C and thenwith Alexa 488-conjugated goat anti-mouse IgG.

Cell death assayTUNEL assays were performed with the ApopTag Fluorescein Direct InSitu Apoptosis Detection Kit (Chemicon International) according to themanufacturer’s protocol.

Neurosphere assayNeurosphere assays were performed as described previously (Ohtsuka etal., 2011). The numbers of primary spheres were counted at day 7.Experiments were repeated three times and the mean and s.e.m. of spherenumbers were calculated. For the neurosphere differentiation assay,primary spheres were cultured for 1 day and plated onto PLL- and laminin-coated Lab-Tek chamber slides (Nalge Nunc) in differentiation medium(DMEM/F-12 supplemented with B-27 and 2% fetal bovine serum). Cellswere examined immunocytochemically 1, 3 and 5 days after plating.

In utero electroporationIn utero electroporation was performed with E17.5 ICR pregnant mice asdescribed previously (Ohtsuka et al., 2011). Neonates at postnatal day (P)2 were harvested and analyzed, as described above.

Quantification and statisticsThe total numbers of marker-positive cells were counted in more than fivesections from at least three different animals per group. The ventricularzone, subventricular zone and cortical plate were defined according to Tbr2staining. The percentage of cells from each compartment was calculated.Statistical differences were examined with Student’s t-test and P<0.05 wasconsidered significant.

Chromatin immunoprecipitation (ChIP)ChIP assays were performed as described previously (Matsui et al., 2010)using the primers and antibodies listed in supplementary material Table S1.

Bisulfite sequencingBisulfite sequencing was conducted using the EpiTect Plus DNA BisulfiteKit (Qiagen). PCR products were cloned and individual inserts weresequenced using BigDye v3.1 chemistry. Sequencing data were analyzedusing QUMA (RIKEN). Primer sequences are listed in supplementarymaterial Table S1.

RESULTSESET is expressed by NPCs in the developing brainIt has been shown that ESET is expressed in ES cells and thepostnatal brain (Ryu et al., 2006; Matsui et al., 2010; Jiang et al.,2010), but expression in the developing brain has not beenexamined extensively. We therefore performed in situ hybridizationand immunohistochemical analyses with mouse embryos atdifferent developmental stages. ESET was expressed in thetelencephalon, otic vesicles, forelimbs and hindlimbs at E9.5 (Fig.1A). At this stage, ESET was highly expressed in the ventricularzone where NPCs reside (Fig. 1B). ESET mRNA expressionoccurred in cells expressing Ki67, a marker for cell proliferation,but was excluded from Tuj1+ neuronal layers (Fig. 1B). At E11.5,ESET expression was found in similar regions (Fig. 1A,B). Allradial glia/apical progenitors (Pax6+) and some basal progenitors(Tbr2+) co-expressed ESET (Fig. 1D,Ea,b), but neurons (Tuj1+) didnot (Fig. 1Ec). ESET mRNA expression in the ventricular zone D

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further decreased at E15.5 (Fig. 1B) and was hardly detectable atE17.5 (Fig. 1B). qRT-PCR also showed that ESET mRNAexpression in purified NPCs decreased as development proceeded(Fig. 1C). Similarly, ESET protein expression also decreased asdevelopment proceeded (supplementary material Fig. S1). Fromthis we infer that ESET is expressed by NPCs (both apical andbasal progenitors), but that the expression decreases over time andis very low by E17.5, when the transition from neurogenesis toastrogenesis in the dorsal telencephalon occurs.

ESET is required for proper regulation of neuraland non-neural gene expressionTo determine whether ESET plays a role in cortical development,we crossed ESET flox mice with Emx2-Cre driver mice to generateforebrain-specific ESET cKO mice. By E11.5, ESET expressionwas mostly abolished in the dorsal telencephalon, but was notaffected in the midbrain of the cKO mice, where the Emx2promoter was inactive (supplementary material Fig. S2A). Thesemutant mice were born but did not survive beyond P10. We alsogenerated ESET cKO mice using Nes-Cre mice, in which Cre isactive in NPCs, and obtained virtually the same results (see below).

Because ESET catalyzes the trimethylation of H3K9 (H3K9me3),a modification that potentially alters gene expression, we examinedwhich genes were misregulated in the absence of ESET bymicroarray expression profiling of the E14.5 dorsal telencephalon.In the ESET cKO brain, 185 genes were upregulated more than 2-fold relative to the WT brain (supplementary material Table S2),whereas 112 genes were downregulated to less than half the level ofthe WT (supplementary material Table S3). The misregulated geneswere categorized into three groups by GeneSpring softwareclustering analysis (Fig. 2A). Cluster 1 included downregulatedgenes, whereas clusters 2 and 3 included upregulated genes; manyof them, particularly the downregulated genes, might be indirectlyregulated by ESET, which functions as a transcriptional repressor.

