3806 RESEARCH ARTICLE
Development 139, 3806-3816 (2012) doi:10.1242/dev.082198
© 2012. Published by The Company of Biologists Ltd
Essential roles of the histone methyltransferase ESET in the
epigenetic control of neural progenitor cells during
development
Siok-Lay Tan1,2, Miyuki Nishi1,3, Toshiyuki Ohtsuka1, Toshiyuki Matsui1, Keiko Takemoto1,
Asuka Kamio-Miura4, Hiroyuki Aburatani4, Yoichi Shinkai1,3 and Ryoichiro Kageyama1,5,*
SUMMARY
In the developing brain, neural progenitor cells switch differentiation competency by changing gene expression profiles that are
governed partly by epigenetic control, such as histone modification, although the precise mechanism is unknown. Here we found
that ESET (Setdb1), a histone H3 Lys9 (H3K9) methyltransferase, is highly expressed at early stages of mouse brain development but
downregulated 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 nonneural gene expression was activated. Furthermore, early neurogenesis was severely impaired, whereas astrocyte formation was
enhanced. We conclude that there is an epigenetic role of ESET in the temporal and tissue-specific gene expression that results in
proper control of brain development.
INTRODUCTION
During cortical development, neural progenitor cells (NPCs) give
rise to various types of neurons – initially deep layer neurons and
then upper layer neurons – and finally differentiate into astrocytes
via 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). These
changes in competency are highly ordered and unidirectional
(Frantz and McConnell, 1996), suggesting that a ‘clock’ is
integrated into the differentiation system of NPCs to ensure that the
cell composition and final size of the brain are characteristically
similar among members of a given species. However, the precise
mechanism by which the differentiation competency of NPCs
switches at the proper time remains largely unknown.
It has been shown that NPCs switch differentiation competency
by changing gene expression profiles that are regulated by
transcription factors. For example, the zinc-finger transcription
factors Fezf2 and Ctip2 (Bcl11b – Mouse Genome Informatics) and
the basic helix-loop-helix (bHLH) transcription factors neurogenin
1 (Ngn1, Neurog1) and Ngn2 regulate the generation of deep layer
neurons (Arlotta et al., 2005; Molyneaux et al., 2005; Chen, B. et
al., 2005; Chen, J. G. et al., 2005; Chen et al., 2008), whereas the
homeodomain transcription factor Pax6 is involved in the
differentiation of upper layer neurons (Fode et al., 2000;
Schuurmans et al., 2004). By contrast, the orphan nuclear receptors
COUP-TFI and COUP-TFII (Nr2f1 and Nr2f2 – Mouse Genome
1
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan. 2Kyoto
University Graduate School of Medicine, Kyoto 606-8501, Japan. 3Kyoto University
Graduate School of Biostudies, Kyoto 606-8502, Japan. 4Research Center for
Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan.
5
Japan Science and Technology Agency, CREST, Shogoin-Kawahara, Sakyo-ku, Kyoto
606-8507, Japan.
*Author for correspondence ([email protected])
Accepted 30 July 2012
Informatics) induce astrocytic differentiation (Naka et al., 2008).
Recent reports suggest that chromatin remodeling and epigenetic
regulation also play important roles in controlling cell
differentiation (Seuntjens et al., 2009; Strobl-Mazulla et al., 2010).
For example, neuron-specific chromatin remodeling complexes
such as BAF53 regulate dendrite development (Lessard et al.,
2007; Yoo et al., 2009), while polycomb complexes such as
Polycomb repressive complex (PRC) 1 and 2, which regulate
histone modification, facilitate the switching from neurogenesis to
astrogenesis by repressing the proneural gene Ngn1 (Hirabayashi
et al., 2009). Histone modifications provide fast and reliable
regulation of gene expression, but their significance in neural
development has not been fully investigated.
The histone H3 lysine 9 (H3K9) methyltransferase ESET (also
known as Setdb1 or KMT1E) represses gene expression in
euchromatin by interacting with the co-repressor KAP1 (Trim28)
(Schultz et al., 2002). Loss of ESET accelerates differentiation of
embryonic stem (ES) cells toward the trophectoderm lineage by
derepressing genes encoding developmental regulators such as
Cdx2 and Hand1 (Bilodeau et al., 2009; Yuan et al., 2009; Yeap et
al., 2009). Interestingly, ESET, as well as KAP1, also plays a major
role in repressing the emergence of proviral particles in ES cells
(Matsui et al., 2010; Rowe et al., 2010; Karimi et al., 2011). There
are ~1000 copies of the endogenous retroviral-like sequences
known as the intracisternal A-particle (IAP) and their expression is
partly repressed by ESET in ES cells (Matsui et al., 2010; Karimi
et al., 2011). This regulatory mechanism is of particular importance
because upregulation of IAP expression has been associated with
retrotransposition and increased susceptibility to cancer (Heidmann
and Heidmann, 1991; Howard et al., 2008). Because ESET ablation
leads to embryonic lethality before implantation (Dodge et al.,
2004), the functions of ESET in the development of
postimplantation embryos, such as neural development, have not
been examined. To circumvent this problem, we investigated the
role of ESET in brain development using ESET conditional
knockout (cKO) mice.
DEVELOPMENT
KEY WORDS: ESET, Astrocyte, Epigenetic control, Histone methyltransferase, Neural progenitor
ESET in neural development
Mouse lines
ESET flox (f) (Matsui et al., 2010), Emx2-Cre (Kimura et al., 2005) and
Nes-Cre (Isaka et al., 1999) mice were described previously. Emx2Cre;ESET(f/+) or Nes-Cre;ESET(f/+) male mice were crossed with
ESET(f/f) female mice, and the day when a vaginal plug was observed was
scored as embryonic day (E) 0.5.
