From the University of Texas Medical Branch, Division of Cardiology and Sealy Center for Molecular Cardiology, Galveston, Texas 77555-1064
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ABSTRACT |
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KDR/flk-1, the receptor for vascular endothelial
growth factor, is required for normal vascular development.
KDR/flk-1 is a TATA-less gene, containing four upstream Sp1
sites and a single transcription start site, although analysis of the
start site sequence discloses only weak similarities with the consensus
initiator element (Inr) sequence. In vitro transcription
assays, however, demonstrate that the region from Two families of transmembrane tyrosine kinase receptors that are
expressed uniquely in endothelial cells and are essential for the
development of the embryonic vascular system have recently been
identified (1). One family includes the vascular endothelial growth
factor (VEGF)1 receptors,
flt-1 (VEGFR-1) and KDR/flk-1 (VEGFR-2) (2, 3). The other family
contains the two members of the Tie receptor family, Tie1 and Tie2
(also known as Tek) (4). Of known endothelial cell markers, KDR/flk-1
is the first to be expressed during endothelial cell development,
appearing as early as day 7.0 post-conception in the mouse embryo (5).
Importantly, deletion of KDR/flk-1 by homologous recombination in mice
blocks endothelial cell development; mice lacking functional KDR/flk-1
fail to form any vascular structures and die in utero
between days 8.0 and 9.0 post-conception (6). These and other data
indicate that KDR/flk-1 is required for the transformation of
endothelial cells from their multipotent precursors and place it
upstream of the other endothelial receptor tyrosine kinases in the
cascade of endothelial cell development.
In addition to its developmental role, KDR/flk-1 and its ligand VEGF
participate in physiologic and pathologic processes with angiogenic
components, such as wound healing, bone remodeling, inflammatory
arthritis, myocardial infarction, peripheral vascular disease, and
tumor growth (reviewed in Ref. 7). These effects of VEGF are mediated
by induction of KDR/flk-1 autophosphorylation and activation of
multiple signal transduction pathways (8-10). In contrast with our
increasing understanding of signaling events downstream of KDR/flk-1,
there remains a paucity of information regarding the upstream events
regulating the expression of this receptor during endothelial cell
development and in angiogenesis.
Given the importance of KDR/flk-1 in endothelial cell biology, we have
cloned the entire human KDR/flk-1 gene (11) and have sought
to define the mechanisms governing its transcriptional regulation. The
human KDR/flk-1 gene is TATA-less and has a core promoter
containing tandemly repeated GC boxes (12). We have shown that
ubiquitously expressed Sp1 binds these sites only in endothelial cells
through a mechanism that involves changes in nucleosome positioning
(13). The identity of nuclear factors that maintain this chromatin
structure, and hence endothelial cell-restricted expression, remains
elusive. Likewise, we do not yet understand how transcriptional
complexes are assembled at this promoter. Even though the
KDR/flk-1 promoter lacks a TATA box and contains GC-rich
regions (a promoter structure that frequently produces transcripts
initiated at multiple, poorly defined nucleotides), ribonuclease
protection and primer extension assays have shown that the gene
contains only one transcription start site (12). The mechanisms by
which assembly of transcription complexes occurs at such a promoter are
not completely understood.
Transcription initiation is a complex process requiring the assembly of
many transcription factors on the promoter. In general, two methods
exist by which basal transcription initiates from a defined site. Most
commonly, mammalian protein-encoding genes contain a TATA box, which
recruits TFIID to the promoter and positions the transcription start
site at a 25-30-bp downstream nucleotide (14). Less frequently, genes
contain a so-called initiator element (Inr), which overlaps the
transcription start site and positions the basal transcription
machinery (15); many genes in the latter group, KDR/flk-1
included, lack a TATA-like upstream sequence altogether. A loose
consensus sequence for Inr-dependent transcription, YYA+1NTYY, has been defined (16), although exceptions to
this rule have been reported (17). The mechanisms whereby Inr-mediated basal transcription occur are at present incompletely understood, although it is clear that initiation complex formation and
transcription-associated factor requirements differ between Inr- and
TATA-mediated transcription (18, 19); indeed, the functional
consequences of a requirement for an Inr for accurate transcription
initiation are also not known, since so few Inr-dependent
promoters have been adequately characterized. To increase the
complexity of the situation, several different functional Inr-binding
proteins have been identified (including TFII-I, YY1, and USF
(20-23)), raising the possibility that different Inr-binding proteins
regulate Inr-dependent transcription under different circumstances.
Based on our observation that the human KDR/flk-1 gene is
TATA-less yet has a single transcription start site, we hypothesized that KDR/flk-1 contains a functional Inr responsible for
transcription initiation, even though the KDR/flk-1
transcription start site sequence corresponds poorly to the Inr
consensus sequence. In the present study, we demonstrate in in
vitro transcription assays that the sequence from In Vitro Transcription Assays--
For in vitro
transcription, the reaction mixture containing 300 ng of supercoiled
template DNA, 4 µl of HeLa nuclear extract (approximately 50 µg of
nuclear protein), and 250 µM each ribonucleoside diphosphate was incubated for 1 h at 30° C, as described (15). For some reactions, Plasmid Construction--
Plasmid pGL2-Et1 was created by
cloning a fragment from Mutagenesis--
Oligonucleotide-directed mutagenesis was
performed as described previously (26). The plasmid pGL2-225+268,
containing the firefly luciferase reporter gene under
control of the wild-type human KDR/flk-1 promoter, has been
described previously (12) and was used as the template. The mutagenic
oligonucleotide 5'-CAGCTCCCACCCTGGTGCGAGTCCCGGGACCCC-3' was
used to generate the construct pGL2INR-MUT.
