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University of Groningen
From microenvironment to epigenetics in endothelial
cellsMaleszewska, Monika
DOI:10.1016/j.imbio.2012.05.026
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Citation for published version (APA):Maleszewska, M. (2015).
From microenvironment to epigenetics in endothelial cells.
University ofGroningen.
https://doi.org/10.1016/j.imbio.2012.05.026
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https://doi.org/10.1016/j.imbio.2012.05.026https://research.rug.nl/en/publications/from-microenvironment-to-epigenetics-in-endothelial-cells(0e2dcc80-c365-4745-a2a7-d38daa4e28cc).htmlhttps://doi.org/10.1016/j.imbio.2012.05.026
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CHAPTERCHAPTER
Identification of candidate oxidative stress response genes
regulated by EZH2 and fluid shear stress
V
Monika Maleszewska, Raphael Kaeriyama e Silva, Guido Krenning,
Martin C. Harmsen
Cardiovascular Regenerative Medicine Research Group, Department
of Pathology and Medical Biology, University of Groningen,
University Medical Center Groningen, Groningen, The Netherlands
In preparation
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ABSTRACT
The exposure of blood vessels to a variety of risk factors, in
particular those that induce oxidative stress, causes endothelial
dysfunction and predisposes to cardiovascular disease. In arteries
the high level of fluid shear stress suppresses endothelial
oxidative stress, while in areas with low or disturbed flow
endothelial cells are prone to dysfunction. The epigenetic
mechanisms that suppress endothelial dysfunction are poorly
understood. As EZH2 methyltransferase is involved in the regulation
of endothelial gene expression and plays a role in endothelial
response to FSS, we hypothesized that EZH2 is also involved in the
regulation of the expression of oxidative stress-responsive genes
under FSS. RNA-seq analysis revealed that expression of genes
within the GO cluster “Response to oxidative stress” is regulated
by EZH2 or by FSS. Of those, 32 genes were regulated by both EZH2
and FSS, inn HUVEC. Four genes whose expression increased more than
2-fold upon both EZH2-depletion and FSS-exposure were HMOX1, PTGS1,
PTK2B and GPX3. EZH2 was present in the proximal promoter regions
of these genes, as determined through the analysis of ENCODE HUVEC
ChIP-seq data. The two genes with the relatively lowest expression,
PTK2B and GPX3, consistently showed high levels of H3K27me3 around
their promoter regions in the ENCODE dataset, suggesting they might
be direct targets of activity of EZH2. The 5’-UTRs of these four
genes shared putative transcription factor binding sites of the
repressive factors ZNF263 and FOXP1.This study revealed four
candidate genes that are under the repressive control of EZH2 and
are activated by FSS, including a well-known oxidative
stress-responsive gene HMOX1. It further explores the generic
epigenetic make-up around promoters of these genes and suggests
regulation by two candidate transcription factors. These results
shed new light on the mechanisms behind the anti-oxidant effects of
high FSS and corroborate the role of EZH2 as an epigenetic
regulator of endothelial gene expression.
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INTRODUCTION
Endothelium, the inner layer of all blood vessels, serves
several functions essential for vascular homeostasis. Chronic
endothelial dysfunction contributes to the development of
cardiovascular disease (CVD). Many risk factors of CVD are known to
also cause endothelial dysfunction by eliciting the oxidative
stress; these include cigarette smoke, chronic inflammation,
oxidized low density lipoprotein (OxLDL), among others.1, 2Reactive
oxygen species (ROS) are an integral part of cellular function:
they are produced in the metabolic reactions in the cytoplasm and
mitochondria and are part of signaling pathways such as NFkB.3
However, their excessive production and chronic presence are
detrimental to the cellular homeostasis. ROS may damage cellular
components through oxidation which may causemitochondrial
dysfunction and cell death. In the cell, multiple biochemical
mechanisms counteract excessive ROS,4 including the oxidative
stress-responsive expression of protective genes, such as heme
oxygenase-1 (HMOX1) or superoxide dysmutases (SOD). The products of
the activated genes are enzymes or scavenger molecules which
neutralize ROS.In the body, endothelial cells (ECs) are constantly
exposed to the blood flow, which exerts the force known as fluid
shear stress (FSS) on the blood vessel wall. Disturbed blood flow
exerts on average low levels of FSS and is associated with
endothelial dysfunction.5-7 Cells exposed to disturbed flow produce
relatively high levels of ROS and are thus exposed to oxidative
stress.8, 9 On the other hand, high FSS protects ECs, preserves
endothelial function 5-7 and associates with reduced levels of
oxidative stress.8, 9Enhancer of Zeste Homolog-2 (EZH2), the
methyltransferase of the Polycomb complex of epigenetic
repressors,10 regulates a large number of genes that are also
affected by FSS in endothelial cells (Maleszewska M et al.,
submitted). High FSS causes decreased EZH2 expression, which
reduces endothelial proliferation and promotes quiescence. In this
study, we investigated whether some of the anti-oxidant effects of
FSS could be explained by the decrease in EZH2 expression resulting
in differential expression of the oxidative stress-responsive
genes.
