From the Renal Division and Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, Ontario M5S 1A8, Canada
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ABSTRACT |
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Understanding transcription initiation of the
endothelial nitric-oxide synthase (eNOS) gene appears pivotal to
gaining a comprehensive view of NO biology in the blood vessel wall.
The present study therefore focused upon a detailed dissection of the
functionally important cis-DNA elements and the multiprotein complexes
implicated in the cooperative control of constitutive expression of the
human eNOS gene in vascular endothelium. Two tightly clustered
cis-regulatory regions were identified in the proximal enhancer of the
TATA-less eNOS promoter using deletion analysis and linker-scanning
mutagenesis: positive regulatory domains I ( Endothelial nitric-oxide synthase
(eNOS)1 is the enzyme
responsible, in major part, for endothelial-derived NO (1). Targeted inactivation of the murine eNOS locus by homologous recombination and
physiologic assessment of ( Regulation of eNOS at the biochemical and enzymatic level is now better
understood. eNOS is a peripheral membrane protein that is localized to
specialized cell surface microdomains implicated in signal transduction
known as plasmalemmal caveolae (5). N-Myristoylation and
palmitoylation are necessary for efficient targeting and membrane
insertion (6). Increases in intracellular calcium following endothelial
activation facilitate interactions between the eNOS apoenzyme and
calmodulin. This enhances NADPH-dependent electron flux
through eNOS dimers resulting in the 5-electron oxidation of
L-arginine and release of NO (7). Surprisingly, the
molecular chaperone Hsp90 is also recruited to eNOS following cellular activation.
Recent studies have highlighted the important contributions of changes
in steady-state eNOS mRNA transcripts to the regulated expression
of NO in disease states in vivo and in models of endothelial activation in vitro. For instance, impairment in the
bioactivity of endothelial-derived NO may be mediated, in part, through
decreased expression of the mRNA and protein for eNOS in
atherosclerotic human blood vessels (8). Lysophosphatidylcholine (9),
shear-stress (10-12), and transforming growth factor- In situ cRNA hybridization studies performed in a wide
variety of human tissues revealed that eNOS mRNA transcripts are
relatively endothelial cell-specific (8, 18). This contrasts with the broad tissue distribution of other known members of the human NOS gene
family, namely neuronal NOS and inducible NOS. As is the case with
nearly all cell-restricted transcripts, some exceptions to the
endothelial-restricted expression of eNOS mRNA transcripts have
been noted: syncytiotrophoblast of human placental villi, pyramidal
cells of the CA1 region of the brain, and cardiac myocytes, among
others. When compared with other genes expressed in vascular endothelium such as preproendothelin-1, endothelin converting enzyme-1,
CD31/PECAM, or von Willebrand factor, the mRNA for eNOS is very
endothelial cell-specific. mRNAs known to be even more restricted
to the vascular endothelium than eNOS are uncommon, but include the
endothelial receptor tyrosine kinases Flk-1/KDR, Flt-1, Tie-1,
Tie-2/Tek, and the cytokine-inducible adhesion molecule E-selectin
(19). In the latter case, the human E-selectin promoter has been
identified as a useful model for biochemical and functional characterization of transcriptional regulation of inducible mRNAs in vascular endothelium.
In contrast to the mechanistic details emerging from studies of
inducible gene expression, few constitutively expressed endothelial cell-restricted genes have been exhaustively dissected. One well studied human gene is preproendothelin-1 (20). Functional studies showed that GATA-2 and AP-1 synergistically activate the
preproendothelin-1 promoter. GATA-2, the major GATA-binding protein
expressed in endothelial cells (21), also has functional importance in
the transcription of other endothelial genes such as human von
Willebrand factor, P-selectin, VCAM-1, and ICAM-2. However, given the
broad tissue distribution of the GATA-2, it cannot be the sole
molecular determinant for the cell type-specific expression of these
genes. Studies have also investigated the cis-acting DNA elements and trans-acting factors that regulate the transcription of Flt-1, Flk-1/KDR, and Tie-2. cAMP response element and Ets motifs cooperate in
activating the Flt-1 promoter (22). Three regions have been identified
within the 5'-flanking sequences of the Flk-1/KDR gene containing
putative Sp1, AP-2, NF- Recently, this laboratory and others reported the isolation and
characterization of complementary and genomic clones for eNOS (18, 25,
26). The human eNOS gene is present as a single copy in the haploid
human genome and has been localized to 7q35-36 (26, 27). Structural
characterization of genomic DNA revealed that the 4052-nt mRNA is
derived from 26 exons distributed over 21 kilobases of human genomic
DNA (26, 27). Even though analysis of 5'-flanking regions failed to
define a canonical TATAA motif a single major transcription initiation
site was defined by primer extension, S1 nuclease protection, and
5'-RACE to be 22 nt upstream of the translational start site (26). The
early characterization of human eNOS genomic clones and 5'-flanking
regions, by us and others, suggested a trivial model. Studies suggested
a prominent role for Sp1 and GATA cis-elements in constitutive
transcription initiation (26-29).
