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HIF-2a Regulates NANOG Expression in Human Embryonic Stem Cells following Hypoxia and Reoxygenation through the Interaction with an Oct-Sox Cis Regulatory Element

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Low O 2 tension is beneficial for human embryonic stem cell (hESC) maintenance but the mechanism of regulation is unknown. HIF-2a was found to bind directly to predicted hypoxic response elements (HREs) in the proximal promoter of OCT4, NANOG and SOX2 only in hESCs cultured under hypoxia (5% O 2). This binding induced an array of histone modifications associated with gene transcription while a heterochromatic state existed at atmospheric O 2 . Interestingly, an enhanced euchromatic state was found when hESCs were exposed to hypoxia followed by 72 hours reoxygenation. This was sustained by HIF-2a which enhanced stemness by binding to an oct-sox cis-regulatory element in the NANOG promoter. Thus, these data have uncovered a novel role of HIF-2a as a direct regulator of key transcription factors controlling self-renewal in hESCs but also in the induction of epigenetic modifications ensuring a euchromatic conformation which enhances the regenerative potential of these cells. Copyright: ß 2014 Petruzzelli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files., FDH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
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HIF-2aRegulates NANOG Expression in Human
Embryonic Stem Cells following Hypoxia and
Reoxygenation through the Interaction with an Oct-Sox
Cis Regulatory Element
Raffaella Petruzzelli, David R. Christensen, Kate L. Parry, Tilman Sanchez-Elsner,
Franchesca D. Houghton*
Centre for Human Development, Stem Cells and Regeneration, Faculty of Medicine, University of Southampton, Southampton, United Kingdom
Abstract
Low O
2
tension is beneficial for human embryonic stem cell (hESC) maintenance but the mechanism of regulation is
unknown. HIF-2awas found to bind directly to predicted hypoxic response elements (HREs) in the proximal promoter of
OCT4, NANOG and SOX2 only in hESCs cultured under hypoxia (5% O
2
). This binding induced an array of histone
modifications associated with gene transcription while a heterochromatic state existed at atmospheric O
2
. Interestingly, an
enhanced euchromatic state was found when hESCs were exposed to hypoxia followed by 72 hours reoxygenation. This
was sustained by HIF-2awhich enhanced stemness by binding to an oct-sox cis-regulatory element in the NANOG
promoter. Thus, these data have uncovered a novel role of HIF-2aas a direct regulator of key transcription factors
controlling self-renewal in hESCs but also in the induction of epigenetic modifications ensuring a euchromatic conformation
which enhances the regenerative potential of these cells.
Citation: Petruzzelli R, Christensen DR, Parry KL, Sanchez-Elsner T, Houghton FD (2014) HIF-2aRegulates NANOG Expression in Human Embryonic Stem Cells
following Hypoxia and Reoxygenation through the Interaction with an Oct-Sox Cis Regulatory Element. PLoS ONE 9(10): e108309. doi:10.1371/journal.pone.
0108309
Editor: Sonia Rocha, University of Dundee, United Kingdom
Received April 14, 2014; Accepted August 28, 2014; Published October 1, 2014
Copyright: ß2014 Petruzzelli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This study was supported by the Gerald Kerkut Charitable Trust, http://www.southampton.ac.uk/,gktrust/, FDH & TS, and the UK Medical Research
Council (G0701153 and G1000406), http://www.mrc.ac.uk, FDH. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: F.D.Houghton@soton.ac.uk
Introduction
hESCs represent a unique source of cells for tissue replacement
and regenerative medicine [1,2]. However, to realize the full
potential of hESCs, the delicate balance between self-renewal and
early differentiation need to be fully elucidated. Recent studies
have found that a physiological O
2
concentration (2–5%) is
beneficial for the maintenance of hESCs in an undifferentiated
state [3–7].
Maintenance of O
2
homeostasis is mediated by hypoxia
inducible factors (HIFs). These heterodimers are formed of a
constitutively expressed HIF-1b(ARNT) subunit and one of the
three different HIF-asubunits (HIF-1a, HIF-2a, HIF-3a) [8,9].
Under normoxia, HIF-asubunits are hydroxylated by prolyl
hydroxylase domain enzymes [10], allowing their recognition by
the von Hippel Lindau tumor suppressor protein (VHL) initiating
HIF-adegradation through the ubiquitin/proteasome complex
[11]. In hypoxic conditions, HIFs are stabilised, translocate to the
nucleus, bind HIF-1band induce expression of hypoxia-responsive
genes [12]. All three HIF-asubunits bind a canonical recognition
sequence (A/G)CGTG termed a hypoxic response element
(HRE), in the proximal enhancer or promoter of HIF target
genes [13].
In hESCs, HIF-1awas only transiently expressed for approx-
imately 48 h at low oxygen tension [7] while HIF-2awas
responsible for the long term response to hypoxia and shown to
be an upstream regulator of OCT4 [7,14], SOX2 and NANOG
[7], transcription factors required to maintain self-renewal as well
as GLUT1, regulating glucose transport [15]. HIF-2ahas also
been found to enhance stemness in hESCs following ischemia/
reperfusion [16] but the mechanisms which regulate these effects
are unknown.