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MATERIALS AND METHODS
Cell culture
Human Umbilical Vein Endothelial Cells (HUVEC) were purchased
from Lonza (Basel, Switzerland) and used between passages 4 and 9.
Cells were cultured in endothelial culture medium (ECM) as
described before,11 with 5.5 mM glucose and 10% fetal calf serum
(FCS; Lonza, Basel, Switzerland). L-α-lysophosphatidylcholine (LPC;
Sigma-Aldrich, St. Louis, MO, USA) was used at the concentration of
25µM in serumfree ECM for 24h; control cells were incubated with
the same concentration of L-α-phosphatidylcholine (PC;
Sigma-Aldrich, St. Louis, MO, USA). Oligomycin (Sigma-Aldrich, St.
Louis, MO, USA) was used at 20µg/ml concentration for 2 days in 5%
FCS ECM, control cells were incubated with DMSO. EZH2 inhibitor
GSK126 (Cellagen Technology, San Diego, CA, USA, C4126-2s) was used
at 1µM concentration for 4 days in ECM with 5% FCS and control
cells were incubated with DMSO. Medium was refreshed daily.Fluid
shear stress (FSS) of 20 dyne/cm2 was applied to confluent HUVEC
cultures in ECM with 5% FCS in Ibidi 0.4 µ-Luer slides with use of
the Ibidi pump system (Ibidi, Planegg/Martinsried, Germany), for 3
days, in standard cell culture conditions. Static controls were
incubated along in the same incubator and medium was refreshed
daily.Human Embryonic Kidney (HEK) cells were cultured in DMEM
(Lonza, Basel, Switzerland) supplemented with 1%
penicillin/streptomycin (Gibco/Thermo Fisher Scientific, Wiltham,
MA, USA), 2mM L-glutamine (Lonza, Basel, Switzerland) and 10%
FCS.
ROS measurement
Cells were detached with accutase (PAA Laboratories, Pasching,
Austria), collected in culture medium into FACS tubes and
centrifuged. Pellets were suspended in 500µl of 20µM solution of
2’,7’-dichlorodihydrofluorescein-diacetate (DCFH-DA; Sigma-Aldrich,
St. Louis, MO, USA) in medium. Cells were incubated at 37°C for
30min. Cells were then analyzed at FACSCaliburTM (BD Biosciences,
San Jose, CA, USA). Further data analysis was performed using
FlowJo software.
