1272 RESEARCH ARTICLE
Development 140, 1272-1281 (2013) doi:10.1242/dev.087379
© 2013. Published by The Company of Biologists Ltd
BRG1 promotes COUP-TFII expression and venous
specification during embryonic vascular development
Reema B. Davis1,2, Carol D. Curtis1 and Courtney T. Griffin1,2,*
SUMMARY
Arteries and veins acquire distinct molecular identities prior to the onset of embryonic blood circulation, and their specification is
crucial for vascular development. The transcription factor COUP-TFII currently functions at the top of a signaling pathway governing
venous fate. It promotes venous identity by inhibiting Notch signaling and subsequent arterialization of endothelial cells, yet nothing
is known about what regulates COUP-TFII expression in veins. We now report that the chromatin-remodeling enzyme BRG1 promotes
COUP-TFII expression in venous endothelial cells during murine embryonic development. Conditional deletion of Brg1 from vascular
endothelial cells resulted in downregulated COUP-TFII expression and aberrant expression of arterial markers on veins. BRG1 promotes
COUP-TFII expression by binding conserved regulatory elements within the COUP-TFII promoter and remodeling chromatin to make
the promoter accessible to transcriptional machinery. This study provides the first description of a factor promoting COUP-TFII
expression in vascular endothelium and highlights a novel role for chromatin remodeling in venous specification.
INTRODUCTION
Arteries and veins are structurally and functionally distinct vessels
that play crucial roles in circulating blood to and from sources of
oxygen. Arteries carry blood from the heart under high pressure and
are surrounded by multiple layers of smooth muscle cells and
extracellular matrix components that provide strength and elasticity
to their vascular walls. Veins, by contrast, have thinner and less
elastic vascular walls, and rely on specialized valves to return blood
under low pressure to the heart. Arterial-venous identity is specified
during embryonic development and is influenced by hemodynamic
forces. Moreover, experimental manipulation of blood flow can alter
the fate of arteries and veins, indicating that vessel identity and
function are plastic properties (Garcia-Cardeña et al., 2001; le Noble
et al., 2004).
Despite the important roles that hemodynamic forces play in
differentiating arteries from veins, it is also clear that genetic factors
contribute significantly to their identity. Molecular markers that
distinguish arteries from veins are expressed before the onset of
blood circulation during embryonic development (Wang et al.,
1998; Zhong et al., 2000; Herzog et al., 2001). In addition, genetic
mutations can induce changes in arterial/venous marker expression
even when hemodynamic forces are unaltered (Jones et al., 2008).
The first genetic markers shown to be differentially expressed on
arteries and veins were the transmembrane ligand ephrin B2
(EFNB2) and its cognate tyrosine kinase receptor EPHB4 (Wang et
al., 1998). These markers are respectively expressed on arterial and
venous endothelial cells of the developing murine yolk sac prior to
blood flow, and their genetic ablation results in failed vascular
remodeling and embryonic lethality by embryonic day 11 (E11.0)
(Wang et al., 1998; Adams et al., 1999; Gerety et al., 1999).
1
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation,
Oklahoma City, OK 73104, USA. 2Department of Cell Biology, University of
Oklahoma Health Sciences Center, Oklahoma City, OK 73126, USA.
*Author for correspondence ([email protected])
Accepted 6 January 2013
However, EFNB2 and EPHB4 are not determinants of arterialvenous specification, as their deletion does not switch vessel identity
(Gerety et al., 1999). Rather, Notch signaling determines arterial
specification (Lawson et al., 2001; Zhong et al., 2001; Duarte et al.,
2004; Krebs et al., 2004). Loss-of-function mutations in Notch
pathway components result in downregulated Efnb2 expression in
arteries (Duarte et al., 2004; Fischer et al., 2004; Krebs et al., 2004;
Kokubo et al., 2005; Koo et al., 2005), whereas overexpression of
Notch signaling components causes veins to ectopically express
Efnb2 (Trindade et al., 2008).
Venous specification was originally thought to occur by default
in the absence of Notch signaling (Thurston and Yancopoulos,
2001). But it is now recognized that the orphan nuclear receptor
chicken ovalbumin upstream promoter-transcription factor II
(COUP-TFII, also known as NR2F2) actively promotes venous
specification by inhibiting Notch signaling in a subset of endothelial
cells (You et al., 2005; Chen et al., 2012). Conditional deletion of
COUP-TFII in embryonic endothelial cells upregulates Notch
signaling in venous endothelium and leads to aberrant arterialization
of veins. COUP-TFII currently functions at the top of the venous
specification pathway, and nothing is known about what regulates
its expression and activity in veins.
ATP-dependent chromatin-remodeling complexes modulate
expression of their target genes by altering accessibility of
transcriptional machinery to gene regulatory regions (Hargreaves
and Crabtree, 2011). Each complex contains a catalytic ATPase,
which generates energy required for altering chromatin accessibility.
Mammalian SWITCH/sucrose non-fermentable (SWI/SNF)-like
complexes contain one of two mutually exclusive ATPases: brahma
(BRM, also known as SMARCA2) or brahma-related gene 1
(BRG1, also known as SMARCA4). Conditional deletion of Brg1
has revealed its importance in a variety of developmental processes,
including embryonic vascular development (Curtis et al., 2012).
We now present evidence that the chromatin-remodeling enzyme
BRG1 epigenetically regulates COUP-TFII expression in veins
during embryonic development. BRG1 remodels chromatin within
the COUP-TFII promoter, impacting the ability of transcriptional
machinery to access the promoter. Genetic depletion of Brg1 results
DEVELOPMENT
KEY WORDS: SWI/SNF, Chromatin remodeling, Veins, Mouse, NR2F2
BRG1 and venous specification
MATERIALS AND METHODS
Mice
Brg1-floxed mice (Brg1fl/fl) (Gebuhr et al., 2003), β-catenin-floxed mice
(Catnbfl/fl) (Brault et al., 2001), Tie2-Cre+ transgenic mice (Kisanuki et al.,
2001), Efnb2LacZ mice (Wang et al., 1998) and BAT-gal transgenic mice
(Maretto et al., 2003) were maintained on a mixed genetic background at the
Oklahoma Medical Research Foundation animal facility. All animal use
protocols were approved by the Institutional Animal Care and Use
Committee.