Cell Culture--
Primary culture human umbilical vein
endothelial cells (HUVEC) were obtained from Clonetics Corp. and were
grown in M199 medium supplemented with 20% heat-inactivated fetal calf
serum, 60 µg/ml endothelial cell growth supplement, 50 µg/ml
heparin, 100 units/ml penicillin, and 100 µg/ml streptomycin as
described (12). Bovine aortic endothelial cells (BAEC) were isolated as
described (12) and grown as monolayers in Dulbecco's modified Eagle's
medium with 10% fetal calf serum and 600 µg/ml glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Nuclear extracts
were prepared as described (27).
Transient Transfections--
BAEC from passages 4-8 were used
for transient transfection experiments. BAEC were used for transient
transfection experiments rather than HUVEC because they can be
reproducibly transfected with higher efficiency. Cells were grown to
40-60% confluence in 6-well plates and then transfected using
Lipofectin (Life Technologies, Inc.), as described (28). The
appropriate reporter plasmid (2 µg) was transfected with 0.8 µg of
the secreted alkaline phosphatase expression vector pSEAP2
(CLONTECH) to correct for variability in
transfection efficiency. To determine the effect of TFII-I on reporter
gene activity, 1 µg of pEBG-TFII-I (25), containing the TFII-I
cDNA in the expression plasmid pEBG, or pEBG alone, were
cotransfected with the reporter plasmid and pSEAP2. (pEBG and
pEBG-TFII-I were provided by Ananda Roy.) Extracts were prepared 48 h after transfection by a detergent lysis method (Promega). Luciferase activity was measured in duplicate for all samples with a
Packard top-count scintillation counter and the Promega Luciferase
Assay system. Secreted alkaline phosphatase activity was measured
utilizing the Great EscAPe system (CLONTECH). The ratio of luciferase activity to alkaline phosphatase activity in each
sample served as a measure of relative luciferase activity. All
constructs were tested in at least four independent transfection experiments, and results are expressed as the mean ± S.E.
Relative luciferase activity among experiments was compared by a
factorial analysis of variance followed by Fisher's least significant
difference test. Statistical significance was accepted at
p < 0.05.
Electrophoresis Mobility Shift Assays (EMSA)--
EMSA was
performed as described (13). The probe consisted of annealed synthetic
20-bp complementary oligonucleotides corresponding to bp Sequence Analysis of the KDR/flk-1 Transcription Start
Site--
We have previously cloned and sequenced the mouse and human
KDR/flk-1 5'-flanking regions and have characterized the
proximal human KDR/flk-1 promoter in detail (12, 13). The
promoters for both species are TATA-less and are characterized by the
presence of multiple GC boxes. Functional characterization of the human promoter demonstrates that four tandemly repeated GC boxes are bound by
Sp1 in endothelial but not in non-endothelial cells and that these GC
boxes lie 39 bp upstream of a single transcription start site at the A
residue of a 5'-CA-3' dinucleotide pair (Fig. 1A). The presence of a single
transcription start site in a TATA-less gene led us to hypothesize that
KDR/flk-1 should contain a functional Inr. Comparison of the
region from The Human KDR/flk-1 Promoter Contains a Functional Inr--
To
address whether the sequence surrounding the KDR/flk-1
transcription start site contains a functional Inr, we measured its
effect on transcript initiation in vitro using a soluble
cell-free system (15). The plasmid pSp1, which contains two 21-bp GC
box repeats but no TATA box upstream of its multiple cloning site, was
used in these assays. Transcription from pSp1 is dependent on the
insertion of sequences with functional Inr activity. We synthesized
oligonucleotides from
We were able to detect pSp1 transcripts by in vitro
transcription, and we mapped the transcription start sites by primer
extension. (We used HeLa nuclear extract in these experiments because
we were not able to consistently produce nuclear extracts from primary culture endothelial cells with sufficient transcriptional activity. As
mentioned below, binding activity to the KDR/flk-1 Inr is
broadly expressed, and therefore studies from cell types such as HeLa should be relevant to the analysis of this promoter under these circumstances.) As expected, the TdT Inr directed
transcription predominantly from a single start site (lane
2, Fig. 2B), whereas in the absence of the Inr no
transcription occurred (lane 1). Plasmid pSp1-KDRE (+),
containing the sequence Characterization of the KDR/flk-1 Inr by Mutagenesis--
Having
identified a functional Inr in the KDR/flk-1 gene, we sought
to determine the nucleotides necessary for Inr activity, with the
expectation that these experiments might help to understand why the
KDR/flk-1 Inr departs from the typical Inr consensus
sequence. A series of constructs were created by introducing mutations
between bps Effect of Inr Disruption on KDR/flk-1 Promoter Activity in in Vivo
Transient Transfection Assays--
The transcription assays discussed
above demonstrate that the sequences spanning the human
KDR/flk-1 transcription start site increase initiation
in vitro. In order to demonstrate a functional role for
these sequences in vivo, in the context of the native promoter, we compared mutant and wild-type KDR/flk-1 Inr
sequences in transfection assays. We created the luciferase reporter
construct pGL2INR-MUT by incorporating the nonfunctional mutations
(CACT to GTGC between Identification of Nuclear Protein Complexes Interacting with the
KDR/flk-1 Inr--
A variety of nuclear proteins, including USF, YY1,
TFII-I, and members of the transcription-associated factor family, have been shown to interact directly with Inrs of various genes (reviewed in
Ref. 19). As a first step to determine whether specific nuclear proteins interact with the KDR/flk-1 Inr, we examined
whether primary culture HUVEC contain a binding activity that will
associate with the fragment from
In an effort to identify proteins that participate in complex A, we
assayed for the presence of known Inr-binding proteins. Our initial
experiments in this regard focused on well described Inr-binding
proteins, TFII-I and YY1 (20, 32). We first tested the ability of
unlabeled fragments to compete away the formation of complex A (Fig.