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ATP measurement
ATP levels were measured using the “ATP Bioluminescence Assay
Kit CLS II” (Roche, Basel, Switzerland), following the
manufacturer’s protocol. Cells were washed with PBS and detached
using accutase. Upon centrifugation, pellets were suspended in
medium and cells were counted. Cells were then centrifuged and
suspended at 2x106 cells/ml (oligomycin experiments) or 1x106
cells/ml (LPC experiments). One volume of each cell suspension was
combined with 9 volumes of boiling buffer (100mM Tris, 4mM EDTA,
pH=7.75). Samples were incubated for 2 min at 100°C, cooled on ice
and centrifuged at 1,000x g for 1 min. Supernatants were collected
and stored on ice. ATP standard was prepared in Milli-Q water by
tenfold serial dilution in the range of 10-4 to 10-10 M. Samples or
standard solutions were combined with luciferase solution 1:1, in
96-well flat-bottom black plates. Both ATP standard and luciferase
were part of the kit and were prepared accordingly with the
manufacturer’s protocol. Luminescence was measured with the
LuminoskanTM Ascent (Thermo Scientific, Wiltham, MA, USA). Linear
standard curves were plotted using log-transformed values and the
standard curve equations were used to determine the concentration
of ATP in the samples.
RNA isolation and qRT-PCR
Cells were washed with PBS and lysed with either TriZOL
(Invitrogen, Carlsbad, CA, USA) or RNA-Bee (Tel-TEST, Inc.,
Friendswood, TX, USA, Invitrogen, Carlsbad, CA, USA). RNA was
isolated with standard chloroform/phenol extraction and
precipicated with 2-propanol. RNA pellets were washed 2 times with
ice-cold 75% ethanol, dried, and suspended in RNAse-free water. RNA
concentrations were measured using Nanodrop (Thermo Scientific,
Wiltham, MA, USA) and cDNA was synthetized using the RevertAidTM
First Strand cDNA synthesis kit (Thermo Scientific, Wiltham, MA,
USA). Real-time qPCR was performed at the ViiA7 Real Time PCR
machine (Applied Biosystems, Foster City, CA, USA).SYBR-Green
chemistry was used (Bio-Rad, Hercules, CA, USA), with 10ng cDNA
input per reaction. Data were analyzed with the ViiA7 software.
Samples were normalized with ΔCt method, using the geometrical mean
of β-actin and GAPDH as control. Fold change in gene expression
versus control was calculated using ΔΔCt method. The following
primers were used (5’-3’; Forward, Reverse): β-actin:
CCAACCGCGAGAAGATGA, CCAGAGGCGTACAGGGATAG,
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EZH2: GCGAAGGATACAGCCTGTGCACA, AATCCAAGTCACTGGTCACCGAAC , GAPDH:
AGCCACATCGCTCAGACAC, GCCCAATACGACCAAATCC, HMOX1:
GGGTGATAGAAGAGGCCAAGA, AGCTCCTGCAACTCCTCAAA, p16:
CTTCCTGGACACGCTGGT, GCAGGTACCGTGCGACAT.
Lentiviral transduction
To produce lentiviral particles, HEK cells were transfected with
lentiviral pLKO.1 vectors containing shRNA against EZH2 (shEZH2) or
scrambled control shRNA (SCR), along with the 2nd generation
lentiviral vector plasmids pCMVΔR8.91 and pVSV-G. To this end,
EndofectinTM-Lenti (Gene Copoeia, Rockville, MD, USA, EFL-1001-01)
was used, according to the manufacturer’s guidelines. Subconfluent
HUVEC cultures were transduced 2 times at 24h intervals, using
0.45µm-filtered HEK supernatants, in ECM with 10% FCS. Every first
transduction was executed with an addition of 4 µg/ml of polybrene.
Cells were selected at day 5 post-first transduction in medium with
2µg/ml of puromycin (Invitrogen). Surviving cells were allowed to
regenerate for 24h prior to lysis at day 7 post-first
transduction.
Bioinformatical analysis
RNA-seq datasets acquired previously (Maleszewska M et al.,
submitted) were analyzed here. Details of the experimental set-up
and analysis methods were described before (ibidem). The list of
genes associated with the Gene Ontology (GO) term “Response to
oxidative stress”, GO:0006979, was obtained from the GO repository
using AmiGO2 (as of 24.09.2014).12 The list was used to retrieve
the differential gene expression data from our RNA-seq datasets,
using CummeRbund (v. 0.1.3)13 in R-studio 0.98. Network analysis
was performed using the String 9.1 database.14 Intersection of
lists of genes was performed with BioVenn15 and visualized with
Venn Diagram R-package. Data presented in Supplementary Table 2 was
obtained from Ensembl,16 using BioMart.17The visualization of the
selected ENCODE HUVEC ChIP-seq data (Broad Institute, Bernstein
lab)18 was performed with Integrative Genomics Viewer (IGV 2.3).19,
20 GEO accesors: GSM733688 (H3K27me3), GSM733673 (H3K4me3),
GSM733691 (H3K27ac), GSM1003517 (H3K9me3), GSM1003518 (EZH2).