Genotyping
PCR genotyping of Brg1-floxed, Tie2-Cre+ and BAT-gal transgenic embryos
and mice was performed as described previously (Griffin et al., 2011). βcatenin-floxed mice and embryos were PCR genotyped using the following
primers: forward (5⬘-AAGGTAGAGTGATGAAAGTTGTT-3⬘) and
reverse (5⬘-CACCATGTCCTCTGTCTATTC-3⬘). These primers amplified
a 221 bp fragment of the wild-type allele and a 324 bp fragment of the
floxed allele. The PCR was performed at an annealing temperature of 60°C.
Efnb2LacZ mice were genotyped by whole-mount X-gal staining for βgalactosidase activity on ear punches.
Primary endothelial cell isolation and maintenance
Endothelial cells from individual murine yolk sacs and embryos were
isolated using anti-PECAM-1-conjugated magnetic beads, as described
previously (Griffin et al., 2011). Primary human umbilical vein endothelial
cells (HUVECs) were isolated and maintained as previously described (Yao
et al., 1996). Primary human aortic endothelial cells (ATCC, Manassas, VA,
USA; PCS-100-011) were maintained using the Endothelial Cell Growth
Kit-BBE (ATCC; PCS 100-040) and Vascular Cell Basal Medium (ATCC;
PCS 100-030). HUVECs and primary aortic endothelial cells were passaged
no more than twice.
Cell culture and transfections
C166 yolk sac endothelial cells (ATCC; CRL-2581) were maintained and
transfected with 100 nM BRG1 siGENOME SMART pool or nonspecific
control small interfering RNA (siRNA) oligonucleotides (Dharmacon,
Lafayette, CO, USA; M-041135-01 and D-001210-01, respectively), as
described previously (Griffin et al., 2011). After 24 hours, protein was
harvested in Laemmli buffer [62.5 mM Tris (pH 6.8)/10% glycerol/5%
SDS/0.01% bromophenol blue] plus 0.2 M dithiothreitol for western
blotting. Alternatively, RNA was harvested after 24 hours in TRIzol
(Invitrogen, Grand Island, NY, USA) for transcript analysis. For the BRG1
overexpression studies, a BRG1 expression plasmid was constructed by
excising a 5338 bp murine Brg1 cDNA from IMAGE clone 30533489
(ATCC; 10698217) with SalI and XbaI, and inserting it into the multiple
cloning site (XhoI-XbaI) of the mammalian expression vector pcDNA3.1
(Invitrogen). The resulting plasmid (or empty pcDNA3.1 vector) was
transfected into C166 endothelial cells at various concentrations for 24 hours
with Lipofectamine 2000 (Invitrogen). For the COUP-TFII rescue
experiment, 200 ng of a COUP-TFII expression plasmid (pCNX-COUPTFII; obtained from Dr Sophia Tsai, Baylor College of Medicine, Houston,
TX, USA) was co-transfected with 100 nM BRG1 siRNA into C166
endothelial cells for 24 hours with Lipofectamine 2000.
Western blot
Total protein was harvested from siRNA-transfected C166 cells after
24 hours knockdown, fractionated in a 9% SDS polyacrylamide gel, and
transferred to a PVDF membrane for western blot analysis using antibodies
to BRG1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-17796),
COUP-TFII (R&D Systems, Minneapolis, MN, USA; PP-H7147-00) and
GAPDH (Sigma, St. Louis, MO, USA; 9545). Band intensity was
determined for each protein using ImageJ software (National Institutes of
Health, Bethesda, MD, USA).
Quantitative real-time PCR (qPCR)
To analyze transcript levels, total RNA from primary yolk sac or embryonic
endothelial cells or C166 siRNA-transfected cells was harvested in TRIzol
(Invitrogen), and RNA was prepared using the RNeasy Mini Kit (Qiagen,
Valencia, CA, USA) according to the manufacturer’s instructions. cDNA
was prepared using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA,
USA), and qPCR was performed using RT2 Fast SYBR Green qPCR Master
Mix (SABioscience, Valencia, CA, USA) and the CFX96 detection system
(Bio-Rad) with gene-specific primers.
qPCR primers
qPCR primers are listed in supplementary material Table S1.
qPCR analysis
The relative fold changes in transcript levels were determined using the
comparative threshold cycle (CT) method with the β-actin and Gapdh
housekeeping genes as internal controls. Data from three independent
experiments were combined and are presented as mean±s.e.m. Statistical
differences were detected using a two-tailed Student’s t-test.
Whole-mount yolk sac and placental staining
For whole-mount yolk sac immunostaining, E9.5 Brg1fl/fl:Tie2-Cre+
embryos with attached yolk sacs were dissected from maternal tissue, fixed
for 10 minutes in 3% paraformaldehyde (PFA), permeabilized, blocked and
stained as described (Griffin et al., 2011). Primary anti-PECAM-1 (1:100;
BD Biosciences, San Jose, CA, USA; 557355) and secondary donkey-antirat-Cy3 IgG (1:250; Jackson ImmunoResearch, West Grove, PA, USA;
9712-165-153) antibodies were used. The tissues were then washed and
post-fixed as described previously (Griffin et al., 2011) and transferred to
glass slides. On the glass slides, the embryos were dissected away from the
yolk sacs while maintaining the orientation of the vitelline arteries and veins
within the yolk sacs. The yolk sacs were then flat-mounted and coverslipped
with DABCO mounting media [2.5% 1,4-diazabicyclo[2.2.2]octane in 9:1
glycerol/PBS (pH 8.6)] for microscopic examination. Embryos were
simultaneously digested for genotyping.
Whole-mount X-gal staining for β-galactosidase activity was performed
on BAT-gal and Efnb2LacZ embryos attached to their yolk sacs and placentae.
Tissues were fixed in a 2% PFA/0.2% glutaraldehyde solution, washed and
X-gal stained as described (Griffin et al., 2008) for 48 hours at room
temperature. The stained yolk sacs were dissected away from embryos and
placentae and flat-mounted in DABCO mounting media on glass slides as
described above.
