From Sangamo BioSciences Incorporated, Richmond, California 94804
Received for publication, December 12, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have mapped conserved regions of enhanced
DNase I accessibility within the endogenous chromosomal locus of
vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger
protein (ZFP) transcription factors were designed to target DNA
sequences contained within the DNase I-hypersensitive regions. These
ZFPs, when fused to either VP16 or p65 transcriptional activation
domains, were able to activate expression of the VEGF-A gene as assayed
by mRNA accumulation and VEGF-A protein secretion through a range
exceeding that induced by hypoxic stress. Importantly, multiple splice
variants of VEGF-A mRNA with defined physiological functions were
induced by a single engineered ZFP transcription factor. We present
evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for
dissecting the requirements for gene regulation at a distance without
altering the DNA sequence of the endogenous target locus.
A major focus in the study of gene regulation involves the
mechanisms by which the cell achieves specific activation of endogenous chromosomal genes. In addressing this issue, rationally designed components of the transcriptional machinery can provide powerful tools
for testing our understanding of gene regulation (1-3). In particular,
artificial transcription factors, targeted to novel sequences within a
given locus and bearing functional domains of the experimenter's
choosing, may prove especially useful since they offer the prospect of
complete recapitulation of any given activation process using totally
defined components. Artificial transcription factors may also provide
practical benefits in areas such as medicine and biotechnology.
The DNA-binding motif of choice that has emerged for achieving specific
recognition of predetermined, desired DNA sequences is the
Cys2-His2 zinc finger. Over the past decade,
selection and design studies have demonstrated the adaptability of this motif and have yielded simple, powerful strategies for designing zinc
finger proteins (ZFPs)1 that
can bind specifically to virtually any DNA sequence (4-15). More
recently, ZFPs with engineered DNA sequence specificities have begun to
be used as artificial transcription factors to regulate endogenous
chromosomal genes (16-18). The growing use of these designed ZFPs has
led to an appreciation of the role of chromatin structure in the
ability of designed transcription factors to successfully access their
target sequences and to function as transcriptional regulators at
endogenous loci. In one recent study, a clear discrepancy was
noted between the ability of ZFPs to activate transcription from naked
reporter DNA and an endogenous chromosomal locus, with only a subset of
reporter-active ZFPs successfully up-regulating the endogenous gene
(17). Subsequent analysis of chromatin structure suggested site
accessibility as the cause of this difference in behavior (17). This
study, along with the growing awareness of the role of chromatin
remodeling in transcriptional regulation (for example, see Refs.
19-21), highlights the necessity of considering chromatin structure in
any use of artificial transcription factors.
Our goal for this study was to identify a panel of ZFPs that could
activate the endogenous gene for vascular endothelial growth factor A
(VEGF-A). VEGF-A is an endothelial cell-specific mitogen that is a key
inducer of new blood vessel growth, both during embryogenesis and in
adult processes such as wound healing (for recent reviews, see Refs.
22-24). Its central roles in both vasculogenesis and angiogenesis
apparently necessitate the control of VEGF-A expression levels by
precise regulatory mechanisms. Mouse studies have highlighted VEGF-A as
a clear example of a gene whose haplo-insufficiency causes embryonic
lethality (25, 26). Furthermore, several studies have suggested that
proper VEGF-A function requires expression of appropriate relative
levels of the major splice variants produced by this gene (27, 28). A
diversity of conditions and transcription factors have been implicated
as inducing VEGF-A expression (29-35), of which perhaps the best
characterized is the hypoxic response, mediated by
hypoxia-inducible factor 1 (36-40). Consistent with its highly
regulated nature, VEGF-A dysregulation plays a role in a variety of
medical conditions, including tumor growth, diabetic retinopathy, and
ischemic heart and limb diseases. Consequently, VEGF-A would appear to
provide an attractive target for both pro- and anti-angiogenic gene
therapies using designed artificial transcription factors.
In this study, we have made use of engineered
Cys2-His2 ZFPs and knowledge of chromosomal
structure to achieve activation of the endogenous chromosomal locus
containing the gene for VEGF-A. We first used DNase I hypersensitivity
mapping analysis to identify accessible regions of the VEGF-A locus
conserved across a variety of cell lines from man and rat. This
analysis identified four distinct DNase I-accessible regions, of which
three were present in the HEK293 cells used for activation studies.
Novel ZFPs targeted to sites within the HEK293-specific DNase
I-accessible regions were then characterized with respect to their
DNA-binding properties and ability to activate VEGF-A expression. We
found that each of our designed ZFPs bound its intended target with an
apparent Kd of <3 nM and, when linked
to the VP16 transcriptional activation domain (41), activated
transcription of both the endogenous VEGF-A gene and a transiently
transfected native reporter construct containing ~3 kilobases of the
VEGF-A promoter. We also linked our ZFPs to the activation domain from
the p65 subunit of NF- We found that this strategy yielded eight distinct ZFPs targeted to
seven 9-bp sites that activated VEGF-A expression and that, for certain
ZFPs, linkage to activation domains from either VP16 or p65 provided
differing levels of activation depending on the chromosomal site
that is targeted. Furthermore, when certain combinations of our VP16-
and p65-linked ZFPs targeted to distinct chromosomal sites were
cotransfected, the observed VEGF-A activation was more than additive
relative to the activation levels of the individual ZFPs. Finally, we
show that the levels of activation achieved by these engineered
transcription factors exceeded VEGF-A levels attained during the
hypoxic response and that the relative proportions of VEGF-A splice
variants produced by this activation closely approximated normal splice
variant ratios seen in these cells.
Mapping of DNase I-accessible Chromatin Regions in the VEGF-A
Locus--
The immortalized cell lines used in these studies (HEK293,
Hep3B, and H9c2(2-1)) were obtained from American Type Culture Collection, and human primary skeletal muscle cells were obtained from
Clonetics Corp. Each line was maintained essentially as recommended by
the suppliers. Rat primary cardiac myocytes were recovered from the
hearts of day 1 neonatal Wistar-Han rats (Charles River Laboratories)
via dissociation with a solution of 115 units/ml type II collagenase
and 0.08% pancreatin. They were then purified on a discontinuous
Percoll gradient, resuspended in plating medium containing 15% serum,
and plated on gelatin-coated plates for 24 h. Cells were
maintained in serum-free medium for 24-48 h prior to use in DNase I
mapping studies.
Nuclei were isolated and treated with DNase I (Worthington) essentially
as described (17), except that DNase I digests were carried out for 1.5 min at 22 °C, and the concentrations of DNase I used were as
indicated in the legend to Fig. 1. Genomic DNA isolation, restriction
enzyme digestion, and Southern blot analysis were then performed
essentially as described (17), except that enzymes and probes were as
indicated in Fig. 1.