4B). As shown previously, complex A could be competed by
10-fold excess of a specific but not 100-fold excess of a nonspecific
competitor. This complex could also be efficiently competed by 10-fold
excess of the TdT Inr, which can compete for binding by
TFII-I in these assays (21), although this Inr may bind and compete for
other general transcription factors as well. By comparison, a YY1
element did not compete for formation of complex A (not shown). As a
more precise test of the components in complex A, we preincubated
nuclear extracts with specific antibodies prior to binding reactions.
When nuclear extract was incubated with an anti-TFII-I antibody (which
is known to block TFII-I binding without producing a supershifted band
(30)) before the binding reaction, formation of the specific
DNA-protein complex A was significantly reduced, instead enhancing
formation of complex B. In contrast, anti-YY1 antibody had no effect on
complex A formation (Fig. 4B) nor did preimmune serum (data
not shown).
The competition and immunodepletion experiments strongly suggested that
TFII-I was present in HUVEC nuclear extracts and participated in
nuclear protein complex formation with the KDR/flk-1 Inr.
Consistent with these observations, we detected TFII-I mRNA and
protein expression in HUVEC (not shown). To prove more definitively
that TFII-I bound the KDR/flk-1 Inr, we examined the
interaction of purified native TFII-I to the KDR/flk-1 Inr,
and we compared complex formation with that of unfractionated HUVEC
nuclear extract by EMSA (Fig. 4C). Under the conditions
employed, a stable complex could form between TFII-I and the
KDR/flk-1 Inr, which was identical in size to a complex
formed by HUVEC nuclear extract alone. (The strong band migrating just
above the probe in this experiment was variably present in our nuclear
extract preparations and may represent a proteolytic fragment with DNA
binding activity.) The TFII-I·Inr complex could be blocked by
preincubation of purified native TFII-I with an anti-TFII-I antibody.
These experiments do not determine the stoichiometry of TFII-I binding
to the KDR/flk-1 Inr, nor do they preclude the possibility
that other nuclear proteins may also interact with this sequence.
However, taken together, these binding studies indicate that TFII-I
participates in complex formation with the KDR/flk-1 Inr,
and they provide a rationale for examining whether TFII-I regulates
KDR/flk-1 promoter activity in vivo and Inr
activity in vitro.
TFII-I Specifically Transactivates the Human KDR/flk-1 Promoter in
Transient Transfection Assays--
To demonstrate the possible role of
TFII-I in activating the KDR/flk-1 promoter in
vivo, the reporter plasmids pGL2 Promoter, pGL2-Et1, or
pGL2 KDR/flk-1 Inr Activity Is Dependent on TFII-I--
By having shown
that TFII-I bound the KDR/flk-1 Inr and transactivated the
promoter in vivo in an Inr-dependent fashion, we addressed whether TFII-I was necessary for the Inr activity observed in
our in vitro studies. By using the in vitro
transcription/primer extension system, we examined the effect of
immunodepletion of nuclear extracts with an anti-TFII-I antibody on Inr
activity (Fig. 6). In comparison with
reactions performed with untreated extracts (lane 2) or with
extracts treated with preimmune serum (lane 5), extracts
depleted of TFII-I prior to addition of template failed to support
transcription (lane 3). Adding back purified native TFII-I
to immunodepleted extracts prior to transcription rescued
transcription, providing further evidence that this effect was due
specifically to depletion of TFII-I (lane 4). This effect was similar to that observed with the TdT Inr (lanes 6-8).
We cannot exclude the possibility that the TFII-I fraction used in these experiments, although purified to homogeneity as assessed by
silver staining, contains one or more factors other than TFII-I that
may account in part for the ability of this fraction to rescue transcript initiation after TFII-I immunodepletion in this assay. However, the results of these experiments are consistent with our
in vivo observations that TFII-I regulates
KDR/flk-1 transcriptional activity through the Inr.
In this report, we demonstrate that the gene encoding KDR/flk-1, a
receptor for VEGF that is essential for vascular endothelial development and for endothelium-dependent processes such as
angiogenesis, contains a functional Inr, although important differences
exist between the nucleic acid sequence of this Inr and the well
established general consensus Inr sequence. In addition, we demonstrate
that the DNA-protein complexes that form on this Inr in EMSA
experiments contain the Inr-binding protein TFII-I and that TFII-I,
through interactions with the Inr sequence described here, is necessary for KDR/flk-1 promoter activity both in vitro and
in vivo.
In order to define and characterize the Inr function of the
KDR/flk-1 promoter, we have employed a well established
in vitro transcription assay in which transcriptional
activity is totally dependent on Inr activity (15). This system allows
for the simultaneous analysis of transcriptional activity and start
site selection. Based on several lines of evidence, the data in Fig. 2
support our hypothesis that KDR/flk-1 contains a functional
Inr and that this assay is measuring specific Inr activity rather than,
for instance, nonspecific nucleation of transcription by the
introduction of a 5'-CA-3' dinucleotide. First, transcription initiates
from an A residue within the KDR/flk-1 Inr in plasmid
pSp1-KDRE (+), rather than from 5'-CA-3' sequences located immediately
upstream or downstream (Fig. 2). Second, disruption of the 5'-CA-3'
sequence in plasmid pSp1-KDRE (+)-M3 does not affect transcriptional
activity or start site selection (Fig. 3). Third, insertion of 5'-CA-3' in a nonspecific or purine-rich context, which occurs fortuitously in
plasmids pSp1-KDRE ( Perhaps the most striking feature of the KDR/flk-1 Inr is
the difference in its nucleic acid sequence compared with the
previously reported and well characterized consensus sequence for Inrs,
YYA+1NTYY (16). The human KDR/flk-1 gene differs
from this degenerate consensus at 3 out of 6 sites (G at The presence of a TATA By having demonstrated that an intact Inr is necessary for constitutive
activity of the KDR/flk-1 promoter, we sought to identify the nuclear proteins interacting with the Inr to understand better the
role of Inr-dependent transcription in KDR/flk-1
expression. We initially attempted to address this question inductively
by affinity purifying KDR/flk-1 Inr-interacting proteins
from HUVEC by binding site selection; however, we were not successful
in recovering fractions of suitable purity for protein sequencing (not
shown). We therefore assayed for the presence of known Inr-binding proteins in our DNA-protein complexes detected by EMSA (Fig. 4). Five
lines of evidence suggest that TFII-I functionally interacts with the
KDR/flk-1 Inr as follows: (i) DNA-protein interactions with the Inr can
be competed by the TdT Inr, which can bind TFII-I (note that
since other nuclear proteins may bind the TdT Inr, this is a
necessary but not sufficient test for the presence of TFII-I); (ii)
DNA-protein interactions can be disrupted by a specific anti-TFII-I
antibody; (iii) purified TFII-I can bind the
KDR/flk-1 Inr; (iv) TFII-I transactivates the
KDR/flk-1 promoter in an Inr-dependent fashion;
and (v) TFII-I is necessary for KDR/flk-1 Inr activity in vitro.