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Each cluster was subjected to Gene Ontology (GO) analysis witha P<0.05 cut-off. Cluster 1 GO analysis revealed enrichment forneurogenesis, neuronal differentiation and signal transmission (P-values of 0.014, 0.014 and 0.031, respectively; Fig. 2B). Genes inthese categories included Reln, Etv1, Nr4a2, Emx2, Satb2, Rorb,Neurog1, Ntrk2 and other important genes required for the properregulation of neuronal differentiation, maturation, as well as themaintenance of neuronal functions. For some of these genes, thechanges in expression were validated by qRT-PCR of E14.5 andE16.5 cortical samples, and similar results were obtained at bothstages (supplementary material Fig. S2B,C).

Interestingly, the GO terms of cluster 1 overlapped substantiallywith those of downregulated genes in ESET-null ES cells (Karimiet al., 2011), suggesting that ESET could be commonly involved inneuronal differentiation in these cells. Cluster 2 included genesinvolved in ossification, such as Sox9 (P0.015; Fig. 2C). Sox9 isknown to regulate gliogenesis in the brain (Stolt et al., 2003), andother glia-related genes such as Tspo and Ppp1r3c were alsoincluded in cluster 2 (supplementary material Table S2). Theseresults suggest that glial differentiation was activated whereasneuronal differentiation was impaired in the absence of ESET.Cluster 3 contained strongly upregulated genes and revealedenrichment for spermatogenesis (P0.044), M phase of the meioticcell cycle (P0.00041), and chromosome organization involved inmeiosis (P0.00039) (Fig. 2D). This cluster included IAP, Mnd1,Tcfl5, Rec8, Rnf17, Sycp3 and Mael. These genes are strictlyexpressed by germ cells in a narrow time frame when meiosis orhomologous recombination takes place. In the WT brain, many ofthese genes must be silenced so as to prevent genomic instability.For instance, IAP belongs to the endogenous retroviruses (ERVs),which could potentially bring about genetic disorders byretrotransposition (Mietz et al., 1987; Qin et al., 2010), whereasMnd1, Rec8 and Sycp3 are the major players in facilitating propercrossing over during homologous recombination in meiosis.

Fig. 1. Expression of ESET mRNA in themouse embryonic brain. (A,B,D,E) Insitu hybridization on whole-mounts (A)and sections (B) with an ESET-specificprobe and immunohistochemistry for ESET(D,Ea-c), Pax6 (D,Ea), Ki67 (B), Tuj1 (B,Ec)and Tbr2 (Eb). DAPI staining was alsoconducted (B,D,E). The right-hand columnin E shows a merge of each row. (C)ESETmRNA levels in neural progenitor cells(NPCs) sorted from the dorsaltelencephalon of pHes1-d2EGFP transgenicmice at E11.5, E13.5, E15.5 and E17.5were examined by microarray and qRT-PCR. Mean ± s.e.m. was quantified at eachtime point (n3). ESET is specificallyexpressed by NPCs, and this expression isgradually downregulated duringdevelopment. Scale bars: 1 mm in A;100m in B; 20m in E.

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Upregulated expression of cluster 2 and cluster 3 genes in thecKO brain was also validated by qRT-PCR (Fig. 3A;supplementary material Fig. S2D) and western blot analyses (Fig.3B). Upregulation of these genes, such as IAP, was also observedin Nes-Cre-driven ESET cKO mice (supplementary material Fig.S3A). The GO terms of clusters 2 and 3 mostly differed from thoseof the upregulated genes in ESET-null ES cells, which includedplasma membrane, vitamin binding and regulation of cell death(Karimi et al., 2011). In the ESET cKO brain, some genes, such asIAP and Mnd1, were ectopically expressed by neurons, whereasothers, such as Sox9, were expressed by NPCs and some migratingcells (Fig. 3C, arrow), suggesting that the onset of ectopicexpression differs from gene to gene. Together, these resultsindicated that ESET is required for the proper expression ofneuronal genes and for the suppression of non-neuronal genes (Fig.2E).