Chromatin organization plays an important role in maintaining
self-renewal and pluripotency of hESCs as perturbation of the
epigenetic profile of key genes can have profound consequences on
stem cell behaviour and fate [17]. Indeed, a high order chromatin
structure has been found to regulate OCT4, SOX2 and NANOG
in an autoregulatory circuit [18]. Furthermore, a complex
genomic interactome might be involved in the regulation of
pluripotency-associated genes in order to sustain pluripotency
[19]. An improved understanding of the transcriptional regulation
of HIFs in hESCs may allow the discovery of a ‘‘molecular sensor’’
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that enhances the self-renewal and therapeutic potential of these
cells.
In this study, we report that HIF-2aregulates hESC pluripo-
tency by binding directly to the OCT4, NANOG and SOX2
proximal promoters in hESCs cultured at low O
2
tension.
Interestingly, this binding and increased expression was also
sustained following 72 hours of reoxygenation leading to activated
chromatin structure within the HRE of these transcription factors
facilitating an enhanced stemness state. Moreover, ChIP assays
revealed that HIF-2awas able to bind an oct-sox cis-regulatory
element only in the NANOG proximal promoter following
reoxygenation. These data suggest that HIF-2acontributes to
the formation of a multiprotein complex bringing into physical
proximity key pluripotency factors to enhance NANOG expres-
sion.
Materials and Methods
Cell culture
Hues-7 hESCs (Howard Hughes Medical Institute/Harvard
University) [29] were cultured as previously described [15]. Cells
were cultured under feeder-free conditions on Matrigel coated
plates at either 20% O
2
,or5%O
2
for a minimum of 3 passages
before use. All cells were removed from the incubator and quickly
passaged under atmospheric conditions. Reoxygenation experi-
ments were performed on hESCs cultured at 5% O
2
for a
minimum of 3 passages before being transferred to 20% O
2
for 72
hours. NT2 cells were maintained in DMEM, 10% fetal bovine
serum and 1% penicillin/streptomycin (Invitrogen) at 37uC, 5%
CO
2
and 5% O
2
.
RT-qPCR
Quantitative Real Time PCR was performed using an ABI
7900 HT Fast Real Time System (Applied Biosystems) in a 384
well plates using a 5 ml reaction containing 1 mg cDNA, 2.5 ml2x
Taqman Universal PCR Master Mix (Applied Biosystems) and
0.25 ml of TaqMan Gene expression Assay Probes (Applied
Biosystems) (Table S1 in File S1).
Immunocytochemistry
hESCs were fixed in 4% paraformaldehyde for 15 min and
incubated with 100 mM Glycine for 10 min. Cells were
permeabilized in 0.2% Triton-X for 10 min and blocked in 10%
fetal calf serum for 30 min before the addition of primary
antibodies diluted in 0.6% BSA in PBS. Primary antibodies used
were HIF-1a(BD Biosciences) 1:100 and HIF-2a(Novus
Biologicals) 1:100 and incubated for 90 min. Staining with the
secondary antibody was performed with anti-mouse conjugated-
FITC (Sigma) 1:100 or goat anti-rabbit Alexa 488 (Molecular
Probes) 1:700 for 1 hour in 0.6% BSA. Cells were mounted in
vectashield containing DAPI (Vecta Laboratories, Peterborough,
UK).
Chromatin Immunoprecipitation assays
ChIP assays were performed as described previously (Forristal et
al., 2013) using the following antibodies: HIF-2a(Novus Biolog-
icals), rabbit IgG (Santa Cruz), H3K9me3-Abcam (Abcam),
H3K4me3 (Abcam), H3K36 me3 (Abcam), OCT4 (Santa Cruz)
and SOX2 (Millipore). Recovered DNA was amplified with
custom Taqman Assays (Applied Biosystems) spanning the
predicted HRE sites (Tables S2 and S3 in File S1).
Vector Constructs
A pcDNA3.1 vector containing HIF-2a(EPAS1) (NCBI
Reference Sequence: NM_001430.4) gene was kindly provided
by Prof. David Russell from the Southwestern Medical Centre
(Texas, USA). A region of 630 bp from 2437 to +207 bp from the
transcription start site of NANOG promoter was amplified with
specific primers that contained XhoI and HINDIII restriction sites
(NanogXho Forward 59CTCGAGCGGCTGGTTT-
CAAACTCCTGA-39and NanogHINDIII Reverse 59-
TTCGAACCGGATGCTTCAAAGC-39). The product was
cloned into a TOPO vector (Invitrogen), and removed using
XhoI/HINDIII restriction enzymes. The fragment was then
cloned into the XhoI/HINDIII sites in a pGL3 Control vector
(Promega). This construct was named pGL3-NANOG. Mutagen-
esis was performed using QuikChange site-directed mutagenesis
(Stratagene) following the manufacturer’s procedure and using
specific primers (Table S4 in File S1). Luciferase activity was
measured using the dual luciferase reporter assay system
(Promega) following the manufacturer’s instructions. Normaliza-
tion was performed using PRL-SV40 Renilla luciferase vector
(Promega).
Transfection and reporter assays
NT2 cells were seeded in 12 well plates (8610
4
cells/well) at 5%
O
2
and transiently transfected using SuperFect transfection
Reagent (Qiagen) following the manufacturers procedure. pcDNA
3.1 and pGL3 empty vectors were co-transfected to check
pcDNA3.1-HEPAS (HIF-2a) activity on the HRE of NANOG
proximal promoter harboured by the pGL3-NANOG vector.