Synthesis, Purification, and Gel Shift Analysis of Zinc Finger
Proteins--
Genes encoding our VEGF-A-targeted ZFPs were assembled,
cloned, and purified as previously described (17). Briefly,
oligonucleotides encoding
Binding studies were performed essentially as described (17), except
that the binding reactions contained ~10 pM labeled target site, and the buffer composition was as follows: 17 mM Tris, 170 mM KCl, 1.7 mM
MgCl2, 3.5 mM dithiothreitol, 0.033 mM ZnCl2, 15% glycerol, 300 µg/ml bovine
serum albumin, and 0.03% IGEPAL. In addition, ZFP
concentrations for these studies were determined directly by measuring
the DNA-binding activity of each ZFP preparation using conditions under
which binding is essentially stoichiometric (concentrations of ZFP and
target site > 50 × Kd). Using this
modified protocol, SP1 exhibits a significantly higher apparent
affinity than was determined in previous studies (17), and it is likely
that the use of both activity-based estimates of ZFP concentration and
the new binding buffer in these studies contributed to the difference
in apparent Kd values.
Zinc Finger Protein Expression Constructs Used for Cell Culture
Studies--
ZFPs with distinct binding properties were assembled and
cloned into the pcDNA3 mammalian expression vector (Invitrogen) as described previously (17). A cytomegalovirus promoter drove the
expression of all the ZFPs in mammalian cells. All ZFP constructs contained an N-terminal nuclear localization signal
(Pro-Lys-Lys-Lys-Arg-Lys-Val) from SV40 large T antigen, a ZFP
DNA-binding domain, an activation domain, and a FLAG peptide
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). ZFP-VP16 fusions contained the
herpes simplex virus VP16 activation domain from amino acids 413-490
(17, 41). ZFP-p65 fusions contained the human NF- Assay for Human VEGF-A Reporter Activation--
The effect of
ZFPs on human VEGF-A promoter activity was measured using a luciferase
reporter construct containing the human VEGF-A promoter. The human
VEGF-A-luciferase reporter pGLPVFH was made by inserting a genomic DNA
fragment containing 3318 bp of the human VEGF-A promoter and its
flanking sequences (nucleotides Assay for Activity of ZFP Fusions for Endogenous VEGF-A
Activation in Human Cells by Transient Transfection--
HEK293 cells
were plated in 24-well plates at a density of 160,000 cells/well and
grown as described above. One day later, plasmids encoding ZFP-VP16
fusions were transfected into the cells via LipofectAMINE reagent
according to the manufacturer's recommendations, using 1.5 µl of
LipofectAMINE reagent and 0.3 µg of ZFP plasmid DNA per well. The
medium was removed and replaced with fresh medium 16 h after
transfection. Forty hours after transfection, the culture medium and
the cells were harvested and assayed for VEGF-A expression. VEGF-A
protein contents in the culture medium were assayed using a human
VEGF-A ELISA kit (R&D Systems) according to the manufacturer's protocol.
For Western analysis of ZFP protein expression, cells were lysed with
Laemmli sample loading buffer, and the lysates were analyzed via
electrophoresis on a 10% polyacrylamide gel (Bio-Rad) followed by
Western blotting using anti-FLAG M2 monoclonal antibody (Sigma), which
recognizes the FLAG epitope tag of the engineered ZFPs. The Western
blots were visualized by ECL (Amersham Pharmacia Biotech) as described
previously (17).
For quantitative RT-PCR analysis of VEGF-A mRNA levels, the cells
were lysed, and total RNA was prepared using the RNeasy total RNA
isolation kit with in-column DNase treatment (QIAGEN Inc.). RNA (25 ng)
was used in real-time quantitative RT-PCR analysis using Taqman
chemistry on an ABI 7700 SDS machine (PerkinElmer Life Sciences) as
described previously (17). Briefly, reverse transcription was performed
at 48 °C for 30 min using MultiScribe reverse transcriptase
(PerkinElmer Life Sciences). Following a 10-min denaturation at
95 °C, PCR amplification using AmpliGold DNA polymerase was
conducted for 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. The results were analyzed using SDS Version 1.6.3 software. The
primers and probes used for Taqman analysis are listed in Table
I. The primers and probes used for
human VEGF-A recognize all known splice variants.
Northern and Splice Variants Analyses--
HEK293 cells were
grown in 150-mm dishes and transfected with the pcDNA3 control
vector or ZFP using LipofectAMINE reagent according to the
manufacturer's recommendations. Cells and conditioned medium were
harvested 40 h after transfection. Total RNA was extracted from
the cells using Trizol reagent (Life Technologies, Inc.) followed by an
RNeasy total RNA isolation midi-prep system (QIAGEN Inc.). For Northern
analysis of VEGF-A mRNA expression, various total RNA samples (30 µg) were resolved on a 1.2% formaldehyde-agarose gel and blotted
onto a Nytran SuperCharge membrane (Schleicher & Schüll). The
membrane was hybridized to a 32P-labeled human VEGF-A-165
cDNA antisense riboprobe at 68 °C in UltrahybTM
hybridization buffer (Ambion, inc.). After washing with 0.1× SSC and
0.1% SDS at 68 °C, the membrane was exposed to film. The same
membrane was stripped by boiling in 0.1% SDS and rehybridized with a
human
For analysis of multiple splice variants, total RNA samples (0.5 µg)
were subjected to 20-cycle RT-PCR using a TitanTM one-tube
RT-PCR system (Roche Molecular Biochemicals). The primers used were
5'-ATGAACTTTCTGCTGTCTTGGGTGCATT-3' and 5'-TCACCGCCTCGGCTTGTCACAT-3'. The PCR products were resolved on a 3% Nusieve 3:1 agarose gel (FMC
Corp. BioProducts), blotted onto a Nytran SuperCharge membrane, and
analyzed by Southern hybridization to a 32P-labeled human
VEGF-A cDNA probe that recognizes all splice variants. The expected
PCR product sizes for VEGF-A-189, VEGF-A-165, and VEGF-A-120 were 630, 576, and 444 bp, respectively.
Constitutive and Cell-specific Regions of Accessible Chromatin in
the VEGF-A Locus--
Our chromatin mapping studies encompassed a
variety of cell types from man and rat, including both tumor lines and
primary cells. The scope of this survey, as well as our choices for
some of the cell types tested, was motivated by our goal of developing candidate ZFPs for a variety of pro- and anti-angiogenic gene therapies. We wished to identify any constitutive accessible chromatin regions in the VEGF-A promoter, as well as those specific to medically relevant target tissues and model organisms. We observed a total of
four distinct hypersensitive regions. Two of these, centered on
approximately bp
In addition to the constitutive hypersensitive regions found at bp
Design and Biochemical Characterization of ZFPs Targeted to Open
Chromatin Regions of VEGF-A--
Design and selection studies of zinc
finger-DNA recognition have yielded a diverse collection of fingers
with characterized triplet specificities (4-14, 16, 48, 49).