Derepression of ERVs is responsible for theupregulation of many genes in the ESET cKO brainGenome database analysis indicated that many of the upregulatedgenes are located near ERVs such as IAP sequences, suggestingthat activation of the long terminal repeat (LTR) of ERVs leads toectopic upregulation of neighboring genes in the absence of ESET.Indeed, one of the most highly upregulated genes in cluster 3,Mnd1 (Fig. 3A,C), has an IAP sequence in the first intron. PCR andsequence analysis showed that the major Mnd1 transcript startedfrom the intronic IAP in the absence of ESET (Fig. 3D,E;supplementary material Fig. S3B,C). RNA deep sequencinganalyses revealed that, among the upregulated genes, six were

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chimeric transcripts with IAPs (supplementary material Table S4)and three (Mnd1, Slc20a2 and Sec14l4) were verified as fusionswith IAP by PCR and sequencing analyses (Fig. 3D,E;supplementary material Fig. S3D,E). Among these six chimerictranscripts with IAPs, three (Adk, Gramd1c and Slc20a2) wereshown to be astrocyte-enriched genes (Cahoy et al., 2008). RNAdeep sequencing additionally revealed the IAP-Capn11 chimerictranscript, which was also verified by PCR and sequencinganalyses (supplementary material Fig. S3F). Other derepressedERVs, such as MMERVK, also formed five upregulated chimerictranscripts (supplementary material Table S4). Therefore, amongthe 185 upregulated genes, a total of 11 were expressed as chimerictranscripts with ERVs in the ESET cKO brain (supplementarymaterial Table S4). In addition, 60 other upregulated genes werefound to have ERVs within 10 kb of the transcription initiation site(supplementary material Table S4). Because deep sequencinganalyses detected enhanced transcription over 10 kb regions fromsome activated ERVs in the absence of ESET (data not shown),these results raised the possibility that as many as 71 of theupregulated genes could be influenced by nearby ERVs,representing ~38% of the upregulated genes in the ESET cKObrain. This possibility remains to be analyzed, and the actualnumber of genes activated by derepressed ERVs could be smaller.

ESET maintains global and localized H3K9me3Because ESET is an H3K9 methyltransferase, we next examinedthe H3K9me3 level in the ESET cKO brain. In the absence ofESET (Fig. 4A), the global H3K9me3 mark in the chromatin wasslightly reduced at E14.5 and E18.5 (Fig. 4B; supplementary

Fig. 2. ESET deficiency leads to misregulation ofgene expression. (A)Microarray analysis of wild-type(WT) and ESET conditional knockout (cKO) mousebrains at E14.5 (n3). Clustering analysis identifiedthree groups (clusters 1, 2 and 3) of genes thatshowed substantial changes in expression levels in theabsence of ESET. (B-D)Cluster 1 highlightsdownregulated genes, whereas clusters 2 and 3include upregulated genes. Gene Ontology (GO)terms among genes that exhibited the highestchanges in expression are shown. (E)A workinghypothesis based on the microarray analysis.

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material Fig. S4A), whereas the H3K9me2 mark was unaffected(Fig. 4C; supplementary material Fig. S4A). Because themicroarray data suggested that there was no overwhelming globalupregulation of gene expression, it is likely that the role of ESETin H3K9me3 regulation could be, to a certain extent, specific orlocalized. Hence, we investigated whether ESET binds directly to,and if so whether it modifies the H3K9me3 status of, the chromatinof two representative misregulated genes: Sox9 from cluster 2 andIAP from cluster 3 (P-values of 0.00136 and 0.001213,respectively, by Student’s t-test of microarray data).

Sox9 regulates the differentiation of many cell types, includingosteoblasts and glial cells (Stolt et al., 2003; Liu et al., 2007), andit is normally expressed in the developing brain, but wassignificantly upregulated in the absence of ESET (Fig. 3A-C). Asdescribed above, IAP element expression was mostly negative inthe WT brain but was derepressed in the absence of ESET (Fig.3A-C). ChIP assay of E14.5 cortical samples showed that ESETbound to the promoters of Sox9, IAP and other genes (Fig. 4D;supplementary material Fig. S4B). In addition, upon ESETdepletion, the H3K9me3 level at these loci was significantlydecreased (Fig. 4E; supplementary material Fig. S4C), whereas theH3K9ac level was enhanced (supplementary material Fig. S4C).These results indicate that ESET directly binds to these genes andrepresses their expression via elevated H3K9me3 modification.

Although there are ~1000 copies of IAP sequences in the mousegenome, only six genes were activated to form chimeric transcriptswith IAPs (supplementary material Table S4), suggesting thatderepression of IAPs is not global but localized. It was previouslyshown that CpG sequences in the IAP LTR regions are highlymethylated, and that demethylation of CpG sequences byinactivation of DNA methyltransferase 1 (Dnmt1) leads to robustIAP expression (Walsh et al., 1998). Therefore, we examinedwhether the DNA methylation status of the IAP LTR was affected

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in the ESET cKO brain. Bisulfite sequencing analysis revealed thatalthough global DNA demethylation was not observed in the globalIAP LTR sequences (94% in the cKO versus 92% in the WT; Fig.4F), CpG sequences of the IAP in Mnd1 and other derepressedgenes were partially demethylated in the ESET cKO braincompared with the WT (87.4% in the cKO versus 99% in the WT;Fig. 4G; supplementary material Fig. S4D,E). Thus, in addition toESET loss, demethylation of CpG might be required for activationof IAP, suggesting that this additional process might contribute todelayed onset of IAP expression in neurons.