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The 5’-UTR sequences of 1,200bp upstream of the first exon of
each analyzed gene were obtained from Ensembl (GRCh38).16 The motif
analysis was performed with Multiple Em for Motif Elicitation
(MEME, v. 4.9.1).21 The resulting significant motifs (E
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and PTK2B, encoding heme oxygenase-1, prostaglandin G/H
synthase-1 (also known as COX-1, Cyclooxygenase-1), glutathione
peroxidase-3, and protein tyrosine kinase-2β (also known as Pyk2),
respectively.
Regulation of EZH2 by changes in cellular ROS and ATP levels
We and others found that action of EZH2 can be affected by
changes in its expression.23, 24 To assess the influence of ROS on
EZH2 expression, we treated
Figure 1. Oxidative stress response genes regulated by EZH2 in
HUVEC. A – Heatmap representation of the relative expression of
genes differentially regulated in HUVEC upon knock-down of EZH2
(static-scrambled control vs. static-SH-EZH2). Log10(FPKM+1) values
are shown in the colour scale, with dark-green indicating high, and
light-green/yellow indicating low expression. B – String
9.1-derived network of the products of genes differentially
regulated in HUVEC upon knock-down of EZH2, organized into 4
clusters (colour-coded). Depicted is the evidence view, showing
different types of interactions between the products of the genes
that can be found in the literature. The lines in different colours
indicate different types of the interaction, as explained in the
Figure.
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Regulation of the oxidative stress response genes
V
HUVEC with LPC, a component of OxLDL species, that induce
oxidative stress and cause endothelial dysfunction. Treatment of
HUVEC with LPC increased intracellular ROS levels (Supplementary
Fig. 1A and B). The gene expression of ROS-responsive HMOX1 and of
EZH2 was elevated, while the expression of EZH2 target gene p16
seemed to decrease, however none of these effects reached
statistical significance (Supplementary Fig. 1C through E). High
levels of oxidative stress lead to mitochondrial dysfunction and a
decreased ATP production 25. ATP levels tended to decrease in
LPC-treated HUVEC
Figure 2. Oxidative stress response genes regulated by FSS in
HUVEC. A – Heatmap representation of the relative expression of
genes differentially regulated in HUVEC upon the exposure to FSS,
20 dyne/cm2, 72h (static-scrambled control vs. FSS-scrambled
control). Log10(FPKM+1) values are shown in the colour scale, with
dark-green indicating high, and light-green/yellow indicating low
expression. B – String 9.1-derived network of the products of genes
differentially regulated in HUVEC upon the exposure to FSS,
organized into 5 clusters (colour-coded), evidence view. The lines
in different colours indicate different types of the interaction
found for the products of these genes in literature.
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Figure 3. Identification of genes regulated by both EZH2 and
FSS. A – Venn diagram showing the numbers of genes regulated only
by the knock-down of EZH2 (24 out of 56 differentially regulated
genes in the comparison of static-scrambled control vs.
static-SH-EZH2), or by FSS only (26 out of 58 differentially
regulated genes in the comparison of static-scrambled control vs.
FSS-scrambled control), or by both (the common field including 32
genes). B – Venn diagram showing the numbers of genes that were
2-times or more upregulated by the knock-down of EZH2 only (4 out
of 8), the exposure to FSS only (3 out of 7), or by both (the
common 4). C – Heatmap representation of relative gene expression
of the 4 genes that were upregulated by both the knock-down of EZH2
and the exposure to FSS. Log10(FPKM+1) values are shown in the
colour scale, with dark-green indicating high, and
light-green/yellow indicating low expression.