TFII-I was originally purified (21) and subsequently cloned (22) based
on its ability to interact with and support transcription from the
AdML Inr. TFII-I is a 120-kDa, uniquely structured
transcription factor containing six directly repeated 90-residue
elements, each of which possesses potential helix-loop-helix
protein-protein interaction motifs. A functional role for TFII-I has
been demonstrated in transcription of the Inr-containing T cell
receptor V TFII-I has previously been shown to bind to at least three different
types of DNA sequences: typical Inrs, E box motifs, and serum response
elements (21, 34, 44, 45). Notably, the serum response elements with
which TFII-I interacts overlap typical Inr motifs, although they do not
have Inr function in vivo. In contrast, the E box motif
bound by TFII-I, CACGTG, does not match the Inr consensus, and as
pointed out above, the KDR/flk-1 Inr matches poorly with the
Inr consensus. The nature of TFII-I-binding site selectivity is not
well understood. On the basis of our results and those of others, it is
possible that either TFII-I contains multiple DNA binding domains or
that TFII-I-binding site selection is influenced by factors such as DNA
structure that may not be readily apparent by analysis of primary
nucleic acid sequence.
The precise role of Inr-binding proteins such as TFII-I in Inr function
and transcriptional regulation is only recently being clarified. A
persistent concern in previous analyses has been the possibility that
these proteins are actually transcriptional activators with binding
sites near or overlapping functional Inrs, rather than true Inr-binding
proteins with the capacity for recruitment of the basal transcriptional
machinery. In the case of YY1 and E2F, data suggest this may be the
case (16). The close correlation between in vitro
KDR/flk-1 Inr activity and TFII-I-mediated transcriptional activation in vivo, as well as the dependence of Inr
activity on TFII-I in vitro argue against this possibility.
At present, KDR/flk-1 is the only gene aside from the T cell
receptor V Since TFII-I-independent pathways have also been described for
transcription initiation (47), we favor the hypothesis that TFII-I
mediates Inr function in a subset of Inr-containing genes, possibly as
the result of common transcriptional requirements. One shared
characteristic of the V10 to +10 relative
to the start site contains Inr activity that is orientation- and
position-dependent, and mutagenesis of the
KDR/flk-1 Inr reduces promoter activity to 28% of the
wild-type promoter in transient transfection assays. Gel shift assays
confirm that nuclear proteins specifically bind the Inr, and
competition experiments demonstrate that TFII-I, a multifunctional
Inr-binding nuclear protein, is a component of these DNA-protein
complexes. TFII-I transactivates the wild-type KDR/flk-1
promoter, but not a promoter containing a mutated Inr, in transient
transfection assays. Immunodepletion of TFII-I from nuclear extracts
prior to in vitro transcription assays abolishes transcription from the KDR/flk-1 Inr, an effect that can be
rescued by adding back purified TFII-I, reflecting the importance of
TFII-I in KDR/flk-1 Inr activity. These experiments
demonstrate that the KDR/flk-1 gene contains a functional
Inr that is bound by TFII-I and that both the functional Inr and TFII-I
activity are essential for transcription.
INTRODUCTION
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Abstract
Introduction
References
10 to +10 in
the human KDR/flk-1 promoter contains Inr activity and that
Inr activity is orientation- and position-dependent.
Moreover, mutation of this element in the context of the native
KDR/flk-1 promoter dramatically decreases promoter activity
in vivo in transient transfection assays. This sequence has
specific nuclear protein binding activity and is bound by the
multi-functional transcription factor TFII-I present in nuclear
extracts or by purified TFII-I. Furthermore, we show that TFII-I
transactivates the KDR/flk-1 promoter in vivo and that TFII-I is necessary for transcription initiation in
vitro. These studies demonstrate the importance of the Inr in
KDR/flk-1 transcription and support a role for TFII-I in the
regulation of this gene and hence in the developmental processes
mediated by KDR/flk-1.
EXPERIMENTAL PROCEDURES
-amanitin (2 µg/ml) was added to test for RNA
polymerase II dependence of the final reaction products. RNA transcripts produced in this manner were treated with DNase, as described (24), and annealed with 100 fmol of 32P-labeled
SP6 primer in 1× primer extension buffer (50 mM Tris (pH
8.3), 50 mM KCl, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM of each dNTP, and 0.5 mM spermidine) for 1 h at 42° C. The duplex was
extended by adding 5 units of avian myeloblastosis virus reverse transcriptase for 30 min at 42° C. Extended DNA products were analyzed by polyacrylamide gel electrophoresis and visualized by
autoradiography. Labeled primers were present in excess in these
experiments to minimize the effects of small differences in primer
concentrations. Immunodepletion experiments were performed by
incubation of nuclear extract with either 1 µl of anti-TFII-I antibody or preimmune serum for 10 min at 30° C prior to adding template for in vitro transcription. Rescue experiments were
performed by the addition of 2 µl of purified native TFII-I as
indicated after immunodepletion, prior to starting in vitro
transcription. The anti-TFII-I antibody and TFII-I protein used in
these experiments and in gel shift assays were generous gifts of Ananda
Roy (Tufts University, Boston) and have been previously described and
characterized (22, 25).