In situ hybridization, immunohistochemistry and antibodies
Embryos were dissected, fixed in 4% paraformaldehyde (PFA)/PBS
overnight and cryoprotected with 20% sucrose for at least 10 hours. On the
following day, embryos were embedded in OCT compound (Tissue-Tek)
and frozen at –80°C. Fixed samples were sectioned at 16 m with a
cryostat and dried for 2 hours. In situ hybridization was then performed as
previously described (Imayoshi et al., 2008). Primers used to produce in
situ hybridization probes are listed in supplementary material Table S1. For
immunohistochemistry, embryos at different ages were fixed in 4% PFA
for 2 hours, equilibrated and sectioned as described above. Sections were
blocked with 5% normal goat serum or donkey serum in 0.1% Triton X100/PBS and incubated with primary antibodies at 4°C overnight. The
following 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, Duke
University, 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, FriedrichAlexander 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 488conjugated mouse, rabbit, rat or goat and Alexa 594-conjugated mouse,
rabbit or rat (1:250, Molecular Probes). Immunofluorescence images were
obtained with a Zeiss Axiophot confocal microscope.
Acquisition and analysis of microarray data
The dorsal telencephalon of E14.5 wild-type (WT) and Emx2-Cre;ESET(f/f)
(ESET cKO) mouse embryos were subjected to RNA extraction and
expression profiling using Affymetrix GeneChip Mouse Genome Array
(n3) as previously described (Shimojo et al., 2008). Analysis of microarray
data was performed using GeneSpring GX version 11. Microarray data are
available at Gene Expression Omnibus under the following accession
numbers: GSM990998, GSM990999, GSM991000, GSM991001,
GSM991002 and GSM991003 (for telencephalon WT 1-3 and cKO 1-3,
respectively).
RNA preparation and quantitative (q) RT-PCR
RNA 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 precipitated
according to the manufacturer’s protocol. RNA samples were subjected to
reverse transcription as previously described (Shimojo et al., 2008). qRTPCR was performed with Thunderbird SYBR Green PCR Mix (TOYOBO)
using the primers listed in supplementary material Table S1.
RNA deep sequencing
Total RNA was isolated from E14.5 WT and ESET cKO embryonic
telencephalon (male), and an RNA integrity number (RIN) was confirmed to
be greater than 8.0 using a 2100 Bioanalyzer (Agilent Technologies). Total
RNA (1 g) was used for generation of poly(A)+ RNA Seq libraries. The
sequencing libraries were prepared using the Illumina TruSeq RNA Sample
Preparation Kit following the manufacturer’s protocol with the slight
modification that fragmentation time was reduced to 2 minutes. After
amplification for 15 cycles, PCR products were purified with Agencourt
AMPure XP magnetic beads (Beckman Coulter), and their size distribution
and concentration were evaluated using a DNA 1000 Series II Assay on the
2100 Bioanalyzer. cDNA at 4.2 pM/lane was applied to the flow cell and
paired-end 75 nt reads were sequenced on an Illumina GAIIx following the
manufacturer’s instructions (Wang et al., 2011). RNA sequencing results were
presented using Integrative Genomics Viewer (Broad Institute) version 2.0.
Bromodeoxyuridine (BrdU) labeling
BrdU (2.5 mg; 5 mg/ml) was given to each pregnant mouse by
intraperitoneal injection every 4 hours for a total of 12 hours at the
indicated embryonic stage. Brains were fixed with 4% PFA, and sections
were incubated in 2 M HCl for 30 minutes at 37°C, followed by
neutralization in 0.1 M sodium tetraborate buffer for 10 minutes. Sections
were incubated with mouse anti-BrdU antibody overnight at 4°C and then
with Alexa 488-conjugated goat anti-mouse IgG.
Cell death assay
TUNEL assays were performed with the ApopTag Fluorescein Direct In
Situ Apoptosis Detection Kit (Chemicon International) according to the
manufacturer’s protocol.
Neurosphere assay
Neurosphere assays were performed as described previously (Ohtsuka et
al., 2011). The numbers of primary spheres were counted at day 7.
Experiments were repeated three times and the mean and s.e.m. of sphere
numbers were calculated. For the neurosphere differentiation assay,
primary spheres were cultured for 1 day and plated onto PLL- and laminincoated Lab-Tek chamber slides (Nalge Nunc) in differentiation medium
(DMEM/F-12 supplemented with B-27 and 2% fetal bovine serum). Cells
were examined immunocytochemically 1, 3 and 5 days after plating.
In utero electroporation
In utero electroporation was performed with E17.5 ICR pregnant mice as
described previously (Ohtsuka et al., 2011). Neonates at postnatal day (P)
2 were harvested and analyzed, as described above.
Quantification and statistics
The total numbers of marker-positive cells were counted in more than five
sections from at least three different animals per group. The ventricular
zone, subventricular zone and cortical plate were defined according to Tbr2
staining. The percentage of cells from each compartment was calculated.
Statistical differences were examined with Student’s t-test and P<0.05 was
considered 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 sequencing
Bisulfite sequencing was conducted using the EpiTect Plus DNA Bisulfite
Kit (Qiagen). PCR products were cloned and individual inserts were
sequenced using BigDye v3.1 chemistry. Sequencing data were analyzed
using QUMA (RIKEN). Primer sequences are listed in supplementary
material Table S1.
