Post on 03-Feb-2022
Epigenetic regulation in induced orexin neuron
1
Epigenetic switching by the metabolism-sensing factors in the generation of
orexin neurons from mouse embryonic stem cells
Koji Hayakawa1, Mitsuko Hirosawa
1, Yasuyuki Tabei
1, Daisuke Arai
1, Satoshi Tanaka
1, Noboru
Murakami2, Shintaro Yagi
1, and Kunio Shiota
1*
1Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences/Veterinary Medical Sciences,
The University of Tokyo.
2Laboratory of Physiology, Department of Veterinary Physiology, Faculty of Agriculture, University of
Miyazaki.
Running title: Epigenetic regulation in induced orexin neuron
*To whom correspondence should be addressed: Kunio Shiota, Laboratory of Cellular Biochemistry,
Department of Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 1138657, Japan, Tel: +81-3-5841-5472; Fax: +81-3-5841-8189; Email:
ashiota@mail.ecc.u-tokyo.ac.jp
Keywords: DNA methylation, Histone acetylation, Embryonic stem cell, Histone acetylase, Neurogenesis,
O-glcnacylation, Ogt, Sirt1, Mgea5, Epigenetics
Background: Orexin plays a central role in the
integration of sleep/wake states and feeding behaviors.
Result: Orexin neurons were induced from pluripotent
stem cells by supplementation of ManNAc.
Conclusion: ManNAc induced switching of epigenetic
factors from Sirt1/Ogt to Mgea5 at Hcrt gene locus.
Significance: This study will be useful to investigate
molecular mechanism in the orexin system and
development of regenerative medicine.
SUMMARY
The orexin system plays a central role in
the integration of sleep/wake and feeding behaviors
in a broad spectrum of neural-metabolic physiology.
Orexin-A and orexin-B are produced by the
cleavage of prepro-orexin, which is encoded on the
Hcrt gene. To date, methods for generating other
peptide neurons could not induce orexin neurons
from pluripotent stem cells. Considering that the
metabolic status affects orexin expression, we
supplemented the culture medium with a nutrient
factor, ManNAc, and succeeded in generating
functional orexin neurons from mouse ES cells
(mESCs). Since DNA methylation inhibitors and
HDAC inhibitors could induce Hcrt expression in
mESCs, the epigenetic mechanism may be involved
in this orexin neurogenesis. DNA methylation
analysis showed the presence of a tissue-dependent
differentially methylated region (T-DMR) around
http://www.jbc.org/cgi/doi/10.1074/jbc.M113.455899The latest version is at JBC Papers in Press. Published on April 26, 2013 as Manuscript M113.455899
Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
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the transcription start site of the Hcrt gene. In the
orexin neurons induced by supplementation of
ManNAc, the T-DMR of the Hcrt gene was
hypomethylated in association with higher H3/H4
acetylation. Concomitantly, the histone
acetyltransferases p300, CBP, and Mgea5 (also
called O-GlcNAcase) were localized to the T-DMR
in the orexin neurons. In non-orexin-expressing
cells, H3/H4 hypoacetylation and hyper-O-GlcNAc
modification were observed at the T-DMRs
occupied by Ogt and Sirt1. Therefore, the results of
the present study suggest that the glucose
metabolite, ManNAc, induces switching from the
inactive state by Ogt-Sirt1 to the active state by
Mgea5, p300, and CBP at the Hcrt gene locus.
Orexin-expressing neurons (orexin
neurons) are localized in the lateral hypothalamus
(LH), and the orexin system is involved in a broad
spectrum of neurometabolic physiology where it
plays a central role in the integration of sleep/wake
states and feeding behaviors (1, 2). Disorganization
and deficiencies in the orexin system are believed to
cause sleep disorders, e.g., narcolepsy, and
metabolic diseases (3, 4). For the development of
drugs and regenerative strategies to address for brain
injuries, the generation of neural cells from
pluripotent stem cells, including embryonic stem
cells (ESCs), is an essential tool (5, 6). Induced
neural cells from pluripotent cells, e.g., GABAergic
(7), dopaminergic (8), and hypothalamic peptide
neurons, including oxytocin, thyrotropin-releasing
hormone (TRH) and neuropeptide Y (NPY) neurons
(9), allow not only for development of medical
applications but also for analysis of molecular
events of cellular function and differentiation. To
date, orexin neurons have not been established from
pluripotent cells and their developmental processes
are still unclear.
Glucose is metabolized through several
pathways: glycolysis, glycogen synthesis, pentose
phosphate pathway, and hexosamine biosynthesis
pathway (HBP). The HBP integrates the metabolism
of glucose, glutamine, acetyl-CoA, and
uridine-diphosphate into the synthesis of
UDP-N-acetyl-glucosamine (UDP-GlcNAc), which
is metabolized to sialic acid, N-acetylneuraminic
acid (Neu5Ac), through the intermediate
N-acetyl-D-mannosamine (ManNAc) (10, 11).
Glucosamine (GlcN) enters the HBP by passing
glutamine/fructose-6- phosphate amidotransferase,
which is the first and limiting enzyme (12-14).
These metabolites are integrated into numerous
cellular functions as regulators of gene expression.
Acetyl-CoA is a donor of protein
acetylation. NAD+ is a critical regulator of Sirtuins
(15). Sirt1, a member of the sirtuin family, functions
as histone deacetylase (HDAC) and is recognized as
a nutrient sensor because a fasting condition or
reduced calorie intake up-regulates its expression
and activity (16).
UDP-GlcNAc is a donor of
O-GlcNAcylation of cytoplasmic as well as nuclear
proteins, including transcription factors, epigenetic
factors such as polycomb group (PcG), and core
histones (17-20). O-GlcNAc transferase (Ogt)
catalyzes the addition of O-GlcNAc to Ser or Thr
residues of target proteins, and O-GlcNAcase (Oga)
removes O-GlcNAc (11, 21). Ogt is known to
interact with other nuclear proteins such as PcG
through TRP domain (21). The Oga gene is
annotated as meningioma expressed antigen 5
(Mgea5) and contains a putative histone
acetyltransferase (HAT) domain (22), suggesting its
involvement in the epigenetic system. Therefore, we
hypothesized that glucose metabolites may have an
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impact on orexin neurogenesis, which is mediated
by the epigenetic system.
The epigenetic system underlies not only
the in vivo development but also the in vitro
differentiation of pluripotent stem cells to
various-type cells (23-25). Epigenetic alterations
such as changes in the DNA methylation status and
histone modifications result in chromatin
remodeling of strictly regulated developmental
genes (26-29). Numerous tissue-dependent
differentially methylated regions (T-DMRs) have
been identified in the mammalian genome (23, 25,
30). Hypermethylated T-DMRs associate with silent
loci, while hypo-methylated T-DMRs associate with
active loci (30, 31). In combination with the DNA
methylation status of T-DMRs, histone
modifications create the multilayered epigenetic
control of long-term gene activity (27, 28, 32-34).
