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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Role of SCL/Tal-1, GATA, and Ets transcription factor binding sites for the
regulation of Flk-1 expression during murine vascular development
Andreas Kappel, Thorsten M. Schlaeger, Ingo Flamme, Stuart H. Orkin, Werner Risau, and Georg Breier
The receptor tyrosine kinase Flk-1 is essential for embryonic blood vessel development and for tumor angiogenesis. To
identify upstream transcriptional regulators of Flk-1, the gene regulatory elements that mediate endothelium-specific
expression in mouse embryos were characterized. By mutational analysis, binding sites for SCL/Tal-1, GATA, and Ets
transcription factors located in the Flk-1
enhancer were identified as critical elements for the endothelium-specific Flk-1
gene expression in transgenic mice. cEts1, a transcription factor that is coexpressed with Flk-1 during embryonic development and tumor angiogenesis,
activated the Flk-1 promoter via 2 binding
sites. One of these sites was required
for Flk-1 promoter function in the embryonic vasculature. These results provide
the first evidence that SCL/Tal-1, GATA,
and Ets transcription factors act upstream of Flk-1 in a combinatorial fashion
to determine embryonic blood vessel formation and are key regulators not only of
the hematopoietic program, but also of
vascular development. (Blood. 2000;96:
3078-3085)
© 2000 by The American Society of Hematology
Introduction
The formation of a functional vascular system is a prerequisite to
fulfill the metabolic demands of the growing vertebrate embryo and
of many solid tumors. During development, a primitive vascular
system is formed by the differentiation of mesodermal precursor
cells into vascular endothelial cells, a process termed vasculogenesis.1 During the subsequent process of angiogenesis, endothelial
cells proliferate and sprout to form capillaries, which eventually
differentiate into mature blood vessels.2 Although vasculogenesis
and angiogenesis are orchestrated by a plenitude of genes, the
receptor tyrosine kinase Flk-1 plays a pivotal role in both processes.1,2 This has been demonstrated by a targeted null mutation of
Flk-1 that completely prevents the differentiation of endothelial
cells from their precursors in vivo3 and by overexpression of a
dominant-negative form of Flk-1 that inhibits tumor angiogenesis.4,5 Flk-1 is a high-affinity receptor for the endothelial cell
mitogen vascular endothelial growth factor (VEGF) and for certain
other VEGF family members.6 VEGF and its receptors Flt-1
(fms-like tyrosine kinase-1, VEGF receptor-1) and Flk-1 (fetal
liver kinase-1, VEGF receptor-2) represent the first endothelial
cell–specific signal transduction system known to be activated
during embryonic development, as the inactivation of any of these
genes leads to defective vasculogenesis.3,7-9 In addition to its
function in endothelial cell differentiation, Flk-1 is required for
primitive and definitive hematopoiesis.10 This reflects the common
origin of endothelial and blood cells from a population of
bipotential precursor cells, the hemangioblasts.1,11 The onset of
Flk-1 expression in mesodermal precursor cells is thought to mark
the establishment of the hemangioblastic lineage during embryonic
development.
Flk-1 is expressed almost exclusively on endothelial cells and
their precursors during vasculogenesis and angiogenesis.12,13 Recently, the transcriptional regulatory elements of the murine Flk-1
gene and its human homologue KDR have been cloned and
characterized in vitro14,15 and in vivo.16 So far, the transcription
factors Sp1 and hypoxia-inducible factor-2␣ (HIF-2␣) (also known
as HRF, EPAS-1, and HLF) have been identified by in vitro studies
as regulators of the promoters for Flk-1 or its human homologue,
kinase-insert domain containing receptor (KDR).16-18 Because the
Flk-1 promoter alone is not capable of directing endotheliumspecific reporter gene expression in transgenic mice,16 it is unlikely
that the endothelium specificity of Flk-1 expression is directed by
Sp1 and HIF-2␣ only. Thus, the transcriptional mechanisms that
govern the distinct expression of Flk-1 in the embryonic vasculature remain unknown.
Several transcription factors such as c-Ets1, GATA-2, SCL/
Tal-1, and HIF-2␣ are predominantly expressed in endothelial cells
or their precursors and are therefore candidate regulators of Flk-1
expression.19-25 SCL/Tal-1 is coexpressed with Flk-1 in hemangioblasts11,26 and regulates Flk-1 expression in zebrafish.27 This
functional link between SCL/Tal-1 and Flk-1 was not observed in
SCL/Tal-1⫺/⫺ mice.28 GATA-2 has been shown to regulate the
promoter activity of several endothelium-specific genes such as
platelet endothelial cell adhesion molecule-1 (PECAM-1) and
endothelin-1 in vitro29-31; however, the in vivo significance of the
GATA binding sites that were identified in these studies remained
unclear. Putative Ets binding sites are involved in the endotheliumspecific expression of the Tie-1 and Tie-2 genes.32,33 Moreover,
c-Ets1 has been shown to regulate the promoter of Flt-1 in vitro.34
From the Max-Planck-Institute for Physiological and Clinical Research, Bad
Nauheim, Germany; the Center for Molecular Medicine, University of Köln,
Köln, Germany; and the Howard Hughes Medical Institute, Harvard Medical
School, Boston, MA.
Reprints: Georg Breier, Max-Planck-Institute for Physiological and Clinical
Research, Parkstrasse 1, 61231 Bad Nauheim, Germany; e-mail:
[email protected].
Submitted January 3, 2000; accepted June 30, 2000.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported in part by the Bundesministerium für Bildung und Forschung,
Deutsche Krebshilfe, Sonderforschungsbereich 397, and the Howard Hughes
Medical Institute.