Ablation of ESET impairs neurogenesisBecause many neuronal and non-neuronal genes weremisregulated, we examined the effects of ESET loss on neuraldevelopment. At E11.5, the number of radial glia/apicalprogenitors (Pax6+) was not significantly affected, whereas basalprogenitors (Tbr2+) were significantly reduced (supplementarymaterial Fig. S5A). At E12.5, Ctip2+ early-born layer V and VIneurons and Tbr2+ basal progenitors were markedly reduced innumber in the ESET cKO cortex compared with the control (Fig.5A,B). Production of Ctip2+ layer V and VI neurons, Tbr1+ layerVI neurons and Tbr2+ basal progenitors was also reduced in theESET cKO cortex compared with the control at E14.5 and E16.5(Fig. 5D-G; supplementary material Fig. S5B). By contrast, thenumber of Brn2+ layer II-III neurons increased in the ESET cKOcortical plate at E14.5 and E16.5 (supplementary material Fig.S5C,D, arrowheads). At E18.5, when the neurogenesis phaseceases, the populations of Ctip2+, Fezf2+, Tbr1+, FoxP2+ andNurr1+ deep layer neurons significantly decreased in the ESETcKO (Fig. 5H,I), implying that ESET ablation impaired earlyneurogenesis and severely affected the generation of deep layerneurons. By contrast, the population of Cux1+ and Satb2+ upperlayer neurons increased in the E18.5 cKO (Fig. 5H,I), suggesting

Fig. 3. Misregulated gene expression in the ESET cKObrain. (A)qRT-PCR analysis of misregulated geneexpression in the WT and ESET cKO mouse brain at E14.5(n6). Values were normalized to that of Gapdh, and aratio relative to the WT level ± s.e.m. was calculated.***P<0.001, t-test. (B)Expression of intracisternal A-particle (IAP), Sox9 and Tbr2 protein was examined in WT,heterozygote (Het) and ESET cKO by western blot withE14.5 whole-cell lysate from the dorsal telencephalon. Ineach case, tubulin provides a loading control.(C)Expression of IAP gag protein, Mnd1 mRNA and Sox9mRNA was examined in coronal sections from both WTand cKO brains. IAP and Mnd1 were expressed by neuronsof the ESET cKO brain, whereas Sox9 mRNA wasexpressed by both NPCs and migrating neurons (arrow).(D)RNA deep sequencing revealed emergence of IAP-Mnd1 chimeric transcripts in the ESET-ablated cortex. Redbar indicates the position of IAP. (E)Conventional PCR wasperformed with IAP- and Mnd1-specific primers usingE14.5 cortical cDNA. PCR products were detectable in theESET cKO samples but not in the WT. Scale bars: 100m.

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that the population of upper layer neurons might expand at theexpense of deep layer neurons in the ESET cKO brain.

In order to determine whether late-born upper layer neuronsdifferentiate prematurely, we performed a BrdU birthdating assayat E13.5 and E16.5 and analyzed BrdU co-labeling with variouslayer-specific markers at E18.5. The percentage of Cux1+ upperlayer neurons born at E13.5 was significantly increased whereasthat of Tbr1+ and FoxP2+ deep layer neurons was significantlydecreased in the cKO (Fig. 5J), revealing that the production oflate-born upper layer neurons was enhanced whereas that of early-born deep layer neurons was prematurely decelerated (Fig. 5J).Approximately 20-30% of Cux1+ and Brn2+ upper layer neuronswere still generated at E16.5 in the WT, whereas only 12-15% ofsuch neurons were born at the same stage in the cKO (Fig. 5K).These results suggest that the production of upper layer neuronswas accelerated at E13.5 at the expense of deep layer neurons butprematurely decelerated at E16.5.

At E18.5, GABAergic neurons, which are derived from themedial ganglionic eminence, tangentially migrated to the dorsaltelencephalon. However, this migration was somewhat reduced inthe ESET cKO brain compared with the WT (supplementarymaterial Fig. S6A), a defect that could be due to a lack of propersignals from excitatory neurons (Lodato et al., 2011).