The epigenetic system regulates the metabolism as
shown by our previous finding, i.e., there are
numerous T-DMRs at loci of nuclear-encoded
mitochondrial proteins (31).
In the present study, by using a neural cell
culture protocol, we found that addition of ManNAc
promotes the expression of the Hcrt gene, and
demonstrated how the epigenetic regulation of the
expression of the Hcrt gene by Sirt1, Ogt, and
Mgea5. Thus, we successfully generated functional
orexin neurons from mouse ESCs (mESCs).
EXPERIMENTAL PROCEDURES
Mono-saccharides and inhibitors-
D-(+)-glucosamine hydrochloride (GlcN), EX-527,
and benzyl 2-acetamido-2-deoxy-α-D-galacto-
pyranoside (BADGP) were purchased from Sigma.
Thiamet-G was purchased from Tocris.
5-Aza-2’-deoxycytidine (5-Aza-dC), Zebularine and
Trichostatin A (TSA) were purchased from Wako.
GlcNAc, ManNAc, and Neu5Ac were purchased
from Tokyo Chemical Industry Co., Sanyo Fine Co.,
and Food & Bio Research Center Inc., respectively.
mESC culture- The mESC line J1, derived
from 129S4/SvJae mouse embryos, was cultured on
a gelatin-coated dish (Sigma-Aldrich) in D-MEM
(Wako) supplemented with 5% FBS, 15%
KnockOUT Serum Replacement (KSR; Invitrogen),
100 mM β-mercaptoethanol (Invitrogen), 2 mM
L-glutamine (Wako), 1 mM non-essential amino acid
(NEAA; Wako), and 1,500 U/mL LIF (ESGRO;
Millipore). Sirt1-/- mESCs and wild type mESCs
(R1 line) were kindly provided by Dr. Michael W.
McBurney (35), and cultured under the same
conditions.
Neural differentiation from mESCs-
Neural differentiation by using the SDIA and
SDIA+BMP4 methods was carried out as described
in previous reports (36). We cultured mESCs (1.7 ×
103 cells/cm
2) on PA6 feeder cells in Glasgow MEM
(Invitrogen) supplemented with 10% KSR, 0.1 mM
NEAA, and 0.1 mM β-mercaptoethanol. PA6 cells
were provided by the RIKEN BRC through the
National Bio-Resource Project of the MEXT, Japan.
The culture medium was changed on day 4 and
every 2 days thereafter. In case of the SDIA+BMP4
method, 5 nM BMP4 (Wako) was added to the
medium from day 4. The gfCDM/SFEBq
differentiation culture was performed as previous
reported but with minor modifications (9). mESCs
were dissociated to a single cell solution in 0.25%
trypsin-EDTA and quickly re-aggregated in growth
factor-free CDM (3,000 cells per 200 μL per well),
which contained Iscove’s modified Dulbecco’s
medium/Ham’s F-12 1:1 (Invitrogen), 1 ×
chemically defined lipid concentrate (Invitrogen),
450 μM monothioglycerol (Wako), and purified
BSA (Sigma) using 96-well low cell-adhesion plates
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(NUNC). After day 7 of culture, spheres were
dissociated by using 0.25% trypsin-EDTA, quickly
re-aggregated using low cell-adhesion 96-well
culture plates (5,000 cells per well), and cultured in
DMEM/F12 supplemented with 38.8 mM glucose
and 10% KSR. On day 10, half of the medium was
replaced with DFBN which contained DMEM/F12
supplemented with 38.8 mM glucose, N2 (Wako),
B27 (Invitrogen), and 10 ng/mL CNTF (Wako). On
day 13, spheres were dissociated by using 0.25%
trypsin-EDTA and plated onto poly
D-lysine/laminin-coated dishes (BD) at a density of
8.5 × 104 cells/cm
2 in DFNB supplemented 50
ng/mL BDNF (Wako) and 50 ng/mL NT3 (Wako)
until day 25.
Neurosphere culture- Pregnant C57BL/6N
mice were euthanized, and fetuses at embryonic day
14.5 were recovered in ice-cold PBS containing
0.6% glucose. For neurosphere culture, cells derived
from telencephalons were suspended in DMEM/F12
(1:1) supplemented with 5.5 mM HEPES, 2 mM
L-glutamine, B27, 20 ng/mL EGF (Sigma), 20
ng/mL bFGF (PeproTech), and 5 μg/mL heparin
(Sigma). Next, 3 × 104 cells were seeded onto a low
cell binding dish (NUNC) and cultured for 10 days,
replacing half of the medium with fresh medium at
every 3 day. To induce differentiation, cells were
dispersed, suspended in the absence of growth
factors, and seeded onto poly-L-lysine- and
laminin-coated dishes (BD).
Tissue collection- Adult mice (C57BL/6N)
were purchased from Charles River Japan and
maintained on a 12-h light/12-h dark schedule with
free access to food and water. The hypothalamus
was recovered by separation from the whole brain of
13-week-old male mice using fine forceps. The
collected tissues were stored at -80°C until use for
RNA and DNA extraction. All experiments using
mice were carried out according to the institutional
guidelines for the care and use of laboratory animals
(Graduate School of Agriculture and Life Sciences,
The University of Tokyo).
RT-PCR and - quantitative PCR- Total
RNA was isolated from cells and tissues with the
RNeasy Plus Mini Kit (Qiagen) according to the
manufacturer’s instructions. First-strand cDNA was
synthesized from 3 μg of total RNA by using
oligo(dT)20 primers and the SuperScript III
First-Strand Synthesis System (Invitrogen). RT-PCR
was conducted with the LA Taq DNA polymerase
(Takara) using 10 ng of cDNA per reaction. PCR
reactions were performed under the following
conditions: denaturation at 95°C for 3 min and the
appropriate number of cycles, each cycle consisted
of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 15
sec. PCR products were subjected to agarose gel
electrophoresis and stained using GelRed (Biotium).
The primer sequences used are listed in
supplemental Table1. Each qPCR was performed
with 10 ng of cDNA and Thunderbird Syber qPCR
Mix (Toyobo) using the ABI7500 thermal cycler
(Applied Biosystems). PCR was performed with the
following thermocycling conditions: denaturation at
95°C for 1 min and 40 cycles, each cycle consisted
of incubation at 95°C for 10 sec and 60°C for 1 min.
Data were normalized to the expression of Actb.