The eNOS gene is constitutively expressed by the vascular endothelium;
however, the transcriptional mechanisms have not been thoroughly
investigated thus far. A comprehensive understanding of the
interdependent protein-DNA and protein-protein interactions that
reciprocally interact with co-activators and the general transcription
machinery on the native eNOS promoter is necessary for developing
further insight into perturbations of eNOS expression in the diseased
blood vessel wall. Based upon this premise, the present study focused
upon a detailed dissection of the functionally important cis-DNA
elements and the multiprotein complexes implicated in the cooperative
control of constitutive expression of the human eNOS gene. Two tightly
clustered activator regions were identified in proximal enhancer
regions of the TATA-less eNOS promoter using deletion analysis and
linker-scanning mutagenesis: positive regulatory domain I (PRD I)
( Cell Culture--
Bovine aortic endothelial cells (BAEC) and
human umbilical vein endothelial cells (HUVEC) were isolated and
characterized as described previously (16, 18). Schneider's
Drosophila line 2 (SL2) were obtained from ATCC (Rockport,
MD), propagated in Schneider's Drosophila medium
supplemented with 10% fetal bovine serum and maintained at 23 °C
with atmospheric CO2. Cell culture reagents were obtained
from Life Technologies, Inc.
Promoter/Reporter Constructs--
Restriction and modifying
enzymes were from New England Biolabs (NEB) (Beverly, MA), Boehringer
Mannheim (Mannheim, Germany), and Pharmacia (Uppsala, Sweden). Plasmid
DNA was prepared using two rounds of gradient-sedimentation
ultracentrifugation in ethidium bromide-saturated cesium chloride
cushions. Multiple independent preparations were employed in
transfection experiments. Comparable findings were evident when plasmid
DNA was prepared in dam Deletion Mutants--
An EMBL3 phage clone containing human eNOS
genomic sequences was isolated using Southern blot hybridization (26).
A 3.6-kb ApaI fragment, extending from Drosophila Eukaryotic Expression Constructs--
Expression
cassettes for Sp1, Sp3 variants, Elf-1, Ets-1, and MAZ were based upon
pPacUO, a transient episomal vector which contains the 2.6-kb
Drosophila actin 5C promoter, a 0.7-kb 5'-UTR Ultrabithorax
(Ubx) internal ribosome entry site, the first eight codons of the Ubx
open reading frame and 1.1-kb of 3'-UTR from the actin 5C gene. The
latter provides polyadenylation signal sequences. pPacUSp1 and pPacUO
was kindly provided by R. Tjian (Howard Hughes Medical Institute,
Berkeley, IL) and has been described previously (30, 31). pPacUSp3 was
provided by G. Suske (Institut fur Molekularbiologie und Tumorfurchung,
Marburg, Germany) (32). For construction of pPacUSp3( Transient Transfection Assays--
For transient transfections
DNA concentrations were independently determined using a DyNA Quant
200 Fluorometer (Hoefer Pharmacia Biotech Inc., Uppsala, Sweden),
an Ultrospec Plus UV/Visible Spectrophotometer (Pharmacia
Biotech, Uppsala, Sweden) and analytical gel electrophoresis. All
transient transfections were carried out using the Lipofectin reagent
(Life Technologies, Inc.). BAEC and HUVEC cultures were plated at
3.3 × 104 cells/ml (3.5 ml) and grown on 60-mm dishes
48 h prior to transfection. Transfection conditions were optimized
using the SV40 promoter/enhancer luciferase control plasmid,
pGL2-Control, to confirm that increasing amounts of templates resulted
in proportional increases in reporter activity. Endothelial cells were
co-transfected with 1.0 µg of promoter/reporter construct, 0.5 µg
of pRSV- Luciferase Assays--
48 h after transfection, cells were
harvested with 300 µl of lysis buffer (0.1 M potassium
phosphate buffer (pH 7.8), 1% Triton X-100, 1 mM DTT, 2 mM EDTA). Protein extracts were centrifuged at 10,000 × g for 2 min to pellet residual cellular debris and stored
at Single-stranded Conformation Polymorphism Analysis of eNOS
Promoter Regions--
A focused SSCP screen of the core promoter
consisted of four overlapping amplicons, averaging 180 nt in size,
extending from Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
lysates were collected as described (34). Confluent endothelial cells
were harvested using trypsin-EDTA, washed twice with PBS at 4 °C,