Immunofluorescence
Brg1fl/fl:Tie2-Cre+ and littermate controls were cryoembedded and sectioned
(10-12 µm). Double immunostaining using anti-PECAM-1 (1:500; BD
Biosciences, 553370) and anti-BRG1 (1:100; Santa Cruz Biotechnology,
SC-17796) antibodies was performed as described previously (Curtis and
Griffin, 2012). Double immunostaining for PECAM-1 and αSMA in
embryonic sections was performed as follows: cryosections were thawed
and blocked in blocking solution [3% normal donkey serum (Jackson
ImmunoResearch)/3% BSA/0.3% Triton X-100/PBS] for 2 hours at room
temperature. Sections were then co-incubated overnight at 4°C in antiPECAM-1 (1:500) and Cy3-conjugated anti-αSMA (1:200; Sigma, C6198)
diluted in blocking solution. After washing three times (3 minutes each) in
0.1% Triton X-100/PBS, sections were incubated for 1 hour at room
temperature in Alexa 488-donkey anti-rat IgG (1:500; Invitrogen) and 1:500
Hoechst (20 µg/ml) diluted in blocking solution. Finally, sections were
washed as above and mounted with DABCO mounting media and glass
coverslips. Double immunostaining for PECAM-1 and either COUP-TFII,
NRP1, DLL4 or EPHB4 was performed similarly with the following
exceptions: cryosections were blocked for 1 hour at room temperature and
co-incubated overnight at 4°C in anti-PECAM-1 (1:500) and either antiCOUP-TFII (1:100; R&D Systems, PP-H7147-00), anti-NRP1 (1:100;
R&D Systems, AF566), anti-DLL4 (1:100; R&D Systems, AF1389) or antiEPHB4 (1:100; R&D Systems, AF446) diluted in blocking solution. For
secondary antibodies, Cy3-donkey anti-rat IgG (1:500; Jackson
ImmunoResearch) and Alexa 488-goat anti-mouse IgG or Alexa 488donkey anti-goat (1:500; Invitrogen) were used.
DEVELOPMENT
in downregulated COUP-TFII and aberrant arterial marker
expression in developing veins. Our data provide important new
insight into an upstream epigenetic regulatory mechanism for the
venous specification cascade.
RESEARCH ARTICLE 1273
1274 RESEARCH ARTICLE
Development 140 (6)
Microscopy and image acquisition
Fluorescent images were obtained with a Nikon Eclipse 80i microscope
using 4× (NA 0.13), 10× (NA 0.3) and 20× (NA 0.5) objectives, an X-cite
120Q light source, and a Nikon DS-Qi1Mc camera. NIS-Elements AR3.0
(Nikon, Melville, NY, USA) software was used for all fluorescent image
acquisition and assembly. ImageJ software was used to quantify
fluorescence intensity of immunostained tissue sections. Gross yolk sac
images were obtained with a Nikon SMZ800 stereomicroscope and Nikon
DS-Fi1 camera and monitor.
Chromatin immunoprecipitation (ChIP)
ChIP qPCR primers
Primers used are listed in supplementary material Table S2.
RESULTS
Brg1fl/fl:Tie2-Cre+ mutant yolk sac veins are
morphologically abnormal
We previously showed that deletion of Brg1 from developing
endothelium using a Tie2-Cre transgene results in abnormal
angiogenesis in yolk sac vessels owing, in part, to misregulated Wnt
signaling (Griffin et al., 2011). To examine angiogenesis defects in
Brg1fl/fl:Tie2-Cre+ yolk sacs more thoroughly, we immunostained
E9.5 littermate control and Brg1fl/fl:Tie2-Cre+ yolk sacs with an
endothelial-specific (anti-PECAM-1) antibody and flat-mounted the
yolk sacs on slides while maintaining the orientation of vitelline
arteries and veins (Fig. 1). We found that angiogenesis defects were
more exaggerated in Brg1fl/fl:Tie2-Cre+ yolk sac veins than in
arteries (Fig. 1B). Brg1fl/fl:Tie2-Cre+ yolk sac veins were poorly
remodeled, and many veins were thin, blunt-ended or failed to
interconnect (Fig. 1D). This venous phenotype was unexpected as
BRG1 is expressed in both arterial and venous endothelial cells and
is excised efficiently from arteries and veins in Brg1fl/fl:Tie2-Cre+
extra-embryonic vessels (supplementary material Fig. S1).
Nevertheless, our observations indicate that Brg1fl/fl:Tie2-Cre+ yolk
sac veins exhibit more phenotypic abnormalities than mutant
arteries.
Brg1fl/fl:Tie2-Cre+ veins express arterial markers
and characteristics
To examine Brg1fl/fl:Tie2-Cre+ yolk sac venous abnormalities
further, we crossed control and Brg1fl/fl:Tie2-Cre+ embryos onto an
Fig. 1. Brg1fl/fl:Tie2-Cre+ yolk sac veins are morphologically
abnormal. (A-D) Anti-PECAM1 staining on flat-mounted E9.5 yolk sacs
revealed Brg1fl/fl:Tie2-Cre+ veins were more abnormal than arteries.
Whereas the arterial sides of control and Brg1fl/fl:Tie2-Cre+ yolk sacs
contained branching networks of vessels, the venous side of Brg1fl/fl:Tie2Cre+ yolk sacs failed to undergo normal branching and development.
(C,D) Magnified views of the boxed regions in A,B, respectively, including
vitelline veins (V.V.). Arrows in D indicate Brg1fl/fl:Tie2-Cre+ veins that failed
to interconnect or underwent aberrant regression. Asterisks indicate
round, non-vascular spaces that are characteristic of failed vascular plexus
remodeling. Scale bars: 500 μm.
arterial reporter line in which LacZ is knocked into the Efnb2 locus
(Efnb2LacZ) (Wang et al., 1998). Upon whole-mount X-gal staining
of E9.5 yolk sacs, we saw Efnb2LacZ reporter activity in control and
Brg1fl/fl:Tie2-Cre+ arteries, as expected (Fig. 2A,B). However,
Brg1fl/fl:Tie2-Cre+ yolk sacs also showed ectopic Efnb2LacZ reporter
activity in veins (Fig. 2B).
We performed immunostaining on cross-sections of E9.75 control
and Brg1fl/fl:Tie2-Cre+ embryos to determine whether BRG1 also
influences arterial marker expression in the embryo proper.
Brg1fl/fl:Tie2-Cre+ cardinal vein endothelial cells aberrantly
expressed the arterial markers neuropilin 1 (NRP1) (Fig. 2D) and
DLL4 (Fig. 2F) at this developmental timepoint. Therefore, both
extra-embryonic and embryonic venous endothelial cells express
arterial markers in Brg1fl/fl:Tie2-Cre+ mutants.