Immunofluorescence assay- Cells
cultured in 4-well dishes were fixed with 4%
paraformaldehyde (Wako) and permeabilized with
0.2% Triton X-100 (Wako) followed by blocking
with 5% BSA/0.1% Tween20/PBS (Sigma) for 1 h
at room temperature (RT) and incubating with the
primary antibody overnight at 4°C. The secondary
antibody was added and the incubation was
continued for 1 h at RT. Nuclei was stained with
DAPI (1 μg/mL; Dojindo). The primary antibodies
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used are listed in supplemental Table 2. The
following secondary antibodies were used: donkey
anti-goat Alex-Flour 488, rabbit anti-mouse Alexa
Flour 594, and chicken anti-rabbit Alexa-Flour 594
(1:1000; Invitrogen). Fluorescence images were
acquired with a microscope (BZ-8000; KEYENCE).
Immuno- and DAPI-stained cells were counted in at
least 30 randomly chosen areas by using the Image J
software (http://rsb.info.nih.gov/ij/). Percentages
indicate the mean of a ratio of an Orexin-A or
-B-positive area to a DAPI-positive area in three
independent cultures.
Orexin-A releasing assay by using
ELISA- mESCs were cultured under the
SDIA+BMP4 condition for 10 days in 4-well dishes
and were subjected to the following analyses. For
the KCl assay, cells were incubated in 500 μL of
aCSF medium (124 mM NaCl, 3 mM KCl, 26 mM
NaHCO3, 2 mM CaCl2, 1 mM MgSO4, 1.25 mM
KH2PO4, and 10 mM D-glucose, pH 7.4) for 10 min
at 37°C, followed by stimulation with aCSF plus
100 mM KCl medium for an additional 10 min. For
measurement of neural peptide sensitivity, neural
differentiated mESCs were incubated in 500 μL of
medium of the SDIA condition supplemented with
leptin (Wako), ghrelin (Wako), or TRH (Wako) at
the appropriate concentrations at 37°C. After 3 h of
incubation, the supernatants were collected and the
orexin-A concentration was measured using the
Orexin-A Fluorescent EIA Kit (Phoenix
Pharmaceuticals) according to the manufacturer’s
instructions.
DNA methylation analysis using the
bisulfite method- Genomic DNA was extracted from
cells and tissues as described previously (30).
Bisulfite conversion was performed using the EZ
DNA Methylation-Gold Kit (Zymo Research). The
EZ DNA Direct-Methylation Kit (Zymo Research)
was used to analysis single colony under the
SDIA+BMP4 condition with 1 mM ManNAc. The
orexin-A-postive and -negative colonies were picked
up using fine pipet after immunostaining by using an
anti-Orexin-A antibody. For each bisulfite PCR,
BIOTAQ HS DNA polymerase (Bioline) was used
to catalyze the amplification. PCR was performed
with the following thermocycling conditions:
denaturation at 95°C for 10 min and 40 cycles, each
cycle consisting of incubation at 95°C for 30 sec,
60°C for 30 sec, and 72°C for 30 sec, followed by a
final extension for 5 min at 72°C. For sequencing,
the PCR fragments were cloned into the pGEM-T
Easy vector (Promega). The vectors were sequenced
by BigDye sequencing (Applied Biosystems).
Chromatin immunoprecipitation assay-
The ChIP assay was performed with 1 × 106 cells
per assay using the ChIP-IT Express Enzymatic Kit
(Active Motif) according to the manufacturer’s
instructions. Briefly, fixed cells were lysed and
mixed with an enzymatic shearing cocktail for 10
min. Antibodies, which were used for IP, are listed
in supplemental Table 2. After IP, DNA was
recovered by using an elution buffer (10% SDS, 300
mM NaCl, 10 mM Tris-HCl, and 5 mM EDTA, pH
8.0) at 65°C for 6 h and then collected using the
Chromation IP DNA Purification Kit (Active motif).
PCR reactions with LA Taq DNA polymerase were
performed under the following conditions:
denaturation at 95°C for 3 min and 32 cycles, each
cycle consisting of 95°C for 30 sec, 60°C for 30 sec,
and 72°C for 15 sec. PCR products were subjected
to agarose gel electrophoresis and stained using
GelRed.
Western blotting- Nuclear and cytoplasmic
fractions of each sample were collected using the
Nuclear Extract Kit (Active Motif) according to the
manufacturer’s protocols. The proteins were
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fractionated by 5–20% SDS-PAGE (XV PANTERA
Gel; DRC), blotted onto nitrocellulose membranes
(Millipore), and incubated at 4°C overnight with the
Primary antibody diluted in 5% BSA/0.1%
Tween20/TBS (Supplemental table 2). Protein bands
were detected using secondary antibody conjugated
with horseradish peroxidase (Jackson
ImmunoResearch) and SuperSignal West Pico or
Femto (Thermo).
Construction of overexpression vectors
and transfection- The sequences of all primers used
for plasmid construction are listed in Supplemental
Table 1. DNA of 3×Flag-fused mouse Ogt and
Mgea5 were generated by PCR amplification from
cDNA of mESCs using PrimeSTAR HS DNA
Polymerase (Takara) and cloned into the
pENTR/D-TOPO vector (Invitrogen). Point
mutations of Mgea5 at position 175 (D->A) or 891
(Y->F) of the amino acid sequence were generated
by using the PrimeSTAR mutagenesis Basal Kit
(Takara) and the pENTR/D-TOPO vector-cloned
3×Flag-Mgea5 as template. The resulting constructs
were confirmed by BigDye sequencing.
3×Flag-fused genes were subcloned into a
pCAG-DEST vector, which was generated by using
a combination of the Gateway Vector Conversion
System (Invitrogen) and pCAGEN (Addgene) and
Gateway LR Clonase (Invitrogen). For transient
overexpression using these vectors, mESCs were
cultured in 10-cm dishes under the SDIA+BMP4
condition with 1 mM ManNAc. At day 7 of culture,
the cells were then transfected with 24 μg of plasmid
and 30 μL of Lipofectamine 2000 (Invitrogen) per
dish. Twenty-four hours after transfection, medium
was changed and transfected cells were collected at
day 10 for the subsequent experiments.
The experiments described in the present
study were repeated, at least, three times with
similar results in each case. The results shown are
representative for all repeated experiments.
RESULTS
ManNAc treatment promotes generation
of orexin neurons from mouse ES cells- The gene
expression of hypothalamic peptides and
transcription factors was induced in mESCs by
employing two known in vitro differentiation
methods, i.e., SDIA+BMP4 and gfCDM/SFEBq (9,
36) (Fig. 1A). The Hcrt gene, however, could not be
induced by using either of these methods. We then
investigated the effect of supplementation with
GlcN, GlcNAc, ManNAc, and Neu5Ac at a
concentration of 1 mM, and found that only
ManNAc could induce Hcrt expression in cells
produced by either in vitro differentiation methods
(Fig. 1B and 1D). On the other hands, other
hypothalamic peptide genes, Npy and Gnrh1 were
not affected by ManNAc supplementation (Fig. 1B).