The ESET cKO cortex was smaller than that of the WT at E18.5(Fig. 5C) and P7 (supplementary material Fig. S6B). At P7, bywhich time neurogenesis and migration were completed, thenumber of Ctip2+ layer V and VI neurons was severely reduced inthe ESET cKO cortex compared with the control, whereas Brn1+

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layer II-III neurons (supplementary material Fig. S6Ca-d) andCux2+ layer II-IV neurons were not significantly affected, and Id2expression was slightly enhanced in the ESET cKO (supplementarymaterial Fig. S6Ce-h). These results showed that production ofdeep layer neurons was most severely impaired, whereas upperlayer neurons were only modestly affected in the absence of ESET.

ESET promotes neuronal survival andproliferationTo further investigate the reason for the smaller cortex of ESETcKO mice (supplementary material Fig. S6B) and the severe lossof deep layer neurons in the ESET cKO cortex (supplementarymaterial Fig. S6C), we examined whether increased apoptosis,reduced proliferation, or a combination of both could be involved.Apoptotic cells, as indicated by TUNEL and cleaved caspase 3staining, significantly increased in number from E13.5 in the cKObrain (Fig. 6A,B). Furthermore, these apoptotic cells were mostlylocated in layers V-VI (Fig. 6B, graph), and many of them co-expressed the deep layer neuronal marker Ctip2 (Fig. 6B,enlargements), suggesting that apoptosis preferentially occurred indeep layer neurons.

Expression of the basal progenitor marker Tbr2, which isrequired for the production of a sufficient number of neurons(Sessa et al., 2008), was reduced in the ESET cKO brain (Fig.5A,D,F; supplementary material Fig. S5A) and, in agreement withthis observation, the number of dividing cells [phosphohistone H3(PH3)+] was reduced in the subventricular zone of the mutant brain,where basal progenitors reside (Fig. 6C,D). By contrast, expression

Fig. 4. ESET directly regulates H3K9 trimethylation at specific genes. (A)ESET protein is almost completely ablated in the cortex of E14.5 ESETcKO mouse embryos (n3). (B,C)The global H3K9me3 level was slightly decreased in the absence of ESET (n3). However, the H3K9me2 level wasnot significantly affected (n3). (D)Cross-linked ChIP assay was performed with IgG or ESET antibody on E14.5 WT cortical samples. Binding activity(relative to the input) of ESET on Sox9 and IAP promoter regions was quantified with s.e.m. (n4). (E)Native ChIP assay was performed withH3K9me3 antibody on E14.5 WT and ESET cKO cortical samples. Binding activity (relative to the input) of H3K9me3 on Sox9 and IAP promoterregions was quantified with s.e.m. ns, not significant; **P<0.01, ***P<0.001, t-test. (F)DNA methylation status of global IAP long terminal repeat(LTR) amplified with common primers did not differ between WT and cKO. (G)Significant demethylation of DNA was observed in the specific IAPLTR located in the Mnd1 gene, where IAP expression was derepressed.

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of the radial glial marker Pax6 was not noticeably changed(supplementary material Fig. S5A) and the number of dividingcells in the ventricular zone was not significantly affected in theESET cKO brain (Fig. 6C,D). Primary neurosphere culture assayswith NPCs extracted from E14.5 WT and ESET cKO dorsaltelencephalon showed that mutant NPCs were less likely todevelop into neurospheres of greater than 50 m diameter than WTNPCs within 7 days of culture (Fig. 6E,F), indicating that theproliferation potential of NPCs was compromised in the absence ofESET.

Together, these results suggest that the decrease in basalprogenitors from early stages and the increase in apoptosiscontribute to the size reduction of the ESET cKO cortex, withparticular effect on the deep layer populations.

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Astrocyte formation is accelerated in the absenceof ESETOur BrdU birthdating and Tbr2 immunostaining analyses showedthat the neurogenic phase seemed to have ended prematurely in theESET cKO brain, and we therefore examined whether the timingof astrocyte formation was affected. We performed immunostainingof the astrocyte marker Gfap at E18.5 and various postnatal stages.At E18.5, only a small number of Gfap+ cells appeared in the WTmedial telencephalon, but their number was significantly increasedin the ESET cKO cortex (Fig. 7A). The enhanced expression ofGfap continued at later stages (Fig. 7A). Upregulation of Gfap alsooccurred in the ESET cKO driven by Nes-Cre (supplementarymaterial Fig. S7A). Furthermore, expression of the astrocytemarkers Gfap, Aldh1l1, Aqp4 and Slc14a1 (Cahoy et al., 2008) was