RESULTS
ESET is expressed by NPCs in the developing brain
It has been shown that ESET is expressed in ES cells and the
postnatal brain (Ryu et al., 2006; Matsui et al., 2010; Jiang et al.,
2010), but expression in the developing brain has not been
examined extensively. We therefore performed in situ hybridization
and immunohistochemical analyses with mouse embryos at
different developmental stages. ESET was expressed in the
telencephalon, otic vesicles, forelimbs and hindlimbs at E9.5 (Fig.
1A). At this stage, ESET was highly expressed in the ventricular
zone where NPCs reside (Fig. 1B). ESET mRNA expression
occurred 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). All
radial glia/apical progenitors (Pax6+) and some basal progenitors
(Tbr2+) co-expressed ESET (Fig. 1D,Ea,b), but neurons (Tuj1+) did
not (Fig. 1Ec). ESET mRNA expression in the ventricular zone
DEVELOPMENT
MATERIALS AND METHODS
RESEARCH ARTICLE 3807
3808 RESEARCH ARTICLE
Development 139 (20)
further decreased at E15.5 (Fig. 1B) and was hardly detectable at
E17.5 (Fig. 1B). qRT-PCR also showed that ESET mRNA
expression in purified NPCs decreased as development proceeded
(Fig. 1C). Similarly, ESET protein expression also decreased as
development proceeded (supplementary material Fig. S1). From
this we infer that ESET is expressed by NPCs (both apical and
basal progenitors), but that the expression decreases over time and
is very low by E17.5, when the transition from neurogenesis to
astrogenesis in the dorsal telencephalon occurs.
ESET is required for proper regulation of neural
and non-neural gene expression
To determine whether ESET plays a role in cortical development,
we crossed ESET flox mice with Emx2-Cre driver mice to generate
forebrain-specific ESET cKO mice. By E11.5, ESET expression
was mostly abolished in the dorsal telencephalon, but was not
affected in the midbrain of the cKO mice, where the Emx2
promoter was inactive (supplementary material Fig. S2A). These
mutant mice were born but did not survive beyond P10. We also
generated ESET cKO mice using Nes-Cre mice, in which Cre is
active 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 examined
which genes were misregulated in the absence of ESET by
microarray expression profiling of the E14.5 dorsal telencephalon.
In the ESET cKO brain, 185 genes were upregulated more than 2fold relative to the WT brain (supplementary material Table S2),
whereas 112 genes were downregulated to less than half the level of
the WT (supplementary material Table S3). The misregulated genes
were categorized into three groups by GeneSpring software
clustering analysis (Fig. 2A). Cluster 1 included downregulated
genes, whereas clusters 2 and 3 included upregulated genes; many
of them, particularly the downregulated genes, might be indirectly
regulated by ESET, which functions as a transcriptional repressor.
Each cluster was subjected to Gene Ontology (GO) analysis with
a P<0.05 cut-off. Cluster 1 GO analysis revealed enrichment for
neurogenesis, neuronal differentiation and signal transmission (Pvalues of 0.014, 0.014 and 0.031, respectively; Fig. 2B). Genes in
these categories included Reln, Etv1, Nr4a2, Emx2, Satb2, Rorb,
Neurog1, Ntrk2 and other important genes required for the proper
regulation of neuronal differentiation, maturation, as well as the
maintenance of neuronal functions. For some of these genes, the
changes in expression were validated by qRT-PCR of E14.5 and
E16.5 cortical samples, and similar results were obtained at both
stages (supplementary material Fig. S2B,C).
Interestingly, the GO terms of cluster 1 overlapped substantially
with those of downregulated genes in ESET-null ES cells (Karimi
et al., 2011), suggesting that ESET could be commonly involved in
neuronal differentiation in these cells. Cluster 2 included genes
involved in ossification, such as Sox9 (P0.015; Fig. 2C). Sox9 is
known to regulate gliogenesis in the brain (Stolt et al., 2003), and
other glia-related genes such as Tspo and Ppp1r3c were also
included in cluster 2 (supplementary material Table S2). These
results suggest that glial differentiation was activated whereas
neuronal differentiation was impaired in the absence of ESET.
Cluster 3 contained strongly upregulated genes and revealed
enrichment for spermatogenesis (P0.044), M phase of the meiotic
cell cycle (P0.00041), and chromosome organization involved in
meiosis (P0.00039) (Fig. 2D). This cluster included IAP, Mnd1,
Tcfl5, Rec8, Rnf17, Sycp3 and Mael. These genes are strictly
expressed by germ cells in a narrow time frame when meiosis or
homologous recombination takes place. In the WT brain, many of
these 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 by
retrotransposition (Mietz et al., 1987; Qin et al., 2010), whereas
Mnd1, Rec8 and Sycp3 are the major players in facilitating proper
crossing over during homologous recombination in meiosis.
DEVELOPMENT
Fig. 1. Expression of ESET mRNA in the
mouse embryonic brain. (A,B,D,E) In
situ hybridization on whole-mounts (A)
and sections (B) with an ESET-specific
probe and immunohistochemistry for ESET
(D,Ea-c), Pax6 (D,Ea), Ki67 (B), Tuj1 (B,Ec)
and Tbr2 (Eb). DAPI staining was also
conducted (B,D,E). The right-hand column
in E shows a merge of each row. (C)ESET
mRNA levels in neural progenitor cells
(NPCs) sorted from the dorsal
telencephalon of pHes1-d2EGFP transgenic
mice at E11.5, E13.5, E15.5 and E17.5
were examined by microarray and qRTPCR. Mean ± s.e.m. was quantified at each
time point (n3). ESET is specifically
expressed by NPCs, and this expression is
gradually downregulated during
development. Scale bars: 1 mm in A;
100m in B; 20m in E.
