epigenetic switching by the metabolism-sensing factors in the generation of orexin neurons from

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:

[email protected]

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. 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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