To investigate whether Brg1fl/fl:Tie2-Cre+ extra-embryonic and
embryonic veins acquire other arterial characteristics, E10.5 control
and Brg1fl/fl:Tie2-Cre+ umbilical vessels were cross-sectioned and
immunostained with an antibody against the smooth muscle cell
marker alpha-smooth muscle actin (αSMA). In control sections,
αSMA-positive cells predominantly surrounded umbilical arteries
rather than umbilical veins (Fig. 3A). By contrast, Brg1fl/fl:Tie2Cre+ umbilical veins recruited αSMA-positive cells to a similar
extent as umbilical arteries (Fig. 3B). Likewise, evidence of aberrant
αSMA-positive cell recruitment to cardinal veins was evident in
E10.5 embryos (Fig. 3D). These results indicate that Brg1fl/fl:Tie2-
DEVELOPMENT
Subconfluent C166 yolk sac endothelial cells, HUVECs or primary human
aortic endothelial cells were used for ChIP with the MAGnify Chromatin
Immunoprecipitation System (Invitrogen) according to the manufacturer’s
instructions. A mixture of BRG1-specific antibodies (Millipore, 07-478;
Abcam, ab4081) was used to immunoprecipitate protein-DNA complexes.
Isotype-matched IgG antibodies (Invitrogen) were used as a negative
control. For total histone H3 ChIP, chromatin was harvested from C166
cells transfected with nonspecific or BRG1-specific siRNAs and
immunoprecipitated using the histone H3-specific and negative-control
antibodies supplied in the ChIPAb+ Histone H3 kit (Millipore, Billerica,
MA, USA; 17-10046). For RNA polymerase II ChIP, chromatin was
harvested as described for H3 ChIP using an anti-RNAPolII antibody
(Abcam, Cambridge, MA, USA; ab5408) and negative control antibodies
from the H3 kit. For H3K9Ac ChIP, chromatin was harvested as described
for H3 ChIP using an anti-histone H3K9Ac antibody (Active Motif,
Carlsbad, CA, USA; 39137), and isotype-matched IgG antibodies
(Invitrogen) were used as a negative control. Real-time quantitative PCR
was performed using RT2 Fast SYBR green qPCR master mix
(SABiosciences) and the CFX96 detection system (Bio-Rad) with specific
primers. Data from three or four independent experiments were combined
and presented as n-fold levels of enrichment over the negative
control±s.e.m. Statistical differences were detected using a two-tailed
Student’s t-test.
BRG1 and venous specification
RESEARCH ARTICLE 1275
Fig. 3. Brg1fl/fl:Tie2-Cre+ veins aberrantly recruit smooth muscle cells.
Tissues from E10.5 littermate control and Brg1fl/fl:Tie2-Cre+ mutants were
sectioned and immunostained for the endothelial cell marker PECAM1
(green), the smooth muscle cell marker α-smooth muscle actin (αSMA)
(red) and the nuclear marker Hoechst (blue). (A,B) Extra-embryonic
umbilical vessels, including umbilical veins (U.V.) and umbilical arteries
(U.A.), were sectioned and stained. In control vessels, αSMA-positive cells
predominantly accumulated around umbilical arteries (A). However,
αSMA-positive cells accumulated around both umbilical arteries and
veins in Brg1fl/fl:Tie2-Cre+ mutants (B). (C,D) Sections of embryos
containing a dorsal aorta (D.A.) and cardinal vein (C.V.) were
immunostained. αSMA-positive cells were detected around the
Brg1fl/fl:Tie2-Cre+ C.V. (arrowheads in magnified inset of D) but not around
the control C.V. (C). Scale bars: 100 μm.
Cre+ extra-embryonic and embryonic veins acquire arterial
characteristics at midgestation and represent the first evidence of a
role for BRG1 in embryonic blood vessels outside of the developing
heart (Curtis et al., 2012).
COUP-TFII expression is downregulated in BRG1deficient endothelial cells
The nuclear receptor and transcription factor COUP-TFII functions
at the top of the known venous specification signaling cascade (Lin
et al., 2011). In order to determine whether COUP-TFII is properly
expressed in Brg1fl/fl:Tie2-Cre+ veins, we immunostained extraembryonic and embryonic vessels with anti-COUP-TFII antibody.
COUP-TFII is a BRG1 target gene
To test whether BRG1 directly regulates COUP-TFII expression,
we assessed BRG1 binding to the murine COUP-TFII promoter in
endothelial cells. We used a comparative sequence alignment
program (www.DCODE.org) to identify conserved regions of the
COUP-TFII promoter in various species (Fig. 5A). We then
designed chromatin immunoprecipitation (ChIP) primers towards
two highly conserved promoter regions (−4.7 kb and −1.2 kb) and
one nonconserved promoter region (−2.5 kb) located upstream of
the COUP-TFII transcription start site (TSS). We also designed
ChIP primers immediately upstream of the TSS at −0.3 kb. Our
ChIP experiments in C166 endothelial cells showed BRG1 bound
the −0.3 kb region immediately upstream of the COUP-TFII TSS as
well as the two highly conserved regions at −1.2 kb and −4.7 kb
DEVELOPMENT
Fig. 2. Brg1fl/fl:Tie2-Cre+ veins express arterial markers. (A,B) Control
and Brg1fl/fl:Tie2-Cre+ embryos were crossed onto an Efnb2LacZ arterial
reporter line and stained with X-gal solution to reveal sites of Efnb2 (LacZ)
expression (blue). Flat-mounted E9.5 yolk sacs displayed aberrant Efnb2
expression in Brg1fl/fl;Efnb2LacZ:Tie2-Cre+ veins (B) compared with control
veins (A). (C-F) E9.75 littermate control and Brg1fl/fl:Tie2-Cre+ embryos were
cross-sectioned, and sections containing a dorsal aorta (D.A.) and cardinal
vein (C.V.) were immunostained for arterial markers. (C,D) Sections were
stained for the endothelial cell marker PECAM1 (red), the arterial marker
NRP1 (green) and the nuclear marker Hoechst (blue). NRP1 was aberrantly
upregulated on endothelial cells within Brg1fl/fl:Tie2-Cre+ cardinal veins
(arrowheads in D). (E,F) Sections were stained for PECAM1 (red), DLL4
(green) and Hoechst (blue). Arterial DLL4 was likewise upregulated on
Brg1fl/fl:Tie2-Cre+ cardinal veins (see arrowheads in F). Scale bars: 100 μm.