Collectively, these fingers provide a "directory" of
triplet-binding modules that may be mixed and matched to obtain
multifinger proteins with the desired binding properties. In
particular, we have found that when care is taken to preserve finger
context, the preferred targets of the resultant ZFPs are typically a
composite of the component finger triplet specificities. Using this
approach, we have designed ZFPs that recognize targets within the
Genes for our ZFPs were assembled using previously described methods
(17), and each protein was expressed in recombinant form (Fig. 2,
A-C). We then characterized the DNA-binding affinity of each
ZFP using a gel shift assay. Procedures for these studies were similar
to those described previously (17) (for details, see "Experimental
Procedures"). We found that our designed ZFPs exhibited a range of
apparent Kd values for their intended DNA targets
from 0.005 to 3 nM (Fig. 2E). For comparison,
under these conditions, SP1, the parent ZFP for our designs, exhibited an apparent Kd of 0.25 nM for its DNA
target (Fig. 2E). These studies demonstrated that each
designed ZFP recognizes its target site with high affinity.
Activation of the Human VEGF-A Gene Promoter by ZFPs--
We chose
to use HEK293 cells for our initial studies of VEGF-A activation with
our designed ZFPs since these cells offered an especially favorable
combination of high transfectability (typically >70%) and low
background expression levels of VEGF-A. We first tested for the ability
of the designed ZFPs to activate the VEGF-A promoter reporter. The ZFPs
were fused to the minimal activation domain of the herpes simplex virus
transcription factor VP16 with a C-terminal FLAG epitope tag and tested
for their activity in cells. To test for human VEGF-A gene promoter
activity, a reporter plasmid was constructed to contain firefly
luciferase gene under the control of the human VEGF-A promoter. When
cotransfected transiently with the reporter plasmid, all of the
designed ZFP-VP16 fusions were able to activate the reporter (Fig.
3B). The level of activation ranged between 3- and 15-fold. The activation was
ZFP-dependent; a fusion of green fluorescent protein (50)
with VP16 was unable to activate the reporter. This result showed that
all of the designed ZFPs were active on a naked DNA template. There
also appeared to be no strong correlation between the binding affinity
of each ZFP and its capacity to transiently activate the VEGF-A
promoter reporter from distant sites in the locus.
Transcriptional Activation of the Endogenous Human VEGF-A Gene
Using ZFPs--
To test whether these ZFP-VP16 fusions were also
active in regulating VEGF-A gene transcription from the endogenous
chromosomal locus, the designed ZFP-VP16 fusions were transiently
transfected into HEK293 cells, and their effect on endogenous VEGF-A
gene expression was analyzed. HEK293 cells produce relatively low
levels of VEGF-A in the absence of any ZFP constructs. As shown in Fig. 3C, expression of the ZFP-VP16 fusions targeted to the open
chromatin regions resulted in the secretion of elevated levels of
VEGF-A into the medium as determined by ELISA. The range of activation varied between 2- and 15-fold, with ZFP VZ+434b being the most active.
The increased VEGF-A protein production induced by ZFP was correlated
with a 2-10-fold increase in the level of VEGF-A mRNA as
determined by quantitative PCR (Fig. 3D). In contrast to
their variable activities, the ZFPs were found to be expressed to
similar levels as determined by Western blotting (Fig. 3E) and by Taqman for mRNA expression (Fig. 3F). Therefore,
the differential activity of the ZFP-VP16 fusions most likely is not
related to differential ZFP expression.
We also compared the behavior of ZFPs targeted to the accessible
regions of VEGF-A with two ZFPs targeted to inaccessible regions. Data
for these studies are shown in Fig. 4.
Whereas all four ZFPs activated the naked reporter construct to
approximately equal levels, a clear discrepancy was seen regarding
activity against the endogenous VEGF-A gene. The accessible
region-targeted ZFPs activated VEGF-A by factors of 4-5, whereas the
ZFPs targeted to sites outside of the accessible regions showed no
appreciable increase in VEGF-A expression levels.
Activation of the Human VEGF-A Gene by ZFPs Fused with Different
Activation Domains--
To achieve a higher level of VEGF-A activation
by ZFPs in human cells, we tested the performance of other activation
domains against that achieved by VP16 (data not shown). We found that the activation domain from the p65 subunit of NF- Cooperation between ZFPs for Activation of the Human VEGF-A
Gene--
The availability of a set of activating ZFPs targeted to
diverse regions of the VEGF-A gene promoter and fused to different activation domains provided an opportunity to investigate whether combinations of ZFPs with different activation domains could achieve an
enhanced activation of VEGF-A gene expression. To test this possibility, various ZFP-VP16 and ZFP-p65 fusions were cotransfected into HEK293 cells and tested for VEGF-A activation by ELISA and Taqman.
As shown in Fig. 6, ZFPs VZ+434b-VP16 and
VZ Comparison with Levels of VEGF-A Produced by Hypoxia--
To
assess how our ZFP-activated levels of VEGF-A expression compared with
the levels produced by physiologically relevant processes, we compared
VEGF-A production induced by ZFPs with that induced by hypoxia. For
many ZFPs tested, e.g. ZFP VZ+434b, we found that the ZFPs
were capable of activating VEGF-A expression to a level higher than
that induced by hypoxia. As shown in Fig. 7, HEK293 cells growing under hypoxic
conditions had a 5-fold higher steady-state VEGF-A mRNA level than
under normoxic conditions (Fig. 7B) and accumulated VEGF-A
protein to nearly 400 pg/ml in medium (a 10-fold increase) (Fig.
7A). ZFP VZ+434b with a p65 activation domain was able to
activate the VEGF-A gene 5-10 times further than that induced by
hypoxia with an accumulation of VEGF-A protein in the culture medium to
nearly 4000 pg/ml (Fig. 7A) and a 20-fold increase in the
VEGF-A mRNA level (Fig. 7B). This observation was also
confirmed by Northern blot analysis (Fig. 7C).
Activation of Multiple Splice Variants Using a Single
ZFP--
Several splice variants of human VEGF-A have been discovered,
each one comprising a specific exon addition (24). The major forms
produce polypeptides with 121, 165, 189, and 206 amino acids, although
VEGF-A-206 is rarely expressed and has been detected only in fetal
liver. Because our ZFPs activated gene transcription from the natural
promoter on the chromosome, we predicted that they would activate all
of the different VEGF-A transcripts equally, preserving their relative
proportions. To test this notion, we analyzed the ability of ZFP
VZ+434b to activate multiple transcripts from the same promoter. To
distinguish the various splice variants, RT-PCR was performed using
primers flanking the splice regions and producing distinct PCR products
for each splice variant. The PCR products were then analyzed by
Southern hybridization using a VEGF-A-165 probe. Three splice variants,
VEGF-A-189, VEGF-A-165, and VEGF-A-121, were detected in HEK293 cells,
with VEGF-A-165 being the predominant form. As demonstrated in Fig.
7D, the introduction of a single ZFP, VZ+434b as either a
VP16 or p65 fusion, resulted a proportional increase in all of the
splice variants.