By using an immunofluorescence assay, we detected
Orexin-A- and Orexin-B-positive cells (6.2±1.3%
and 7.1±1.9%, respectively, of total cells) in the
colonies of Tubb3- and Ncam-positive neural cells
differentiated in the presence of ManNAc (Fig. 1C).
In addition, Dynorphin-A, which is known to be
expressed with orexin neuron (37), was co-localized
with orexin-A signals (Fig. 1C). Furthermore, we
examine the expression profiles of eight orexin
neural markers by RT-PCR on the basis of recent
reports (38). All of the marker genes were detected
in ManNAc-treated cells (Fig. 1E).
At the early differentiation stage (days 0–4
and 0–7), ManNAc supplementation was less
effective on the induction of orexin neurons than at
the late stage (days 4–10 and 7–10) (Fig. 1F).
Considering that neural progenitor cells (NPCs) first
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appear around day 4 under the culture conditions
employed (36), we conclude that ManNAc acts at
the later stage of neural differentiation, i.e., after the
neuronal fate commitment of progenitor cells.
Indeed, ManNAc supplementation could induce
Hcrt expression in neurospheres (Nsph) derived
from fetal mouse telencephalons (embryonic day
14.5) (Fig. 1G).
Orexin neurons respond physiologically to
leptin, and ghrelin (39, 40). Secretion of Orexin-A in
the presence of high levels of KCl suggested the
involvement of leaky K+ channels in glucose
sensing of induced orexin neurons (41) (Fig. 1H).
Physiological stimulants such as ghrelin and TRH
dose-dependently stimulated Orexin-A secretion
(Fig. 1I) in ManNAc-induced cells, which was
inhibited by leptin. These data provide futher
support for conclusion that supplementation of an
intermediate metabolite of glucose, ManNAc,
enables the generation of functional orexin neurons
from mESCs.
DNA methylation status of T-DMRs at the
Hcrt gene locus- To explore epigenetic mechanisms
that underlie the differentiation of orexin neurons,
we treated mESCs with 5 μM 5-Aza-dC or 100 μM
Zebularine, inhibitors of DNA methyltransferase,
and/or 200 nM TSA, an inhibitor of histone
deacetylase. Both inhibitors and their combination
induced Hcrt expression (Fig. 2A). These data
suggested that DNA methylation and histone
deacetylation are involved in Hcrt gene silencing.
Orexin-A and -B are produced by cleavage of a
polypeptide, prepro-orexin, encoded by the Hcrt
gene, whose promoter has 2 putative orexin
regulatory elements, ORE1 and ORE2 conserved
among animal species (42). We, therefore, examined
the DNA methylation status around the transcription
start site (TSS) containing ORE1.
Bisulfite sequencing identified a T-DMR
upstream (T-DMR-U, -778~-10 bp) and downstream
(T-DMR-D, +5~+665 bp) of the TSS (Fig. 2B).
T-DMR-U, which includes ORE1, was heavily
methylated in mESCs (98%), while the methylation
status was 84~89% in mESC-derived neural cells
produced by employing the SDIA+BMP4 culture
method. In contrast, ManNAc treatment caused a
decrease in the CpG methylation status to 78% (Fig.
2B). Furthermore, the colony of Orexin-A-positive
cells showed hypomethylation at T-DMR-U (40%)
compared to the negative colony (81%). ORE1
contained binding sites for Nr6a1 and Ebf2 (so
called O/E3), which are important for Hcrt
expression (43, 44). Because these binding sites
show no CpG sequences, methylation of T-DMR-U
would not directly inhibit the binding of these
factors. However, the accessibility of these factors
could be restricted by condensed chromatin, which
is induced by DNA methylation of T-DMR-U.
T-DMR-D, which is located in the 1st
intron of the Hcrt gene, was 46% methylated in
ManNAc-treated cells and 71% in mESCs (Fig. 2B).
Similarly, cells differentiated for 4 days in vitro
showed hypomethylation at T-DMR-D (Fig. 2B). In
contrast, Neu5Ac treatment promoted methylation at
T-DMR-D in mESC-derived neurons. These data
indicated that there are T-DMRs that are affected by
supplementation with intermediates of HBP.
ManNAc caused hyperacetylation of H3
and H4 at T-DMRs of Hcrt- We examined the
epigenetic status at these T-DMRs by ChIP analysis
for molecules related to histone acetylation. ChIP
analysis of region 1 in T-DMR-U and region 2 in
T-DMR-D revealed that acetylation of H3K9,
H3K14, H3K27, H3K56, H4K8, and H4K16 as well
as trimethylation of H3K4 were markedly elevated
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by ManNAc treatment (Fig. 2C). In accordance with
the acetylation status, histone acetyltransferases
(HAT) p300 and CBP, which are responsible for
pan-H3Ac and -H4Ac, are increased at the T-DMRs.
Furthermore, Mgea5, which has putative histone
acetyltransferase activity responsible for H3K14Ac
and H4K8Ac, accumulated at the T-DMR (Fig. 2C).
In repressive factors, Sin3A, and Ezh2
were decreased at the regions in ManNAc-treated
cells by ChIP analysis (Fig. 2C), corresponding with
the decrease in repressive histone marks, H3K9me3
and H3K27me3. An increase in HDACs in
Neu5Ac-treated cells suggested that Neu5Ac might
induce hyper-repression of Hcrt in combination with
hypermethylation of DNA.
Western blot analysis of nuclear and
cytoplasmic proteins of treated cells indicated that
ManNAc induced subcellular de-localization of
Sirt1 from the nucleus to the cytoplasm (Fig. 2D). In
contrast, p300, CBP and Mgea5 were increased in
nucleus of ManNAc-treated cells. Concomitantly,
levels of histone acetylation were also increased (Fig.
2E). Immunofluorescence assay, focusing on orexin
neuron, confirmed that Mgea5 was mainly located in
nuclear, while Sirt1 was in cytoplasm (Fig. 2F).
These data suggested that with regard to
the epigenetic control of the Hcrt gene, p300, CBP,
and Mgea5 contributed in the active state, while
Sirt1, Sin3A, and Ezh2 contributed in the inactive
state.
Generation of orexin neurons by using a
Sirt1 inhibitor- The involvement of Sirt1 in orexin
neural differentiation was investigated using
Sirt1-knockout (Sirt1-/-) mESCs. RT-PCR revealed
Hcrt expression in differentiated Sirt1-/- mESCs
when using the SDIA+BMP4 method, even in the
absence of ManNAc (Fig. 3A). Orexin-A- and
orexin-B-positive cells were observed among
differentiated Sirt1-/- mESCs (Fig. 3B). Acetylation
levels of H3K9, K14, K27, K56, H4K8, and H4K16
at regions 1 and 2 were higher in differentiated
Sirt1-/- cells compared to wild type cells (Fig. 3C).