In memoriam Werner Risau (1953-1998).
3078
© 2000 by The American Society of Hematology
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
Despite the expression of Ets, GATA, or SCL/Tal-1 transcription
factors in endothelial cells or their progenitors, their role in early
murine vascular development is poorly understood. Gene targeting
experiments have revealed their function in hematopoiesis35-37 but
did not provide evidence of their involvement in embryonic blood
vessel differentiation, perhaps because of the functional redundancy of related family members. However, transgenic rescue of
the hematopoietic defects in SCL/Tal-1–deficient mice revealed
that this factor is also critical for developmental angiogenesis in the
yolk sac.28 We therefore investigated whether these transcription
factors are involved in early embryonic vascular development by
regulating the expression of Flk-1, the earliest known marker for
endothelial cells.
We have recently identified gene regulatory elements of Flk-1
that reproduced most properties of the endogenous Flk-1 expression in transgenic mice.16 The endothelial cell type specificity of
these sequences was mediated by an autonomous endotheliumspecific enhancer located in the first intron of Flk-1. In contrast, the
Flk-1 promoter was not sufficient for endothelium-specific reporter
gene expression, but rather contributed to a strong and positionindependent reporter gene expression in transgenic mice. In the
present study, we have characterized critical transcription factor
binding sites located in the Flk-1 intron enhancer. A minimal
sequence of 430 bp from the Flk-1 first intron was necessary and
sufficient for endothelium-specific reporter gene expression in
transgenic mouse embryos. Two SCL/Tal-1 motifs in this minimal
enhancer were required for high-level and uniform endothelial
expression in transgenic mice. The mutation of a GATA site
rendered the enhancer completely inactive in vivo. Analysis of
protein–DNA interactions on the Flk-1 intron enhancer demonstrated a specific binding of SCL/Tal-1 and of a GATA factor to
these sites. Moreover, cell type–specific interaction of nuclear
proteins with the putative Ets binding site that was required for the
cell-type specificity of the enhancer during embryonic development was observed. In addition, c-Ets1 activated the Flk-1 promoter via 2 previously undiscovered Ets sites, one of them being
functional during embryonic development.
Materials and methods
Plasmid construction and DNA sequence analysis
The pGL-2 (Promega, Madison, WI)-based LacZ reporter gene construct
containing the Flk-1 promoter from bp ⫺640 to bp ⫹299 relative to the
transcriptional start site was constructed as described previously.16 Deletion
fragments of the 510-bp SwaI/BamHI fragment from the Flk-1 first intron
were amplified by polymerase chain reaction (PCR), as described,15 using
the oligonucleotide enhfor as a forward primer. For amplification of the
225-bp, 321-bp, or 430-bp intron fragments (Figure 1A), the primers
⫺225rev, ⫺321rev, or ⫺430rev were used as reverse primers, respectively.
Primers used were as follows: enhfor, 5⬘-GGGGATCCTA AATGTGCTGT
CTTTAGAAGCC-3⬘; ⫺225rev, 5⬘-CCCGTCGACC CACTGACATT TCTTGTAAAGC-3⬘; ⫺321rev, 5⬘-CCCGTCGACG TGGAGTTCCT GTTTTCCTGCG-3⬘; and ⫺430rev, 5⬘-CCCGTCGACG GATTGACTTT GCCCCAGTCCC-3⬘. The PCR products were inserted into the SalI and
BamHI sites of the pGL-2–based LacZ reporter gene construct containing
the Flk-1 promoter, as described previously.16 The 324-bp intron fragment
was a BglII/BamHI fragment derived from the 510-bp SwaI/BamHI
fragment from the Flk-1 first intron and was inserted by blunt-end cloning
into the blunted BamHI site of the LacZ reporter gene construct. pKSenh
was constructed by insertion of the 430-bp intron fragment into the BamHI
and SalI sites of pBSIIKS(⫹) (Stratagene, La Jolla, CA).
Flk-1 promoter deletion fragments were amplified by PCR as described15 using the following forward primers: wt, 5⬘-GGGGTACCTT
REGULATION OF Flk-1 IN MURINE VASCULAR DEVELOPMENT
3079
Figure 1. A 430-bp fragment from the Flk-1 first intron is sufficient for endotheliumspecific reporter gene expression in transgenic mice. (A) Partial structure and
restriction-enzyme map of the murine Flk-1 locus. Exons are represented by shaded
boxes. The positions of the 939-bp promoter fragment and the 510-bp SwaI/BamHI
enhancer fragment are indicated. Abbreviations for restriction enzymes are Xh, XhoI;
Sw, SwaI; B, BamHI. (B) Structure of reporter gene constructs. The LacZ reporter
gene (blue) is flanked by a Flk-1 promoter fragment spanning bp ⫺640 to bp ⫹299
(red) and subfragments of the 510-bp SwaI/BamHI intron fragment (green). Transcription terminates at a simian virus (SV40) polyadenylation signal (yellow). (C) Structure
of intron fragments and their position in the 510-bp SwaI/BamHI intron fragment.
Fragment lengths are indicated. (D) Whole-mount LacZ-stained embryo. This
embryo is transgenic for a reporter gene construct in which the LacZ gene is under
control of the 939-bp Flk-1 promoter and the 430-bp enhancer fragment.