PRL-SV40 Renilla luciferase vector was used to normalize the
transfection efficiency. Luciferase expression was measured using
the Luciferase Reporter Assay System (Promega) with a D-20/20
luminometer (Turner Biosystems).
Statistical analysis
An Anderson-Darling normality test was performed to deter-
mine whether data were normally distributed. Relative gene
expression differences between cells cultured at 5% and 20%
oxygen tension were analysed using a 1-sample t-test. Percentage
of Input (non-immunoprecipitated chromatin) was calculated as
10062
[Ct (Input)–Ct (IP)]
for each sample. Differences in chromatin
relative enrichment between cells cultured at 5% and 20% oxygen
tension were analysed using a Student’s t-test. A value of P,0.05
was considered significant. Differences between HIF-2abinding to
the oct-sox element or intermediate region were determined using
an ANOVA test followed by a Fisher’s test. All data are presented
as a mean 6SEM.
Results
HIF-2abinds in vivo to the OCT4, NANOG and SOX2
proximal promoters only in hypoxic conditions in hESCs
In agreement with Forristal et al. (2010), hESCs cultured at 5%
O
2
tension display an increased expression of OCT4, SOX2 and
NANOG compared to those maintained at 20% O
2
(Figure S1 in
File S1). To verify whether endogenous HIF-2ainteracts with the
predicted HREs in the promoter region of OCT4, NANOG and
SOX2 genes, ChIP analysis was performed on hESCs cultured
either at 5% or 20% O
2
. Amplification of a putative HRE in both
the OCT4 and NANOG promoter revealed a 4-fold enrichment
(P,0.01) in cells cultured at 5% O
2
compared to the IgG control.
In contrast, hESCs cultured at 20% O
2
revealed no significant
enrichment compared to the IgG control (Fig. 1A). For the SOX2
proximal promoter, 2 different HREs situated at 21450 bp and 2
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1100 bp from the transcriptional start site were analysed. These
HREs differ by the presence of the A nucleotide (A)CGTG
(SOX2A) or a G nucleotide (G)CGTG (SOX2G). ChIP samples
showed a 6 fold (P,0.05) and 3 fold (P,0.05) increase in the level
of HIF-2abinding in the promoter of SOX2A and SOX2G
respectively compared to the IgG control (Fig. 1A). To further
verify the specificity of HIF-2abinding, a negative control probe
specific for the FOXP3 promoter was used. This probe was
designed to amplify a region in the proximal promoter which does
not contain an HRE but instead was situated between 2 potential
HREs at 2670 bp and +104 bp from the transcription start site
(Fig. 1A). qPCR on three independent ChIP experiments on
chromatin derived from hESCs cultured at either 5% or 20% O
2
revealed no significant enrichment for HIF-2ain this FOXP3
promoter region (P.0.05). These data indicate that HIF-2abinds
directly to HREs in the proximal promoter of OCT4, NANOG
and SOX2 only in hypoxic conditions in hESCs.
Functional Analysis of the HRE in the NANOG proximal
promoter
To verify the novel, direct binding of endogenous HIF-2ain the
HRE (2301 bp) to the NANOG proximal promoter and to
interrogate whether this was functional and able to activate
transcription, luciferase reporter assays were performed in NT2
cells. By cloning a fragment of the proximal NANOG promoter (2
437 bp to +207 bp from the transcription start site), that includes
the HRE site for HIF-2a(pGL3-NANOG 2301 bp) into a PGL3
vector, a significant promoter-driven increase in transcription was
observed (P,0.001) only when NT2 cells were co-transfected with
a HIF-2aexpression vector (Fig. 1B). Furthermore, a significant
reduction (P,0.001) of NANOG transcription was observed when
a mutant Renilla plasmid vector (pGL3-Mut NANOG 2301 bp),
where the HRE sequence had been abrogated, was co-transfected
with HIF-2a, when compared to the wild type vector (Fig. 1B).
This confirms that HIF-2adirectly binds the HRE at 2301 bp
from the start site in the NANOG promoter and is functionally
responsible for NANOG transcription.
Reoxygenation affects the mRNA profile of genes
regulating self-renewal
We next investigated whether reoxygenation following hypoxia
affects OCT4, SOX2 and NANOG expression in hESCs. Using
RT-qPCR, NANOG expression was significantly increased after
24 hours of reoxygenation and remained highly expressed
following exposure to reoxygenation for 72 hours (Fig. 2A).
Similarly, OCT4 mRNA showed a more gradual but significant
increase in expression between 24–72 hours of reoxygenation. In
contrast, there was no difference in SOX2 mRNA expression
upon reoxygenation. These data were intriguing and suggested
that reoxygenation could also have an influence on the epigenetic
state of hESCs.