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V
(Supplementary Fig. 1F). Also cells depleted of ATP by means of
oligomycin treatment (Supplementary Fig. 2A) had approximately
2-fold higher levels of EZH2 mRNA (Supplementary Fig. 2B).
Validation of HMOX1 regulation
Of the four genes upregulated by EZH2-depletion and
FSS-exposure, HMOX1 had the relatively highest expression in
control HUVEC, yet it was further upregulated by EZH2-depletion,
FSS-exposure, and both (Fig. 3C). Independent validation confirmed
that HMOX1 gene was upregulated upon the knock-down of EZH2 (Fig.
4A) and the exposure to FSS (Fig. 4B). Also the inhibition of EZH2
activity in HUVEC led to an increase in HMOX1 expression (Fig.
4C).
Chromatin landscape of HMOX1, PTGS1, GPX3 and PTK2B genes
To better understand the regulation of HMOX1, PTGS1, GPX3 and
PTK2B gene expression, we used the ENCODE ChIP-seq data from HUVEC
cells, which can be regarded as equivalent to our control condition
(i.e. to the scrambled shRNA static control cells). In the ENCODE
dataset, EZH2 was present in proximity of the gene body and 5’-UTR
of HMOX1, but the H3K27me3 was absent, likely replaced by high
levels of H3K27ac (Fig. 5A). The high level of marks associated
with active transcription (H3K4me3 and H3K27ac) and low level of
repressive marks (H3K27me3) suggests that the HMOX1 gene is
actively expressed already in control HUVEC, which corroborates the
relatively high expression of HMOX1 in our experiment in control
conditions (Fig. 3C). Interestingly, another gene with relatively
high expression in control conditions, PTGS1 (Fig. 3C), had no
H3K27me3 present in its proximity, similarly to HMOX1, despite the
presence of EZH2 peaks (Fig. 5B). On the
Figure 4. HMOX1 gene expression is regulated by EZH2 and FSS. A
– Gene expression of HMOX1 upon 7-day knock-down of EZH2 in HUVEC.
B – Gene expression of HMOX1 upon 3-day exposure to FSS, 20
dyne/cm2. C – Gene expression of HMOX1 upon treatment with EZH2
inhibitor GSK126 for 4 days. *p
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Regulation of the oxidative stress response genes
VFi
gure
5. C
hrom
atin
land
scap
e ar
ound
the
gene
bod
y an
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-UT
R o
f HM
OX
1, P
TG
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and
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enes
(A, B
C a
nd D
, res
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The
track
s dep
ict t
he C
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and
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rom
the
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OD
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ta (B
road
Inst
itute
), vi
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he e
xper
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hile
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the
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f EZH
2 or
the
hist
one
mod
ifica
tion
in q
uest
ion.
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V
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other hand, the genes with the lowest basal expression (Fig.
3C), GPX3 and PTK2B, had both EZH2 and H3K27me3 in their proximity
(Fig. 5C and D, respectively), indicative of a repressed state.
Identification of shared putative TFBSs in 5’-UTRs of HMOX1,
PTGS1, GPX3 and PTK2B
Genes regulated in response to the same stimuli are likely to
share similarities in their transcriptional control, including
common transcription factors driving their expression. The four
candidate genes identified here are regulated in response to
oxidative stress. We therefore checked whether they share any
common motifs within their 5’-UTRs (1200bp upstream of the first
exon), which could be potential transcription factor binding sites
(TFBSs). The most significantly enriched motif, motif A (Fig. 6A)
was found in the 5’-UTRs of all four genes (Fig. 6C and
Supplementary Fig. 3A) and was significantly similar to the motif
bound by the transcription factor ZNF263 (Fig. 6E). The second
identified motif, motif B (Fig. 6A), was only shared by HMOX1 and
GPX3 (Fig. 6D and Supplementary Fig. 3B), and was similar to the
motif bound by FOXP1 transcription factor (Fig. 6F).