CTGGACCGAC CCAGCCAGG-3⬘; delets1, 5⬘-GGGGTACCCA ACCGAAATGT CTTCTAGGG-3⬘; delets2, 5⬘-GGGGTACCCC GCCCGGCACA GTTCCGGGG-3⬘; delets3, 5⬘-GGGGTACCGC GTGGGAAACC
GGGAAACCC-3⬘; delets4, 5⬘-GGGGTACCAA ACCTGGTATC CAGTGGGGG-3⬘; delets5, 5⬘-GGGGTACCGG GGGGCGTGGC CGGACGCAG-3⬘; and delets6, 5⬘-CCGGTACCAC GCAGGGAGTC CCCACCCC3⬘. The primer ⫹16rev (5⬘-GGGAAGCTTG ACTCAGGGCA
GAAAGAGAGC-3⬘) was included as a reverse primer. PCR products were
cloned into the Acc65I and HindIII sites of pGL-2 containing the luciferase
reporter gene. The PCR product generated with primers wt and ⫹16 rev was
also cloned into the Acc65I and HindIII sites of pGL-2 containing the LacZ
reporter gene, and the 430-bp intron fragment was cloned into the BamHI
and SalI sites.
To generate reporter constructs containing the thymidine kinase (TK)
promoter, the 145-bp XhoI/NcoI fragment from ptk-32 spanning the TK
promoter was inserted by blunt-end cloning into the blunted XhoI site of
pGL-2 containing either the luciferase or LacZ reporter gene cassettes to
generate pTKLuc and pTKLacZ, respectively. pTKEnh was generated by
cloning the 430-bp Flk-1 intron fragment into the SalI and BamHI sites
of pTKLacZ.
The chicken c-Ets1 cDNA expression vector was pSG5c-Ets1p68.38
Mouse c-Ets2 cDNA was amplified by PCR as described for the HIF-2␣
cDNA16 using primers ets2 5⬘ (5⬘-GGGGATCCGG CGCGATGAAT
GACTTTGG-3⬘) and ets2 3⬘ (5⬘-CCCTCGAGTC TTCTGTATCA
GGCTGG-3⬘). The PCR product was cloned into the BamHI and XhoI sites
of pcDNA3 (Invitrogen, Carlsbad, CA).
The nucleotide sequence of all constructs was determined on an Applied
Biosystems 373 automated sequencer (PE Biosystems, Weiterstadt, Germany). The search for potential transcription factor binding sites was
performed using the on-line software MatInspector (http://transfac.gbfbraunschweig.de). Site-directed mutagenesis of reporter gene constructs
was performed with the QuikChange Site-Directed Mutagenesis kit
(Stratagene).
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3080
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
KAPPEL et al
Table 2. Summary of the in vivo activity of Flk-1 enhancer fragments
Cell culture and transfection analysis
All cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. For cotransfection assays, A293 cells
were split 1:2 and transfected 18 hours later with 2.5 ␮g of DNA,
complexed with 10 ␮L of Superfect (Qiagen, Hilden, Germany). For the
analysis of luciferase constructs containing Flk-1 promoter sequences only,
1 ␮g of the luciferase reporter construct and 0.5 ␮g of pTKLacZ were
cotransfected with 1 ␮g of c-Ets1 or c-Ets2 expression vectors, respectively,
or pcDNA3 as control. For the analysis of LacZ constructs containing Flk-1
enhancer sequences, 1 ␮g of the LacZ reporter construct and 0.5 ␮g of
pTKLuc were cotransfected with 1 ␮g of GATA-2, c-Ets1, or c-Ets2
expression vectors, respectively, or pcDNA3 as control. After 24 hours,
reporter gene activities were determined as described.16 For each extract,
the reporter gene activity corresponding to the construct containing Flk-1
regulatory sequences was normalized to the activity corresponding to
pTKLuc or pTKLacZ, respectively. The normalized reporter gene activity
of the control transfections was arbitrarily set at 1. Each cotransfection
experiment was performed at least 6 times.
Nuclear extracts of bovine aortic endothelial (BAE) cells,39 TK1,40 and endothelioma cells41 were prepared as described.42,43 For the electrophoretic mobility
shift assay, 5 ␮L of e.END40 (a murine embryonic endothelioma cell line; kindly
provided by Dr Urban Deutsch) nuclear extract (approximately 10 ␮g) was
preincubated for 30 minutes on ice in 19 ␮L of buffer (20 mmol/L HEPES, pH
7.3, 0.5 mmol/L dithiothreitol, 10% glycerol, 1 ␮g poly-dIdC, 0.025% NP40)
either without or with competitor (2 pmol) or with 1.5 ␮L of anti-SCL/Tal-1
antiserum.44 After this, 1 ␮L of 32P-labeled probe (10 fmol) was added, and after
incubation for 30 minutes at room temperature, the complexes were resolved by
electrophoresis in a 5% polyacrylamide gel (29:1; 0.5 ⫻ Tris-bovate). Oligonucleotides used were as follows (only the upper strands are shown; see Table 1 for the
changes in the corresponding mutant oligonucleotides): G72-W, 5⬘-CACAGCATGA TAAAAGACAA; S232-W, 5⬘-AGATCATCAG ATGGAGGTTC;
S358-W, 5⬘-TTGTGACCAT CTGCCCATTC; SCL-W (Geneka, Montreal,
Canada), 5⬘-ACCTGAACAG ATGGTCGGCT; SCL-M, 5⬘-ACCTGAATTG
ATGGTCGGCT; and GATA-W (Santa Cruz Biotech, Santa Cruz, CA), 5⬘CACTTGATAA CAGAAAGTGA TAACTCT. In vitro DNAseI footprinting on
supercoiled DNA was performed as described45 using pKSenh as plasmid DNA,
10 to 100 ␮g of nuclear extracts or bovine serum albumin (BSA; Serva,
Heidelberg, Germany), and 10 ng of DNAseI (Amersham Pharmacia Biotech,
Uppsala, Sweden). DNAseI cleavage products were detected by PCR as
described45 using the 32P-radiolabeled primer enhfor. Parallel sequencing of
pKSenh with the radiolabeled primer enhfor was performed with the Sequenase
kit (United States Biochemical, Cleveland, OH). The products of the footprint
and sequencing reaction were detected on a sequencing gel, as described.45
Table 1. Summary of the in vivo activity of different mutated
enhancer constructs
Wild-type
sequence
Mutant
sequence
TG
EC
TG
EC
324
8
0
225
9
1
321
8
0
430
15
6
The number of embryos is indicated. Embryos were transgenic for LacZ reporter
gene constructs containing the 939-bp Flk-1 promoter spanning bp ⫺640 to bp ⫹299,
and intron fragments depicted in Figure 1C. TG indicates number of transgenic
embryos analyzed; EC, number of transgenic embryos showing endothelium-specific
reporter gene expression.