COUP-TFII was downregulated in endothelial cells lining E10.5
Brg1fl/fl:Tie2-Cre+ umbilical veins (Fig. 4B) and E9.75 Brg1fl/fl:Tie2Cre+ cardinal veins (Fig. 4D). To examine COUP-TFII transcript
levels in Brg1fl/fl:Tie2-Cre+ endothelial cells, we isolated RNA from
E10.5 yolk sac and embryonic endothelial cells and performed
quantitative real-time PCR (qPCR). We found COUP-TFII mRNA
levels were significantly reduced in Brg1fl/fl:Tie2-Cre+ endothelial
cells compared with control endothelial cells (Fig. 4E,F). We also
knocked down BRG1 in the C166 murine yolk sac-derived
endothelial cell line (Wang et al., 1996) and found COUP-TFII
mRNA and protein levels were significantly downregulated
(Fig. 4G,H; supplementary material Fig. S2). These expression data
indicate that COUP-TFII expression is diminished in BRG1deficient endothelial cells both in vivo and in vitro.
1276 RESEARCH ARTICLE
Development 140 (6)
Fig. 4. COUP-TFII expression is downregulated in Brg1-deficient
endothelial cells. (A-D) Cryosections of littermate control and
Brg1fl/fl:Tie2-Cre+ tissues were immunostained for the endothelial cell
marker PECAM1 (red), COUP-TFII (green) and the nuclear marker Hoechst
(blue). (A,B) Cross-sectioned E10.5 umbilical vessels were immunostained,
and although COUP-TFII was expressed in umbilical vein (U.V.) endothelial
cells in the control section (A), it was significantly diminished in
Brg1fl/fl:Tie2-Cre+ venous endothelial cells (B). U.A., umbilical artery.
(C,D) E9.75 embryos were cross-sectioned and immunostained. COUP-TFII
was expressed in endothelial cells of the cardinal vein (C.V.) in the control
section (C) but was downregulated in Brg1fl/fl:Tie2-Cre+ C.V. endothelial
cells (D). D.A., dorsal aorta. For A-D, insets show magnified views of the
boxed regions and arrowheads indicate individual endothelial cells. Scale
bars: 100 μm. (E,F) Primary endothelial cells (ECs) were isolated from E10.5
control and Brg1fl/fl:Tie2-Cre+ tissues, RNA was purified and cDNA was
synthesized. Samples from individual littermate control and Brg1fl/fl:Tie2Cre+ yolk sacs (E) or embryos (F) were processed for qPCR analysis of Brg1
and COUP-TFII expression. Data from three independent experiments
were combined and are presented as relative fold change over the
expression levels in control cells±s.em. Significant differences were
calculated using a two-tailed Student’s t-test (*P<0.05). (G,H) C166
endothelial cells were transfected with nonspecific (NS) or BRG1-specific
siRNA for 24 hours. (G) RNA was isolated, cDNA was synthesized and qPCR
for Brg1 or COUP-TFII was performed. Data from three independent
experiments were combined and are presented as relative fold change
over the expression levels in NS siRNA-treated cells±s.e.m. Significant
differences were calculated using a two-tailed Student’s t-test (*P<0.05).
(H) Protein samples were subjected to western blot analysis with
antibodies that recognize BRG1, COUP-TFII or GAPDH.
(Fig. 5B). We also assessed BRG1 binding to the COUP-TFII
promoter in human umbilical vein endothelial cells (HUVECs) and
in human aortic endothelial cells. Unlike the C166 cell line, which
BRG1 mediates chromatin remodeling at the
COUP-TFII promoter
In order to determine the functional consequence of BRG1 binding
to the COUP-TFII promoter, we knocked down BRG1 in C166
endothelial cells and performed ChIP with an antibody to total
histone H3 (H3). H3 binding correlates with nucleosome density
and indicates whether chromatin is arranged in a closed or open
conformation. In the absence of BRG1, H3 was enriched at the −0.3
kb and the −1.2 kb regions of the COUP-TFII promoter, indicating
chromatin was highly compacted in those regions (Fig. 6A).
Conversely, H3 occupancy was decreased at the −4.7 kb promoter
region upon BRG1 knockdown, implying that chromatin was
decompacted in that region (Fig. 6A). These results suggest BRG1
differentially remodels chromatin in various regions of the COUPTFII promoter. To determine whether BRG1 impacts the ability of
transcriptional machinery to bind the COUP-TFII promoter, we next
assessed the ability of RNA polymerase II (RNAPolII) to ChIP to
the COUP-TFII promoter in the absence of BRG1. We found
RNAPolII binding near the COUP-TFII TSS was significantly
decreased in BRG1 knockdown endothelial cells (Fig. 6B).
We also assessed enrichment of lysine 9 acetylation on histone
H3 (H3K9Ac) within the COUP-TFII promoter in the presence and
absence of BRG1. This covalent epigenetic mark is typically
associated with chromatin decompaction and transcriptional
activation when found close to a transcription start site (Nishida et
al., 2006). ChIP analysis revealed significantly diminished H3K9Ac
enrichment at the −0.3 kb and −1.2 kb regions of the COUP-TFII
promoter upon BRG1 knockdown in C166 endothelial cells
(supplementary material Fig. S4). This finding is even more striking
when the increased nucleosome density in these regions following
BRG1 knockdown is taken into account (Fig. 6A). It is also
consistent with the decreased RNAPolII binding we see near the
COUP-TFII TSS after BRG1 knockdown (Fig. 6B). Interestingly,
H3K9Ac is enriched at the −4.7 kb region of the COUP-TFII
promoter after BRG1 knockdown (supplementary material Fig. S4).
Considering this enrichment is seen in conjunction with chromatin
decompaction (Fig. 6A), H3K9 acetylation per nucleosome is
strikingly high. Although BRG1 has no histone acetytransferase or
deacetylase activities on its own, it can associate with or recruit coregulatory complexes containing these functions (Fry and Peterson,
2001; Trotter and Archer, 2008). Therefore, H3K9 acetylation and
deacetylation at the sites we examined within the COUP-TFII
promoter may occur simultaneously with or secondarily to
chromatin-remodeling mediated by BRG1.
Altogether, our data show BRG1 binds and remodels the COUPTFII promoter at three sites, impacting the ability of transcriptional
DEVELOPMENT
we found expressed a mixture of arterial and venous markers, these
primary cells differentially expressed COUP-TFII and other venous
and arterial markers (data not shown). We found BRG1 did not bind
to the −4.7 kb COUP-TFII promoter region in HUVECs or aortic
endothelial cells, but it bound to the −1.2 kb region in both human
cell types (supplementary material Fig. S3). Furthermore, BRG1
significantly bound the −0.3 kb promoter region in HUVECs but
not in aortic endothelial cells. Although the difference in BRG1
binding to this region between the two cell types was small, our
findings may indicate the −0.3 kb region plays an important role in
differentiating COUP-TFII expression in venous and arterial
endothelial cells. Altogether, our results demonstrate that BRG1
associates with COUP-TFII regulatory regions in human and
murine endothelial cells, and indicate that COUP-TFII is a direct
BRG1 target gene.