Furthermore, treatment with EX-527, a Sirt1
inhibitor, resulted in Hcrt expression in
differentiated wild type cells (Fig. 3A) and an
increase in histone acetylation of the same residues
of neural differentiated Sirt1-/- cells (Fig. 3C).
Therefore, inhibition of Sirt1 at the Hcrt locus,
which was induced by ManNAc supplementation, is
a key event in the differentiation of orexin neurons.
Another mechanism of the effect of
ManNAc on differentiation of orexin neurons was
suggested by the observation that ManNAc
treatment caused a further increase in Hcrt
expression in cells derived from Sirt1-/- mESCs as
well as in EX-527-treated neural cells derived from
mESCs (Fig. 3A). The increased expression of Hcrt
by ManNAc treatment was associated with an
elevated acetylation at H3K9, K14, K27, K56,
H4K8, and K16 and ManNAc-induced accumulation
of Mgea5, p300, and CBP at T-DMRs of Hcrt (Fig.
2C). Therefore, both steps, i.e., deletion of Sirt1 and
accumulation of Mgea5, p300, and CBP could be
responsible for Hcrt gene activation during orexin
neurogenesis.
Loss of O-GlcNAcylation at T-DMRs
promotes Hcrt expression- Mgea5 has dual
enzymatic activity, i.e., HAT and Oga activities (22).
Thus, in addition to histone acetylation,
O-GlcNAcylation might also be involved in the
regulation of the Hcrt gene. ChIP analysis using an
RL2 antibody, which recognizes O-GlcNAc
modifications, revealed that there are O-GlcNAc
signals at regions 1 and 2 in non-, GlcNAc-, and
Neu5Ac-treated cells in contrast to ManNAc-treated
cells (Fig. 4A). ManNAc treatment caused an
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increase in Mgea5 and decrease in Ogt (Fig. 2C and
4A), which is in contrast to the results of Neu5Ac
treatment. This reciprocal change of Mgea5 and Ogt
is well matched with low and high O-GlcNAc levels
at regions 1 and 2 in ManNAc-treated and
Neu5NAc-treated cells, respectively.
Treatment with Thiamet-G, an Oga
inhibitor, diminished and treatment with BADGP, an
Ogt inhibitor, augmented the expression of
ManNAc-induced Hcrt expression (Fig. 4B).
Therefore, O-GlcNAcylation plays a suppressive
role in Hcrt gene activation. Overexpression of Ogt
inhibited Hcrt gene expression, and that of Mgea5
increased Hcrt gene expression in cultures with
ManNAc supplementation (Fig. 4C). In these
experiments, increased or decreased O-GlcNAc
modification levels were observed at T-DMRs in
cells overexpressing Ogt and Mgea5, respectively
(Fig. 4D), confirming the involvement of O-GlcNAc
modification in Hcrt gene activation by Ogt and
Mgea5.
O-GlcNAc modification and Ogt are
co-localized with Sirt1, Sin3A, and Ezh2 in the
inactive state of the Hcrt gene- O-GlcNAc
modification increased at T-DMRs of the Hcrt gene
where Sirt1, Sin3A, and Ezh2 accumulated (Fig. 2C
and 4A). Re-ChIP analysis using RL2 as the 1st
antibody showed co-localization of Sirt1, Sin3A,
and Ezh2 with O-GlcNAc modification at regions 1
and 2 in Hcrt non-expressing cells (Fig. 4E). This is
in contrast to the results of ManNAc-treated cells, in
which the signal for these repressive molecules was
substantially reduced (Fig. 4E). Again, in the
Re-ChIP analysis with an anti-Ogt antibody, the RL2
signal as well as those of Sirt1, Sin3A, and Ezh2
was strongly detected in the Hcrt inactive state,
especially in Neu5Ac-treated cells.
Mgea5/Oga activity links to histone H3
and H4 acetylation- Mgea5 accumulated by
ManNAc treatment at T-DMRs, and Thiamet-G
treatment not only decreased the acetylation levels
of H3K14 and H4K8 and other histone acetylation
levels but also increased the levels of Ogt and
O-GlcNAc (Fig. 5A). These findings suggested that
reciprocal modifications between O-GlcNAc
modification and histone acetylation could be
important for the regulation of the Hcrt gene. Under
these circumstances, the absence of increase in Sirt1
suggested that this reciprocal change occurs after
clearance of Sirt1 at regions 1 and 2.
We hypothesized that Mgae5 could play a
role in both Oga and HAT processes. Therefore, we
prepared constructs for 3×Flag-fused Mgae5; wild
type (WT), a mutation at the Oga domain (D175A),
and a mutation at HAT domain (Y891), respectively
(22, 45) (Fig. 5B), and introduced them in neural
differentiated mESCs. In western blot analyses of
whole nuclear extracts, WT-overexpressing neural
differentiated mESCs showed an increase in
H3K14Ac and H4K8Ac, indicating HAT activity of
Mgea5 (Fig. 5C). More importantly, acetylated
histone levels were diminished in both mutants
(D175A and Y891F) compared to the WT (Fig. 5C),
suggesting that O-GlcNase activity is important for
HAT activity of Mgea5. As indicated by the above
findings, at regions 1 and 2 of the T-DMRs,
H3K14Ac and H4K8Ac were increased in the WT
and decreased in both mutants (D175A and Y891F)
(Fig. 5D).
In the D175A mutant, O-GlcNAc
modification was increased, while the WT and
Y891F mutant showed lower O-GlcNAc
modification levels. The expression of the Hcrt gene
was increased in WT-Mgea5 over-expressing cells,
and both mutants did not show any activity (Fig. 5E).
Based on these results, we conclude that a dual
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function of Mgae5, consisting of Oga and HAT,
plays an important role in the activation of the Hcrt
gene.
DISCUSSION
This is the first report on the generation of
orexin neurons from ESCs. The epigenetic status at
T-DMRs of the Hcrt locus exhibited a unique feature,
i.e., involvement of DNA methylation, histone
acetylation, and O-GlcNAcylation. We found that
treatment with ManNAc induced hypo-CpG
methylation and hyperacetylation of H3 and H4 at
Hcrt T-DMRs by regulating the localization of the
metabolic sensing molecules Sirt1, Ogt, and Mgea5.
Thus, an epigenetic switch on histones from a
hypoacetylated state with unidentified
O-GlcNAcylated nuclear proteins to a
hyperacetylated state at T-DMRs is the key events in
the generation of orexin neurons. Furthermore, the
hyperacetylation of the T-DMRs was associated with
trimethylation of H3K4. The mESC-derived orexin
neurons are equipped with the physiological
response to neurotransmitter peptides such as TRH,
ghrelin, and leptin, as demonstrated in previous
findings by using brain tissue slices of experimental
animals (39, 40). Thus, our findings provide novel
and important information for research into the
orexin system.