and resuspended gently in 400 µl of Buffer A (10 mM HEPES
(pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM
phenylmethylsulfonyl fluoride) and incubated on ice for 15 min. To
disrupt cytoplasmic membranes, 25 µl of ice-cold 10% Nonidet P-40
was added and the mixture was gently vortexed. Nuclei were pelleted
(10,000 × g, 5 min) at 4 °C and resuspended in
ice-cold Buffer C (20 mM HEPES (pH 7.9), 400 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
DTT, 1 mM phenylmethylsulfonyl fluoride) and rocked
vigorously for 15 min at 4 °C. The mixture was subjected to
centrifugation (10,000 × g) for 5 min at 4 °C, and
the supernatant was stored in aliquots at Data Analysis--
Unless otherwise indicated, data are
expressed as the mean ± S.E. obtained from at least three
independent transfection experiments, each done in triplicate.
Comparisons were made with analysis of variance, followed by the
Student-Newman-Keuls test. The level of statistically significant
difference was defined as p < 0.05.
Functional Analysis of Human eNOS 5'-Flanking Regions--
To
analyze the mechanisms of transcriptional regulation important in the
human eNOS gene and to define functionally important cis-DNA elements
in the 5'-flanking region of this gene, 13 eNOS 5'- to 3'-deletion
promoter/reporter constructs were generated. All had a variable 5'- end
but a common 3'- end, ending at +109 relative to the start site of
transcription. Luciferase expression was assayed following transient
transfections of BAEC or HUVEC and normalized using
A further series of transient transfections were designed to examine
the regions between
To gain a more thorough understanding of the mechanisms implicated in
the regulated expression of the eNOS gene in endothelial cells, a
systematic and comprehensive mutational analysis of eNOS proximal
promoter regions was conducted by mutating consecutive 10-bp sequences
across the 5'-flanking region. All mutants contained a 10-bp sequence
not known to contain functionally important cis-regulatory elements
(5'-GCAGATCCGC-3'). Such studies allow functionally important regions
downstream of
The same 10-bp linker-scanning mutations were also incorporated into
the pGL2
Functional characterization of the proximal eNOS promoter utilized
cis-DNA sequences previously reported by this, and other, laboratories
(26, 27). To determine if the reported findings were more broadly
applicable, SSCP was utilized to determine whether any common allelic
variants exist in a healthy general population. Characterization of
eNOS sequences spanning Nucleoprotein Complexes Formed by PRD I--
A 30-mer
double-stranded DNA probe ( Nucleoprotein Complexes Formed by PRD II--
A series of
protein-DNA EMSA complexes were observed with the labeled
YY1 is a C2H2-type zinc finger DNA-binding
protein known to bind to consensus DNA sequences evident in PRD II
(5'-CCATT-3') (36, 37). To test whether YY1 protein bound to PRD II,
the
A second PRD II probe corresponding to eNOS promoter sequence spanning
MAZ (for myc-associated zinc finger) is a zinc-finger
transcription factor that displays protean roles in transcription
initiation, interference, and termination (39). MAZ was originally
identified in the c-myc P2 promoter and is known, through
binding site selection assays, to bind to GGGAGGG- or CCCTCCC (CT
elements) (39, 40). Putative MAZ-binding CT elements were identified in
the eNOS promoter at Sp1 and Ets Family Members Are Essential Activating Components of
PRD I and PRD II--
Functional promoter analyses in endothelial
cells revealed that mutating activator regions encompassing high and
low affinity Sp1/Sp3 recognition sequences ( MAZ Exhibits a Negative Effect on eNOS Functional Promoter
Activity--
MAZ can both activate and inhibit transcription
initiation (39). The functional contribution of MAZ to eNOS
promoter/reporter was assessed in Drosophila Schneider cells
activity using co-transfection experiments. Cotransfection of
increasing amounts of MAZ expression plasmid alone, over the range
1-250 ng/plate, did not have any effect on activity of the Functional Studies Evaluating the Effect of
NH2-terminal Deleted Sp3 in Drosophila Schneider
Cells--
In mammalian cells the nucleoprotein Sp3 exists as 3 protein isoforms as a result of alternate usage of translational