204 bp to +170 bp of the human
endothelin-1 promoter (gift of Dr. Thomas Quertermous,
Stanford University School of Medicine) upstream of the firefly
luciferase gene into the vector pGL2 Basic. Plasmids pSp1
(containing two 21-bp SV40 Sp1 repeats upstream of the polylinker in
vector pSP72) and pSp1/TdT (containing the 18-bp terminal
deoxynucleotidyltransferase (TdT) Inr inserted between the
SacI and BamHI sites of pSp1) were generous gifts
from Steven T. Smale (University of California, Los Angeles). pSp1-KDRE
(+), pSp1-KDRE (
), and pSp1-KDRE (+) mutants were constructed by
insertion of double-stranded oligonucleotides into the EcoRI
site of plasmid pSp1 using standard ligation techniques. pSp1-KDRS (+)
and pSp1-KDRS (
) were similarly constructed by insertion of
oligonucleotides into the SmaI site of plasmid pSp1. Names
of synthetic oligonucleotides and constructs generated indicate the
insertion sites (E for EcoRI and S for SmaI) and
orientation (either forward (+) or reverse (
)) relative to the native
KDR/flk-1 gene and are listed as follows: pSp1-KDRE (+) and
pSp1-KDRS (+), 5-CCCACCCTGCACTGAGTCCC-3'; pSp1-KDRE (
) and pSp1-KDRS
(
), 5'-GGGACTCAGTGCAGGGTGGG-3'; pSp1-KDRE (+)-M1,
5'-CCCACAATGCACTGAGTCCC-3'; pSp1-KDRE (+)-M2,
5'-CCCACCCGTCACTGAGTCCC-3'; pSp1-KDRE (+)-M3,
5'-CCCACCCTGACCTGAGTCCC-3'; pSp1-KDRE (+)-M4,
5'-CCCACCCTGCAAGGAGTCCC-3'; pSp1-KDRE (+)-M5,
5'-CCCACCCTGCACTTCGTCCC-3'; pSp1-KDRE (+)-M6,
5'-CCCACCCTGGTGCGAGTCCC-3'.
10 to +10 of
the KDR/flk-1 promoter sequence (12). A typical binding
reaction contained 50,000 cpm DNA probe, 0.5 µg of
poly(dI-dC)·poly(dI-dC), 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 5% glycerol, and 5 µg of nuclear extract in a final volume of 25 µl. In reactions in which purified TFII-I was used in place of nuclear extract, 0.1 µg of
poly(dI-dC)·poly(dI-dC) was used. The reaction mixture was incubated
at room temperature for 20 min and fractionated on a 5% native
polyacrylamide gel in 0.5× TBE buffer. To determine the specificity of
the DNA-protein complexes, we performed competition assays using a
molar excess of the unlabeled double-stranded KDR/flk-1
oligonucleotide (specific inhibitor) or excess of an unrelated
double-stranded oligonucleotide of comparable length (nonspecific
inhibitor) or the TdT Inr (15). To characterize specific
DNA-binding proteins, we incubated nuclear extracts with anti-TFII-I or
anti-YY1 antibody (Santa Cruz Biotechnology) for 3 h at 4 °C
before adding probe.
RESULTS
10 to +10 in the human 5'-flanking sequence with the
analogous sequence in the mouse promoter (12, 29) demonstrates that
there is a high degree of conservation across species (Fig.
1B). Remarkably, the A residue at +1 in the human promoter
is substituted with a C residue in the mouse promoter, whereas the 8 residues downstream of +1 are identical. The human transcription start
site sequence meets the minimal Inr criteria by virtue of having an A
at +1 and a T at +3, since these residues are the most critical for
determining the level of Inr activity (16). However, these residues,
although in a relatively pyrimidine-rich context, are not accompanied
by a sequence meeting the consensus for Inr activity,
YYA+1NTYY, which has been determined empirically (16) and
by comparison with other well characterized Inrs (15, 21, 30).
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Fig. 1.
Structure of the human KDR/flk-1
core promoter. A, depiction of the topology of
the KDR/flk-1 promoter. The arrow denotes the
transcription start site. Numbers represent the position of
nucleotides in relationship to the transcription start site.
B, comparison of the human KDR/flk-1 Inr with the
homologous sequence in the mouse KDR/flk-1 promoter and with
other well characterized Inr sequences, including that for the Inr
consensus sequence.
10 to +10 (5'-CCCACCCTGCACTGAGTCCC-3') and
inserted the annealed oligonucleotides into the EcoRI and SmaI sites of vector pSp1 in both orientations (Fig.
2A). The recombinant plasmids
were named according to the insertion sites and orientation. Plasmid
pSp1/TdT, containing the TdT Inr, was used for comparison as
a positive control.
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Fig. 2.
Measurement of KDR/flk-1 Inr
activity by in vitro transcription. A,
Inr-containing constructs used in in vitro transcription
experiments and the position of inserted oligonucleotides in
relationship to upstream sequences. Known transcription start sites are
identified by arrows. 5'-CA-3' dinucleotide pairs present in
plasmids pSp1-KDRE (+), pSp1-KDRE ( ), and pSp1-KDRS (
) are
underlined. B, the Inr-containing constructs listed above
were incubated with nuclear extract. RNA transcripts synthesized in
this manner were analyzed by primer extension and visualized by
autoradiography. The large arrow denotes a single transcript
initiating from +1 of the KDR/flk-1 promoter in plasmid
pSp1-KDRE (+). The small arrow marks a specific transcript
from the TdT Inr contained in plasmid pSp/TdT. NS
identifies a region rich in nonspecific fragments. Some reactions were
performed in the presence of
-amanitin to inhibit RNA polymerase II
as indicated.