Hypoxia alters the histone modification profile in the
proximal promoter of key transcription factors regulating
hESC self-renewal
To determine the impact of hypoxia on chromatin modifica-
tions around the predicted HRE site in OCT4, NANOG and
SOX2 proximal promoters, ChIP assays using H3K4me3 and
H3K36me3, markers of transcriptional activation, and H3K9me3
a marker of gene silencing were performed. Pie charts were used to
represent the percentage input precipitated by each modified
histone as a proportion of the total, for each gene of interest
whereas bar charts were used to show comparison of individual
epigenetic marks and are shown in the Supplementary data. Using
probes designed to cover the HRE site at 21956 bp in the OCT4
proximal promoter, a significant proportion of the H3K36me3
histone marker was found in hESCs cultured under hypoxic
conditions compared to 20% O
2
(Fig. 2B and Figure S2 in File
S1). A similar trend of histone modification levels were obtained
when the HRE at 2301 bp in the NANOG proximal promoter
and the HREs at 21450 bp and 21100 bp from the start site in
the SOX2 proximal promoter were amplified in hESCs cultured
under hypoxic conditions (Fig. 2B and Figure S2 in File S1).
hESCs maintained at 20% O
2
displayed high H3K9me3 levels
and significantly reduced H3K4me3 and H3K36me3 within the
NANOG and SOX2 HREs compared to cells cultured under
hypoxic conditions. These data confirm that the chromatin state in
hESCs cultured at 20% O
2
tension is more heterochromatic and
inaccessible to transcription factors or chromatin remodelling
factor binding (Fig. 2B and Figure S2 in File S1). Overall, at 5%
O
2
a significant increase of the H3K36me3 levels was observed for
OCT4, SOX2 and NANOG. These trends are consistent with a
more open conformational chromatin under low levels of O
2
and
indicate active changes that correlate with transcriptional activity.
Hypoxia/reoxygenation induces a euchromatic state
within the HREs of OCT4, SOX2 and NANOG proximal
promoters
To investigate whether the epigenetic profile within the HRE
site was affected following reoxygenation, ChIP assays were
performed on hESCs subjected to hypoxia followed by 72 hours of
reoxygenation. Surprisingly, an overall increased proportion of
H3K4me3 and H3K36me3 and a dramatic reduction in the
proportion of H3K9me3 bound was found within the HRE in
OCT4, SOX2A promoter compared with chromatin of hESCs
cultured at 20% O
2
(Fig. 2B and Figure S2 in File S1).
Interestingly, these differences were particularly marked for the
HRE within the NANOG promoter (Fig. 2B and Figure S2 in File
S1) suggesting that, when cells were exposed to hypoxia/
reoxygenation chromatin retains a more open conformational
state, compared to normoxic conditions (Fig. 2B).
HIF-2adirectly interacts with the HRE of pluripotency
genes after 3 days reoxygenation
We next considered that nuclear HIF-2amay not be degraded
following reoxygenation but instead may bind to the HRE of all
core pluripotency genes and could be responsible for these
epigenetic changes. ChIP assays were performed on hESCs
collected after 72 hours reoxygenation. HIF-2ashowed a
significant enrichment in the HRE within the OCT4 (P,0.04),
NANOG (P,0.001), SOX2A (P,0.05) and SOX2G (P,0.001)
promoter compared with chromatin isolated from hESCs cultured
at 20% O
2
(Fig. 3A). Thus, our data indicates that at least nuclear
HIF-2aprotein is stabilised and not degraded following hypoxia
and 72 hours of reoxygenation as was further confirmed using
immunocytochemistry (Figure S3 in File S1). Interestingly, when
compared to hESCs maintained at 5% O
2
, there was a significant
enrichment of HIF-2abinding only to the NANOG promoter (P,
0.01) (Fig. 3B). These data reveal that HIF-2ais able to sustain
higher NANOG transcriptional activation together with moderate
levels of OCT4 and SOX2 after an oxidative insult. Moreover,
HIF-2amRNA expression was found to be significantly higher on
each day post-reoxygenation compared to cells cultured at 20%
O
2
(Fig. 3C). Interestingly, this increased HIF-2aexpression was
similar to that observed in cells cultured under hypoxic conditions
(Fig. 3D). These data suggest that HIF-2ais transcriptionally
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Figure 1. HIF-2adirectly binds to HREs in the OCT4, NANOG and SOX2 proximal promoters in hESCs cultured under hypoxia. (A)
ChIP analysis of HIF-2abinding a predicted HRE site in the proximal promoters of OCT4, NANOG, SOX2A and SOX2G on chromatin isolated from
hESCs cultured either at 20% or 5% O
2
. Amplification of FOXP3 was used as a negative control. DNA enrichment is expressed as a percentage of
Input. An average of 3 independent experiments is represented (*P,0.05, **P,0.01; NS: no significant difference). (B) Schematic representation of
the HIF-2aexpression vector (pcDNA3.1-HIF-2atop panel). The luciferase reporter construct driven by NANOG promoter showed a significant
increase (**P,0.01) in luciferase activity compared to the control. Mutation in the predicted HRE caused a significant decrease in the luciferase
activity (**P,0.01) compared to the control. An average of 4 independent experiments is shown.
doi:10.1371/journal.pone.0108309.g001
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regulated and sustains high nuclear protein levels following
ischaemic preconditioning.
We also wanted to investigate the effect of hypoxia following 72
hours of reoxygenation on HIF-1ausing immunocytochemistry.
As expected, HIF-1aprotein was present in hESCs after 24 hours
of hypoxia but was absent in cells maintained for at least 3
passages at 5% O
2
(Figure S4 in File S1) [7]. HIF-1awas also not
expressed in hESCs following hypoxia and 72 hours of reoxygena-
tion (Figure S4 in File S1). This confirms that HIF-1asustains only
the first adaptation to hypoxia and highlights the unique role of
HIF-2ain regulating hESC self-renewal not only under hypoxia
but also following reoxygenation.