initiation sites (55). One isoform with an apparent molecular mass of
110 kDa represents the full-length Sp3 protein, whereas the other two
isoforms with apparent molecular masses of 80 and 78 kDa represent internal AUG-initiated variants of Sp3. These smaller Sp3 variants, which lack the NH2-terminal transactivation domain, are
still capable of binding Sp1 recognition elements but may have a
repressive effect on transcription depending upon the number of binding
sites within the cellular promoter (55). The molecular characterization of these Sp3 variants presumably accounts for the faster migrating DNA
complexes evident in a variety of mammalian cell lines using EMSA (55).
Given the above findings that EMSA using endothelial nuclear extracts
demonstrate protein-DNA complexes consistent with these Sp3 variants,
we sought to evaluate the functional consequences of removing the Sp3
trans-activation domain on eNOS promoter function. As shown in Fig.
8A (n = 3, triplicate determinations), NH2-terminal deleted Sp3
expression cassette (Sp3 Functional Studies Evaluating the Effect of Elf-1 on eNOS Promoter
Activity in Drosophila Schneider and Endothelial Cells--
Given the
demonstration that Elf-1 accounts, in part, for endothelial PRD II
nucleoprotein complexes we determined the functional properties of
Elf-1 in Drosophila Schneider co-transfection experiments. Increasing amounts of Elf-1 expression construct alone, over the range
1-250 ng/plate, minimally enhanced eNOS promoter activity (Fig.
9A) (n = 3, triplicate determinations). Surprisingly, co-transfection of Elf-1
elicited a concentration-dependent repressive effect on the
functional activity of half-maximal amounts of Sp1 (15-40 ng) (Fig.
9B) (n = 3, triplicate determinations). In
other words, Elf-1 exerted a negative effect on the ability of Sp1
protein to activate the eNOS promoter in SL2 cells. In contrast, Elf-1 failed to repress eNOS promoter activity in the presence of Ets-1. Even
100 ng of Elf-1 expression cassette failed to inhibit the cooperativity
evident when the Two tightly clustered activator regions were identified in
proximal regions of the human eNOS promoter using deletion analysis and
linker-scanning mutagenesis: PRD I ( Sp1, Sp2, Sp3, and Sp4 are closely related members of a gene family
encoding zinc-finger (His2Cys2) transcription
factors. Sp1, Sp2, and Sp3 are ubiquitously expressed, whereas Sp4
protein is primarily expressed in certain cell types of the brain. All four proteins contain a highly conserved DNA binding zinc-finger domain
close to the COOH terminus, and two glutamine- and
serine/threonine-rich domains near the NH2 terminus. The
latter domains evidence less sequence identity. Functional analyses
reveal that Sp1 and Sp4 transcription factors are strong activators in
mammalian cell lines, whereas the structure and function of Sp3 is more
complex. Complicating this further, Sp1 self-interaction and
protein-protein interaction between Sp1 family members is well
documented. Domain swapping experiments have highlighted important
differences in Sp1 and Sp3 structural elements consistent with the view
that interactions with other transcription factors, co-activators, and
the general transcription machinery may differ between Sp1 family
members (32). In the current studies, EMSA and functional promoter
analysis in endothelial and Drosophila Schneider cells provided clear evidence for the important and complex contributions of
Sp1 family members to eNOS promoter activity. PRD I corresponds to a
high affinity Sp1 site (5'-GGGGCGGGGC-3') located at 104/
95 relative to
transcription initiation) and II (
144/
115). Analysis of
trans-factor binding and functional expression studies revealed a
surprising degree of cooperativity and complexity. The nucleoprotein
complexes that form upon these regions in endothelial cells contained
Ets family members, Sp1, variants of Sp3, MAZ, and YY1. Functional
domain studies in Drosophila Schneider cells and
endothelial cells revealed examples of positive and negative
protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1,
Elf-1, and MAZ. Therefore, multiprotein complexes are formed on the
activator recognition sites within this 50-base pair region of the
human eNOS promoter in vascular endothelium.