Generation and analysis of transgenic mice
The generation, genotyping, and whole-mount LacZ staining of transgenic
mouse embryos were performed as described.16,46
Results
Nuclear extract preparation, electrophoretic mobility shift
assay, and in vitro DNAseI footprint
Mutated site
enhancer
Enhancer fragment (bp)
ET
wt
wt
wt
15
6
4
GATA bp 43
AGATAC
AGgggC
10
3
3
GATA bp 72
TGATAA
TGgggA
9
0
4
GATA bp 264
GGATAC
GGgggC
9
5
2
SCL/Tal-1 bp 232
TCAGAT
TCcccT
12
7
6
SCL/Tal-1 bp 358
GCAGAT
GCcccT
11
5
5
Ets bp 308
AGGAAC
AcGcgC
10
0
3
Ets bp 260
GTCCCG
GcgcgG
13
4
0
The number of embryos is indicated. To exclude stage-specific effects, transgenic
embryos from at least 2 different stages between E10 and E12 were analyzed. Wt indicates
wild type; TG, number of transgenic embryos analyzed; EC, number of transgenic embryos
showing endothelium-specific reporter gene expression; ET, number of transgenic embryos showing ectopic reporter gene expression. Embryos were transgenic for LacZ
reporter gene constructs containing the 939-bp Flk-1 promoter and the 430-bp minimal
enhancer bearing mutations in putative transcription factor binding sites. The position of
putative transcription factor binding sites in the 430-bp enhancer is depicted in Figure 2.
A 430-bp minimal enhancer contained in the first intron of Flk-1 is
sufficient for endothelial cell–specific reporter gene expression
We have recently demonstrated that 5⬘-flanking sequences of
Flk-1 alone are not sufficient for endothelium-specific reporter
gene expression in vivo.16 However, a 510-bp SwaI/BamHI
fragment from the first intron of the Flk-1 gene conferred
endothelium-specific gene expression to the 939-bp Flk-1
promoter in transgenic mouse embryos. The intron sequences
acted as an endothelial cell–type specific autonomous enhancer.
To characterize the minimal sequences contained in the 510-bp
enhancer that are required for endothelium-specific reporter
gene expression in vivo, we created LacZ reporter constructs
containing the 939-bp Flk-1 promoter and subfragments of the
510-bp enhancer (Figure 1A,B). Transgenic mouse embryos
were generated with the reporter gene constructs and were
analyzed for LacZ expression during the critical phase of
embryonic blood vessel growth between embryonic day 10 and
day 12 (E10-E12). Among the enhancer subfragments tested
(Figure 1C), only a 430-bp fragment could reproducibly target
LacZ expression to vascular endothelial cells (Figure 1D; Table
1). Reporter gene activity was observed in blood vessels that
originated by vasculogenesis, such as the liver vasculature, and
vessels that originated by angiogenesis, such as brain vessels
(Figure 1D). The deletion of 186 bp spanning the 5⬘ end of the
430-bp fragment rendered the reporter gene construct inactive in
vivo, as did the deletion of 109 bp at the 3⬘ end of the 430-bp
fragment (Table 2), indicating a requirement of these regions for
endothelial cell–specific transcription mediated by the Flk-1
regulatory elements. Therefore, the regulatory elements sufficient for endothelium-specific reporter gene expression in
transgenic mouse embryos are contained in a 430-bp minimal
enhancer from the Flk-1 first intron.
Importance of SCL/Tal-1 and GATA transcription factor binding
sites for the in vivo function of the 430-bp Flk-1
minimal enhancer
One hallmark of the Flk-1 minimal enhancer is the presence of
multiple putative binding sites for GATA and SCL/Tal-1 transcription factors (Figure 2). These factors have been proposed to
play a role in vasculogenesis and angiogenesis21,24-28 and are
therefore candidate regulators of Flk-1 expression. To study the
function of these GATA and SCL/Tal-1 sites in Flk-1 expression,
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
REGULATION OF Flk-1 IN MURINE VASCULAR DEVELOPMENT
3081
72 of the minimal enhancer can functionally interact with
GATA-2 or other members of the GATA family.