ESET in neural development
RESEARCH ARTICLE 3809
Upregulated expression of cluster 2 and cluster 3 genes in the
cKO 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 observed
in Nes-Cre-driven ESET cKO mice (supplementary material Fig.
S3A). The GO terms of clusters 2 and 3 mostly differed from those
of the upregulated genes in ESET-null ES cells, which included
plasma membrane, vitamin binding and regulation of cell death
(Karimi et al., 2011). In the ESET cKO brain, some genes, such as
IAP and Mnd1, were ectopically expressed by neurons, whereas
others, such as Sox9, were expressed by NPCs and some migrating
cells (Fig. 3C, arrow), suggesting that the onset of ectopic
expression differs from gene to gene. Together, these results
indicated that ESET is required for the proper expression of
neuronal genes and for the suppression of non-neuronal genes (Fig.
2E).
Derepression of ERVs is responsible for the
upregulation of many genes in the ESET cKO brain
Genome database analysis indicated that many of the upregulated
genes are located near ERVs such as IAP sequences, suggesting
that activation of the long terminal repeat (LTR) of ERVs leads to
ectopic 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 and
sequence analysis showed that the major Mnd1 transcript started
from the intronic IAP in the absence of ESET (Fig. 3D,E;
supplementary material Fig. S3B,C). RNA deep sequencing
analyses revealed that, among the upregulated genes, six were
chimeric transcripts with IAPs (supplementary material Table S4)
and three (Mnd1, Slc20a2 and Sec14l4) were verified as fusions
with IAP by PCR and sequencing analyses (Fig. 3D,E;
supplementary material Fig. S3D,E). Among these six chimeric
transcripts with IAPs, three (Adk, Gramd1c and Slc20a2) were
shown to be astrocyte-enriched genes (Cahoy et al., 2008). RNA
deep sequencing additionally revealed the IAP-Capn11 chimeric
transcript, which was also verified by PCR and sequencing
analyses (supplementary material Fig. S3F). Other derepressed
ERVs, such as MMERVK, also formed five upregulated chimeric
transcripts (supplementary material Table S4). Therefore, among
the 185 upregulated genes, a total of 11 were expressed as chimeric
transcripts with ERVs in the ESET cKO brain (supplementary
material Table S4). In addition, 60 other upregulated genes were
found to have ERVs within 10 kb of the transcription initiation site
(supplementary material Table S4). Because deep sequencing
analyses detected enhanced transcription over 10 kb regions from
some activated ERVs in the absence of ESET (data not shown),
these results raised the possibility that as many as 71 of the
upregulated genes could be influenced by nearby ERVs,
representing ~38% of the upregulated genes in the ESET cKO
brain. This possibility remains to be analyzed, and the actual
number of genes activated by derepressed ERVs could be smaller.
ESET maintains global and localized H3K9me3
Because ESET is an H3K9 methyltransferase, we next examined
the H3K9me3 level in the ESET cKO brain. In the absence of
ESET (Fig. 4A), the global H3K9me3 mark in the chromatin was
slightly reduced at E14.5 and E18.5 (Fig. 4B; supplementary
DEVELOPMENT
Fig. 2. ESET deficiency leads to misregulation of
gene expression. (A)Microarray analysis of wild-type
(WT) and ESET conditional knockout (cKO) mouse
brains at E14.5 (n3). Clustering analysis identified
three groups (clusters 1, 2 and 3) of genes that
showed substantial changes in expression levels in the
absence of ESET. (B-D)Cluster 1 highlights
downregulated genes, whereas clusters 2 and 3
include upregulated genes. Gene Ontology (GO)
terms among genes that exhibited the highest
changes in expression are shown. (E)A working
hypothesis based on the microarray analysis.
3810 RESEARCH ARTICLE
Development 139 (20)
Fig. 3. Misregulated gene expression in the ESET cKO
brain. (A)qRT-PCR analysis of misregulated gene
expression in the WT and ESET cKO mouse brain at E14.5
(n6). Values were normalized to that of Gapdh, and a
ratio relative to the WT level ± s.e.m. was calculated.
***P<0.001, t-test. (B)Expression of intracisternal Aparticle (IAP), Sox9 and Tbr2 protein was examined in WT,
heterozygote (Het) and ESET cKO by western blot with
E14.5 whole-cell lysate from the dorsal telencephalon. In
each case, tubulin provides a loading control.
(C)Expression of IAP gag protein, Mnd1 mRNA and Sox9
mRNA was examined in coronal sections from both WT
and cKO brains. IAP and Mnd1 were expressed by neurons
of the ESET cKO brain, whereas Sox9 mRNA was
expressed by both NPCs and migrating neurons (arrow).
(D)RNA deep sequencing revealed emergence of IAPMnd1 chimeric transcripts in the ESET-ablated cortex. Red
bar indicates the position of IAP. (E)Conventional PCR was
performed with IAP- and Mnd1-specific primers using
E14.5 cortical cDNA. PCR products were detectable in the
ESET cKO samples but not in the WT. Scale bars: 100m.
in the ESET cKO brain. Bisulfite sequencing analysis revealed that
although global DNA demethylation was not observed in the global
IAP LTR sequences (94% in the cKO versus 92% in the WT; Fig.