INTRODUCTION
Top
Abstract
Introduction
References
/
) off-spring has reinforced the
viewpoint that NO in blood vessels plays a quintessential role in
regulation of local blood vessel tonus (2, 3), in remodeling of the
vascular wall in response to changes in flow or distending hydrostatic
pressure and in modulation of hemostatic pathways (4). As our
understanding of the contributory roles of NO in the blood vessel wall
evolves, so does the need to firmly understand the basic principles
governing the regulated expression of the eNOS mRNA transcript and enzyme.
(13)
represent important examples of exogenous stimuli known to modify eNOS
gene transcription. An intriguing facet of the control of steady-state
eNOS mRNA expression in vascular endothelium is the contribution of
post-transcriptional regulation (14-17). The eNOS mRNA transcript
normally has a very long half-life in vascular endothelium, usually
greater than 24-48 h. Tumor necrosis factor-
(14, 18), hypoxia
(15), entry into the cell cycle (16), and oxidized low density
lipoprotein (17) change the metabolic fate of eNOS mRNA
transcripts, decreasing the half-life of the mRNA to a few hours.
The regulation of eNOS mRNA stability in response to exogenous
stimuli, especially the mechanism by which the transcript is degraded,
is an evolving story.
B, and E-box elements important for
functional activity in endothelial cells (23). More recent studies with
the Tie-2 gene (24) identified negative (region I) and positive
regulatory elements (regions U and A), although the trans-factors
remain to be examined (24). In each of these examples, detailed studies
that seek a comprehensive dissection of the functionally important
cis-DNA elements and the multiprotein complexes implicated in the
cooperative control of constitutive expression in endothelial cells are
slowly emerging.
104/
95 relative to transcription initiation) and PRD II
(
144/
115). Analysis of trans-factor binding and functional expression studies revealed a surprising degree of cooperativity and
complexity. Through analysis of nucleoprotein complexes in endothelial
cells and functional domain studies in Drosophila Schneider
cells and endothelial cells, we demonstrate positive and negative
protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1,
Elf-1, MAZ, and YY1.
MATERIALS AND METHODS
,
dcm
bacteria.
3500 to +113
relative to the major transcription start site, was subcloned into the
ApaI site of pBluescript SK I (
) (Stratagene, La Jolla,
CA). The 3.6-kb ApaI fragment was isolated by preparative
gel electrophoresis, blunt-ended with Klenow enzyme, and ligated to
12-bp SalI linkers (NEB). The 3.6-kb SalI
fragment was then subcloned into the XhoI site of the
promoter-less reporter construct, pGL2-Basic (Promega, Madison, WI),
and designated as pGL2
3500/+109. pGL2
3500/+109 was used as a
template to generate pGL2
1900/+109, pGL2
1193/+109, pGL2
1001/+109, pGL2
743/+109, pGL2
265/+109, pGL2
49/+109, and pGL2
14/+109 by means of restriction enzyme digestion. The integrity of
promoter/reporter gene constructs was assessed with DNA sequence analysis. PCR was used to generate pGL2
217/+109, pGL2
185/+109, pGL2
151/+109, pGL2
133/+109, and pGL2
92/+109. Oligonucleotides were synthesized using a Beckman Oligo 1000 DNA synthesizer (Beckman Instruments, Fullerton, CA). Constructs generated with PCR were sequenced using an automated ABI Prism 377 DNA sequencer (Perkin-Elmer Applied Biosystems Canada Inc., Mississauga, ON) to monitor for PCR-associated nucleotide incorporation errors.