BRG1 and venous specification
RESEARCH ARTICLE 1277
Fig. 5. BRG1 binds to the COUP-TFII promoter in endothelial cells. (A) Alignment of the murine COUP-TFII promoter region with sequences from
zebrafish, frog, opossum, dog, chimpanzee and human genomes from the NCBI DCODE website (http://www.dcode.org). Peak heights indicate degree
of sequence homology; pink bars above peaks denote evolutionarily conserved regions; yellow represents the COUP-TFII 5⬘ untranslated region; blue
indicates COUP-TFII exon 1. Boxed regions were selected for further analysis of BRG1 binding. Numbers above boxed regions denote approximate
distances upstream of the COUP-TFII transcription start site (TSS). (B) Chromatin immunoprecipitation (ChIP) assays were performed on C166 endothelial
cells using antibodies against BRG1 or isotype-matched non-specific IgG as a negative control. DNA was isolated and amplified by qPCR to determine
whether BRG1 bound to various COUP-TFII promoter regions. Significant BRG1 binding was detected at the −0.3 kb, the −1.2 kb and the −4.7 kb
promoter regions. A region upstream of the Fzd5 promoter (Fzd5 UP) was used as a negative control BRG1-binding region, and the Adamts1 promoter
served as a positive control BRG1-binding region, as previously described (Griffin et al., 2011). Data from four independent experiments were combined
and are presented as fold enrichment over the level of ChIP with negative control IgG antibodies±s.e.m. Significant differences were calculated using a
two-tailed Student’s t-test (*P<0.05).
Wnt signaling does not contribute to extraembryonic venous specification
The highly conserved −4.7 kb COUP-TFII promoter region we
found bound by BRG1 in C166 endothelial cells (Fig. 5B) has
previously been shown to be bound by the central Wnt signaling
mediator β-catenin in preadipocytes (Okamura et al., 2009).
Because BRG1 promotes Wnt signaling and directly co-activates
certain Wnt target genes in yolk sac vasculature (Griffin et al.,
2011), we questioned whether BRG1 and β-catenin co-activate
COUP-TFII expression in extra-embryonic veins. Using a Wnt
signaling reporter transgenic mouse line (BAT-gal) (Maretto et al.,
2003), we found yolk sac vitelline veins and umbilical veins had
more reporter activity than vitelline and umbilical arteries
(supplementary material Fig. S5A-D). This unexpected finding
suggests that Wnt signaling plays an important role in extraembryonic veins. However, when we crossed vascular β-catenin
loss-of-function mice (Catnbfl/fl:Tie2-Cre+) onto the arterial
Efnb2LacZ reporter line, Catnbfl/fl:Tie2-Cre+ vitelline veins did not
show the same aberrant reporter activity as seen in Brg1fl/fl:Tie2Cre+ yolk sac veins (compare supplementary material Fig. S5F and
Fig. 2B). Furthermore, cross-sections of E10.5 control and
Catnbfl/fl:Tie2-Cre+ umbilical veins showed comparable levels of
COUP-TFII expression upon immunostaining (supplementary
material Fig. S5G,H). Finally, qPCR analysis revealed COUP-TFII
mRNA levels were not significantly changed in β-catenin
knockdown C166 endothelial cells compared with control cells
(supplementary material Fig. S5I). Our combined results indicate
Wnt signaling is not crucial for venous specification or COUP-TFII
expression in extra-embryonic veins, so the role of elevated Wnt
signaling in these vessels remains unknown.
Notch pathway genes are upregulated and Ephb4
is downregulated in Brg1 mutant endothelial cells
COUP-TFII inhibits multiple components of the Notch signaling
pathway to promote venous specification in endothelial cells (You
et al., 2005; Chen et al., 2012). Nrp1 and Foxc1, two upstream
mediators of Notch signaling, and Hey2, a downstream effector of
Notch signaling, are directly targeted by COUP-TFII in human
umbilical vein endothelial cells (HUVECs) (Chen et al., 2012). In
order to determine whether these and other components of the Notch
signaling pathway are impacted by Brg1 deletion, we performed
qPCR on primary endothelial cells isolated from E10.5 control and
Brg1fl/fl:Tie2-Cre+ embryos. As predicted, we saw significant
upregulation of the Notch pathway genes and arterial markers Hey1,
Dll4, Hey2 and Foxc1 (Fig. 7A). We did not see transcriptional
changes in the upstream Notch signaling mediator and COUP-TFII
target gene Nrp1, but this finding is consistent with published
findings from COUP-TFII knockdown HUVECs (Chen et al.,
2012). Therefore, deletion of Brg1 results in upregulation of
multiple arterial Notch signaling pathway genes, as would be
predicted in endothelial cells with downregulated COUP-TFII.
As arterial-venous malformations (AVMs) are associated with
misregulated vascular Notch signaling (Lawson et al., 2001; Duarte
et al., 2004; Krebs et al., 2004; Carlson et al., 2005; Kim et al., 2008;
Krebs et al., 2010), we also examined Brg1fl/fl:Tie2-Cre+ mutants
for abnormal arterial-venous fusion events. We found no evidence
of AVMs in serial sections of Brg1fl/fl:Tie2-Cre+ mutants. However,
Coup-TFIIfl/fl:Tie2-Cre+ mutants likewise show no evidence of
arterial-venous fusions (You et al., 2005), so the lack of AVMs in
our Brg1fl/fl:Tie2-Cre+ mutants is not unexpected.
We also analyzed transcriptional changes in the venous marker
Ephb4. Ephb4 was significantly downregulated in Brg1fl/fl:Tie2-
DEVELOPMENT
machinery to access the TSS (Fig. 6C). In the presence of BRG1, the
−1.2 kb and −0.3 kb sites are decompacted, allowing RNAPolII to
bind the promoter and activate transcription of COUP-TFII.
Conversely, BRG1 appears to facilitate the compaction of the −4.7
kb COUP-TFII promoter site. We speculate that BRG1 may act at
this region to prevent a repressor protein from binding and inhibiting
COUP-TFII expression in venous endothelial cells.