4F), CpG sequences of the IAP in Mnd1 and other derepressed
genes were partially demethylated in the ESET cKO brain
compared with the WT (87.4% in the cKO versus 99% in the WT;
Fig. 4G; supplementary material Fig. S4D,E). Thus, in addition to
ESET loss, demethylation of CpG might be required for activation
of IAP, suggesting that this additional process might contribute to
delayed onset of IAP expression in neurons.
Ablation of ESET impairs neurogenesis
Because many neuronal and non-neuronal genes were
misregulated, we examined the effects of ESET loss on neural
development. At E11.5, the number of radial glia/apical
progenitors (Pax6+) was not significantly affected, whereas basal
progenitors (Tbr2+) were significantly reduced (supplementary
material Fig. S5A). At E12.5, Ctip2+ early-born layer V and VI
neurons and Tbr2+ basal progenitors were markedly reduced in
number in the ESET cKO cortex compared with the control (Fig.
5A,B). Production of Ctip2+ layer V and VI neurons, Tbr1+ layer
VI neurons and Tbr2+ basal progenitors was also reduced in the
ESET cKO cortex compared with the control at E14.5 and E16.5
(Fig. 5D-G; supplementary material Fig. S5B). By contrast, the
number of Brn2+ layer II-III neurons increased in the ESET cKO
cortical plate at E14.5 and E16.5 (supplementary material Fig.
S5C,D, arrowheads). At E18.5, when the neurogenesis phase
ceases, the populations of Ctip2+, Fezf2+, Tbr1+, FoxP2+ and
Nurr1+ deep layer neurons significantly decreased in the ESET
cKO (Fig. 5H,I), implying that ESET ablation impaired early
neurogenesis and severely affected the generation of deep layer
neurons. By contrast, the population of Cux1+ and Satb2+ upper
layer neurons increased in the E18.5 cKO (Fig. 5H,I), suggesting
DEVELOPMENT
material Fig. S4A), whereas the H3K9me2 mark was unaffected
(Fig. 4C; supplementary material Fig. S4A). Because the
microarray data suggested that there was no overwhelming global
upregulation of gene expression, it is likely that the role of ESET
in H3K9me3 regulation could be, to a certain extent, specific or
localized. Hence, we investigated whether ESET binds directly to,
and if so whether it modifies the H3K9me3 status of, the chromatin
of two representative misregulated genes: Sox9 from cluster 2 and
IAP 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, including
osteoblasts and glial cells (Stolt et al., 2003; Liu et al., 2007), and
it is normally expressed in the developing brain, but was
significantly upregulated in the absence of ESET (Fig. 3A-C). As
described above, IAP element expression was mostly negative in
the WT brain but was derepressed in the absence of ESET (Fig.
3A-C). ChIP assay of E14.5 cortical samples showed that ESET
bound to the promoters of Sox9, IAP and other genes (Fig. 4D;
supplementary material Fig. S4B). In addition, upon ESET
depletion, the H3K9me3 level at these loci was significantly
decreased (Fig. 4E; supplementary material Fig. S4C), whereas the
H3K9ac level was enhanced (supplementary material Fig. S4C).
These results indicate that ESET directly binds to these genes and
represses their expression via elevated H3K9me3 modification.
Although there are ~1000 copies of IAP sequences in the mouse
genome, only six genes were activated to form chimeric transcripts
with IAPs (supplementary material Table S4), suggesting that
derepression of IAPs is not global but localized. It was previously
shown that CpG sequences in the IAP LTR regions are highly
methylated, and that demethylation of CpG sequences by
inactivation of DNA methyltransferase 1 (Dnmt1) leads to robust
IAP expression (Walsh et al., 1998). Therefore, we examined
whether the DNA methylation status of the IAP LTR was affected
ESET in neural development
RESEARCH ARTICLE 3811
Fig. 4. ESET directly regulates H3K9 trimethylation at specific genes. (A)ESET protein is almost completely ablated in the cortex of E14.5 ESET
cKO mouse embryos (n3). (B,C)The global H3K9me3 level was slightly decreased in the absence of ESET (n3). However, the H3K9me2 level was
not significantly affected (n3). (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. (n4). (E)Native ChIP assay was performed with
H3K9me3 antibody on E14.5 WT and ESET cKO cortical samples. Binding activity (relative to the input) of H3K9me3 on Sox9 and IAP promoter
regions 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 IAP
LTR located in the Mnd1 gene, where IAP expression was derepressed.
layer II-III neurons (supplementary material Fig. S6Ca-d) and
Cux2+ layer II-IV neurons were not significantly affected, and Id2
expression was slightly enhanced in the ESET cKO (supplementary
material Fig. S6Ce-h). These results showed that production of
deep layer neurons was most severely impaired, whereas upper
layer neurons were only modestly affected in the absence of ESET.
ESET promotes neuronal survival and
proliferation
To further investigate the reason for the smaller cortex of ESET
cKO mice (supplementary material Fig. S6B) and the severe loss
of deep layer neurons in the ESET cKO cortex (supplementary
material 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 3
staining, significantly increased in number from E13.5 in the cKO
brain (Fig. 6A,B). Furthermore, these apoptotic cells were mostly
located in layers V-VI (Fig. 6B, graph), and many of them coexpressed the deep layer neuronal marker Ctip2 (Fig. 6B,
enlargements), suggesting that apoptosis preferentially occurred in
deep layer neurons.
Expression of the basal progenitor marker Tbr2, which is
required 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 with
this 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
DEVELOPMENT
that the population of upper layer neurons might expand at the
expense of deep layer neurons in the ESET cKO brain.