In this study, we have successfully designed a panel of ZFPs that
bind with high affinity to diverse DNA sequences present within the
VEGF-A locus (Fig. 2) and that are capable of activating expression of
the endogenous human chromosomal VEGF-A gene (Figs. 3-7). Our
experimental approach incorporated information regarding the chromatin
structure of the VEGF-A locus such that proteins were designed to
sequences present within the DNase I-hypersensitive regions (Fig. 1),
and this contributed to the large number of locations that we have
identified from which designed transcription factors may activate
VEGF-A (Figs. 3 and 4) relative to single targets described in previous
studies (16-18). We propose that ZFP targeting strategies that
incorporate information regarding chromatin structure will provide a
more efficient means for identifying artificial transcription factors
capable of specifically regulating endogenous genes compared with
strategies based on ZFP design principles alone. We found that several
of these designed ZFPs are quite potent, yielding VEGF-A levels that
exceed those induced by hypoxia (Fig. 7). We speculate that the
regulation of multiple VEGF-A splice variants will be of utility in
somatic gene therapy approaches to cardiovascular disease (51-56).
Our panel of artificial transcription factors targeted to diverse sites
in VEGF-A provides a unique tool for assessing the structural
determinants of transcriptional activation at an endogenous locus, and
several of our results may bear the signature of the transcriptional
effects of chromatin structure. We found, for example, that our panel
of ZFPs exhibits quite different patterns of activation when tested
using the endogenous VEGF-A locus versus a naked promoter
reporter construct (Fig. 3, compare B and C). They also differ depending on whether the site is within a DNase I-accessible region or not (Fig. 4). Furthermore, we observed that the
ability of the p65 activation domain to outperform VP16 also varies in
a target site-dependent manner, with relative activation levels varying over a factor of 3 depending on the location of the ZFP
target within the VEGF-A locus (Fig. 5). These effects highlight the
role of structural context that is imposed by chromatin and the binding
of other regulatory proteins on the capacity of transcription factors
to activate an endogenous locus and also reemphasize the distinct
regulatory and steric requirements of different activation domains to
achieve optimal performance.
The capacity to generate a panel of designed transcription factors
targeted to diverse sites in an endogenous locus also provides practical advantages in a variety of applications. In studying the
effects of up- or down-regulation of a target locus, for example, conclusions regarding gene function will be strongest if a given effect
is observed repeatedly using multiple different regulators. In
addition, for potential medical uses, the availability of multiple ZFP
candidates provides a greater likelihood of finding one that yields
optimal benefits with minimal side effects. Perhaps the most exciting
possibilities for use of these proteins, however, lie in the study of
transcriptional regulation. For example, the ability to target multiple
activation domains to arbitrary sites in the same locus may have
applications in the study of synergy. In this study, we have taken the
first steps to examine this issue and have demonstrated enhanced
effects on transcriptional activation mediated by cotransfected VP16
and p65 activation domain-bearing ZFPs. It seems reasonable to
speculate that, by using larger combinations of appropriately targeted
functional domains, our ZFPs may offer the prospect for total
reconstitution of activation processes using completely defined components.
An additional observation of these studies is that ZFPs fused with the
268-amino acid activation domain of p65 (42) activate the endogenous
VEGF-A gene as well as or better than the 78-amino acid activation
domain of the herpes simplex virus VP16 (41). Although both VP16 and
NF- Finally, we have observed that our designed ZFPs up-regulate each major
splice variant of VEGF-A proportionally. This is important because
recent studies suggest that proper isoform balance is crucial for
VEGF-A function (27, 28). In particular, the 165, 189, and 206 splice
variants have increasingly stronger heparin-binding domains, which help
them bind to the extracellular matrix and are involved in presentation
to VEGF-A receptors. The heparin-binding ability is a critical
determinant of VEGF-A potency, resulting in different biological
activities for different splice variants. Currently, most VEGF-A gene
therapy trials involve the application of just a single VEGF-A splice
variant protein or cDNA (51-55), and it has been suggested that an
ideal gene therapy agent should be able to recapitulate natural ratios
of various different VEGF-A isoforms. Activation of VEGF-A using our
designed ZFPs may therefore offer advantages in this regard, and
VEGF-A-specific ZFPs could provide key components of the next
generation of pro-angiogenic gene therapy agents.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (42) and tested for the capacity to activate
transcription of endogenous VEGF-A both alone and in certain
combinations with our VP16-linked ZFPs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix and
-sheet regions of each
three-finger protein were assembled using PCR (see Fig. 2, A
and B), and each resultant ZFP gene was cloned into the
pMal-c2 plasmid (New England Biolabs Inc.) as a fusion with DNA
encoding maltose-binding protein. Maltose-binding protein-ZFP fusions
were then expressed and affinity-purified using an amylose resin (New
England Biolabs Inc.).
B transcription
factor p65 subunit (amino acids 288-548) as the activation domain
(42).
2279 to +1039) into the pGL3-basic
vector (Promega) between the KpnI and NcoI sites.
The translation start codon ATG of the VEGF-A gene was directly fused
with the luciferase gene in this construct. HEK293 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum in a 5% CO2 incubator at 37 °C. Cells were
plated in 24-well plates at a density of 160,000 cells/well 1 day
before transfection. The VEGF-A reporter construct and ZFP-VP16 fusion
plasmid were cotransfected into the cells via LipofectAMINE reagent
(Life Technologies, Inc.) according to the manufacturer's
recommendations, using 1.5 µl of LipofectAMINE reagent, 260 ng of the
VEGF-A reporter construct, 30 ng of plasmid DNA encoding ZFP-VP16, and
10 ng of the pRL-CMV control plasmid (Promega). The medium was removed
and replaced with fresh medium 16 h after transfection. Forty
hours after transfection, the medium was removed, and the cells were
harvested and assayed for luciferase reporter activity using the
dual-luciferase assay system (Promega) according to the manufacturer's protocol.
Nucleotide sequences of the primers and probes used for Taqman analysis
-actin antisense riboprobe.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
550 and +1 of the VEGF-A promoter, were invariably observed in every cell type tested. Fig.
1 shows typical experimental results
identifying these regions in both man (Fig. 1A) and rat (Fig. 1B). Both regions appeared as doublets when viewed at
higher resolution (e.g. Fig. 1, A and
C). The discovery of an open region centered on bp +1
was somewhat expected, as hypersensitive regions are often observed in
the vicinity of sites for transcription initiation (for a review, see
Ref. 43), and this region also contains conserved regulatory elements
that have been shown to be important for VEGF-A activation, including
targets for SP1 and AP-2 (44). The observation in both man and rat of
accessible chromatin in the
550 region was somewhat more surprising,
as no regulatory elements have thus far been mapped to this area of the
VEGF-A promoter. Interestingly, this region exhibits a high degree of
sequence conservation across all species for which VEGF-A promoter
sequence is available (man, mouse, and rat) (Fig. 1F,
gray trace), which becomes more pronounced when conservation is considered in terms of regulatory element-sized sequence blocks (Fig. 1F, black trace). This suggests the
possibility that as yet unidentified regulators of VEGF-A transcription
may operate via binding to elements in this region. Initial studies in
which a DNA fragment containing this region was fused to the promoter of a luciferase reporter indicated that this region exhibited cis-acting regulatory functions (data not shown). We
excluded the possibility that this DNA sequence itself was a more
favored substrate for DNase I digestion by performing a control mapping study using purified naked genomic DNA, which indicated no enhanced cutting of this region by DNase I (data not shown).