DISCUSSION
Main findings
In this study we identify four candidate genes associated with
the response to oxidative stress, which can be regulated by the
decrease in EZH2 under FSS in endothelial cells: HMOX1, PTGS1, GPX3
and PTK2B. We further propose two candidate transcription factors
which can be involved in the regulation of these genes based on the
shared sequence motifs in the 5’-UTRs: ZNF263 and FOXP1.
Figure 6. Identification of common motifs in the 5’-UTR regions
of human HMOX1, GPX3, PTGS1 and PTK2B genes. A and B – Sequence
logos of the two significantly enriched motifs from the 5’-UTRs of
HMOX1, GPX3, PTGS1 and PTK2B. C – The localization of the motif A
within the 1200bp of 5’-UTRs of HMOX1, GPX3, PTGS1 and PTK2B,
obtained from MEME. D – The localization of the motif B within the
1200bp of 5’-UTRs of HMOX1 and GPX3, obtained from MEME. E – The
sequence logo (upper) of the ZNF263 transcription factor, the most
significantly similar to the motif A (lower), derived through
TOMTOM analysis using JASPAR database. F – The sequence logo
(upper) of the FOXP1 transcription factor, the most significantly
similar to the motif B (lower), derived through TOMTOM analysis
using JASPAR database.
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The role of HMOX1, PTGS1, GPX3 and PTK2B in oxidative stress and
endothelial cells
GPX3 is connected to the resolution of the oxidative stress, as
it is an enzyme facilitating the reaction of glutathione with ROS.
GPX3 is normally excreted to plasma and is required for the
antithrombotic properties of the endothelium.26 PTGS1, well known
as COX-1, is one of the enzymes in the prostaglandin synthesis,
converting arachidonic acid to prostaglandin H2. It is crucial for
prostacyclin (PGI2) production in endothelial cells in response to
oxidative challenge.27,28 In addition, endothelial secreted PGI2 is
important to maintain vascular homeostasis as it inhibits platelet
activation. PTK2B seems to be the least directly associated with
the oxidative stress, yet it is activated by ROS. Moreover, PTK2B
mutations are associated with hypertension in the Japanese
population,29 but little is known on its own anti-oxidant
properties. HMOX1 is an oxidative stress-responsive gene, crucial
for cellular resistance to ROS. The HMOX1 enzyme is responsible for
catabolism of heme to biliverdin, ferrous ion and carbon monoxide.
In the condition of oxidative stress, free heme can catalyze
reactions creating free radicals, and the induction of HMOX1
expression is pivotal to counteract this harmful phenomenon. This
role of HMOX1, along with the protective properties of the products
of its enzymatic reaction, is essential for cellular homeostasis,
and yields HMOX1 as an antiapoptotic and cytoprotective molecule.30
HMOX1 is also crucial for the function of endothelial cells.31, 32
The single known human case of HMOX1 deficiency demonstrated with
severe endothelial damage due to unresolved oxidative stress.33
Genetic variation in HMOX1 promoter, reflected by differential
expression of HMOX1, affects some aspects of the CVD, and directly
modulates the endothelial function.34
The repressive regulation of HMOX1, PTGS1, GPX3 and PTK2B
The HMOX1, PTGS1, GPX3 and PTK2B genes are regulated by EZH2. It
is corroborated by the fact that the depletion of EZH2 correlates
with an increase in their expression. Moreover, the ENCODE results
show that EZH2 is present around their promoter regions, and the
low-expressed GPX3 and PTK2B also bear the repressive H3K27me3
mark. The relatively higher expressed HMOX1 and PTGS1 have low
levels of H3K27me3, but high levels of H3K27ac, indicative of
active transcription. Interestingly, both of the candidate
transcription factors identified here, ZNF263 and FOXP1, are in
fact transcriptional repressors.35-37 This reinforces
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Regulation of the oxidative stress response genes
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the concept that the promoters of HMOX1, PTGS1, GPX3 and PTK2B
are regulated by repression. Next to the repressive H3K27me3 mark,
these promoters also bore the H3K4me3 mark, indicative of active
transcription. In budding yeast, H3K4me3 is found at the 5’ end of
genes, often along with the polymerase II in its active state.38 In
mammalian cells, K4me3 decorates histones 3 close to transcription
start sites, mapping right after the DNAse I hypersensitivity sites
(DHSs), and thereby marking the transition points between a gene
promoter and transcribed region.39 The ENCODE data used here showed
that the active H3K4me3 mark is present along with EZH2 and
H3K27me3 repressive mark in case of GPX3 and PTK2B It suggests that
the removal of H3K27me3 could lead to activation of transcription.