Among the glucose metabolites
involved in the HBP that we examined, it is
interesting that only ManNAc, but not GlcN,
GlcNAc, and Neu5Ac, was effective to generate
orexin neurons. In a PC12 cells, both Neu5Ac
and ManNAc stimulate the differentiation of
neurons, and overexpression of UDP-GlcNAc
2-epimerase/ManNAc kinase (GNE), which
immediately converts ManNAc into Neu5Ac,
causes a similar effect on PC12 cells, prompting
us to consider ManNAc as a precursor for sialic
acids (46). In the present study, however, only
ManNAc but not Neu5Ac could induced
orexine neurons, suggesting that ManNAc
exerts its effect other than Neu5Ac production.
Indeed, ManNAc causes dislocation of
Ogt/Sirt1/Sin3A/Ezh2 from T-DMRs of the
Hcrt gene locus and recruitment of p300, CBP,
and Mgea5 in the present study. These data
suggest a unique role for ManNAc as an
element of a signaling pathway that contributes
to modifications of the epigenetic status at the
Hcrt gene locus to induce orexin neurons.
UDP-GlcNAc is a precursor for
ManNAc and is synthesized via the HBP from
approximately 3% of the total glucose (47, 48).
It is reported that GlcN enters the HBP and
subsequently causes an increase in
UDP-GlcNAc (12, 13), and then UDP-GlcNAc
regulates Ogt activity by increasing the
availability of the substrate (49, 50).
ManNAc is incorporated into mammalian cells
easily rather than Neu5AC in culture condition
(51). In the cells, at least, two molecules
recognize ManNAc; GNE and GlcNAc
2-epimerase. GlcNAc 2-epimerase catalyzes the
inter-conversion of GlcNAc and ManNAc
(52-54). By analogy to the effect of GlcN (12,
13), ManNAc might also affect the level of
UDP-GlcNAc in the cells by changing the
activities of GNE, GlcNAc 2-epimerase or
unidefined molecules.
Between the inactive and active status of
the Hcrt gene, a dynamic change was observed in
the histone acetylation level at T-DMRs. In the
active state of Hcrt gene expression, histone H3 and
H4 were hyperacetylated. Mgea5, p300, and CBP
were localized at Hcrt T-DMRs, which were
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occupied by Sirt1 and Ogt in the inactive state.
Considering the dynamic changes that occur in
conversion from a hypoacetylated to a
hyperacetylated status, these are the molecules
believed to be responsible for histone acetylation at
T-DMRs. Importantly, Mgea5 has both Oga and
HAT activities (22, 45). The zinc finger-like motif of
Mgea5 is responsible for the recognition of the
acetyl-substrate (55). Mgea5 showed HAT activity
and the capability to modify all core histones in vitro,
which include H3K14 and H4K8. In the present
study, overexpression of Mgea5 induced the
hyperacetylation of H3K14 and H4K8 while the
mutant Mgea5 (Y891F) did not show this activity,
indicating that Mgea5 works as HAT intracellularly.
Treatment with an Oga-inhibitor (Thiamet-G) and
overexpression of both Mgea5 mutants (D175A and
Y891F) did not induce histone acetylation and Hcrt
gene expression. Thus, Mgea5 must exert its dual
function of Oga and HAT in the establishment of an
active epigenetic state on the Hcrt gene locus. We
conclude that Mgea5 is the primary factor for the
epigenetic change at the T-DMR to express the Hcrt
gene and induce the differentiation of orexin
neurons.
The epigenetic status of each T-DMR is
regulated by the interplay between DNA
methyltransferases, histone modification enzymes,
histone subtypes, non-histone nuclear proteins,
non-coding RNAs, and other factors (28). Many
non-histone proteins such as Sin3A and Ezh2
function as the links between DNA methylation and
histone modification. At each T-DMR, the DNA
methylation status locally correlates with the histone
modification status and vice versa (28). Therefore,
decrease in the levels of Sin3A and Ezh2 at the
T-DMR of Hcrt following ManNAc treatment may
be a molecular link between histone modification
and induction of DNA demethylation.
Re-ChIP experiments using antibodies
against O-GlcNAc modification and Ogt revealed a
protein complex (O-GlcNAc complex) consisting of
Sirt1, Ogt, Sin3A, and Ezh2 at the T-DMRs.
Considering that there are hundreds of
O-GlcNAcylated proteins (10), the components of
the O-GlcNAc complex are also likely to be
O-GlcNAcylated. Sin3A, which is a core component
of several transcriptional co-repressor complexes,
including Sin3A/HDAC, has been shown to recruit
Ogt to promoters to repress the transcription (56),
and Sin3A itself is modified by O-GlcNAc (57).
While there is no report on such a modification of
Sirt1 and Ezh2, localization of the
Polycomb-repressive complex 2, including Ezh2,
has significant similaritiy to the O-GlcNAc
modification on the Drosophila melanogaster
genome (17). The remaining strong O-GlcNAc
signal at T-DMRs following treatment with
ManNAc and an Oga inhibitor (Thiamet-G)
prompted us to consider another unidentified
O-GlcNAcylated protein involved in the repression
mechanisms of Hcrt, because this treatment removes
the O-GlcNAc complex from T-DMRs. Possible
other targets of O-GlcNAcylation are core histone
proteins. To date, O-GlcNAcylated H2B, H3, and
H4 have been reported by MASS analysis (18-20).
The O-GlcNAcylation sites of core histones are also
assumed on the basis of analogy to non-histone
proteins of which Thr/Ser residues are modified by
mutually exclusive phosphorylation and
O-GlcNAcylation (58). Future studies using induced
orexin neurons will allow us to address these issues.
The induced orexin neurons will also be
useful to investigate molecular mechanisms in the
orexin system. Because Sirt1 has been recognized as
neuro-protective molecule (59), the inhibitory role in
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the differentiation of orexin neurons was unexpected.
This finding is contradictory to the previous findings
demonstrating co-localization of orexin and Sirt1 in
mouse LH and decreased orexin levels in adult Sirt1
KO mice (60), indicating that Sirt1 is necessary for
the orexin expression. We believe that the molecular
mechanism of differentiation should be considered
separately from the responses observed in
differentiated neurons. Induced orexin neurons, in
particular, will provide a strong tool for the
development of medical applications: molecules
identified in this study could be target molecules for
the evaluation and screening for diseases related to
the orexin system.
ManNAc exhibited novel effects on
epigenetic processes, including DNA demethylation,
histone acetylation, and O-GlcNAcylation (Fig. 5F).
In mESCs and neural precursor cells, T-DMRs are
hypermethylated and H3/H4 are hypoacetylated.
Hypoacetylation is established by Sirt1. Ogt, Ezh2
and Sin3A are also co-localized with the silencing
complex. Loss of O-GlcNAcylation is a pivotal step
in the transformation from the silent state with
H3/H4 hypoacetylation to the active state with
hyperacetylation at T-DMRs of the Hcrt gene.