HIF-2abinds to an oct-sox cis regulatory element within
the NANOG promoter
Bioinformatic analysis of the NANOG promoter revealed an
oct-sox cis-regulatory element at 2208 bp (TTTGCATTA-
CAATG) from the transcription start site and in close proximity
(93 bp) to the HRE (Fig. 4A). We considered the possibility that
HIF-2acould be responsible for a three-dimensional chromatin
structure that keeps transcription factors more closely together
when cells are cultured at either 5% O
2
, 20% O
2
or following
hypoxia/reoxygenation. Using ChIP assays, HIF-2awas found to
bind to the oct-sox element of the NANOG promoter only when
hESCs were subjected to hypoxia followed by 72 hours
reoxygenation (Fig. 4B). In contrast, no binding to this element
was observed in hESCs cultured at either 5% (Fig. 4C), or 20% O
2
Figure 2. Hypoxia and reoxygenation enhance the expression of pluripotency genes through a euchromatic state within the HRE
site. (A) RT-qPCR analysis for OCT4, NANOG and SOX2 mRNA in hESCs subjected to 24, 48 and 72 hours reoxygenation compared to those
maintained at 20% O
2
. All data have been normalized to UBC and to 1 for hESCs maintained at 20% O
2
. Values are mean of 5 independent
experiments 6SEM (*P,0.05, **P,0.01, ***P,0.001 significantly different from 20% O
2
). (B) Pie charts showing ChIP analysis of histone modification
markers H3K4me3, H3K36me3 and H3K9me3 binding a predicted HRE site in the proximal promoters of OCT4, NANOG, SOX2A and SOX2G in hESCs
exposed to 20% O
2
,5%O
2
, and hypoxia reoxygenation respectively. DNA enrichment is expressed as a percentage of Input relative to the IgG
control. An average of 3 to 4 independent experiments is represented.
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(Fig. 4D). Moreover, no interaction was found within the oct-sox
element in the OCT4 promoter (Fig. 4E) and SOX2 intron
(Fig. 4F) in hESCs subjected to hypoxia followed by 72 hours
reoxygenation. The observed interaction of HIF-2aand the oct-
sox element could be experimentally dependent on the fact that
both HRE and oct-sox are in the same DNA molecule and
relatively close (93 bp), which could potentially lead to both sites
precipitating together as an artifact. To rule this possibility out, we
designed a control probe that covers the 93 bp sequence between
the HRE site and the oct-sox element in the NANOG promoter
(called intermediate region) and expected this region not to be
present in the immunoprecipitated samples (Fig. 4A, B). qPCR on
four independent ChIP experiments on chromatin derived from
hESCs subjected to hypoxia and reoxygenation for 72 hours and
immunoprecipitated with HIF-2arevealed no significant binding
to the intermediate region compared to the oct-sox element and
the IgG control. (Fig. 4B). These results show that both the HRE
and oct/sox binding sites are in physical contact with HIF-2aonly
when hESCs are exposed to reoxygenation. This suggests that
HIF-2afunctions as an enhancer to increase the expression of
NANOG under conditions of reoxygenation.
Functional Analysis of the oct-sox element in the NANOG
proximal promoter
To verify the functionality of the HIF-2abinding to the oct-sox
cis regulatory element (2208 bp) in the NANOG proximal
promoter, Luciferase reporter assays were performed in NT2
cells. A 630 bp genomic sequence of the NANOG promoter which
contains the HRE site for HIF-2a(2301 bp) and the oct-sox cis
regulatory element (2208 bp) was cloned into a PGL3 vector. In
addition, two Renilla plasmid vectors were used where the oct-sox
cis element (pGL3-Mut NANOG 2208 bp) or both the HRE and
the oct-sox elements (PGL3-Mut NANOG 2301/Mut 2208 bp)
were mutated in order to disrupt the HIF-2abinding. All plasmids
were co-transfected with a HIF-2aexpression vector (pcDNA3.1-
HIF-2a) in NT2 cells. Luciferase reporter assays showed a
Figure 3. HIF-2asustains the expression of NANOG following hypoxia/reoxygenation. ChIP analysis of HIF-2abinding a predicted HRE site
in the proximal promoters of OCT4, NANOG, SOX2A and SOX2G on chromatin isolated from hESCs subjected to hypoxia followed by 72 hours
reoxygenation. HIF-2abinding to the HRE of pluripotency genes is expressed relative to 20% O
2
(A) or 5% O
2
(B). DNA enrichment is expressed as a
percentage of Input. An average of 4 independent experiments is represented (*P,0.05, ***P,0.001). RT-qPCR for HIF-2aexpression in hESCs
subjected to 24, 48 and 72 hours reoxygenation. All data have been normalized to UBC and to 1 for hESCs cultured at 20% O
2
(C) or 5% O
2
(D). Values
are mean of 3 to 4 independent experiments 6SEM (*P,0.05).