Fig. 6. BRG1 remodels chromatin at the COUP-TFII promoter and
influences accessibility of transcriptional machinery. (A,B) C166
endothelial cells were transfected with nonspecific (NS) or BRG1-specific
siRNA for 24 hours prior to processing for ChIP assays. (A) ChIP with an
antibody against total histone H3 was used to determine nucleosome
density at various regions of the COUP-TFII promoter. Nucleosome density
was significantly decreased at the −4.7 kb region of the COUP-TFII
promoter but was significantly increased at the −1.2 kb and −0.3 kb
promoter regions following BRG1 knockdown. (B) ChIP with an antibody
against RNA polymerase II (RNAPolII) indicated its ability to bind the −0.3
kb region of the COUP-TFII promoter was significantly decreased
following BRG1 knockdown. For A and B, a region upstream of the Fzd5
promoter (Fzd5 UP) was used as a negative control region, and the
Adamts1 promoter served as a positive control region for BRG1-induced
changes in nucleosome density or RNAPolII binding, respectively. Data
from three independent experiments were combined and are presented
as fold enrichment over the levels of ChIP with the H3 or RNAPolII
antibodies in NS siRNA transfected cells±s.e.m. Significant differences
were calculated using a two-tailed Student’s t test (*P<0.05). (C) Model of
how BRG1 epigenetically promotes COUP-TFII expression. In wild-type
endothelial cells, BRG1 binds the −4.7 kb region of the COUP-TFII
promoter, where it mediates chromatin compaction. BRG1 also binds the
−1.2 kb and −0.3 kb regions of the promoter, where it mediates
chromatin decondensation, thereby allowing binding of RNAPolII close to
the COUP-TFII transcription start site. These events promote the
expression of COUP-TFII in wild-type cells. By contrast, in Brg1fl/fl:Tie2-Cre+
endothelial cells, which lack BRG1, the −4.7 kb COUP-TFII promoter region
undergoes chromatin decondensation, potentially allowing for binding of
a transcriptional repressor protein. Likewise, the −1.2 kb and −0.3 kb
regions of the promoter undergo chromatin compaction, thereby
inhibiting efficient RNAPolII binding and diminishing COUP-TFII
expression.
Cre+ endothelial cells (Fig. 7A) and in BRG1 knockdown C166
cells (supplementary material Fig. S6A). EPHB4 protein levels were
likewise diminished in Brg1fl/fl:Tie2-Cre+ cardinal vein endothelial
Development 140 (6)
cells (supplementary material Fig. S6B,C). These results are
reminiscent of the decreased Ephb4 transcripts seen in COUP-TFII
knockdown HUVECs and the decreased EPHB4 expression seen
in COUP-TFIIfl/fl:Tie2-Cre+ embryonic veins (You et al., 2005;
Chen et al., 2012). We also analyzed expression of the venous
marker Nrp2, which is a direct COUP-TFII target gene in
developing lymphatic vessels (Lin et al., 2010), but we saw no
evidence of its downregulation in E10.5 Brg1fl/fl:Tie2-Cre+
endothelial cells (Fig. 7A). Nevertheless, the downregulation of
Ephb4 seen in Brg1-deficient endothelial cells suggested that BRG1
promotes venous specification at midgestation.
To further substantiate our evidence that BRG1 promotes venous
specification, we overexpressed BRG1 in C166 endothelial cells
and assessed transcription of the venous markers COUP-TFII and
Ephb4. Both genes were upregulated with increasing expression of
exogenous BRG1 (Fig. 7B). Together with our evidence from lossof-function experiments showing that COUP-TFII and Ephb4
expression are downregulated in Brg1-deficient endothelial cells,
these gain-of-function experiments support our conclusion that
BRG1 promotes venous specification in vascular endothelium.
The most downstream effector of Notch signaling that is directly
inhibited by COUP-TFII is the Notch target gene and transcription
factor Hey2 (Chen et al., 2012). HEY2 may play a key role in
venous specification as it mediates inhibition of Ephb4 expression
in developing zebrafish veins (Zhong et al., 2001). In addition to
seeing Hey2 transcription upregulated in primary E10.5
Brg1fl/fl:Tie2-Cre+ endothelial cells (Fig. 7A), we also saw Hey2
significantly upregulated in BRG1 knockdown C166 endothelial
cells (Fig. 7C). This upregulation of Hey2 was presumably
secondary to decreased COUP-TFII expression as exogenous
COUP-TFII significantly reduced the aberrant Hey2 expression seen
in BRG1 knockdown cells (Fig. 7C). Our findings corroborate the
current model of how COUP-TFII inhibits Notch signaling to
promote venous specification and add new insight into epigenetic
mechanisms for regulating COUP-TFII expression in veins
(Fig. 7D).
DISCUSSION
COUP-TFII promotes venous specification by suppressing Notch
signaling in endothelial cells (You et al., 2005), but the mechanism
by which it does so has been unclear. Recent work by Chen et al.
shows COUP-TFII directly inhibits transcription of two upstream
mediators of Notch signaling: the transcription factor Foxc1 and
the VEGF co-receptor Nrp1 (Chen et al., 2012). FOXC1 promotes
expression of the Notch ligand Dll4 (Hayashi and Kume, 2008),
and NRP1 mediates VEGF signaling along with VEGFR2 to
promote Notch signaling (Lawson et al., 2002; Swift and
Weinstein, 2009). Therefore COUP-TFII-mediated downregulation
of Foxc1 and Nrp1 suppresses Notch signaling in veins. In
addition, COUP-TFII directly inhibits the Notch target gene Hey2
in venous endothelial cells (Chen et al., 2012). Several pieces of
evidence indicate HEY2 plays a crucial role in arterial
specification. First, when Hey2 and the paralogous gene Hey1 are
simultaneously targeted for deletion in mice, the arterial marker
Efnb2 is significantly reduced in embryonic arteries (Fischer et al.,
2004). Likewise, Nrp1 is reduced in Hey1/Hey2-deficient arteries,
indicating that HEY2 may feed back to regulate upstream
mediators of Notch signaling (Fischer et al., 2004). Furthermore,
HEY2 activates an artery-specific gene expression program when
ectopically expressed in veins (Chi et al., 2003). Altogether,
COUP-TFII-mediated inhibition of Hey2, Foxc1 and Nrp1
profoundly
impacts
arterial/venous
specification
by
DEVELOPMENT
1278 RESEARCH ARTICLE
BRG1 and venous specification
RESEARCH ARTICLE 1279
downregulating Notch signaling in endothelial cells. Despite its
important influence on vessel identity, however, nothing is known
about what lies upstream of COUP-TFII and regulates its
expression in veins. In this study, we present evidence that the
chromatin-remodeling enzyme BRG1 directly promotes expression
of COUP-TFII in venous endothelial cells by remodeling the
COUP-TFII promoter to make it accessible to transcriptional
machinery.