View larger version (30K):
[in a new window]
Fig. 1.
Regions of enhanced chromatin accessibility
in the promoter of VEGF-A. A and B,
constitutive sites. Nuclei from the indicated human (A) or
rat (B) cells were partially digested with DNase I (see
"Experimental Procedures"), followed by Southern blot analysis
using the indicated restriction enzymes and probes. Vertical
bars represent genomic DNA in the VEGF-A promoter region.
Bent arrows denote the transcription start site, and
tick marks indicate units of 100 bp. Positions of
restriction enzyme target sites are indicated in base pairs relative to
the start site of VEGF-A transcription. The migration pattern for a set
of DNA standard fragments is indicated to the right of each
panel, with the size of each fragment given in base pairs.
Arrows highlight the relationship of the observed bands to
the location of accessible chromatin regions relative to the
transcription start sites of VEGF-A. DNase I concentrations were as
follows: A, HEK293 nuclei (lanes 1-4), 0, 7.5, 15, and 60 units/ml; and Hep3B nuclei (lanes 5-8), 0, 7.5, 15, and 30 units/ml. B, rat cardiac myocyte nuclei (lanes
1-4), 0, 3.75, 7.5, and 15 units/ml; and rat H9c2(2-1) nuclei
(lanes 5-8), 0, 15, 30, and 60 units/ml. C,
presence of an accessible region ~1000 bp upstream of the VEGF-A
transcription start site in primary skeletal muscle cells, but not in
HEK293 cells. Details were as described for A and
B, except that nuclei were isolated from HEK293 cells or
human primary skeletal muscle cells. DNase I concentrations were as
follows: HEK293 nuclei (lanes 1-5), 0, 7.5, 15, 30, and 60 units/ml; and human primary skeletal muscle nuclei (lanes
6-10), 0, 3.75, 7.5, 15, and 30 units/ml. D, presence
of an accessible region 500 bp downstream of the VEGF-A transcription
start site in HEK293 cells. Details were as described for A
and B. DNase I concentrations were as follows: 0, 15, 30, 60, and 120 units/ml (lanes 1-5, respectively).
E, summary of DNase I-accessible regions observed in our
studies. The cell types tested are indicated on the left. Observation
of a particular open region in a given cell type is denoted by an
arrow. The 550 and +1 hypersensitive regions occurred in
all cell types tested, whereas the
1000 and +500 sites appeared only
in a subset of tested cells. A schematic representation of the VEGF-A
promoter, encompassing bases
1000 to +1000 relative to the principal
transcription start site, is provided on the bottom. The black
arrow denotes the principal site of transcription initiation,
whereas the position of a reported alternate start site (47) is
highlighted by the white arrow. Key regulatory elements are
also shown (hypoxia response element (HRE)). DNase
I-accessible regions are indicated by gradient-shaded
rectangles. F, sequence conservation among man, mouse,
and rat in the promoter region of VEGF-A. Each point in the
gray profile indicates the fractional conservation of human
VEGF-A sequence in both rat and mouse within a 50-bp window centered on
that point. The black profile is identical, except that it
indicates the fractional conservation of 5-bp blocks.
550 and +1, we identified two other stretches of accessible chromatin
that were apparent in only a subset of the cells used in our studies.
One of these regions encompassed ~300 bp centered on the hypoxia
response element of VEGF-A and was observed in human primary skeletal
muscle cells (Fig. 1C) and in the Hep3B cell line (data not
shown). In contrast, this site was clearly not observed in HEK293 cells
(Fig. 1C). The hypoxia response element encompasses a region
of enhanced sequence conservation in the VEGF-A promoter (Fig.
1F) and has been shown to contain several conserved
regulatory elements that are required for induction of VEGF-A by
hypoxia, including a binding site for hypoxia-inducible factor 1 (38,
39, 46). A final hypersensitive region was observed ~500 bp
downstream of the transcription start site in HEK293 cells (Fig.
1D), primary skeletal muscle cells (data not shown), and rat
primary cardiac myocytes (Fig. 1B). Interestingly, this
region contains a putative SP1-binding site and is adjacent to an
alternate transcription start site (47) (Fig. 1E). This region also displayed the capacity to activate the expression of a
reporter gene in cis (data not shown). In summary, these studies identified four regions of accessible chromatin within the
promoter region of VEGF-A that were conserved in rat and man (Fig.
1E). Three of these, centered at approximately bp
550, +1,
and +500 relative to the transcription start site, were found in HEK293
cells and were targeted for further studies with designed ZFPs.
550, +1, and +500 open chromatin regions observed in HEK293 cells
(Fig. 2D) and assembled genes
for these ZFPs. We also included two control ZFPs targeted to sites
outside of the hypersensitive regions. The designs for our ZFPs are
shown in Table II with each finger design indicated by the amino acid
sequence of positions "
1" through "+6" of its
-helix. We have named the ZFPs
according to their target location relative to the transcription start
site of VEGF-A. Thus, the first bases in the target sequences of
VZ
475 and VZ+590 lie 475 base pairs upstream and 590 nucleotides
downstream, respectively, of the principal site of transcription
initiation. One of our designs has two target sites in this region and
so has been given a complex name to reflect this fact (VZ+42/+530).
Where there are multiple ZFPs targeted to a given sequence, this is
indicated by the use of a lowercase letter suffix at the end of each
name to distinguish between alternate ZFP designs.
View larger version (50K):
[in a new window]
Fig. 2.
Construction scheme and DNA-binding
properties of the VEGF-A targeted ZFPs. A, ribbon
diagram of the x-ray crystal structure of an individual zinc finger
with two -sheets linked to the DNA-binding
-helix (61). The
locations of oligonucleotide primers used for assembly are indicated
relative to the regions of the finger they encode. In our ZFP assembly
scheme, oligonucleotides (Oligo) 1, 3, and 5 comprise the
-sheet regions, and oligonucleotides 2, 4, and 6 comprise the
DNA-binding
-helix regions. B, assembly scheme of the
ZFPs. Six overlapping oligonucleotides were annealed and amplified with
a pair of external oligonucleotides. The PCR products were then cloned
into the KpnI and BamHI sites of the pMal-c2
bacterial expression vector. C, scheme of the
maltose-binding protein-ZFP fusions used for gel shift analysis.
D, schematic representation of the human VEGF-A gene showing
the locations of the principal (black arrow) and a reported
alternate (white arrow) (47) transcription initiation site,
DNase I-accessible regions in HEK293 cells (gradient-filled
rectangles), and target site locations for the ZFPs used in these
studies (white vertical rectangles). The location of the
upstream edge of each ZFP target is indicated by the number below it.
Numbering is relative to the start site of transcription (+1).