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Figure 4. Hypoxia/reoxygenation induces HIF-2abinding to the oct-sox
cis
element in the NANOG promoter. (A) Schematic
representation of the probes designed to cover the HRE, oct-sox cis element and intermediate region on the NANOG proximal promoter. (B) ChIP
analysis of HIF-2abinding to the oct-sox-cis element and the intermediate region in hESCs cultured under hypoxia followed by 72 hours
reoxygenation. An average of 4 independent experiments is represented (*P,0.05, **P,0.01). ChIP analysis of HIF-2abinding the oct-sox cis-
regulatory element in NANOG proximal promoter on chromatin isolated from hESCs cultured at 5% O
2
(C), 20% O
2
(D). ChIP analysis of HIF-2abinding
the oct-sox cis-regulatory element in OCT4 proximal promoter (E) and SOX2 intron (F) on chromatin isolated from hESCs subjected to 72 hours
reoxygenation. DNA enrichment is expressed as a percentage of Input. An average of 3 independent experiments is represented. (NS: no significant
difference).
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significant reduction of the promoter-driven transcription (P,
0.05) when NT2 cells were co-transfected with a HIF-2a
expression vector and a mutant of the HRE site (pGL3-Mut
NANOG 2301 bp) compared to the wild type sequence (pGL3-
NANOG 2301 bp). Upon mutation of the oct-sox element within
the NANOG promoter (pGL3-Mut NANOG 2208 bp), the level
of activation decreased to about half (P,0.01) of that for wild type
NANOG (Fig. 5). Interestingly, when both the HRE and oct-sox
elements were mutated in NANOG (PGL3-Mut NANOG 2301/
Mut 2208 bp), there was an even greater reduction in luciferase
activity (P,0.001; Fig. 5) suggesting that HIF-2ais able to interact
not only with the HRE sequence but also with the oct-sox element
in the NANOG proximal promoter and that this binding is
functionally responsible for NANOG activity.
OCT4 and SOX2 bind to the oct-sox element within the
NANOG promoter in normoxia, hypoxia and following 72
hours of reoxygenation
Given that OCT4 and SOX2 play a key role in controlling
hESC pluripotency through the transcriptional regulation of
NANOG [20], we next considered the effect of oxygen tension
in the binding of OCT4 and SOX2 to the composite oct-sox
element within the NANOG promoter. ChIP assays were carried
out with OCT4 and SOX2 antibodies in hESCs cultured under
normoxia, hypoxia and following 72 hours of reoxygenation. The
ChIP material was amplified by qPCR using a specific probe
which covers the oct-sox element within the NANOG proximal
promoter (2208 bp). ChIP assays showed a significant 15 fold (P,
0.01) and 35 fold (P,0.001) increase of OCT4 and SOX2 binding
within the oct-sox cis regulatory element in the NANOG proximal
promoter when hESCs were maintained at 20% O
2
(Fig. 6).
Interestingly, when hESCs were cultured at 5% O
2
, OCT4 and
SOX2 binding to the oct-sox element dropped to 2 fold (P,0.001)
and 20 fold (P,0.001) respectively compared to the IgG control
while the level of enrichment decreased to about 5 fold (P,0.01)
and 10 fold (P,0.001) when hESCs were exposed to hypoxia
followed by reoxygenation (Fig. 6). These data confirm the
occupancy of OCT4 and SOX2 at the oct-sox element within
the NANOG promoter in undifferentiated hESCs in all the
oxygen conditions tested. Furthermore, these results suggest that
under normoxia NANOG transcription is mainly influenced by
OCT4 and SOX2, while under hypoxia and particularly following
reoxygenation, there is a trend towards reduced binding of these
proteins.
Discussion
Accumulating evidence suggests that resident stem/progenitor
cell populations are able to promote survival in response to
oxidative stress and repopulate damaged tissues. However the
precise ‘‘stemness’’ feature which promotes stem cell survival and
regeneration is not fully understood. This study proposes that
hESCs exposed to hypoxia and reoxygenation maintain their
stemness through a novel mechanism of epigenetic regulation
whereby HIF-2aenhances NANOG expression to protect cells
from oxidative stress. Our data provide evidence that endogenous
HIF-2abinds directly to HREs in the proximal promoter of
OCT4, NANOG and SOX2 in hESCs cultured under hypoxia
but not at atmospheric O
2
tensions. Moreover, a direct
involvement of HIF-2ain NANOG gene expression was observed
not only under hypoxia but also following reoxygenation. As
NANOG has a central role in hESC pluripotency and self-
renewal, but also in cell cycle progression [21–23] HIF-2abinding
could explain the increased rate of proliferation observed in
hESCs under hypoxia [7]. Our ChIP data showed that HIF-2a
preferentially binds to SOX2A compared to the SOX2G. The
biological significance of this result is not known but may reflect
the presence of other transcription factors that can cooperate with
HIF-2ato regulate SOX2 expression, or the difference observed
in the chromatin conformation around the HRE.
Figure 5. HIF-2abinds the oct-sox
cis
-regulatory element and drives NANOG activity. Schematic representation of the HIF-2aexpression
vector (pcDNA3.1-HIF-2atop panel). A pcDNA3.1-HIF-2avector was transiently co-transfected in NT2 cells with a luciferase reporter construct driven
by NANOG promoter with intact HRE and oct-sox element or with different constructs in which either the HRE, the oct-sox or both sites were
mutated. Luciferase activity was significantly decreased when the HRE (P,0.01), the oct-sox element (P,0.01) and both sites (P,0.001) were
mutated compared to the NANOG gene promoter construct with the unmutated HRE and oct-sox element. An average of 4 independent
experiments is shown.