As BRG1 is not a transcription factor, it cannot be the only
regulator of COUP-TFII expression in veins. Indeed, loss of BRG1
did not eliminate COUP-TFII expression completely from primary
or cultured endothelial cells. Moreover, we found BRG1 expressed
in both arteries and veins in vivo, whereas COUP-TFII expression
is restricted to veins (You et al., 2005). Both of these points indicate
that a venous-specific transcription factor or combination of
transcription factors co-regulates COUP-TFII expression with the
help of BRG1. We initially thought Wnt signaling transcription
factors were ideal venous-specific candidates for promoting COUP-
TFII expression in extra-embryonic veins. This hypothesis was
based on our observation that Wnt signaling reporter activity
occurred preferentially in yolk sac and umbilical veins rather than
arteries. It was also based on a published report that Wnt signaling
promotes COUP-TFII expression in preadipocytes (Okamura et al.,
2009). Further support for this hypothesis came from our finding
that BRG1 co-activates expression of certain Wnt target genes in
extra-embryonic vasculature (Griffin et al., 2011). However, we
found no evidence that the central Wnt signaling component βcatenin is required for extra-embryonic COUP-TFII expression or
venous specification. Instead, Wnt signaling appears to play a more
crucial role in promoting arterial specification at midgestation
(Corada et al., 2010). Therefore, the role of the prominent Wnt
signaling we detected in extra-embryonic veins remains unknown,
and the venous-specific transcription factors that work with BRG1
to promote COUP-TFII expression still require identification. The
BRG1-binding sites that we identified within the COUP-TFII
promoter could serve as starting points in experimental searches for
DEVELOPMENT
Fig. 7. BRG1 impacts expression of genes downstream of COUP-TFII signaling. (A) Primary endothelial cells (ECs) were isolated from E10.5 control
and Brg1fl/fl:Tie2-Cre+ embryos, RNA was purified, and cDNA was synthesized. Expression levels of Brg1, the arterial markers (red) Nrp1, Hey1, Dll4, Hey2
and Foxc1, and the venous markers (blue) COUP-TFII, Ephb4 and Nrp2 were measured by qPCR. Data from three independent experiments were
combined and are presented as relative fold change over the normalized expression level of each gene in control cells (dotted line) ±s.e.m. Significant
differences were calculated using a two-tailed Student’s t-test (*P<0.05). (B) C166 cells were transfected with increasing amounts (0.02 ng, 0.2 ng, and 2
ng) of empty vector or comparable amounts of a BRG1 expression plasmid for 24 hours. RNA was isolated, cDNA was synthesized and qPCR for Brg1,
COUP-TFII and Ephb4 was performed. Data from three independent experiments were combined and are presented as relative fold change over the
normalized expression level of each gene in cells transfected with corresponding amounts of empty vector (dotted line). Bars represent ±s.e.m.;
significant differences were calculated using a two-tailed Student’s t-test (*P<0.05). (C) C166 endothelial cells were transfected with nonspecific (NS)
siRNA, BRG1-specific siRNA or BRG1 siRNA plus a COUP-TFII expression plasmid for 24 hours. RNA was isolated, cDNA was synthesized and qPCR for Hey2
was performed. Bars represent ±s.e.m. from three independent experiments; significant differences were calculated using a two-tailed Student’s t-test
(*P<0.05). (D) Model of how BRG1 impacts venous specification. In arterial endothelial cells (ECs), Notch signaling promotes expression of arterial
markers such as Ephrin B2. In venous ECs, BRG1 epigenetically promotes expression of COUP-TFII, presumably in cooperation with an unknown venousspecific co-regulatory protein or transcription factor. COUP-TFII directly inhibits Nrp1 and Foxc1, two upstream mediators of the Notch signaling
pathway. COUP-TFII also directly inhibits the downstream Notch effector Hey2 (Chen et al., 2012). As a result of COUP-TFII-mediated Notch pathway
inhibition, arterial marker expression is suppressed and the venous marker EPHB4 is expressed.
venous-specific transcription factors that regulate COUP-TFII
expression. Alternatively, the BRG1-binding sites may serve as
guideposts for identifying arterial-specific inhibitors of COUP-TFII,
as we found BRG1 binding to be mostly similar in human arterial
and venous endothelial cells.
BRG1-containing SWI/SNF complexes exercise temporally and
spatially specific control over target genes during developmental
processes (Ho and Crabtree, 2010). Therefore, one outstanding issue
is whether BRG1 differentially impacts the timing and location of
COUP-TFII expression in developing vasculature. This research
article demonstrates that BRG1 promotes venous COUP-TFII
expression during early stages of vascular development, but it is not
clear whether BRG1 is required for COUP-TFII maintenance on
more mature veins, as Brg1fl/fl:Tie2-Cre+ embryos die from anemia
by E11.0 (Griffin et al., 2008). To address this issue, vascular Brg1
excision must be induced at later stages of embryonic development.
Later induction of Brg1 excision will also help determine whether
BRG1 plays crucial roles in lymphatic specification and tumor
angiogenesis – two processes for which COUP-TFII expression and
maintenance are required (Lin et al., 2010; Qin et al., 2010;
Srinivasan et al., 2010).
In conclusion, our data indicate that BRG1 promotes venous
specification through COUP-TFII induction during vascular
development. This study delineates a novel role for chromatin
remodeling activity in the regulation of blood vessel identity and
broadens our understanding of how epigenetic processes influence
vascular development.
Acknowledgements
We thank Mr Vijay Muthukumar for technical assistance and mouse colony
maintenance, and Griffin lab members for critiquing this manuscript. We also
thank Dr Hendra Setiadi for HUVECs and Dr Sophia Tsai for the COUP-TFII
expression plasmid, and for sharing pre-published results with us.
Funding
This work was supported by grants from the National Institutes of Health to
C.T.G. [R00HL087621, P20GM103441 and R01HL111178]. Deposited in PMC
for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.087379/-/DC1
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DEVELOPMENT
BRG1 and venous specification