An Ets transcription factor binding site is required for the
endothelium specificity of the Flk-1 minimal enhancer
Figure 2. Nucleotide sequence and putative transcription factor binding sites
of the 430-bp Flk-1 enhancer. Sequences matching known transcription factor
binding sites are underlined. The sequence of the 430-bp Flk-1 minimal enhancer is
deposited in GenBank (accession number AF 153058).
we mutated them individually in a LacZ reporter gene construct
(Figure 1B) containing the 939-bp Flk-1 promoter and the
430-bp minimal enhancer and tested the reporter gene constructs
in transgenic mouse embryos (Table 1). Based on microscopic
inspection of whole-mount LacZ-stained embryos, the mutation
of the SCL/Tal-1 binding motifs located at bp 232 or bp 358 of
the minimal enhancer (Figure 2) led in both cases to nonhomogeneous and reduced reporter gene activity in E11-E12 transgenic embryos (Figure 3A-D) as compared with the activity of
the wild-type construct (Figure 1D). To confirm that SCL/Tal-1
interacts with these sites in endothelial cells, we performed
electrophoretic mobility shift assays using nuclear extracts from
a mouse endothelioma cell line. Complexes formed when
nuclear extracts were incubated with oligonucleotides spanning
SCL/Tal-1 sites at bp 232 or at bp 358 (Figure 4A,B). Complex
formation was mediated by the SCL/Tal-1 sites because it could
be inhibited by an excess of the wild-type but not the mutant
oligonucleotides. Competition was also observed with a wildtype but not a mutant consensus SCL-binding site. Finally, when
the binding reactions were incubated with an antiserum specific
for mouse SCL/Tal-1, the predominant complex (Figure 4A,B;
arrowheads) was supershifted (Figure 4A,B; asterisk). This
indicates that SCL/Tal-1, by interacting with the SCL/Tal-1 sites
in the Flk-1 minimal enhancer, contributes to a strong and
uniform vascular reporter gene expression at least in midgestation mouse embryos. However, the frequency of transgenic
embryos expressing LacZ in the vasculature and the endothelium specificity of reporter gene expression were not altered by
any of the SCL/Tal-1 mutations (Table 1).
Mutation of the GATA sites located at bp 43 and bp 264
(Figure 2; Table 1) did not alter the reporter gene expression
level or frequency (Figure 3E,H; Table 1). Hence, these sites are
not required for the in vivo activity of the minimal enhancer at
the developmental stages examined (E10-E12). In contrast,
mutation of the GATA site located at bp 72 (Figure 2) led to a
complete loss of the endothelium specificity of the minimal
enhancer in vivo, as only a weak and ectopic reporter gene
expression was observed (Figure 3F,G; Table 1). When nuclear
extracts prepared from endothelioma cells were incubated with
an oligonucleotide spanning this GATA motif, a complex was
observed whose formation was inhibited by oligonucleotides
containing a GATA consensus motif but not by an oligonucleotide containing the mutated GATA sequence (Figure 4C).
Moreover, a specific shift was observed with extracts prepared
from A293 cells transfected with an expression vector encoding
GATA-2 (data not shown), a member of the GATA family that is
highly expressed in endothelial cells. Thus, the GATA site at bp
To identify additional functional transcription factor binding sites
on the Flk-1 minimal enhancer that specifically interact with
nuclear proteins from endothelial cells, we performed a DNAseI
footprint analysis of the Flk-1 minimal enhancer sequence. Nuclear
extracts from BAE cells and, as a negative control, from TK1
lymphoma cells were tested for their ability to form nucleoprotein
complexes with the 430-bp Flk-1 minimal enhancer. The only
difference in the binding pattern of both nuclear extracts was
observed on a putative Ets site located at bp 308 of the minimal
enhancer (Figure 5A). Although proteins from both extracts
protected the core sequence of this site from DNAseI digestion,
only proteins from the BAE extract induced a DNAseI-hypersensitive site adjacent to the Ets site (Figure 5A). This indicates a cell
type–specific difference in protein binding to the Ets site. To test
whether this difference reflects a functional requirement of the Ets
site for the endothelium specificity of the minimal enhancer, we
mutated the site in a LacZ reporter gene construct containing the
939-bp Flk-1 promoter and the 430-bp minimal enhancer (Figure
1B). The analysis of embryos transgenic for this construct confirmed that the Ets site is required for the endothelium-specific
Flk-1 expression, as the embryos expressed the reporter gene only
ectopically (Figure 5B,C; Table 1). In contrast, the mutation of a
putative Ets site located at bp 260 of the minimal enhancer (Figure
2) had no effect on the endothelium specificity of reporter gene
expression (Figure 5D; Table 1).
c-Ets1 is expressed in endothelial cells and activates the
promoters of several endothelium-specific genes.19,20,34,47 In
particular, c-Ets1, but also c-Ets2, activates the promoter of the
VEGF receptor tyrosine kinase Flt-1 in vitro.34 We therefore
studied whether the putative Ets site located at bp 308 of the
Figure 3. Mutational analysis of SCL/Tal-1 and GATA motifs in the Flk-1 minimal
enhancer in transgenic mouse embryos. Mutations were introduced into the
indicated transcription factor sites shown in Figure 2, as presented in Table 1. The
LacZ reporter gene is expressed under the control of the 939-bp Flk-1 promoter and
mutants of the 430-bp minimal enhancer. Different representative transgenic embryos are shown after LacZ staining. (A,B) Mutation of the SCL/Tal-1 site (bp 232).
(C,D) Mutation of the SCL/Tal-1 site (bp 358). (E) Mutation of the GATA site (bp 43).
(F,G) Mutation of the GATA site (bp 72). (H) Mutation of the GATA site (bp 264).
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3082
KAPPEL et al
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
Figure 4. Electrophoretic mobility shift assays demonstrating specific binding of SCL/Tal-1 and a GATA
factor to the Flk-1 minimal enhancer. SCL/Tal-1–
containing complexes interact with SCL/Tal-1 motifs at
bp 232 (A) and at bp 358 (B), and a GATA factor interacts
with the GATA motif at bp 72 (C) of the minimal enhancer.