In order to determine whether late-born upper layer neurons
differentiate prematurely, we performed a BrdU birthdating assay
at E13.5 and E16.5 and analyzed BrdU co-labeling with various
layer-specific markers at E18.5. The percentage of Cux1+ upper
layer neurons born at E13.5 was significantly increased whereas
that of Tbr1+ and FoxP2+ deep layer neurons was significantly
decreased in the cKO (Fig. 5J), revealing that the production of
late-born upper layer neurons was enhanced whereas that of earlyborn deep layer neurons was prematurely decelerated (Fig. 5J).
Approximately 20-30% of Cux1+ and Brn2+ upper layer neurons
were still generated at E16.5 in the WT, whereas only 12-15% of
such neurons were born at the same stage in the cKO (Fig. 5K).
These results suggest that the production of upper layer neurons
was accelerated at E13.5 at the expense of deep layer neurons but
prematurely decelerated at E16.5.
At E18.5, GABAergic neurons, which are derived from the
medial ganglionic eminence, tangentially migrated to the dorsal
telencephalon. However, this migration was somewhat reduced in
the ESET cKO brain compared with the WT (supplementary
material Fig. S6A), a defect that could be due to a lack of proper
signals 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, by
which time neurogenesis and migration were completed, the
number of Ctip2+ layer V and VI neurons was severely reduced in
the ESET cKO cortex compared with the control, whereas Brn1+
3812 RESEARCH ARTICLE
Development 139 (20)
of the radial glial marker Pax6 was not noticeably changed
(supplementary material Fig. S5A) and the number of dividing
cells in the ventricular zone was not significantly affected in the
ESET cKO brain (Fig. 6C,D). Primary neurosphere culture assays
with NPCs extracted from E14.5 WT and ESET cKO dorsal
telencephalon showed that mutant NPCs were less likely to
develop into neurospheres of greater than 50 m diameter than WT
NPCs within 7 days of culture (Fig. 6E,F), indicating that the
proliferation potential of NPCs was compromised in the absence of
ESET.
Together, these results suggest that the decrease in basal
progenitors from early stages and the increase in apoptosis
contribute to the size reduction of the ESET cKO cortex, with
particular effect on the deep layer populations.
Astrocyte formation is accelerated in the absence
of ESET
Our BrdU birthdating and Tbr2 immunostaining analyses showed
that the neurogenic phase seemed to have ended prematurely in the
ESET cKO brain, and we therefore examined whether the timing
of astrocyte formation was affected. We performed immunostaining
of the astrocyte marker Gfap at E18.5 and various postnatal stages.
At E18.5, only a small number of Gfap+ cells appeared in the WT
medial telencephalon, but their number was significantly increased
in the ESET cKO cortex (Fig. 7A). The enhanced expression of
Gfap continued at later stages (Fig. 7A). Upregulation of Gfap also
occurred in the ESET cKO driven by Nes-Cre (supplementary
material Fig. S7A). Furthermore, expression of the astrocyte
markers Gfap, Aldh1l1, Aqp4 and Slc14a1 (Cahoy et al., 2008) was
DEVELOPMENT
Fig. 5. ESET deficiency impairs
neural development.
Immunohistochemistry (Ctip2, Tbr1,
Tbr2, Cux1, Satb2 and FoxP2) or in
situ hybridization (Fezf2 and Nurr1) on
sections of the telencephalon of WT
and ESET cKO mice. (A,C)At E12.5 (A)
and E18.5 (C), the numbers of Ctip2+
and Tbr2+ cells were decreased in the
ESET cKO brain compared with the
WT. (D,F)At E14.5 (D) and E16.5 (F),
the numbers of Ctip2+, Tbr1+ and
Tbr2+ cells were decreased in the ESET
cKO brain compared with the WT.
(H)At E18.5, the numbers of Cux1+
and Satb2+ upper layer neurons were
increased in the ESET cKO brain,
whereas Ctip2+, Fezf2+, Tbr1+, FoxP2+
and Nurr1+ deep layer neurons were
decreased in the ESET cKO brain
compared with the WT.
(B,E,G,I) Quantification of neuronal
number with s.e.m. at E12.5 (B), E14.5
(E), E16.5 (G) and E18.5 (I). n3 per
group. **P<0.01, ***P<0.001, t-test.
(J,K)Pulse-chase experiments with
BrdU administered at E13.5 (J) or
E16.5 (K) (n3 for both) followed by
analysis at E18.5. Quantification
shows the percentage with s.e.m. of
cells that were double labeled with
BrdU and layer-specific markers. At
E13.5, a higher proportion of Cux1+
neurons incorporated BrdU, whereas
lower 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:
100m in A,D,F; 1 mm in C; 50m
in H.
ESET in neural development
RESEARCH ARTICLE 3813
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 cKO
mouse brain compared with the WT from E13.5 onwards. (B)Apoptotic cells (cleaved Casp3+) were abundant specifically in the deep layer of the
cortical plate in the ESET cKO brain, whereas there were very few in the WT. Boxed regions are enlarged beneath. Distribution of apoptotic cells in
the ESET cKO brain was quantified (bottom). (C)Immunohistochemical analysis of PH3 and Tbr2. Some basal progenitors (Tbr2+) exhibited PH3 in
the WT, but this rarely occurred in the ESET cKO brain, suggesting that basal progenitors were proliferating in the WT whereas their proliferation
was reduced in the ESET cKO brain. (D)Quantification with s.e.m. of the number of PH3+ cells (n4). Proliferation of basal progenitors (Tbr2+) in the
subventricular zone was significantly decreased, but that of radial glia cells (Pax6+) in the ventricular zone was not affected in the ESET cKO brain
compared with the WT. CP, cortical plate; SVZ, subventricular zone; VZ, ventricular zone. ns, not significant; **P<0.01, t-test. (E)Primary
neurospheres generated from E14.5 WT and cKO brains. (F)The average numbers with s.e.m. of primary neurospheres greater or less than 50m in
diameter as originated from 5000 cells. The cKO produced fewer and smaller neurospheres than the WT. Scale bars: 50m in A,C; 100m in B,E.