E, gel shift assays using the various VEGF-A-targeted ZFPs
binding to their DNA targets. A 2-fold dilution series of each protein
was tested for binding to its DNA target, with the highest
concentration in the second lane and the lowest
concentration in the eleventh lane (from left to
right). The first lane is a control lane containing probe
alone.
Zinc finger designs and apparent Kd values of the human
VEGF-A-targeted ZFPs
View larger version (19K):
[in a new window]
Fig. 3.
Transcriptional activation properties of ZFPs
targeted to DNase I-accessible regions of VEGF-A. A,
schematic representation of the naked VEGF-A promoter reporter
(top) and endogenous chromosomal (bottom) targets
used in this experiment. Coverage of portions of the endogenous
promoter with white ovals indicates occlusion of these
regions with nucleosomes. ZFP targets are indicated by white
vertical rectangles, and arrows connect each target
with the name of its corresponding ZFP below. B, activation
of the naked human VEGF-A promoter reporter by ZFP-VP16 fusions.
ZFP-VP16 fusion plasmids were cotransfected with a VEGF-A reporter
construct containing the luciferase gene under the control of 3.4 kilobases of the human VEGF-A promoter into HEK293 cells, and
the reporter activity was assayed 40 h post-transfection as
described under "Experimental Procedures." A constitutive
Renilla luciferase construct was also cotransfected to serve
as a transfection control for normalization. The fold reporter
activation by the ZFPs was calculated based on the normalized
luciferase reporter activity in comparison with that of a control
vector encoding VP16-FLAG without ZFP. C and D,
activation of the endogenous human VEGF-A gene by ZFP-VP16 fusions. Plasmids encoding ZFP-VP16 fusions were transfected into HEK293
cells via LipofectAMINE reagent as described under "Experimental
Procedures." The control vector expressed VP16-FLAG fused with green
fluorescent protein instead of ZFP. Forty hours after transfection, the
culture medium and cells were harvested and assayed for endogenous
VEGF-A expression. In C, the VEGF-A protein contents in the
culture medium were measured by ELISA using a human VEGF-A ELISA kit.
The VEGF-A protein production induced by the ZFPs was compared with
that of the control vector. The fold activation was plotted. In
D, the steady-state VEGF-A mRNA levels in the
transfected cells was measured by quantitative RT-PCR using Taqman
chemistry as described under "Experimental Procedures." The levels
of VEGF-A mRNA were normalized against glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). E, the ZFP protein
contents in the transfected cells analyzed by Western blotting using
anti-FLAG antibody (Sigma), which recognizes the FLAG epitope tag of
the engineered ZFPs. F, levels of ZFP mRNA in the
transfected cells determined by Taqman and normalized against
glyceraldehyde-3-phosphate dehydrogenase mRNA levels. The primers
and probe were designed to recognize the sequence encoding the VP16
activation domain and FLAG tag.
View larger version (21K):
[in a new window]
Fig. 4.
Differential behavior of ZFPs targeted to
accessible and inaccessible regions of VEGF-A. A,
schematic representation of the naked VEGF-A promoter reporter
(top) and endogenous chromosomal (bottom) targets
used in this experiment. Coverage of portions of the endogenous
promoter with white ovals indicates occlusion of these
regions with nucleosomes. ZFP targets are indicated by white
vertical rectangles, and arrows connect each target
with the name of its corresponding ZFP below. B, activation
of the naked human VEGF-A promoter reporter. The indicated ZFP-VP16
fusion plasmids were cotransfected with the VEGF-A-luciferase reporter
construct as described under "Experimental Procedures." The fold
luciferase reporter activation by the ZFPs was calculated in comparison
with that of a control vector encoding VP16-FLAG without ZFP.
C, activation of the endogenous human VEGF-A gene. Plasmids
encoding the various indicated ZFP-VP16 fusions were transfected into
HEK293 cells, and the VEGF-A protein contents secreted in the culture
medium 40 h after transfection were measured by ELISA as described
for Fig. 3C. The VEGF-A protein production induced by the
ZFPs was compared with that of the control vector, and the -fold
activation was plotted.
B offered higher levels of activation when tested using several of our ZFPs (Fig. 5). In this experiment, some ZFP-p65
fusions, e.g. ZFP VZ+434b, induced a VEGF-A protein
accumulation that was 3-4-fold higher than that induced by ZFP-VP16
fusions (Fig. 5C), although the ZFP-p65 fusion proteins were
expressed and accumulated in cells to similar levels as the ZFP-VP16
fusions (Fig. 5B). The higher level of ZFP-p65
fusion-induced VEGF-A protein production was consistent with a higher
level of VEGF-A mRNA transcription as determined by Taqman analysis
(Fig. 5D). Interestingly, for some ZFPs such as VZ
8, the
p65 and VP16 fusions displayed similar activities, suggesting that VP16
and p65 may have distinct target location-dependent
activation mechanisms.
View larger version (15K):
[in a new window]
Fig. 5.
Activation of the endogenous human VEGF-A
gene by ZFP with different activation domains. Various ZFPs were
fused with VP16 ( ) or the activation domain (AD) from
NF-
B (p65) (
), both with a C-terminal FLAG tag. Their activities
were tested as described in the legend to Fig. 3 and compared.
A, shown is a scheme of the VEGF-A-targeted VP16 and p65
activation domain-linked ZFP fusions. B, the ZFP protein
contents in the transfected cells were analyzed by Western blotting
using anti-FLAG antibody. C, the VEGF-A protein contents in
the culture medium were measured by ELISA. D, the
steady-state VEGF-A mRNA levels in the transfected cells were
measured by Taqman. NLS, nuclear localization signal.
573a-p65 individually activated VEGF-A protein production in HEK293
cells by 8- and 6-fold, respectively. However, when they were
cotransfected at a 1:1 ratio into the cells, the level of VEGF-A gene
activation was enhanced (30-fold by ELISA) compared with the levels
induced by each individual ZFP. A similar cooperation between ZFPs
VZ+434b-p65 and VZ
573a-VP16 was also observed (data not shown).
View larger version (18K):
[in a new window]
Fig. 6.
Cooperativity between ZFPs with different
activation domains in VEGF-A gene activation. Plasmids containing
different ZFP-activation domain fusions were cotransfected at a 1:1
ratio into HEK293 cells. Endogenous VEGF-A activation was measured
40 h after transfection. A, shown is a scheme of the
experiment. B, the VEGF-A protein contents in the culture
medium were assayed by ELISA. C, the VEGF-A mRNA levels
in the transfected cells were measured by Taqman. D, the ZFP
protein contents in the transfected cells were analyzed by Western
blotting using anti-FLAG antibody. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
View larger version (15K):
[in a new window]
Fig. 7.
Activation of the endogenous human VEGF-A
gene by ZFP VZ+434b and by hypoxia. HEK293 cells grown in 150-mm
dishes either were transfected with plasmids encoding ZFP VZ+434b fused
with VP16 or p65 or with a control vector expressing no ZFP or were
grown under hypoxic conditions (0.5% oxygen) for 24 h. Endogenous
VEGF-A gene activation was measured as described in the legend to Fig.