Antis indicates antiserum; comp, competitor oligonucleotide; W, wild-type; M, mutant.
minimal enhancer can functionally interact with c-Ets1 or
c-Ets2. A293 cells were cotransfected with a LacZ reporter gene
construct containing the 939-bp Flk-1 promoter and the minimal
enhancer, and with expression vectors for c-Ets1 or c-Ets2,
respectively, or pcDNA3 as control. Both Ets factors increased
the reporter gene activity (Figure 6A). However, when the Flk-1
promoter sequences in the reporter gene construct were exchanged by the herpes simplex virus TK minimal promoter, the
reporter gene activity was not increased by c-Ets1 or c-Ets2 in
cotransfected A293 cells, but was decreased (Figure 6B). This
indicates that c-Ets1 or c-Ets2 cannot activate the minimal
enhancer in vitro.
Figure 5. The Ets site (bp 308) is required for the endothelium specificity of the
Flk-1 minimal enhancer. (A) DNAseI footprint. The 430-bp Flk-1 minimal enhancer
subcloned into pBluescript KS was incubated with increasing amounts (triangles) of
BSA or nuclear extracts from BAE or TK1 cells, and digested with DNAseI. The
cleavage products were analyzed as described in “Materials and methods” on a
sequencing gel. Sequencing reactions terminated at the indicated bases were loaded
in parallel. The position of the Ets site (bp 308) of the minimal enhancer is indicated.
Arrows indicate changes in the DNAseI cleaving pattern. HS indicates DNAseI
hypersensitive site. (B,C) Different LacZ-stained mouse embryos transgenic for a
construct containing the 939-bp Flk-1 promoter and the 430-bp minimal enhancer
with a mutation in the Ets site at bp 308. (D) LacZ expression in a mouse embryo
transgenic for a construct containing the 939-bp Flk-1 promoter and the 430-bp
minimal enhancer with a mutation in the Ets site at bp 260.
Ets factors regulate Flk-1 promoter activity
We next investigated whether the Flk-1 promoter can be activated
by these Ets factors. A293 cells were cotransfected with a
luciferase reporter gene construct containing the 939-bp Flk-1
promoter including the 5⬘UTR, and with c-Ets1 or c-Ets2 expression plasmids or pcDNA3 as control. Both Ets factors increased the
reporter gene activity of this construct (Figure 6C), indicating that
the 939-bp Flk-1 promoter fragment contains functional Ets
binding sites.
The analysis of this DNA sequence revealed 6 putative Ets
binding sites in the 5⬘ flanking region of Flk-1 (Figure 7A). To
determine which of these putative binding sites are functional, we
generated a series of 5⬘-end deletions of a Flk-1 promoter fragment
extending from bp ⫺640 to bp ⫹16 and fused the resulting
fragments to a luciferase reporter gene in the pGL-2 vector (Figure
7B). A293 cells were cotransfected with the promoter deletion
constructs and with a c-Ets1 expression vector or pcDNA3,
respectively. The deletion of the Ets sites E#3 and E#6 led to a
significant drop of the c-Ets1–mediated stimulation of reporter
gene activity (Figure 7B) from 13.15-fold to 6.64-fold and
5.31-fold to 3.5-fold, respectively. In contrast, deletion of the
motifs E#1, E#2, E#4, and E#5 had no significant influence on the
increase of reporter gene activity mediated by c-Ets1. To confirm
that c-Ets1 can functionally interact with the c-Ets1 motifs E#3 and
E#6, we individually mutated both sites in the Flk-1 promoter
Figure 6. The Flk-1 promoter is activated by c-Ets1 and c-Ets2. A293 cells were
cotransfected with LacZ (A,B) or luciferase (C) reporter gene constructs containing
Flk-1 gene regulatory elements and with expression vectors for c-Ets1, c-Ets2, or
pcDNA3 as control. The structures of the reporter gene constructs are shown in the
bottom panels. (A) Cotransfection with a LacZ reporter gene construct containing the
939-bp Flk-1 promoter and the 430-bp minimal enhancer. (B) Cotransfection with a
LacZ reporter gene construct containing the TK promoter and the 430-bp minimal
enhancer. (C) Cotransfection with a luciferase reporter gene construct containing the
939-bp Flk-1 promoter. Relative promoter activities were determined as described in
“Materials and methods.” The promoter activities of the control transfections were
arbitrarily set at 1. The relative induction levels shown in A and C are not comparable
because of the different stabilities of the different reporter gene products (␤galactosidase versus luciferase).
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BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
REGULATION OF Flk-1 IN MURINE VASCULAR DEVELOPMENT
3083
Figure 7. The Flk-1 promoter contains 2 functional Ets sites. (A) Nucleotide sequence and putative Ets binding sites of the Flk-1 promoter spanning bp ⫺640 to ⫹16.
Sequences matching putative Ets binding sites are underlined. The nucleotide sequence of the Flk-1 promoter spanning bp ⫺640 to bp ⫹299 is deposited in GenBank
(accession number AF 153057). (B) 5⬘-Deletion analysis of the Flk-1 promoter. Luciferase reporter gene constructs were cotransfected with either a c-Ets1 expression vector
(black bars) or pcDNA3 (gray bars) as control. Values that are significantly (P ⬍ .05, Student t test) below the corresponding values of the previous construct are marked with
an asterisk. (C) Mutational analysis of the Ets sites E#3 and E#6. Luciferase reporter gene constructs containing the 939-bp Flk-1 promoter with the wild-type sequence or
mutations in the Ets sites E#3 or E#6 (black cross) were cotransfected with either a c-Ets1 expression vector (black bars) or pcDNA3 (gray bars). The sequence 5⬘-CGGA-3⬘ of
the Ets sites E#3 or E#6 (see Figure 6) was mutated to 5⬘-CccA-3⬘. The activation of both mutant promoters by c-Ets1 was significantly lower (P ⬍ .05; Student t test) than the
activation of the wild-type promoter by c-Ets1. The promoter activities of the pcDNA3 transfections in A and B were arbitrarily set at 1, and promoter activities were determined
as described in “Materials and methods.” The relative induction levels shown in B and C are not directly comparable because the 5⬘-UTR is lacking in the constructs used for the
analysis in B.