Fig. 5. ESET deficiency impairsneural development.Immunohistochemistry (Ctip2, Tbr1,Tbr2, Cux1, Satb2 and FoxP2) or insitu hybridization (Fezf2 and Nurr1) onsections of the telencephalon of WTand ESET cKO mice. (A,C)At E12.5 (A)and E18.5 (C), the numbers of Ctip2+

and Tbr2+ cells were decreased in theESET cKO brain compared with theWT. (D,F)At E14.5 (D) and E16.5 (F),the numbers of Ctip2+, Tbr1+ andTbr2+ cells were decreased in the ESETcKO brain compared with the WT.(H)At E18.5, the numbers of Cux1+

and Satb2+ upper layer neurons wereincreased in the ESET cKO brain,whereas Ctip2+, Fezf2+, Tbr1+, FoxP2+

and Nurr1+ deep layer neurons weredecreased in the ESET cKO braincompared with the WT. (B,E,G,I) Quantification of neuronalnumber with s.e.m. at E12.5 (B), E14.5(E), E16.5 (G) and E18.5 (I). n3 pergroup. **P<0.01, ***P<0.001, t-test.(J,K)Pulse-chase experiments withBrdU administered at E13.5 (J) orE16.5 (K) (n3 for both) followed byanalysis at E18.5. Quantificationshows the percentage with s.e.m. ofcells that were double labeled withBrdU and layer-specific markers. AtE13.5, a higher proportion of Cux1+

neurons incorporated BrdU, whereaslower proportions of Tbr1+ and FoxP2+

neurons incorporated BrdU. At E16.5,lower proportions of Cux1+ and Brn2+

neurons incorporated BrdU. *P<0.05,***P<0.001, t-test. Scale bars:100m in A,D,F; 1 mm in C; 50m in H.

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increased at P7 in the ESET cKO cortex (supplementary materialFig. S7B). These results indicated that astrocyte differentiation wasaccelerated and enhanced in the absence of ESET.

It is possible that increased neuronal death induced astrogliosisin the ESET cKO brain. To rule out this possibility, we performedGfap and TUNEL double staining in P4 brains. Although the totalnumber of TUNEL+ cells increased in the cKO, the majority ofGfap+ cells were present in regions where there were no apoptoticcells (Fig. 7B), suggesting that enhanced Gfap expression was notdue to cell death-induced gliosis. Furthermore, highermagnification images revealed that these Gfap+ cells exhibitedtypical astrocyte morphology (Fig. 7B, lower panels). In addition,we performed an in vitro differentiation assay using NPCs preparedfrom E14.5 WT and cKO dorsal telencephalon to investigate thegliogenic potential of these progenitors. Indeed, NPCs from theESET cKO cortex more readily differentiated into Gfap+ cells thanthe WT NPCs, although the frequency of cell death did not differbetween ESET cKO and WT NPCs (Fig. 7C; supplementarymaterial Fig. S7C). The percentage of Gfap+ colonies was alsosignificantly increased in the absence of ESET on the third dayupon differentiation (Fig. 7D). By contrast, overexpression ofESET by in utero electroporation decreased astrocytedifferentiation (supplementary material Fig. S7D), althoughoverexpression of ESET alone was not able to block astrocytedifferentiation completely (supplementary material Fig. S7D).These results suggest that ESET represses the premature onset ofastrocyte formation in the developing cortex.

3813RESEARCH ARTICLEESET in neural development

To determine whether the Gfap gene is a direct target of ESET,we performed ChIP assays on the Gfap promoter. ChIP assay withE14.5 cortical samples showed that ESET directly binds to theGfap promoter (Fig. 7E,F). In addition, upon ESET depletion, theH3K9me3 level at this promoter was significantly decreased (Fig.7G), whereas the H3K9ac level was enhanced (Fig. 7H). Theseresults indicated that ESET directly binds to the Gfap promoter andrepresses its expression via elevated H3K9me3 marks.

DISCUSSIONEpigenetic regulation is necessary for the establishment andmaintenance of heritable gene expression during development, butvery little is known about the significance of histone modificationssuch as H3K9me3 in neural development. Here, we showed thatthe H3K9 methyltransferase ESET controls the proper expressionof neural genes and suppresses the expression of non-neural genes,thereby regulating brain development.