10 to +10 in the forward orientation with the
transcription start site located 38 bp downstream of the last GC repeat
(similar to the distance from the GC boxes in the endogenous
KDR/flk-1 promoter), was able to initiate transcription from
a single site. This site corresponded to the +1 nucleotide of the
endogenous KDR/flk-1 promoter, producing a fragment slightly
larger than that produced by pSp1/TdT after gel electrophoresis
(lane 3). Most interestingly, transcription initiated from
this site rather than from 5'-CA-3' dinucleotides, produced in the
cloning process, located 7 bp upstream or 9 bp downstream (Fig.
2A). If the transcription start site was situated 52 bp
downstream of the GC boxes (plasmid pSp1-KDRS (+), lane 4)
transcription still occurred; however, the ability to initiate from a
single site was lost. If the oligonucleotide was inserted in the
reverse orientation relative to the GC boxes, either 38 bp (pSp1-KDRE
(
)) or 52 bp (pSp1-KDRS (
)) downstream, no appreciable transcription was evident (lanes 5 and 6). In
every case, primer extension products were blocked by the addition of
-amanitin, confirming that the observed results are dependent on RNA
polymerase activity and are not primer extension artifacts (lanes
7-9). These experiments demonstrate the following: (i) the
sequence overlapping the KDR/flk-1 transcription initiation
site contains a functional Inr; (ii) KDR/flk-1 Inr activity
is orientation-dependent; (iii) Inr activity is determined
by the distance from upstream elements; and (iv) Inr activity is RNA
polymerase II-dependent.
5 to +5 in the Inr in plasmid pSp1-KDRE (+) (Fig.
3A). Introduction of two point
mutations at
5 and
4 (both from C to A) to create plasmid pSp1-KDRE
(+)-M1 did not affect Inr activity (Fig. 3B, lane
3) compared with the activity of pSp1-KDRE (+) (lane
2). Mutation of residues
3 and
2 (from T to G and G to T,
respectively, to create pSp1-KDRE (+)-M2) consistently increased Inr
activity modestly (lane 4), an effect that may be explained
by introducing a pyrimidine at position
2 and therefore establishing
concordance with the Inr consensus at this position (16). Surprisingly, mutations at positions
1 and +1 (from C to A and A to C,
respectively, pSp1-KDRE (+)-M3), which increase divergence from the
consensus sequence, also increased the level of transcription slightly
and did not change the start site position (lane 5). As
discussed below, this mutation provides one line of evidence indicating that KDR/flk-1 Inr activity is not simply the result of a
5'-CA-3' nucleation event. Mutations at positions +2 and +3 (from C to A and T to G, respectively; pSp1-KDRE (+)-M4) not only decreased transcription but also influenced transcription start site usage (lane 6); this was to be expected by removing the two
pyrimidines at these positions. We had anticipated that changing
residues +4 and +5 to pyrimidines (from G to T and A to C,
respectively, pSp1-KDRE (+)-M5) would enhance Inr activity by improving
homology with the Inr consensus at these positions. Surprisingly, both the rate of transcription and start site selectivity were decreased by
these changes. The significance of this result is discussed below.
Finally, complete corruption of the core Inr sequence from positions
1 to +3 (from CACT to GTGC, pSp1-KDRE (+)-M6) resulted in loss of all
Inr activity in the assay (lane 8). Since this last mutation
had the most profound effect on Inr activity, we utilized it as a test
of the role of Inr activity in in vivo assays of
transcriptional activity.
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Fig. 3.
Functional analysis of the
KDR/flk-1 Inr by site-directed mutagenesis.
A, depiction of the mutated Inrs tested in comparison to the
wild-type KDR/flk-1 Inr, and the plasmids containing them.
B, the Inr-containing constructs shown were tested for Inr
activity in in vitro transcription assays. The
arrow indicates the position of transcripts correctly
initiated from the KDR/flk-1 Inr.
1 to +3) into the luciferase reporter plasmid pGL2
225+268; this plasmid contains a fragment of the human
KDR/flk-1 promoter from
225 to +268, which is the smallest
fragment of this promoter with complete activity in transient
transfection assays (12). Reporter constructs were transiently
transfected into BAEC, and luciferase activity was measured.
Mutagenesis of the KDR/flk-1 Inr significantly reduced
promoter activity to 28 ± 5.5% of pGL2-225+268
(p < 0.05, data not shown; see also Fig. 5). These
results are consistent with previous reports demonstrating that
mutation of functional Inrs reduces, but does not abolish, transcription in transient transfection assays (16, 22, 25), and they
indicate that the wild-type KDR/flk-1 Inr sequence is required for maximal transcriptional activity of the
KDR/flk-1 promoter in vivo, as it is in
vitro.
10 to +10 by EMSA, since HUVECs are
known to express KDR/flk-1 mRNA and protein at high levels (12,
31). Incubation of the labeled Inr fragment with nuclear extract
resulted in the formation of two retarded bands, A and
B (Fig. 4A).
Formation of both complexes was efficiently abolished by as little as
10-fold molar excess of unlabeled Inr fragment. Complex B was partially competed by an unlabeled nonspecific competitor, whereas complex A was
not efficiently competed. We should note that the binding activity of
complex B was variably present in HUVEC extracts (Fig. 4C
and data not shown). On the basis of this finding and the competition experiments, we favor the interpretation that this complex represents nonspecific binding to the Inr; however, we cannot exclude the possibility that it represents a specific complex with lower affinity for the Inr in comparison with complex A. Similar retarded bands were
also observed when EMSA was performed with nuclear extracts from a
variety of nonendothelial cell
types,2 indicating that the
protein or proteins involved in their formation are broadly expressed
among cell types.