spanning bp ⫺640 to bp ⫹299. The wild-type or mutant promoter
fragments were fused to the luciferase reporter gene in the pGL-2
vector (Figure 7C). A293 cells were cotransfected with these
reporter gene constructs and with a c-Ets1 expression vector or
pcDNA3. The 31.4-fold increase in promoter activity of the
wild-type construct by c-Ets1 dropped to 17.47-fold in the case of
the E#3 mutant and to 12.54-fold in the case of the E#6 mutant
(Figure 7C). This indicates that both the E#3 and the E#6 Ets sites
can functionally interact with c-Ets1.
To examine whether the Ets sites E#3 and E#6 contribute to the
function of the Flk-1 promoter during embryonic development, we
individually mutated both sites in a LacZ reporter gene construct
containing the Flk-1 promoter spanning bp ⫺640 to bp ⫹299 and
the 430-bp minimal enhancer. Transgenic embryos were generated
with the mutant or wild-type constructs, and the LacZ expression in
transgenic embryos was analyzed at E10.5 or E11.5 (Table 3).
When compared with the construct containing the wild-type
promoter (Figure 8A), the mutation of the Ets site E#3 resulted in a
reduced reporter gene activity in the vasculature of transgenic
embryos (Figure 8B). In contrast, mutation of the Ets site E#6 did
not alter the LacZ expression level at the developmental stages
examined (Figure 8C). These results indicate that the Ets site E#3
in the Flk-1 promoter is required for high-level endothelial gene
expression during embryonic development.
regulating early embryonic vascular development and tumor angiogenesis. Upstream regulators of Flk-1 can therefore be considered
as master regulators of vasculogenesis and angiogenesis. To
elucidate the transcriptional control mechanisms that regulate the
cell type specificity of Flk-1 expression, we have characterized the
gene regulatory elements of Flk-1 in vivo that were isolated in a
previous study.16 A 430-bp fragment of the Flk-1 intron was
sufficient to confer endothelium-specific reporter gene expression
to the 939-bp Flk-1 promoter. Deletions on both ends of this
fragment abolished the cell type–specific enhancer activity, indicating that a complex composition of transcription factor binding sites
is present within this fragment. In combination with the 939-bp
Flk-1 promoter, the 430-bp minimal enhancer could target reporter
gene expression to blood vessels that originated either by vasculogenesis or by angiogenesis. This suggests that the expression of
Flk-1 is mediated by the same regulatory elements during both
processes of blood vessel formation.
Despite the strong activity of the Flk-1 enhancer in the
endothelium of transgenic mice, in which the enhancer is integrated
into chromatin, this element exhibits only little activity in transiently transfected endothelial cells.16 It therefore appears that the
Flk-1 enhancer belongs to a class of enhancers that need to be
integrated into chromatin to exhibit their activity. Transcription
factors binding to this class of enhancers, which includes the CD34
Discussion
Flk-1 is the first endothelial receptor tyrosine kinase known to be
expressed on endothelial cell precursors and plays a central role in
Table 3. In vivo effect of Ets-site mutations in the Flk-1 promoter
Mutated site
Wild-type sequence
Mutant sequence
TG
EC
wt
wt
wt
15
6
E#3
TTCCGG
TTggGG
14
4
E#6
CCGGAC
CCccAC
13
7
The number of embryos is indicated. Embryos were transgenic for LacZ reporter
gene constructs containing the 939-bp Flk-1 promoter with mutations in the indicated
Ets sites and the 430-bp minimal enhancer. Abbreviations are shown in Table 2. The
position of the Ets sites in the Flk-1 promoter is depicted in Figure 6.
Figure 8. The Ets site E#3 is required for the activity of the Flk-1 promoter in
transgenic mouse embryos. (A) An embryo transgenic for a construct containing
the 939-bp Flk-1 promoter and the 430-bp minimal enhancer, showing a uniform and
strong vascular LacZ expression. (B,C) LacZ expression in mouse embryos transgenic for constructs containing the 939-bp Flk-1 promoter with a mutation in either the
Ets site E#3 (B) or in the Ets site E#6 (C), and the 430-bp minimal enhancer.
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3084
BLOOD, 1 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 9
KAPPEL et al
and MyoD enhancers,48,49 act by influencing the chromatin structure rather than by interacting with the basal transcription machinery, the model described for classic enhancers.50 Transcription
factors that bind to the Flk-1 enhancer are therefore expected to
cause only little, if any, increase in the Flk-1 enhancer activity in
transiently transfected cells, making it difficult to study the
transcription factor requirement of the Flk-1 enhancer by transient
cotransfections. In contrast, the in vivo analysis of mutations in
transcription factor binding sites of the Flk-1 enhancer provides a
powerful approach to study the function of transcription factors in
Flk-1 gene expression.