ESET is essential for temporal-specific geneexpressionESET is highly expressed by NPCs at an early stage, but isdownregulated over time, and expression is very low during theneuron-to-astrocyte fate switch. In the ESET cKO brain, generationof early-born deep layer neurons was markedly impaired, whereasgeneration of late-born upper layer neurons was not significantlyaffected (Fig. 5; supplementary material Fig. S6C). By contrast, thegeneration of astrocytes was significantly enhanced in the absence

Fig. 6. ESET promotes cell survival and proliferation. (A)The number of TUNEL+ cells was not affected at E11.5 but increased in the ESET cKOmouse brain compared with the WT from E13.5 onwards. (B)Apoptotic cells (cleaved Casp3+) were abundant specifically in the deep layer of thecortical plate in the ESET cKO brain, whereas there were very few in the WT. Boxed regions are enlarged beneath. Distribution of apoptotic cells inthe ESET cKO brain was quantified (bottom). (C)Immunohistochemical analysis of PH3 and Tbr2. Some basal progenitors (Tbr2+) exhibited PH3 inthe WT, but this rarely occurred in the ESET cKO brain, suggesting that basal progenitors were proliferating in the WT whereas their proliferationwas reduced in the ESET cKO brain. (D)Quantification with s.e.m. of the number of PH3+ cells (n4). Proliferation of basal progenitors (Tbr2+) in thesubventricular zone was significantly decreased, but that of radial glia cells (Pax6+) in the ventricular zone was not affected in the ESET cKO braincompared with the WT. CP, cortical plate; SVZ, subventricular zone; VZ, ventricular zone. ns, not significant; **P<0.01, t-test. (E)Primaryneurospheres generated from E14.5 WT and cKO brains. (F)The average numbers with s.e.m. of primary neurospheres greater or less than 50m indiameter as originated from 5000 cells. The cKO produced fewer and smaller neurospheres than the WT. Scale bars: 50m in A,C; 100m in B,E.

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of ESET. Thus, in the ESET cKO brain, generation of astrocytes isaccelerated at the expense of neurons, most notably early-bornneurons. These results suggest that decreasing the expression ofESET during development might be one of the internal clockmechanisms that regulate the timing of cell fate switches of NPCs.The mechanism of gradual downregulation of ESET duringdevelopment remains to be determined, but both extrinsic andintrinsic factors could be involved. It was reported that someextrinsic factors such as ciliary neurotrophic factors, leukemiainhibitory factor and cardiotrophin 1 promote astrocytedifferentiation (Kahn et al., 1997; Nakashima et al., 1999; Millerand Gauthier, 2007), and these factors could lead to downregulationof ESET expression. It was previously reported that the bHLHgenes Hes1 and Ngn2 are expressed in an oscillatory manner inNPCs (Shimojo et al., 2008), and it has been proposed that thisoscillation is one of the internal clock mechanisms (Kageyama etal., 2007). Interestingly, there are multiple Hes1 binding sites in theESET promoter (data not shown), suggesting that Hes1 oscillationscould lead to gradual downregulation of ESET expression,although further analyses are required to clarify any suchrelationship.

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ChIP assays showed that ESET directly binds the promoters ofthe astrocyte-related genes Gfap and Sox9. Furthermore, in theabsence of ESET, the H3K9me3 mark on these astrocyte-relatedgene loci was reduced, leading to upregulation of astrocyte geneexpression. These results suggest that ESET prevents prematureastrocyte formation by repressing Gfap and other astrocyte-relatedgene expression via H3K9me3. However, overexpression of ESETat E17.5 reduced astrocyte formation but failed to inhibit thisprocess completely. This suggests that additional regulation, suchas DNA methylation (Takizawa et al., 2001), might be required toinhibit astrocyte formation.

The expression of neuronal genes was downregulated in theESET cKO brain, but the precise mechanism of thisdownregulation remains to be determined, especially as ESET isknown to function as a transcriptional repressor. It was previouslyreported that Sox9 represses the expression of neuronal genes (Stoltet al., 2003; Scott et al., 2010). Furthermore, it was shown that theneurogenic and gliogenic programs inhibit each other bysequestering the CBP-Smad1 complex (Sun et al., 2001). Thus, oneof the likely mechanisms for downregulation of neuronal genes isthe upregulation of astrocytic genes.

Fig. 7. Inactivation of ESET enhancesastrocyte formation.(A)Immunohistochemistry for Gfap andDAPI staining on coronal sections of E18.5,P2, P4 and P7 mouse telencephalon. Gfapexpression was upregulated in the ESET cKObrain compared with the WT. (B)Enhancedastrogenesis was observed in the P4 cKOcortex compared with the WT. Highermagnification images show that the majorityof Gfap+ cells in the cKO did not overlapwith TUNEL staining. (C)Neurospheredifferentiation assay. E14.5 NPCs werecultured in differentiation conditions for 1, 3and 5 days and then immunostained withanti-Gfap antibody. Gfap expression wasincreased in the cKO on day 3 and day 5.(D)The proportion with s.e.m. of Gfap+