ManNAc treatment caused de-localization of Sirt1,
Ogt, Ezh2 and Sin3A, and recruitment of Mgea5,
which have Oga and HAT activities. In this active
state, other HATs such as p300 and CBP are also
involved. We find it intriguingly that the process of
generation of orexin neurons, central regulators of
the whole body metabolism, comprises of an
epigenetic mechanism consisting of nutrient-sensing
molecules.
Here, we have demonstrated the
multilayered epigenetic regulation by Sirt1, Ogt and
Mgea5 in orexin neurogenesis. We propose that
induced orexin neurons will provide a valuable tool
in development of regenerative medicine
applications.
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Acknowledgements- We thank Dr. Michael W. McBurney (Ottawa Hospital Research Institute) for
providing us with Sirt1-/- and WT mESCs, and Dr. Bruce Murphy (University of Montreal) and Ms. Ruiko
Tani for discussions and comments during the preparation of this article. This study was supported by the
Advanced research for medical products Mining Program of the National Institute of Biomedical Innovation
(NIBIO), Japan. We acknowledge Dr. Keiji Hirabayashi and Ms. Yukiko Abe for technical assistance. The
authors declare no conflict of interest. The author contributions are as follows: K.H., S.Y., and K.S. designed
this study. The study was discussed with M.H., D.A., S.T., and N. M.. K.H. and Y.T. performed cell culture
and Immunofluorescence experiments. K.H. performed all other experiments. K.H., S.Y., and K.S. prepared
the manuscript. K.S., S.Y., K.H., M.H. and D.A. have a patent (Method for inducing orexin neurons,
PCT/JP2012/075137) related to this work.
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FIGURE LEGENDS
FIGURE 1. Induction of orexin neurons from mESCs by treatment with ManNAc
(A) Gene expression of hypothalamic transcription factors and peptides in the hypothalamus, fetal
brain, and neural cells induced from mESCs by using SDIA, SDIA+BMP4, or gfCDM/SEFBq methods. RT(-)
indicates PCR results for Actb without reverse transcription. (B) Expression of Hcrt mRNA in neural cells
differentiated from mESCs in the culture with SIDA+BMP4 medium containing 1 mM of GlcN, GlcNAc,
ManNAc, or Neu5Ac for 10 days. The mRNA expression was examined by RT-PCR (left) and -qPCR (right).
Data represent the mean ± S.E. of triplicates of cell culture. The value of Hcrt and Npy expression for lane 5
and 1, respectively, were set to 1. Asterisks indicate significant difference from lane 1 (Student’s t-test; **, P <
0.01) (C) Immunofluorescence assay of Orexin-A and -B in differentiated cells from mESCs in
SDIA+BMP4 medium. The cells were probed for Orexin-A (green, upper), Tubb3 (red, upper), Orexin-B
(green, middle), Ncam (red, middle), DynorphinA (red, bottom), and DAPI (blue). Scale bars represent 100
μm. Orexin-A and orexin-B positive cells were 6.2±1.3% (n=90) and 7.1±1.9% (n=90), respectively, of total
cells in SDIA+BMP4 condition with ManNAc. (D) Left, RT-PCR of Hcrt in neural differentiated mESCs
cultured under the gfCDM/SFEBq condition in the presence of 1 mM GlcNAc and ManNAc. Right,
Immunofluorescence assay of Orexin-B in neural differentiated mESCs cultured under the gfCDM/SFEBq
condition in the presence of 1 mM ManNAc. Cells were probed for Orexin-B (green), Ncam (red), and DAPI
(blue) after 25 days of gfCDM/SFEBq culture. Arrowheads indicate orexin-positive neuron. Scale bars denote
100 μm. Orexin-B positive cells were 5.8±1.1% (n=90) of total cells in gfCDM/SFEBq condition with
ManNAc. (E) Gene expression of orexin neural marker genes in neural cells differentiated from mESCs in
SDIA+BMP4 medium containing 1 mM of GlcN, GlcNAc, ManNAc, or Neu5Ac for 10 days. (F) Effects of
GlcNAc, ManNAc, or Neu5Ac on the Hcrt mRNA expression during the differentiation period (early [days
0–4 and 0-7], late [4–10 and 7-10)], or full [day 0–10]) in the SIDA+BMP4 medium. Expression of Hcrt was
examined by RT-PCR. (G) RT-PCR of Hcrt in differentiated-neurospheres treated with 1 mM GlcNAc,
ManNAc, and Neu5Ac. Each monosaccharide was added from the initiation of differentiation culture. (H)
Response of Orexin-A secretion to high KCl levels in neural cells induced from mESCs by using
SDIA+BMP4 medium supplemented with or without ManNAc. Cells were exposed to high KCl levels for 10
min, and Orexin-A in the medium was measured by conducting an ELISA assay. Data represent the mean ±
S.E. of triplicates of cell culture. Asterisks indicate significant difference (Student’s t-test; **, P < 0.01) (I)
Dose-response of orexin-A secretion to the ligands (ghrelin, leptin, and TRH) in neural cells differentiated
from mESCs in SDIA+BMP4 medium. Orexin-A concentrations in the medium were measured by using
ELISA.
FIGURE 2. Epigenetic status of T-DMRs of the Hcrt gene by DNA methylation and histone
modifications
(A) Expression of Hcrt in mESCs following treatment with inhibitors of DNA methylation and histone
deacetylation. mESCs were cultured for 48 h with 5 μM 5-aza-2’-deoxycytidine (5Aza), 200 nM Trichostatin
A (TSA), or 100 μM Zebularine (Zeb). (B) DNA methylation status of T-DMRs of Hcrt gene around the
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transcriptional start site. Top. Schematic diagram of genes. The vertical lines denote the positions of cytosine
residues of CpG sites. The thick horizontal line indicates the region of the ChIP-PCR fragment. Bottom. Open
and filled squares represent unmethylated and methylated cytosines, respectively. ORE1 indicates orexin
regulatory element 1. Neural cells were differentiated from mESCs in SDIA+BMP4 medium.
Orexin-A-positive (OrxA(+)) or orexin-A-negative (OrxA(-)) colony was picked up by pipet after staining
with an anti-orexin-A antibody and then subjected to the bisulfite reaction. The red and green letters indicate
the level of DNA methylation (%) at T-DMR-U (red square) and T-DMR-D (green square), respectively. (C)
Histone modifications and accumulation of epigenetic regulators at T-DMRs of the Hcrt gene locus. ChIP
assay was performed to determine the histone acetylation and methylation status and the accumulation of
histone modification enzymes at regions 1 and 2 (upper diagram of Fig. 2B) in the T-DMR in differentiated
neural cells from mESCs by culturing in SDIA+BMP4 medium in the presence or absence of 1 mM of
GlcNAc, ManNAc, or Neu5Ac. Input (10%) and normal IgG of rabbit and mouse (rIgG and mIgG) were used
as positive and negative control, respectively. (D) Nuclear and cytoplasmic levels of histone modification
enzymes, (E) histone acetylation and methylation. Levels of histone modification enzymes and the histone
modification status were detected by western blotting of nuclear and cytoplasmic extracts prepared from
neural differentiated mESCs cultured under the SDIA+BMP4 condition and treated with 1 mM GlcNAc,
ManNAc, and Neu5Ac. Graphs indicate protein level of Mgea5 and Sirt1 in nuclear and cytoplasmic fractions.