doi:10.1371/journal.pone.0108309.g005
NANOG Regulation by HIF-2ain Reoxygenation
PLOS ONE | www.plosone.org 8 October 2014 | Volume 9 | Issue 10 | e108309
Consistent with these data, a heterochromatic state was
observed under atmospheric O
2
tensions. Since HIF-2ais not
degraded under these conditions but displays a cytoplasmic
localization [7], the high levels of H3K9me3 and absence of
activation markers H3K4me3 and H3K36me3 at the HRE are
likely to explain the lack of binding to OCT4, SOX2 and
NANOG. In contrast, a bivalent chromatin state was observed
under hypoxic conditions in the HRE of pluripotency genes with
moderate levels of H3K9me3 which likely reflect the hypoxic
induction of G9a methyltransferase [24] and increased levels of
H3K36me3 which is associated with gene transcription elongation
[25]. Hence, hESCs may respond rapidly to O
2
changes by
accumulating OCT4, NANOG and SOX2 transcripts. Surpris-
ingly, only a small increase in H3K4me3 was observed in the
predicted HRE site under hypoxic conditions and may be due to a
family of jumonji-domain histone demethylases such as JARID1B,
known to be regulated by HIF-1aand HIF-2a[26].
In order to mimic the effect of an ischemia-reperfusion
environment, we subjected hESCs to reoxygenation. Histone
modification analysis showed that H3K4me3 and H3K36me3
levels remained high while H3K9me3 was dramatically reduced
compared to hESCs cultured at 5% O
2
. These modifications were
associated with a direct interaction of HIF-2awithin the HRE of
all core pluripotency genes but particularly with the NANOG gene
promoter. This finding was intriguing since HIF-2awas not
degraded following reoxygenation suggesting either a different
mechanism of degradation and/or post-transcriptional modifica-
tion. Recently, low p53 and high HIF-2alevels were also found to
be associated with increased NANOG expression upon reoxy-
genation [16]. Our data extends this finding and suggests that
epigenetic modifications associated with HIF-2abinding may
promote NANOG activity, a mechanism which likely protects cells
from oxidative stress.
We identified a novel, specific interaction between HIF-2aand
an oct-sox cis regulatory element in the NANOG promoter. This
interaction occurred only when hESCs were subjected to hypoxia
followed by reoxygenation and might be the result of the
formation of a tight chromatin structure on the NANOG
promoter, bringing together the HRE and oct-sox site through
HIF-2aand other ancillary proteins thereby leading to an
increased expression (Fig. 7 bottom left). Another possibility that
cannot be ruled out is that reoxygenation generates a new binding
site for HIF-2athat then binds to the oct-sox element,
independently of the binding to the HRE site (Fig. 7 bottom
right). These will need further investigation however it is
tantalizing to hypothesize that a tight loop conformation could
form a protective ‘‘pocket’’ that may shield HIF-2afrom
degradation, maintaining its nuclear localization upon reoxygena-
tion. Surprisingly, this binding was not present under hypoxic or
normoxic conditions alone.
The oct-sox element is functionally important for NANOG
expression and establishing a pluripotent phenotype in stem cells
[27] but our data highlight that OCT4 and SOX2 are not the only
proteins that can bind and allow the pluripotent-specific expres-
sion of NANOG. Hence, regardless of the molecular structure of
the promoter, our data suggest that, under reoxygenation, hESCs
activate an alternative mechanism of regulation in which HIF-2a
activity is central to the maintenance of self-renewal, through the
interaction with an oct-sox element. Indeed, binding of HIF-2ato
the oct-sox element is functionally active and able to confer
NANOG transcriptional activation since disruption of the oct-sox
element or both HRE and oct-sox sequences are able to reduce
NANOG activity. Furthermore, we found that OCT4 and SOX2
binding to the oct-sox element was particularly enriched when
hESCs were cultured at 20% O
2
while less binding occurred in
hypoxia and following 72 hours of reoxygenation. These data
allow us to speculate that the presence of HIF-2ain hypoxia and
reoxygenation takes over the activation of NANOG in hESCs
possibly working in combination with OCT4 and SOX2 which
simultaneously associate with the oct-sox element. We also
observed that SOX2 binding was more enriched within the
NANOG oct-sox element compared to OCT4. This will need
further investigation but since SOX2 is known to bend DNA [28],
this transcription factor may in part contribute to the chromatin
conformation within the oct-sox element that leads to recruitment
of HIF-2ain hypoxia and reoxygenation.
In conclusion, we demonstrate that HIF-2ais recruited as a co-
activator to the oct-sox element under reoxygenation, subsequent-
ly enhancing NANOG expression. We therefore propose a novel
mechanism of epigenetic regulation in hESCs whereby HIF-2a
binds the HRE and forms a multiprotein complex together with
OCT4 and SOX2 and other chromatin remodelling factors in
order to enhance NANOG expression following hypoxia/
reoxygenation to protect cells from oxidative stress. This
epigenetic mechanism may be part of a molecular program,
characteristic of resident stem/progenitor cell populations that
repopulate and promote survival during post-injury stress, or in a
wide range of diseases associated with ischemia and reoxygena-
tion.