colonies on day 3. n7 for WT, n6 for cKO.***P<0.001, t-test. (E-H)Cross-linked ChIPassay was performed with IgG or ESETantibody on E14.5 WT cortical samples anddirect binding of ESET on the Gfap promoterregions was observed (F; n4). The amplifiedregions are shown (E). Native ChIP assay wasperformed with H3K9me3 and H3K9acantibodies on E18.5 WT and ESET cKOcortical samples (G,H). The H3K9me3 levelson the Gfap promoter regions weredecreased, whereas the H3K9ac levels wereincreased in the cKO. Binding activity(relative to the input) with s.e.m. on theGfap promoter regions was quantified. ns,not significant; *P<0.05, **P<0.01,***P<0.001, t-test. Scale bars: 100m inA,B top; 50m in C; 10m in B bottom.

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3815RESEARCH ARTICLEESET in neural development

Epigenetic regulation in the nervous systemRecent studies revealed that epigenetic regulation is essential forthe maintenance of neuronal identity. Ablation of the H3K9methyltransferase G9a (Ehmt2 – Mouse Genome Informatics),which catalyzes dimethylation of H3K9 (Tachibana et al., 2001),leads to derepression of non-neuronal genes and NPC-specificgenes in neurons and to abnormal behaviors in mice (Schaefer etal., 2009; Maze et al., 2010). The precise role of ESET in neuronsis not known, but it was shown that overexpression of ESET inneurons leads to the repression of some neuronal genes and toabnormal behaviors in mice, and that ESET expression is highlyupregulated in patients with Huntington’s disease (Ryu et al., 2006;Jiang et al., 2010). Thus, the proper level of ESET is important fornormal neuronal functions, although a loss-of-function analysis forESET in neurons remains to be performed.

ESET is essential for repression of non-neuralgenesIn addition to the regulation of neural genes, we found that ESET isimportant for repressing the endogenous retroelement IAP in somatictissues. Among the upregulated genes in the ESET cKO brain, manywere associated with derepressed IAPs and other ERVs. In particular,genes near derepressed IAPs were strongly upregulated in the ESETcKO brain. One such gene is Mnd1, which regulates meioticrecombination between homologous chromosomes (Petukhova et al.,2005). Normally, Mnd1 is not expressed in the developing nervoussystem, but in the ESET cKO cortex chimeric transcripts consistingof the IAP and Mnd1 sequences were robustly expressed. Althoughit remains to be determined whether any defects occur upon theectopic expression of such chimeric transcripts, these transcriptsmight lead to abnormal chromosomal recombination, which ispossibly prevented by ESET in normal tissues. Similarly, loss ofESET results in derepression of ERVs in ES cells, causing thesesequences to be expressed ectopically, resulting in defects in themaintenance of ES cells (Bilodeau et al., 2009; Yuan et al., 2009;Matsui et al., 2010; Karimi et al., 2011). These results indicate thatone of the major roles of ESET is the permanent suppression of ERVexpression in many cell types.

It was previously shown that methylation of CpG sequences inthe LTR region is required for repression of IAP expression (Walshet al., 1998). Interestingly, the CpG sequences of the IAP LTRregion located in the Mnd1 gene are highly methylated in the WTbrain but hypomethylated in the ESET cKO brain, suggesting thathypomethylation of CpG sequences is involved in the increasedexpression of IAP in the ESET cKO brain. Because ESET is knownto interact with DNA methyltransferases (Li et al., 2006), ESETmight regulate DNA methylation by recruiting DNAmethyltransferases.

IAP sequences can be copied and retrotransposed to otherregions (Heidmann and Heidmann, 1991), which might induceinsertional mutagenesis. However, in our preliminary analysis, aclear increase in the number of IAP copies in the genome of mutantmice was not detectable. Nevertheless, the retrotransposition couldoccur at a low frequency in the absence of ESET, as transcriptionof full-length IAP sequences increased robustly. Thisretrotransposition might cause carcinogenesis and other diseasesinduced by insertional mutagenesis.

Thus, in summary, ESET appears to be very important for themaintenance of neural identity by suppressing non-neural geneexpression and retrotransposition.

AcknowledgementsWe thank Shinichi Aizawa for Emx2-Cre mice; Itaru Imayoshi for in situhybridization probes and discussions; Bryan R. Cullen for anti-gag (IAP)antibody; Michael Wegner for Sox9 antibody; Eiji Yoshihara for technicalassistance; and Iris Manosalva for ovary cDNA.

FundingThis work was supported by Grants-in-aid from the Ministry of Education,Culture, Sports, Science and Technology of Japan. S.L.T. was supported by aMEXT scholarship of the Ministry of Education, Culture, Sports, Science andTechnology of Japan.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.082198/-/DC1

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