3 and compared. A, the VEGF-A protein contents in the
culture medium were measured by ELISA. B, the steady-state
VEGF-A mRNA levels in the cells were measured by Taqman and
normalized against 18 S RNA. C, the VEGF-A mRNA levels
were detected by Northern hybridization using a 32P-labeled
VEGF-A-165 cDNA probe. D, the splice variants for VEGF-A
were analyzed by RT-PCR and detected by Southern hybridization using a
32P-labeled VEGF-A-165 cDNA probe as described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B p65 are strong acidic activation domains and share certain
functional features (for example, recruitment of the ARC/DRIP
complex and facilitated assembly of a preinitiation complex on promoter
DNA (57, 58)), differences in their transactivation mechanism have been
reported. For example, the histone acetyltransferase activity of p300
has been demonstrated to be necessary for activation by NF-
B, but
less essential for activation by VP16 (59, 60). It is possible that the
local availability of p300 or other coactivators in the endogenous
VEGF-A locus may account for the differences we observed.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Reed Hickey and Frank Giordano for providing key protocols and advice for the rat neonatal cardiac myocyte preparations; Brian Johnstone, Priya Sreenivasan, and Yolanda Santiago for helpful discussions and assistance with the mapping of DNase I-accessible regions; Michelle Ha for expert technical support in early cell culture studies; and Monica Miller, Katherine Pasquetti, Anna Vincent, Damon Toroian, and Brad Campos for assembly and initial testing of ZFP constructs. We are also grateful to Edward Lanphier, Peter Bluford, and Carl Pabo for encouragement and support.
![]() |
FOOTNOTES |
---|
* 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.
These authors contributed equally to this work.
§ Present address: Bolder Biotechnology, Inc., Campus Box 347, C.U.-Boulder, Boulder, CO 80309-0347.
¶ To whom correspondence should be addressed: Sangamo BioSciences Inc., Point Richmond Tech Center, 501 Canal Blvd., Suite A100, Richmond, CA 94804. Tel.: 510-970-6000 (ext. 216); Fax: 510-236-8951; E-mail: awolffe@sangamo.com.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M011172200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ZFPs, zinc finger
proteins;
VEGF-A, vascular endothelial growth factor A;
HEK293, human
embryonic kidney 293;
NF-B, nuclear factor
B;
bp, base pair(s);
PCR, polymerase chain reaction;
RT-PCR, reverse
transcription-polymerase chain reaction;
ELISA, enzyme-linked
immunosorbent assay.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Kim, J. S.,
Kim, J.,
Cepek, K. L.,
Sharp, P. A.,
and Pabo, C. O.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3616-3620 |
2. | Chatterjee, S., and Struhl, K. (1995) Nature 374, 820-822[CrossRef][Medline] [Order article via Infotrieve] |
3. | Klages, N., and Strubin, M. (1995) Nature 374, 822-823[CrossRef][Medline] [Order article via Infotrieve] |
4. | Jamieson, A. C., Kim, S. H., and Wells, J. A. (1994) Biochemistry 33, 5689-5695[Medline] [Order article via Infotrieve] |
5. |
Jamieson, A. C.,
Wang, H.,
and Kim, S. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12834-12839 |
6. | Rebar, E. J., and Pabo, C. O. (1994) Science 263, 671-673[Medline] [Order article via Infotrieve] |
7. | Rebar, E. J., Greisman, H. A., and Pabo, C. O. (1996) Methods Enzymol. 267, 129-149[CrossRef][Medline] [Order article via Infotrieve] |
8. | Desjarlais, J. R., and Berg, J. M. (1992) Proteins Struct. Funct. Genet. 13, 272[Medline] [Order article via Infotrieve] |
9. | Desjarlais, J. R., and Berg, J. M. (1992) Proteins Struct. Funct. Genet. 12, 101-104[Medline] [Order article via Infotrieve] |
10. | Desjarlais, J. R., and Berg, J. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2256-2260[Abstract] |
11. |
Greisman, H. A.,
and Pabo, C. O.
(1997)
Science
275,
657-661 |
12. |
Choo, Y.,
and Klug, A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11168-11172 |
13. |
Choo, Y.,
and Klug, A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11163-11167 |
14. |
Segal, D. J.,
Dreier, B.,
Beerli, R. R.,
and Barbas, C. F., III
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2758-2763 |
15. |
Liu, Q.,
Segal, D. J.,
Ghiara, J. B.,
and Barbas, C. F., III
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5525-5530 |
16. |
Bartsevich, V. V.,
and Juliano, R. L.
(2000)
Mol. Pharmacol.
58,
1-10 |
17. |
Zhang, L.,
Spratt, S. K.,
Liu, Q.,
Johnstone, B.,
Qi, H.,
Raschke, E. E.,
Jamieson, A. C.,
Rebar, E. J.,
Wolffe, A. P.,
and Case, C. C.
(2000)
J. Biol. Chem.
275,
33850-33860 |
18. |
Beerli, R. R.,
Dreier, B.,
and Barbas, C. F., III
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1495-1500 |
19. | Archer, T. K., Lefebvre, P., Wolford, R. G., and Hager, G. L. (1992) Science 255, 1573-1576[Medline] [Order article via Infotrieve] |
20. |
Tse, C.,
Sera, T.,
Wolffe, A. P.,
and Hansen, J. C.
(1998)
Mol. Cell. Biol.
18,
4629-4638 |
21. |
Wolffe, A. P.,
and Hayes, J. J.
(1999)
Nucleic Acids Res.
27,
711-720 |
22. | Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J., and Holash, J. (2000) Nature 407, 242-248[CrossRef][Medline] [Order article via Infotrieve] |
23. | Flamme, I., Frohlich, T., von Reutern, M., Kappel, A., Damert, A., and Risau, W. (1997) Mech. Dev. 63, 51-60[CrossRef][Medline] [Order article via Infotrieve] |
24. | Ferrara, N. (1999) J. Mol. Med. 77, 527-543[CrossRef][Medline] [Order article via Infotrieve] |
25. | Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., O'Shea, K. S., Powell-Braxton, L., Hillan, K. J., and Moore, M. W. (1996) Nature 380, 439-442[CrossRef][Medline] [Order article via Infotrieve] |
26. | Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C., Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W., and Nagy, A. (1996) Nature 380, 435-439[CrossRef][Medline] [Order article via Infotrieve] |
27. | Carmeliet, P., Ng, Y. S., Nuyens, D., Theilmeier, G., Brusselmans, K., Cornelissen, I., Ehler, E., Kakkar, V. V., Stalmans, I., Mattot, V., Perriard, J. C., Dewerchin, M., Flameng, W., Nagy, A., Lupu, F., Moons, L., Collen, D., D'Amore, P. A., and Shima, D. T. (1999) Nat. Med. 5, 495-502[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Grunstein, J.,
Masbad, J. J.,
Hickey, R.,
Giordano, F.,
and Johnson, R. S.