Two binding sites for the transcription factor SCL/Tal-1 were
identified as important elements of the 430-bp minimal enhancer
because their individual mutations resulted in a decreased reporter gene
activity and nonhomogeneous LacZ expression pattern in the vasculature of transgenic mouse embryos. These results suggest that the
function of the SCL/Tal-1 sites and their interacting factor is to increase
the transcriptional activity of Flk-1 and that the minimal enhancer is
probably active in a given endothelial cell. This hypothesis is supported
by the observations that the overexpression of SCL/Tal-1 increases the
number of Flk-1–expressing endothelial precursor cells in transgenic
zebrafish embryos and that SCL/Tal-1 specifies vascular progenitors in
zebrafish.26,27 SCL/Tal-1 has also been suggested to function during
hemangioblast differentiation in mice because SCL/Tal-1 is expressed in
both endothelial and blood cell precursors in blood islands, and in
hemangioblasts.11,25 However, although yolk sac vascularization is
defective in SCL/Tal-1⫺/⫺ mice, endothelial cell differentiation proceeds
normally.28 It has therefore been suggested that a functional redundancy
of SCL/Tal-1–related factors might explain the differentiation of endothelial cells in SCL/Tal-1⫺/⫺ mice.35,37 Our results provide the first direct
evidence that SCL/Tal-1 acts upstream of Flk-1 in higher vertebrate
embryos via the Flk-1 minimal enhancer.
The mutation of a GATA site rendered the Flk-1 minimal enhancer
completely inactive, as the mutant enhancer could not confer endothelium-specific reporter gene expression in transgenic mouse embryos. We
provide evidence that a GATA factor can functionally interact with this
GATA site in vitro. This suggests that GATA factors determine the cell
type specificity of Flk-1 expression. GATA factors were initially
identified as regulators of the hematopoietic program.35 However, the
GATA factors GATA-2, GATA-3, GATA-4, and GATA-5 are also
expressed in endothelial cells.21,51-53 In addition, GATA-2 has been
demonstrated to activate the promoters of endothelium-specific genes
for endothelin-1 and PECAM-1 in vitro.29-31 Therefore, GATA-2 has
been suggested to play a role in endothelial cell differentiation.
However, no endothelial target genes of GATA factors have been
identified in vivo. To our knowledge, our data provide the first evidence
that Flk-1 is a target gene for GATA factors during murine embryonic
vascular development.
In addition to the functional GATA site, the endothelium specificity
of the Flk-1 minimal enhancer is also determined by an Ets site. As is the
case for Flk-1, the in vivo activities of the endothelium-specific
regulatory elements of Tie-1 and Tie-2 are also dependent on Ets
sites.32,33 This suggests a common requirement for Ets sites in endothelial gene expression. Several Ets factors such as c-Ets1, NERF, and Fli-1
are highly expressed in endothelial cells19,20,54,55 and are therefore
candidate regulators of endothelial genes such as Flk-1. However, the
nature of the factor that activates the minimal enhancer via the Ets site
remains unknown.
In contrast to the minimal enhancer, the Flk-1 promoter was
activated by c-Ets1 via 2 Ets sites in vitro. However, only one of
these Ets sites was required for high-level reporter gene expression
during embryonic development. Our findings do not preclude the
possibility that the other functional Ets site may be functional
during other developmental stages or under pathologic conditions
such as tumor angiogenesis, in which c-Ets1 is highly expressed in
endothelial cells.56 c-Ets1 is coexpressed with Flk-1 in blood
islands and blood vessels during embryonic development.19,20 Our
experiments suggest that the high activity of the Flk-1 promoter in
transfected endothelial cells15 and in vivo16 is in part determined by
the 2 functional Ets sites in the Flk-1 promoter. This hypothesis is
supported by our finding that the individual mutation of both Ets
sites led to a reduction of Flk-1 promoter activity in transfected
endothelial cells (A. Kappel, unpublished observations).
c-Ets1 has also been shown to activate the promoter of Flt-134 and
the promoters of proteases that are expressed by endothelial cells during
angiogenesis, such as urokinase-type plasminogen activator and matrix
metalloproteinase-1.57 Because VEGF receptors and proteases are
required for angiogenesis, these data suggest that c-Ets1 or related
factors regulate the expression of a group of genes that convert
endothelial cells from a resting to an angiogenic phenotype.
Loss-of-function experiments failed to address a function of
c-Ets1 and GATA transcription factors in the embryonic vasculature, probably because of the redundant function of other members
of these transcription factor families expressed in endothelial
cells.35-37 This redundant expression of transcription factors with
similar functions makes it difficult to address the role of a single
family member in the regulation of a target gene by loss-offunction experiments. In contrast, the mutation of specific binding
sites for these transcription factor families in endothelial target
genes such as Flk-1 prevents the action of all family members. The
in vivo analysis of mutated transcription factor binding sites in the
regulatory elements of key regulator genes of vasculogenesis and
angiogenesis, such as Flk-1, therefore provides a valuable tool to
study the role of transcription factor families during endothelial
cell differentiation and vascular development.
Our results provide the first direct evidence that GATA, Ets, and
SCL/Tal-1 transcription factors are not only key regulators of hematopoiesis,35-37 but also specify endothelial cell differentiation and vascular
development by regulating Flk-1 expression. Hence, similar transcriptional programs might regulate the establishment of the endothelial and
blood cell lineages during embryonic development, reflecting a common origin of endothelial and blood cells.
Acknowledgments
We thank Dr Catherine Porcher for providing SCL/Tal-1 antiserum; Dr
Dietmar von der Ahe, Haemostasis Unit, Kerckhoff Klinik, Bad
Nauheim, Germany, for kindly providing pSG5c-Ets1p68; and Dr Felix
Müller-Holtkamp and Michael Walker for generating transgenic mice.
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2000 96: 3078-3085
Role of SCL/Tal-1, GATA, and Ets transcription factor binding sites for
the regulation of Flk-1 expression during murine vascular development
Andreas Kappel, Thorsten M. Schlaeger, Ingo Flamme, Stuart H. Orkin, Werner Risau and Georg
Breier
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