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Fig. 4.
Identification of TFII-I as a
KDR/flk-1 Inr-binding protein by EMSA.
A, a radiolabeled double-stranded oligonucleotide encoding
the KDR/flk-1 Inr was incubated with HUVEC nuclear extract
(NE) and the indicated molar ratios of either unlabeled
specific or nonspecific competitor (Comp.) oligonucleotide.
DNA-protein complexes were resolved by gel electrophoresis. Shifted
bands A and B are denoted by arrows.
B, EMSA was performed by incubating the KDR/flk-1
Inr with HUVEC nuclear extract. The specificity of complex A formation
(denoted by an arrow) was tested by addition of unlabeled
specific (Sp) or nonspecific (NS)
oligonucleotides or with an oligonucleotide encoding the TdT
Inr (TdT). Components of the DNA-protein complexes formed
were assayed by incubating nuclear extracts with antibodies
(Ab) recognizing TFII-I or YY1 prior to binding reactions.
C, the KDR/flk-1 Inr was incubated with either
HUVEC nuclear extract (NE), with affinity purified TFII-I,
or with TFII-I in the presence of a specific TFII-I antibody in order
to compare complexes produced by unfractionated nuclear extract and
TFII-I alone.
225+268 were cotransfected into BAEC with either the eukaryotic
expression plasmid pEBG-TFII-I or pEBG alone as a control (25).
Cotransfection of TFII-I had no effect on activity of the
SV40 promoter present in pGL2 Promoter nor on the
endothelin-1 promoter (Fig.
5), another endothelial cell-restricted
promoter (33). In contrast, TFII-I significantly increased
KDR/flk-1 promoter activity by approximately 3-fold in
comparison with empty vector (p < 0.05). This
induction may actually underestimate the ability of TFII-I to
transactivate this promoter, since activity of this construct is
already very high (equivalent to the potent SV40 promoter
plus enhancer (12)) and since endothelial cells express endogenous
TFII-I constitutively (data not shown). Since TFII-I can bind to the
KDR/flk-1 Inr, we asked whether an intact and functional Inr
was necessary for TFII-I-mediated transactivation. Consistent with our
previous experiments, mutation of the Inr resulted in a decrease in
promoter activity to 28% of the wild-type vector. It is significant to
note that this level, while low, was 2 orders of magnitude greater than
the activity of the promoterless vector pGL2 Basic and therefore was
well above the limits of detection for this assay. In contrast with its
effect on the wild-type promoter, TFII-I did not transactivate a
promoter with a mutated Inr. Whereas we cannot exclude the possibility
that TFII-I interacts with other sequences within the
KDR/flk-1 promoter (and, indeed, TFII-I has been shown to
interact with E box sequences in addition to the Inr in the
adenovirus major late (AdML) promoter (34)),
these data indicate that an intact and functional Inr is necessary for KDR/flk-1 transactivation by TFII-I.
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Fig. 5.
Transactivation of the KDR/flk-1
Inr by TFII-I. The indicated reporter constructs (2 µg)
and either a mammalian expression vector expressing TFII-I or the same
vector alone (1 µg) were transfected into BAEC. After 48 h,
transfected cells were lysed and luciferase activity was measured.
Results were normalized to secreted alkaline phosphatase activity to
correct for transfection efficiency and expressed as a percentage of
pGL2 225+268 alone. *p < 0.05, compared with
transfection with pEBG.
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Fig. 6.
Functional role of TFII-I in
KDR/flk-1 Inr function. In vitro
transcription assays were performed, as described previously, with the
indicated plasmids. To test for the requirement of TFII-I in Inr
activity, nuclear extracts were immunodepleted by incubation with
antibodies (Ab) recognizing TFII-I or with preimmune serum
(PI) prior to in vitro transcription. The
specificity of this assay for detecting immunodepletion of TFII-I was
tested by reintroduction of TFII-I to the transcription assays after
immunodepletion.
DISCUSSION
) and pSp1-KDRS (
) (Fig. 2A; see
also Ref. 16), does not support transcription in this assay. Therefore, we believe these data indicate the existence of a specific Inr function
in the KDR/flk-1 promoter. Similar to other Inrs (24, 35),
KDR/flk-1 Inr activity is orientation-dependent
and inhibitable by
-amanitin. Transcript initiation is also
influenced in our studies by Inr position in relation to upstream Sp1
sites. (Unfortunately, these positional effects prevent us from making
quantitative comparisons between the activity of the
KDR/flk-1 and TdT Inrs in our experiments, since
the start sites are positioned differently in relation to upstream
sequences.) In the case of KDR/flk-1, placing the Inr 52, rather than 38, bp downstream of Sp1 sites results in transcription initiating at multiple sites, although overall transcriptional activity
seems preserved. Such findings have been reported for other Inrs (15)
and suggest that, at least in this assay, the transcriptional
activation and start site selection properties of an Inr may be
separable and that start site selection may be more susceptible than
transcriptional activation to minor perturbations in promoter
structure. The necessity for, and the mechanisms for maintaining, a
single start site remain enigmas in the general understanding of Inr function.