(2000)
Mol. Cell. Biol.
20,
7282-7291 |
29. | Damert, A., Ikeda, E., and Risau, W. (1997) Biochem. J. 327, 419-423[Medline] [Order article via Infotrieve] |
30. |
Diaz, B. V.,
Lenoir, M. C.,
Ladoux, A.,
Frelin, C.,
Demarchez, M.,
and Michel, S.
(2000)
J. Biol. Chem.
275,
642-650 |
31. |
Cohen, T.,
Nahari, D.,
Cerem, L. W.,
Neufeld, G.,
and Levi, B. Z.
(1996)
J. Biol. Chem.
271,
736-741 |
32. | Chua, C. C., Hamdy, R. C., and Chua, B. H. (1998) Free Radic. Biol. Med. 25, 891-897[CrossRef][Medline] [Order article via Infotrieve] |
33. | Salimath, B., Marme, D., and Finkenzeller, G. (2000) Oncogene 19, 3470-3476[CrossRef][Medline] [Order article via Infotrieve] |
34. | Ladoux, A., and Frelin, C. (1994) Biochem. Biophys. Res. Commun. 204, 794-798[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Ryuto, M.,
Ono, M.,
Izumi, H.,
Yoshida, S.,
Weich, H. A.,
Kohno, K.,
and Kuwano, M.
(1996)
J. Biol. Chem.
271,
28220-28228 |
36. |
Kimura, H.,
Weisz, A.,
Ogura, T.,
Hitomi, Y.,
Kurashima, Y.,
Hashimoto, K.,
D'Acquisto, F.,
Makuuchi, M.,
and Esumi, H.
(2001)
J. Biol. Chem.
276,
2292-2298 |
37. |
Kimura, H.,
Weisz, A.,
Kurashima, Y.,
Hashimoto, K.,
Ogura, T.,
D'Acquisto, F.,
Addeo, R.,
Makuuchi, M.,
and Esumi, H.
(2000)
Blood
95,
189-197 |
38. |
Levy, A. P.,
Levy, N. S.,
Wegner, S.,
and Goldberg, M. A.
(1995)
J. Biol. Chem.
270,
13333-13340 |
39. |
Liu, Y.,
Cox, S. R.,
Morita, T.,
and Kourembanas, S.
(1995)
Circ. Res.
77,
638-643 |
40. | Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., and Semenza, G. L. (1996) Mol. Cell. Biol. 16, 4604-4613[Abstract] |
41. | Sadowski, I., Ma, J., Triezenberg, S., and Ptashne, M. (1988) Nature 335, 563-564[CrossRef][Medline] [Order article via Infotrieve] |
42. | Ruben, S. M., Dillon, P. J., Schreck, R., Henkel, T., Chen, C. H., Maher, M., Baeuerle, P. A., and Rosen, C. A. (1991) Science 251, 1490-1493[Medline] [Order article via Infotrieve] |
43. | Gross, D. S., and Garrard, W. T. (1988) Annu. Rev. Biochem. 57, 159-197[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Milanini, J.,
Vinals, F.,
Pouyssegur, J.,
and Pages, G.
(1998)
J. Biol. Chem.
273,
18165-18172 |
45. | Grant, R. A., Rould, M. A., Klemm, J. D., and Pabo, C. O. (2000) Biochemistry 39, 8187-8192[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Ikeda, E.,
Achen, M. G.,
Breier, G.,
and Risau, W.
(1995)
J. Biol. Chem.
270,
19761-19766 |
47. | Akiri, G., Nahari, D., Finkelstein, Y., Le, S. Y., Elroy-Stein, O., and Levi, B. Z. (1998) Oncogene 17, 227-236[CrossRef][Medline] [Order article via Infotrieve] |
48. | Desjarlais, J. R., and Berg, J. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7345-7349[Abstract] |
49. | Elrod-Erickson, M., Benson, T. E., and Pabo, C. O. (1998) Structure 6, 451-464[Medline] [Order article via Infotrieve] |
50. | Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Science 263, 802-805[Medline] [Order article via Infotrieve] |
51. | Rosengart, T. K., Lee, L. Y., Patel, S. R., Kligfield, P. D., Okin, P. M., Hackett, N. R., Isom, O. W., and Crystal, R. G. (1999) Ann. Surg. 230, 466-470[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Rosengart, T. K.,
Lee, L. Y.,
Patel, S. R.,
Sanborn, T. A.,
Parikh, M.,
Bergman, G. W.,
Hachamovitch, R.,
Szulc, M.,
Kligfield, P. D.,
Okin, P. M.,
Hahn, R. T.,
Devereux, R. B.,
Post, M. R.,
Hackett, N. R.,
Foster, T.,
Grasso, T. M.,
Lesser, M. L.,
Isom, O. W.,
and Crystal, R. G.
(1999)
Circulation
100,
468-474 |
53. |
Hendel, R. C.,
Henry, T. D.,
Rocha-Singh, K.,
Isner, J. M.,
Kereiakes, D. J.,
Giordano, F. J.,
Simons, M.,
and Bonow, R. O.
(2000)
Circulation
101,
118-121 |
54. | Esakof, D. D., Maysky, M., Losordo, D. W., Vale, P. R., Lathi, K., Pastore, J. O., Symes, J. F., and Isner, J. M. (1999) Hum. Gene Ther. 10, 2307-2314[CrossRef][Medline] [Order article via Infotrieve] |
55. | Isner, J. M., Pieczek, A., Schainfeld, R., Blair, R., Haley, L., Asahara, T., Rosenfield, K., Razvi, S., Walsh, K., and Symes, J. F. (1996) Lancet 348, 370-374[CrossRef][Medline] [Order article via Infotrieve] |
56. | Isner, J. M., Baumgartner, I., Rauh, G., Schainfeld, R., Blair, R., Manor, O., Razvi, S., and Symes, J. F. (1998) J. Vasc. Surg. 28, 964-973[Medline] [Order article via Infotrieve] |
57. | Naar, A. M., Beaurang, P. A., Zhou, S., Abraham, S., Solomon, W., and Tjian, R. (1999) Nature 398, 828-832[CrossRef][Medline] [Order article via Infotrieve] |
58. | Rachez, C., Lemon, B. D., Suldan, Z., Bromleigh, V., Gamble, M., Naar, A. M., Erdjument-Bromage, H., Tempst, P., and Freedman, L. P. (1999) Nature 398, 824-828[CrossRef][Medline] [Order article via Infotrieve] |
59. |
Kraus, W. L.,
Manning, E. T.,
and Kadonaga, J. T.
(1999)
Mol. Cell. Biol.
19,
8123-8135 |
60. |
Li, J.,
O'Malley, B. W.,
and Wong, J.
(2000)
Mol. Cell. Biol.
20,
2031-2042 |
61. | Pavletich, N. P., and Pabo, C. O. (1991) Science 252, 809-817[Medline] [Order article via Infotrieve] |