The concomitant presence of H3K4me3 (active mark) and H3K27me3
(repressive mark) is characteristic for so called bivalent domains,
regulating many developmentally important genes. It is believed to
facilitate the timely activation of their transcription during
development and differentiation. Genes regulated by bivalent
domains remain in a poised state, where the balance between
H3K27me3 and H3K4me3 can be modulated, fine-tuning the gene
expression.40 The concomitant presence of H3K4me3 and H3K27me3 at
the promoters of HMOX1, PTGS1, GPX3 and PTK2B might serve a similar
purpose, i.e. constitute the mechanism which holds these genes
poised for upregulation in case of occurrence of oxidative
stress.FSS-induced decrease in EZH2 abundance would therefore
contribute to the decrease in H3K27me3 levels at the promoters of
these genes, allowing for H3K27 acetylation and activation of
transcription of HMOX1, PTGS1, GPX3 and PTK2B, altogether resulting
in more resistant endothelial phenotype (Fig. 7).While the ENCODE
data we used can be considered equivalent to our data from the
control conditions, it might also differ because of different batch
of HUVEC cells used and differences in the cell culture routines,
including cell culture medium. Future verification of the
ENCODE-based observations, included in this chapter, in our culture
system will be required to confirm our conclusions.
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CONCLUSIONS
This study identifies a group of candidate genes of high
importance in the response of endothelial cells to FSS. The
regulation of these genes depends on EZH2 (RNA-seq data), and
likely on its epigenetic mark H3K27me3 (based on the integrated
ENCODE ChIP-seq data). Besides exploring the similarities in the
epigenetic landscape of the promoters of HMOX1, PTGS1, GPX3 and
PTK2B, we further propose two transcriptional factors, ZNF263 and
FOXP1, which could be involved in the repression of these genes,
alongside with EZH2. These findings further corroborate the role
for EZH2 in the repression of FSS-responsive genes in endothelial
cells.
Figure 7. Graphical abstract showing the proposed mechanism of
repressive regulation of HMOX1, PTGS1, GPX3 and PTK2B, and the
modulation by FSS.
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ACKNOWLEDGEMENTS
We would like to thank Dr. V. van den Boom and Prof. Dr. J.J.
Schuringa (Dept. Experimental Hematology, University Medical Center
Groningen) for the lentiviral plasmids (pLKO.1-SCR and
pLKO.1-shEZH2). The authors would like to acknowledge the financial
support of the Netherlands Institute for Regenerative Medicine
(NIRM, #FES0908; M.C.H). This work was additionally supported by
the Jan-Kornelis de Cock foundation (M.M.), the Groningen
University Institute for Drug Exploration (GUIDE; G.K. and M.C.H.)
and the ZonMW/Netherlands Organization for Scientific Research
(NWO) Innovational Research Incentive (#916.11.022; G.K.). The
authors declare no conflicts of interests.
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V
SUPPLEMENTARY MATERIAL
Supplementary Figures
Supplementary Figure 1. EZH2 expression is not regulated by
LPC-induced ROS. A – Representative histogram showing the shift in
MFI between cells treated with L-α-phopshatydilocholine (PC) and
L-α-lysophosphatydilocholine (LPC), corresponding to the increase
in the ROS levels in the cells. B – Increase in ROS levels in HUVEC
treated with LPC as compared to control conditions (PC). ***p
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VSupplementary Figure 2. Cellular ATP levels might affect the
expression of EZH2. A – The ATP concentration in control (DMSO)
cells and oligomycin-treated cells. B – Gene expression of EZH2 in
control cells and cells treated with oligomycin. N=2, error bars
depict the range.
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Regulation of the oxidative stress response genes
VSu
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Chapter V
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V