To determine whether the Gfap gene is a direct target of ESET,
we performed ChIP assays on the Gfap promoter. ChIP assay with
E14.5 cortical samples showed that ESET directly binds to the
Gfap promoter (Fig. 7E,F). In addition, upon ESET depletion, the
H3K9me3 level at this promoter was significantly decreased (Fig.
7G), whereas the H3K9ac level was enhanced (Fig. 7H). These
results indicated that ESET directly binds to the Gfap promoter and
represses its expression via elevated H3K9me3 marks.
DISCUSSION
Epigenetic regulation is necessary for the establishment and
maintenance of heritable gene expression during development, but
very little is known about the significance of histone modifications
such as H3K9me3 in neural development. Here, we showed that
the H3K9 methyltransferase ESET controls the proper expression
of neural genes and suppresses the expression of non-neural genes,
thereby regulating brain development.
ESET is essential for temporal-specific gene
expression
ESET is highly expressed by NPCs at an early stage, but is
downregulated over time, and expression is very low during the
neuron-to-astrocyte fate switch. In the ESET cKO brain, generation
of early-born deep layer neurons was markedly impaired, whereas
generation of late-born upper layer neurons was not significantly
affected (Fig. 5; supplementary material Fig. S6C). By contrast, the
generation of astrocytes was significantly enhanced in the absence
DEVELOPMENT
increased at P7 in the ESET cKO cortex (supplementary material
Fig. S7B). These results indicated that astrocyte differentiation was
accelerated and enhanced in the absence of ESET.
It is possible that increased neuronal death induced astrogliosis
in the ESET cKO brain. To rule out this possibility, we performed
Gfap and TUNEL double staining in P4 brains. Although the total
number of TUNEL+ cells increased in the cKO, the majority of
Gfap+ cells were present in regions where there were no apoptotic
cells (Fig. 7B), suggesting that enhanced Gfap expression was not
due to cell death-induced gliosis. Furthermore, higher
magnification images revealed that these Gfap+ cells exhibited
typical astrocyte morphology (Fig. 7B, lower panels). In addition,
we performed an in vitro differentiation assay using NPCs prepared
from E14.5 WT and cKO dorsal telencephalon to investigate the
gliogenic potential of these progenitors. Indeed, NPCs from the
ESET cKO cortex more readily differentiated into Gfap+ cells than
the WT NPCs, although the frequency of cell death did not differ
between ESET cKO and WT NPCs (Fig. 7C; supplementary
material Fig. S7C). The percentage of Gfap+ colonies was also
significantly increased in the absence of ESET on the third day
upon differentiation (Fig. 7D). By contrast, overexpression of
ESET by in utero electroporation decreased astrocyte
differentiation (supplementary material Fig. S7D), although
overexpression of ESET alone was not able to block astrocyte
differentiation completely (supplementary material Fig. S7D).
These results suggest that ESET represses the premature onset of
astrocyte formation in the developing cortex.
3814 RESEARCH ARTICLE
Development 139 (20)
of ESET. Thus, in the ESET cKO brain, generation of astrocytes is
accelerated at the expense of neurons, most notably early-born
neurons. These results suggest that decreasing the expression of
ESET during development might be one of the internal clock
mechanisms that regulate the timing of cell fate switches of NPCs.
The mechanism of gradual downregulation of ESET during
development remains to be determined, but both extrinsic and
intrinsic factors could be involved. It was reported that some
extrinsic factors such as ciliary neurotrophic factors, leukemia
inhibitory factor and cardiotrophin 1 promote astrocyte
differentiation (Kahn et al., 1997; Nakashima et al., 1999; Miller
and Gauthier, 2007), and these factors could lead to downregulation
of ESET expression. It was previously reported that the bHLH
genes Hes1 and Ngn2 are expressed in an oscillatory manner in
NPCs (Shimojo et al., 2008), and it has been proposed that this
oscillation is one of the internal clock mechanisms (Kageyama et
al., 2007). Interestingly, there are multiple Hes1 binding sites in the
ESET promoter (data not shown), suggesting that Hes1 oscillations
could lead to gradual downregulation of ESET expression,
although further analyses are required to clarify any such
relationship.
ChIP assays showed that ESET directly binds the promoters of
the astrocyte-related genes Gfap and Sox9. Furthermore, in the
absence of ESET, the H3K9me3 mark on these astrocyte-related
gene loci was reduced, leading to upregulation of astrocyte gene
expression. These results suggest that ESET prevents premature
astrocyte formation by repressing Gfap and other astrocyte-related
gene expression via H3K9me3. However, overexpression of ESET
at E17.5 reduced astrocyte formation but failed to inhibit this
process completely. This suggests that additional regulation, such
as DNA methylation (Takizawa et al., 2001), might be required to
inhibit astrocyte formation.
The expression of neuronal genes was downregulated in the
ESET cKO brain, but the precise mechanism of this
downregulation remains to be determined, especially as ESET is
known to function as a transcriptional repressor. It was previously
reported that Sox9 represses the expression of neuronal genes (Stolt
et al., 2003; Scott et al., 2010). Furthermore, it was shown that the
neurogenic and gliogenic programs inhibit each other by
sequestering the CBP-Smad1 complex (Sun et al., 2001). Thus, one
of the likely mechanisms for downregulation of neuronal genes is
the upregulation of astrocytic genes.
