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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
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.
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. c-Ets1, 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) 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 Several transcription factors such as c-Ets1, GATA-2, SCL/Tal-1, and
HIF-2 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 endothelium-specific 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 position-independent 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.
Plasmid construction and DNA sequence analysis
Flk-1 promoter deletion fragments were amplified by PCR as
described15 using the following forward primers: wt,
5'-GGGGTACCTT 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
CCCACCCC-3'. 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 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.gbf-braunschweig.de). Site-directed mutagenesis of
reporter gene constructs was performed with the QuikChange
Site-Directed Mutagenesis kit (Stratagene).
Cell culture and transfection analysis
Nuclear extract preparation, electrophoretic mobility shift assay,
and in vitro DNAseI footprint
Generation and analysis of transgenic mice The generation, genotyping, and whole-mount LacZ staining of transgenic mouse embryos were performed as described.16,46
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, 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 wild-type 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 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 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 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.
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
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
Flk-1 is the first endothelial receptor tyrosine kinase known to be expressed on endothelial cell precursors and plays a central role in 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 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 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-of-function 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.
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.
Submitted January 3, 2000; accepted June 30, 2000.
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).
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.
Reprints: Georg Breier, Max-Planck-Institute for Physiological and Clinical Research, Parkstrasse 1, 61231 Bad Nauheim, Germany; e-mail: g.breier{at}kerckhoff.mpg.de.
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 M. Band, I. Shams, A. Joel, and A. Avivi Cloning and in vivo expression of vascular endothelial growth factor receptor 2 (Flk1) in the naturally hypoxia-tolerant subterranean mole rat FASEB J, January 1, 2008; 22(1): 105 - 112. [Abstract] [Full Text] [PDF]
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M. Khandekar, W. Brandt, Y. Zhou, S. Dagenais, T. W. Glover, N. Suzuki, R. Shimizu, M. Yamamoto, K.-C. Lim, and J. D. Engel A Gata2 intronic enhancer confers its pan-endothelia-specific regulation Development, May 1, 2007; 134(9): 1703 - 1712. [Abstract] [Full Text] [PDF]
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L. J. Patterson, M. Gering, C. E. Eckfeldt, A. R. Green, C. M. Verfaillie, S. C. Ekker, and R. Patient The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrafish Blood, March 15, 2007; 109(6): 2389 - 2398. [Abstract] [Full Text] [PDF]
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Y. Murakami, S. Yamagoe, K. Noguchi, Y. Takebe, N. Takahashi, Y. Uehara, and H. Fukazawa Ets-1-dependent Expression of Vascular Endothelial Growth Factor Receptors Is Activated by Latency-associated Nuclear Antigen of Kaposi's Sarcoma-associated Herpesvirus through Interaction with Daxx J. Biol. Chem., September 22, 2006; 281(38): 28113 - 28121. [Abstract] [Full Text] [PDF]
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J. E. Pimanda, W.Y. I. Chan, I. J. Donaldson, M. Bowen, A. R. Green, and B. Gottgens Endoglin expression in the endothelium is regulated by Fli-1, Erg, and Elf-1 acting on the promoter and a -8-kb enhancer Blood, June 15, 2006; 107(12): 4737 - 4745. [Abstract] [Full Text] [PDF]
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A. H. Licht, F. Muller-Holtkamp, I. Flamme, and G. Breier Inhibition of hypoxia-inducible factor activity in endothelial cells disrupts embryonic cardiovascular development Blood, January 15, 2006; 107(2): 584 - 590. [Abstract] [Full Text] [PDF]
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M. Ema, S. Takahashi, and J. Rossant Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors Blood, January 1, 2006; 107(1): 111 - 117. [Abstract] [Full Text] [PDF]
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J.-R. Landry, S. Kinston, K. Knezevic, I. J. Donaldson, A. R. Green, and B. Gottgens Fli1, Elf1, and Ets1 regulate the proximal promoter of the LMO2 gene in endothelial cells Blood, October 15, 2005; 106(8): 2680 - 2687. [Abstract] [Full Text] [PDF]
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H. Hirai, I. M Samokhvalov, T. Fujimoto, S. Nishikawa, J. Imanishi, and S.-I. Nishikawa Involvement of Runx1 in the down-regulation of fetal liver kinase-1 expression during transition of endothelial cells to hematopoietic cells Blood, September 15, 2005; 106(6): 1948 - 1955. [Abstract] [Full Text] [PDF]
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A. Hegen, S. Koidl, K. Weindel, D. Marme, H. G. Augustin, and U. Fiedler Expression of Angiopoietin-2 in Endothelial Cells Is Controlled by Positive and Negative Regulatory Promoter Elements Arterioscler Thromb Vasc Biol, October 1, 2004; 24(10): 1803 - 1809. [Abstract] [Full Text] [PDF]