Figure 6. OCT4 and SOX2 bind to the oct-sox
cis
element in the NANOG proximal promoter. ChIP analysis of OCT4 and SOX2 binding to
the oct-sox cis element in hESCs cultured at either 5% O
2
, 20% O
2
,or5%O
2
followed by 72 hours reoxygenation. An average of 4 independent
experiments is represented (**P,0.01, ***P,0.001).
doi:10.1371/journal.pone.0108309.g006
NANOG Regulation by HIF-2ain Reoxygenation
PLOS ONE | www.plosone.org 9 October 2014 | Volume 9 | Issue 10 | e108309
Supporting Information
File S1 Supporting information. Figure S1, Pluripotency
markers are reduced in hESCs cultured at 20% O
2
condition
compared to hES cells cultured at 5% O
2
. RT-qPCR analysis of
OCT4, SOX2 and NANOG expression in hESCs cultured at 5%
or 20% O
2
. All data have been normalized to UBC and to 1 for
5% O
2
. Values are mean of 4 independent experiments 6SEM
(*P,0.05). Figure S2, Histone modifications induced within the
HRE of OCT4, NANOG and SOX2 genes in hESCs cultured in
hypoxia and following reoxygenation. ChIP assays H3K4me3,
H3K9me3 or H3K36me3 on chromatin isolated from hESCs
cultured either at 20% O
2
,5%O
2
or 72 hours post-reoxygenation.
Data have been normalized to 5% O
2
for hESCs cultured at 20%
O
2
(A), or either to 20% O
2
(B) or 5% O
2
(C) for hESCs subjected
to reoxygenation. DNA enrichment is expressed as a percentage of
input minus the background IgG. An average of 3 to 4
independent experiments is represented (*P,0.05, **P,0.01;
***P,0.001). Figure S3, Expression of HIF-2ain hESCs cultured
at 5% O
2
followed by 72 hours of reoxygenation (Reoxy). Protein
expression of HIF-2a(A and B), merged with DAPI (B) and the
secondary antibody only negative control (C and D), merged with
DAPI (D) of hESCs cultured on Matrigel under hypoxic
conditions followed by 72 hours of reoxygenation. Scale bar
25 mm. Figure S4, Expression of HIF-1ain hESCs cultured at 5%
O
2
for at least 3 passages (5% O
2
), 5% O
2
followed by 72 h of
reoxygenation (Reoxy), or 20% O
2
followed by 24 hours at 5% O
2
(24 h 5% O
2
). Protein expression of HIF-1a(A–E), merged with
DAPI (B, D, F) and the secondary antibody only negative control
(G, H), merged with DAPI (H) of hESCs culture at 5% O
2
for at
least 3 passages (A–B), 5% O
2
followed by 72 hours reoxygenation
(C–D), or 20% O
2
followed by 24 hours at 5% O
2
(E–F). Scale bar
25 mm. Table S1, Table S2, Table S3, Table S4.
(DOCX)
Acknowledgments
We would like to thank the Gerald Kerkut Charitable Trust and the UK
Medical Research Council (MRC) for funding this research (G0701153
and G1000406).
Author Contributions
Conceived and designed the experiments: FDH TS RP. Performed the
experiments: RP DRC. Analyzed the data: RP. Contributed to the writing
of the manuscript: RP FDH TS. Provided Technical support: KLP.
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Thesis
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To realize the full potential of human embryonic stem cells (hESCs), it is important to develop culture conditions that maintain hESCs in a pluripotent, undifferentiated state. A low O(2) atmosphere (approximately 4% O(2)), for example, prevents spontaneous differentiation and supports self-renewal of hESCs. To identify genes whose expression is sensitive to O(2) conditions, microarray analysis was performed on RNA from hESCs that had been maintained under either 4% or 20% O(2). Of 149 genes differentially expressed, 42 were up-regulated and 107 down-regulated under 20% O(2). Several of the down-regulated genes are most likely under the control of hypoxia-inducing factors and include genes encoding enzymes involved in carbohydrate catabolism and cellular redox state. Although genes associated with pluripotency, including OCT4, SOX2, and NANOG were generally unaffected, some genes controlled by these transcription factors, including LEFTY2, showed lowered expression under 20% O(2), while a few genes implicated in lineage specification were up-regulated. Although the differences between O(2) conditions were generally subtle, they were observed in two different hESC lines and at different passage numbers. The data are consistent with the hypothesis that 4% O(2) favors the molecular mechanisms required for the maintenance of pluripotency.
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To summarize recent reports on the effects of low oxygen on the undifferentiated phenotype and differentiation of embryonic stem cells (ESCs). The oxygen level to which ESCs are exposed is an important environmental parameter. Under conditions maintaining the undifferentiated phenotype, low oxygen reduces spontaneous differentiation of human ESCs but reduces pluripotency gene expression in mouse ESCs, although reports are conflicting. Differentiation under low oxygen increases generation of neurons, cardiomyocytes, hematopoietic progenitors, endothelial cells, and chondrocytes. Many of the effects of low oxygen have been attributed to action by hypoxia inducible factor-1alpha (HIF-1alpha). The oxygen level in the gas phase (pO2gas) is often different than that experienced by the cells (pO2cell) and is unrecognized by investigators, which makes interpretation of the literature difficult. This difference increases with high cell densities, high cellular oxygen consumption rates, and large medium heights. The problem can be addressed by use of oxygen-permeable culture dishes and by estimation of pO2cell with mathematical models. Low oxygen influences aspects of ESC pluripotency and differentiation. A better understanding of its effects and mechanism along with better estimation and control of pO2cell is important for applying low oxygen culture to regenerative medicine applications.