DEVELOPMENT
Fig. 7. Inactivation of ESET enhances
astrocyte formation.
(A)Immunohistochemistry for Gfap and
DAPI staining on coronal sections of E18.5,
P2, P4 and P7 mouse telencephalon. Gfap
expression was upregulated in the ESET cKO
brain compared with the WT. (B)Enhanced
astrogenesis was observed in the P4 cKO
cortex compared with the WT. Higher
magnification images show that the majority
of Gfap+ cells in the cKO did not overlap
with TUNEL staining. (C)Neurosphere
differentiation assay. E14.5 NPCs were
cultured in differentiation conditions for 1, 3
and 5 days and then immunostained with
anti-Gfap antibody. Gfap expression was
increased in the cKO on day 3 and day 5.
(D)The proportion with s.e.m. of Gfap+
colonies on day 3. n7 for WT, n6 for cKO.
***P<0.001, t-test. (E-H)Cross-linked ChIP
assay was performed with IgG or ESET
antibody on E14.5 WT cortical samples and
direct binding of ESET on the Gfap promoter
regions was observed (F; n4). The amplified
regions are shown (E). Native ChIP assay was
performed with H3K9me3 and H3K9ac
antibodies on E18.5 WT and ESET cKO
cortical samples (G,H). The H3K9me3 levels
on the Gfap promoter regions were
decreased, whereas the H3K9ac levels were
increased in the cKO. Binding activity
(relative to the input) with s.e.m. on the
Gfap promoter regions was quantified. ns,
not significant; *P<0.05, **P<0.01,
***P<0.001, t-test. Scale bars: 100m in
A,B top; 50m in C; 10m in B bottom.
Epigenetic regulation in the nervous system
Recent studies revealed that epigenetic regulation is essential for
the maintenance of neuronal identity. Ablation of the H3K9
methyltransferase G9a (Ehmt2 – Mouse Genome Informatics),
which catalyzes dimethylation of H3K9 (Tachibana et al., 2001),
leads to derepression of non-neuronal genes and NPC-specific
genes in neurons and to abnormal behaviors in mice (Schaefer et
al., 2009; Maze et al., 2010). The precise role of ESET in neurons
is not known, but it was shown that overexpression of ESET in
neurons leads to the repression of some neuronal genes and to
abnormal behaviors in mice, and that ESET expression is highly
upregulated in patients with Huntington’s disease (Ryu et al., 2006;
Jiang et al., 2010). Thus, the proper level of ESET is important for
normal neuronal functions, although a loss-of-function analysis for
ESET in neurons remains to be performed.
ESET is essential for repression of non-neural
genes
In addition to the regulation of neural genes, we found that ESET is
important for repressing the endogenous retroelement IAP in somatic
tissues. Among the upregulated genes in the ESET cKO brain, many
were associated with derepressed IAPs and other ERVs. In particular,
genes near derepressed IAPs were strongly upregulated in the ESET
cKO brain. One such gene is Mnd1, which regulates meiotic
recombination between homologous chromosomes (Petukhova et al.,
2005). Normally, Mnd1 is not expressed in the developing nervous
system, but in the ESET cKO cortex chimeric transcripts consisting
of the IAP and Mnd1 sequences were robustly expressed. Although
it remains to be determined whether any defects occur upon the
ectopic expression of such chimeric transcripts, these transcripts
might lead to abnormal chromosomal recombination, which is
possibly prevented by ESET in normal tissues. Similarly, loss of
ESET results in derepression of ERVs in ES cells, causing these
sequences to be expressed ectopically, resulting in defects in the
maintenance of ES cells (Bilodeau et al., 2009; Yuan et al., 2009;
Matsui et al., 2010; Karimi et al., 2011). These results indicate that
one of the major roles of ESET is the permanent suppression of ERV
expression in many cell types.
It was previously shown that methylation of CpG sequences in
the LTR region is required for repression of IAP expression (Walsh
et al., 1998). Interestingly, the CpG sequences of the IAP LTR
region located in the Mnd1 gene are highly methylated in the WT
brain but hypomethylated in the ESET cKO brain, suggesting that
hypomethylation of CpG sequences is involved in the increased
expression of IAP in the ESET cKO brain. Because ESET is known
to interact with DNA methyltransferases (Li et al., 2006), ESET
might regulate DNA methylation by recruiting DNA
methyltransferases.
IAP sequences can be copied and retrotransposed to other
regions (Heidmann and Heidmann, 1991), which might induce
insertional mutagenesis. However, in our preliminary analysis, a
clear increase in the number of IAP copies in the genome of mutant
mice was not detectable. Nevertheless, the retrotransposition could
occur at a low frequency in the absence of ESET, as transcription
of full-length IAP sequences increased robustly. This
retrotransposition might cause carcinogenesis and other diseases
induced by insertional mutagenesis.
Thus, in summary, ESET appears to be very important for the
maintenance of neural identity by suppressing non-neural gene
expression and retrotransposition.
RESEARCH ARTICLE 3815
Acknowledgements
We thank Shinichi Aizawa for Emx2-Cre mice; Itaru Imayoshi for in situ
hybridization probes and discussions; Bryan R. Cullen for anti-gag (IAP)
antibody; Michael Wegner for Sox9 antibody; Eiji Yoshihara for technical
assistance; and Iris Manosalva for ovary cDNA.
Funding
This 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 a
MEXT scholarship of the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.082198/-/DC1
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