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X. Wang, Y. Peng, Y. Ma, and N. Jahroudi Histone H1-like protein participates in endothelial cell-specific activation of the von Willebrand factor promoter Blood, September 15, 2004; 104(6): 1725 - 1732. [Abstract] [Full Text] [PDF]
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N. Petrovic, S. V. Bhagwat, W. J. Ratzan, M. C. Ostrowski, and L. H. Shapiro CD13/APN Transcription Is Induced by RAS/MAPK-mediated Phosphorylation of Ets-2 in Activated Endothelial Cells J. Biol. Chem., December 5, 2003; 278(49): 49358 - 49368. [Abstract] [Full Text] [PDF]
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N. Minegishi, N. Suzuki, T. Yokomizo, X. Pan, T. Fujimoto, S. Takahashi, T. Hara, A. Miyajima, S.-i. Nishikawa, and M. Yamamoto Expression and domain-specific function of GATA-2 during differentiation of the hematopoietic precursor cells in midgestation mouse embryos Blood, August 1, 2003; 102(3): 896 - 905. [Abstract] [Full Text] [PDF]
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J. B. Rance, G. A. Follows, P. N. Cockerill, C. Bonifer, D. A. Lane, and R. E. Simmonds Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites Blood, June 1, 2003; 101(11): 4393 - 4401. [Abstract] [Full Text] [PDF]
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H. Lie-Venema, A. C. Gittenberger-de Groot, L. J.P. van Empel, M. J. Boot, H. Kerkdijk, E. de Kant, and M. C. DeRuiter Ets-1 and Ets-2 Transcription Factors Are Essential for Normal Coronary and Myocardial Development in Chicken Embryos Circ. Res., April 18, 2003; 92(7): 749 - 756. [Abstract] [Full Text] [PDF]
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N. Tomita, R. Morishita, Y. Taniyama, H. Koike, M. Aoki, H. Shimizu, K. Matsumoto, T. Nakamura, Y. Kaneda, and T. Ogihara Angiogenic Property of Hepatocyte Growth Factor Is Dependent on Upregulation of Essential Transcription Factor for Angiogenesis, ets-1 Circulation, March 18, 2003; 107(10): 1411 - 1417. [Abstract] [Full Text] [PDF]
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Y. Peng and N. Jahroudi The NFY Transcription Factor Inhibits von Willebrand Factor Promoter Activation in Non-endothelial Cells through Recruitment of Histone Deacetylases J. Biol. Chem., February 28, 2003; 278(10): 8385 - 8394. [Abstract] [Full Text] [PDF]
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G. Elvert, A. Kappel, R. Heidenreich, U. Englmeier, S. Lanz, T. Acker, M. Rauter, K. Plate, M. Sieweke, G. Breier, et al. Cooperative Interaction of Hypoxia-inducible Factor-2alpha (HIF-2alpha ) and Ets-1 in the Transcriptional Activation of Vascular Endothelial Growth Factor Receptor-2 (Flk-1) J. Biol. Chem., February 21, 2003; 278(9): 7520 - 7530. [Abstract] [Full Text] [PDF]
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H. Hirai, M. Ogawa, N. Suzuki, M. Yamamoto, G. Breier, O. Mazda, J. Imanishi, and S.-I. Nishikawa Hemogenic and nonhemogenic endothelium can be distinguished by the activity of fetal liver kinase (Flk)-1 promoter/enhancer during mouse embryogenesis Blood, February 1, 2003; 101(3): 886 - 893. [Abstract] [Full Text] [PDF]
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S. Pikkarainen, H. Tokola, R. Kerkela, T. Majalahti-Palviainen, O. Vuolteenaho, and H. Ruskoaho Endothelin-1-specific Activation of B-type Natriuretic Peptide Gene via p38 Mitogen-activated Protein Kinase and Nuclear ETS Factors J. Biol. Chem., January 31, 2003; 278(6): 3969 - 3975. [Abstract] [Full Text] [PDF]
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L. Hadri, A. Ozog, F. Soncin, and A.-M. Lompre Basal Transcription of the Mouse Sarco(endo)plasmic Reticulum Ca2+-ATPase Type 3 Gene in Endothelial Cells Is Controlled by Ets-1 and Sp1 J. Biol. Chem., September 20, 2002; 277(39): 36471 - 36478. [Abstract] [Full Text] [PDF]
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E. Lelievre, F. Lionneton, V. Mattot, N. Spruyt, and F. Soncin Ets-1 Regulates fli-1 Expression in Endothelial Cells. IDENTIFICATION OF ETS BINDING SITES IN THE fli-1 GENE PROMOTER J. Biol. Chem., July 5, 2002; 277(28): 25143 - 25151. [Abstract] [Full Text] [PDF]
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J. Gaspar, S. Thai, C. Voland, A. Dube, T. A. Libermann, M.L. Iruela-Arispe, and P. Oettgen Opposing Functions of the Ets Factors NERF and ELF-1 During Chicken Blood Vessel Development Arterioscler Thromb Vasc Biol, July 1, 2002; 22(7): 1106 - 1112. [Abstract] [Full Text] [PDF]
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K.-i. Minehata, Y.-s. Mukouyama, T. Sekiguchi, T. Hara, and A. Miyajima Macrophage colony stimulating factor modulates the development of hematopoiesis by stimulating the differentiation of endothelial cells in the AGM region Blood, April 1, 2002; 99(7): 2360 - 2368. [Abstract] [Full Text] [PDF]
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Y. Peng and N. Jahroudi The NFY transcription factor functions as a repressor and activator of the von Willebrand factor promoter Blood, April 1, 2002; 99(7): 2408 - 2417. [Abstract] [Full Text] [PDF]
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J. Aitsebaomo, M. L. Kingsley-Kallesen, Y. Wu, T. Quertermous, and C. Patterson Vezf1/DB1 Is an Endothelial Cell-specific Transcription Factor That Regulates Expression of the Endothelin-1 Promoter J. Biol. Chem., October 12, 2001; 276(42): 39197 - 39205. [Abstract] [Full Text] [PDF]
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P. Oettgen Transcriptional Regulation of Vascular Development Circ. Res., August 31, 2001; 89(5): 380 - 388. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(HIF-2
/
640 to bp
+299 relative to the transcriptional start site was constructed as described previously.
-galactosidase versus luciferase).