2, G at +4,
and A at +5, see Fig. 1). We have performed mutagenesis at each of
these sites in an effort to gain insight into what differences, if any,
exist between the KDR/flk-1 Inr and Inrs with more typical
sequences (Fig. 3). A change in the G residue at
2 to a pyrimidine
modestly enhances Inr activity, consistent with the notion that Inrs
more closely fitting the consensus sequence have higher activity (16). Surprisingly, changing the A at +1 to C (albeit with insertion of an A
at
1) also increases Inr activity. The dispensability of A at this
position is consistent with the fact that an A residue is wholly
lacking in the analogous mouse KDR/flk-1 sequence. Most remarkably, changing the G and A residues at +4 and +5 to pyrimidines does not enhance Inr activity, as would be expected, and instead decreases transcriptional activity and alters start site selection. These results were not expected and indicate that differences between
the KDR/flk-1 Inr and the consensus YYA+1NTYY in
these positions are necessary for maximal Inr activity. Three possible
explanations exist for these differences. First, the
KDR/flk-1 promoter may require a fundamentally different
mechanism of transcription initiation than that of Inrs bearing the
"classical" consensus, and these differences are reflected in the
KDR/flk-1 promoter sequence. KDR/flk-1 would be
relatively unique in this regard, however, since other Inrs with
similarities to the KDR/flk-1 Inr rather than to the
consensus have not yet been described. Second, the proposed Inr
consensus, despite the extensive empirical and observational data on
which it is based, may not account well for the sequence variations
allowable for "typical" Inrs for TATA
/Inr+ genes. Third,
transcription of KDR/flk-1 may occur in a manner similar to
that of other Inrs from TATA-less genes, and the differences from
consensus may be due to constraints placed on the sequence by factors
independent of initiation complex formation that do not directly
interact with the core initiator sequences themselves; such factors may
be general transcriptional activators, specific but indirect enhancers
of initiation, or may have other functions. The KDR/flk-1
Inr may provide a model whereby such possibilities can be tested to
better understand Inr activity in general.
/Inr+ promoter in a cell type-specific,
developmentally critical gene such as KDR/flk-1 raises the general issue of why such a core promoter exists, in lieu of the more
common and well characterized TATA-containing promoters (36). As
pointed out by Garraway et al. (37), there are three
potential explanations for the presence of Inrs within promoters as
follows: (i) the TATA box may be detrimental to gene expression; (ii)
the Inr may be required for expression; and (iii) the
30 sequence typically occupied by the TATA box may be used by another protein. Whereas indirect evidence exists to support the third possibility in
some cases (38), it seems equally likely that specific transcriptional requirements for a subset of genes mandates either the absence of a
TATA box or the presence of an Inr. Indeed, transcription initiation
from TATA
/Inr+ and TATA+/Inr
promoters are fundamentally different
at the biochemical level (18, 30). Remarkably, a preponderance of
TATA
/Inr+ genes characterized to date encode cell type-specific
and/or developmentally essential proteins (30, 39-42). It is tempting
to speculate that Inr-dependent transcription might in some
way be necessary for the exquisite control in gene regulation needed
under circumstances such as those that must occur during the commitment
of hemangioblastic precursors to the endothelial lineage that is
regulated by KDR/flk-1 expression (6, 36).
and AdML promoters (21, 25, 30, 32)
through interactions with their Inr sequences, and TFII-I physically
associates with the HIV-1, TdT, and
ribonucleotide reductase R1 Inrs as well (17, 43). In
addition to Inr binding activity, TFII-I can associate with the E box
motif, CACGTG (34), and with serum response element sequences (44, 45).
TFII-I interacts functionally with several transcription factors,
including helix-loop-helix proteins USF (21) and Myc (34), homeodomain
protein Phox 1 (44), MADS box protein serum response factor (44, 45),
and STAT1 and STAT3 (45), in order to transactivate gene expression. In
addition, TFII-I is bound and phosphorylated by Bruton's tyrosine
kinase (46) and is also phosphorylated in response to epidermal growth factor stimulation (45). These diverse activities suggest either that
TFII-I has multiple independent molecular functions or that TFII-I
integrates signaling pathways at both basal and regulatory levels to
influence gene expression.
promoter for which both in vitro
and in vivo requirements have been met for TFII-I Inr
function (25).
and KDR/flk-1
promoters is that they mediate exquisitely cell-type-specific,
developmentally regulated expression of functionally indispensable
proteins. Although not itself cell type-specific, TFII-I may integrate
signals from other nuclear proteins to maintain appropriate cell
type-specific transcription initiation. That TFII-I itself is a target
for cell type-specific signaling pathways is consistent with this
possibility (46). In the case of KDR/flk-1, TFII-I might
interact with other, more cell type-restricted transactivators, such as
yet to be characterized homeodomain or MADS box proteins, to initiate
transcription under the appropriate circumstances. In this regard, an
understanding of the role of TFII-I in KDR/flk-1 gene
expression may help to unravel the complexities of endothelial cell
development and cell type-specific gene expression.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Ananda Roy for providing TFII-I antibody and protein and plasmids pEBG and pEBG-TFII-I; to Thomas Quertermous for the endothelin-1 promoter; and to Stephen T. Smale for providing plasmids pSp1 and pSP1/TdT. We acknowledge the editorial assistance of Joann Aaron. We thank Marschall S. Runge for continued support and encouragement and the members of the Patterson laboratory for insightful commentary.
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FOOTNOTES |
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* This work was supported in part by the Sealy-Smith Foundation, by NHLBI Grants HL03658 and HL61656 from the National Institutes of Health, and by a grant-in-aid from the American Heart Association, Texas Affiliate (to C. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of Texas
Medical Branch, Division of Cardiology, 9.138 Medical Research Bldg.,
301 University Blvd., Galveston, TX 77555-1064. Tel.: 409-747-1796; Fax: 409-747-0692; E-mail: cpatters{at}utmb.edu.
The abbreviations used are: VEGF, vascular endothelial growth factor; R, receptor; Inr, initiator element; TdT, terminal deoxynucleotidyltransferase; HUVEC, human umbilical vein endothelial cell(s); BAEC, bovine aortic endothelial cell(s); EMSA, electrophoresis mobility shift assay(s); AdML, adenovirus major late; bp, base pair.
2 Y. Wu and C. Patterson, unpublished observations.
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REFERENCES |
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