The levels were estimated by the intensity of each band. Data represent the mean ± S.E. of three
independent experiments. Asterisks indicate significant difference from lane 1(Student’s t-test; **, P < 0.01; *,
P < 0.05). (F) Immunofluorescence assay of Orexin-A, Mgea5 and Sirt1 in neural cells induced from mESCs
by using SDIA+BMP4 medium supplemented with or without ManNAc. Scale bars denote 10 μm.
FIGURE 3. Sirt1 contributes to the inactive state of the Hcrt gene
(A) Expression of Hcrt mRNA in Sirt KO mESCs (Sirt-/-) and in mESCs in the presence of a Sirt1
inhibitor (EX-527). RT-PCR (upper) and RT-qPCR (bottom) revealed expression of the Hcrt mRNA in
undifferentiated, neural differentiated Sirt1-/-mESCs, and neural cells differentiated from mESCs in
SDIA+BMP4 with or without ManNAc in the presence or absence of EX-527. WT indicates wild type mESCs.
ManNAc (1 mM) and EX-527 (50 nM) were added to the medium on days 0 and 7, respectively. DMSO was a
control of EX-527 treatment. RT-qPCR data represent the results of three independent experiments. Data
represent the mean ± S.E. of three independent experiments. Asterisks indicate significant difference from lane
2 (Student’s t-test; **, P < 0.01). The value of Hcrt expression for lane 2 was set to 1. (B)
Immunofluorescence assay of Orexin-A and -B in neural differentiated Sirt1-/- mESCs cultured under
SDIA+BMP4 conditions. Scale bars represent 100 μm. (C) Histone acetylation status of Hcrt T-DMRs by
ChIP assay in neural differentiated Sirt1-/- mESCs and EX-527-treated neural differentiated mESCs cultured
under SDIA+BMP4 conditions in the presence or absence of ManNAc.
FIGURE 4. O-GlcNAcylation system has a role of repressing Hcrt gene expression
(A) Accumulation of Ogt and O-GlcNAcylation at T-DMRs of Hcrt gene loci. ChIP assays of
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O-GlcNAcylation were performed using an antibody RL2. Rabbit IgG and RL2 absorbed with GlcNAc (200
mM) (Abs.) were negative controls, respectively. (B) Effect of an Ogt-inhibitor and Oga-inhibitor on Hcrt
gene expression induced by ManNAc. RT-PCR and -qPCR of Hcrt in neural differentiated mESCs cultured
under the SDIA+BMP4 condition containing an inhibitor of Ogt (BADGP) or Oga (Thiamet-G). BADGP or
Thiamet-G was added to the medium from day 7. ManNAc (1 mM) was added from day 0. Data represent the
mean ± S.E. of three independent experiments. Asterisks indicate significant difference from lane 1 (Student’s
t-test; **, P < 0.01; *, P < 0.05). The value of Hcrt expression for lane 1 was set to 1. (C) Effects of
overexpression of Ogt and Mgea5 on Hcrt gene expression. RT-qPCR of Hcrt in 3×Flag-fused Ogt-,
Mgea5-overexpressing neural differentiated mESCs cultured under the SDIA+BMP4 condition in the
presence of 1 mM ManNAc. A vector, which expresses only 3×Flag mRNA, was used as control in the
overexpression experiment. On day 7, cells were transfected with the overexpression vectors by using
lipofection. At 10 days of culture, cells were collected, and RNA was isolated. Data represent the results of
three independent experiments. Asterisks indicate significant difference from lane 1 (D) O-GlcNAcylation at
the T-DMR in Ogt or Mgea5 expressing cells. ChIP assay of O-GlcNAcylation by RL2 in 3×Flag-fused Ogt-,
Mgea5-overexpressing neural differentiated mESCs cultured under the SDIA+BMP4 condition in the
presence of 1 mM ManNAc. (E) Co-accumulation of Ogt, Sirt1, Sin3A, and Ezh2 at T-DMR with the
O-GlcNAcylation signal in the non-Hcrt expressing state. Interaction of O-GlcNAcylation was revealed by the
Re-ChIP assay. RL2 or anti-Ogt was used as first antibody.
FIGURE 5. Dual function of Mgea5 as histone acetyltransferase and O-GlcNAcase at Hcrt T-DMRs
(A) ChIP assay of histone acetylation in neural differentiated mESCs cultured under the
SDIA+BMP4 condition in the presence of 5 μM Thiamet-G. (B) Structure of Mgea5 with O-GlcNAcase and
HAT domains. The amino acid residue at position 175 (D->A) or 891(Y->F) was mutated to achieve
deficiency of O-GlcNAcase or HAT activity, respectively. (C) Acetylation status of Mgea5-target residues,
H3K14 and H4K8, in 3×Flag-fused Mgea5 (WT, D175A, and Y891F)-overexpressing neural differentiated
mESCs cultured under the SDIA+BMP4 condition in the presence of 1 mM ManNAc. Histone acetylation
levels were detected by western blotting of nuclear fractions. (D) ChIP assay of O-GlcNAcylation and histone
acetylation of Mgea5-target residues in Mgea5 (WT, D175A, and Y891F)-overexpressing neural differentiated
mESCs cultured under the SDIA+BMP4 condition in the presence of 1 mM ManNAc. (E) Expression level of
Hcrt in Mgea5 (WT, D175A, and Y891F)-overexpressing neural cells differentiated from mESCs. Expression
levels of Hcrt were measured by using RT-PCR (left) and RT-qPCR (right). Data represent the mean ± S.E. of
three independent experiments. Asterisks indicate significant difference from lane 1 (Student’s t-test; **, P <
0.01; *, P < 0.05).The value of Hcrt expression for lane1 was set to 1. (F) Proposed model of the epigenetic
state in orexin and non-orexin neurons
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Noboru Murakami, Shintaro Yagi and Kunio ShiotaKoji Hayakawa, Mitsuko Hirosawa, Yasuyuki Tabei, Daisuke Arai, Satoshi Tanaka,
neurons from mouse embryonic stem cellsEpigenetic switching by the metabolism-sensing factors in the generation of orexin
published online April 26, 2013J. Biol. Chem.
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