1193/+109 Linker Mutants--
Twelve linker-scanning
mutations, containing the sequence 5'-GCAGATCCGC-3', and spanning a
120-bp region of the human eNOS promoter from
164 to
45, relative
to the transcription start site, were created. These mutations were
incorporated into the pGL2
1193/+109 construct. Two types of
amplicons, namely an A amplicon (the 5'-fragment) and a B amplicon (the
3'-fragment), were created using a modified PCR-based method. Primers
used to create the linker-scanning mutants are listed in Table
I. The A amplicon was generated using two
primers: Amain and An. Amain is a gene-specific sense primer that is homologous to
327 to
309 regions of the eNOS promoter. An,
where n corresponds to the location of the specific 10 bp to
be mutated, is a 25-mer, antisense primer containing a 15-bp sequence
homologous to the eNOS promoter and a 10-bp mutation at the 5'-end that
is heterologous to eNOS sequence. Similarly, the B amplicon was
generated using two primers: Bmain and
Bn. Bn is a 25-mer, sense primer containing 15 bp of homologous eNOS sequence and a heterologous 10-bp mutation at the 5'-end of the primer. Bmain is a
21-mer homologous to the luciferase reporter gene sequence in the
pGL2-Basic vector. A and B amplicons were subcloned into the pCR II
vector (Invitrogen Corp., San Diego, CA) and sequenced. The A and B
amplicons were subsequently subcloned between the PstI and
BglII sites of the
1193/+109 pGL2 promoter/reporter
luciferase construct.
Primers pairs used to construct all pGL2-1193/+109 linker-scanning
mutants
265/+109 Linker Mutants--
Each pGL2
1193/+109 linker
mutant construct was digested with SmaI (a 3'-multiple
cloning site within pGL2-Basic vector) and PstI (
265),
blunt-ended with Klenow enzyme, and ligated.
Nterm) the
pPacUSp3 plasmid was subjected to a partial NotI and a
BglII digestion. The 8.9-kb fragment was blunt-ended with
Klenow and an intramolecular ligation performed removing 0.85-kb from
the NH2 terminus of the Sp3 open reading frame leaving
amino acids 297-667 of human Sp3 (Sp3
NH2). pPacUElf-1
was constructed using a full-length human Elf-1 cDNA kindly
provided by J. M. Leiden (Chicago, IL). A 1.9-kb XhoI
fragment was subcloned into the XhoI site of pPacUO. The
resulting plasmid was subjected to a partial PstI
restriction digestion and blunt-ended with Klenow enzyme to remove 4 nt
placing the Elf-1 cDNA in frame with the first 8 codons of the Ubx
open reading frame. pPacUEts-1 was constructed using a full-length
murine Ets-1 cDNA provided by B. J. Graves (University of
Utah, Salt Lake City, UT). A 1.5-kb NdeI-BamHI
fragment was blunt-ended and subcloned into the blunt-ended BamHI site of pPacUO. pCGN-MAZ was kindly provided by Thomas
Shenk (Howard Hughes Medical Institute, Princeton, NJ) (33). For
pPacUMAZ, pCGN-MAZ was cleaved with a partial EcoRI
digestion. The linearized plasmid was blunt-ended and digested with
BamHI. pPacUO was cleaved with XhoI, blunt-ended,
and cleaved with BamHI. The resulting 2.3-kb fragment
containing the MAZ open reading frame was cloned into the
BamHI sites of pPacUO.
-gal, and 1.5 µg of pBluescript II SK(
) DNA.
-Galactosidase activity was used to control for transfection
efficiency, and pBluescript II SK(
) DNA was used to optimize
DNA/Lipofectin ratios and hence transfection efficiency. The formation
of DNA/Lipofectin complexes containing DNA/Lipofectin in a 2:1
(mass:mass) ratio were incubated for 60 min at 22 °C and then added
to cells at 37 °C in serum-free Opti-MEM I. The transfection mix was
replaced at 5 h with RPMI 1640 supplemented with 15% bovine serum
and antibiotics. Each transfection experiment was performed in
triplicate and repeated a minimum of three times. pGL2-Control vector
was used as a positive control. The pGL2-Basic vector lacking both a
eukaryotic promoter and enhancer sequences was used as a negative
control. For Drosophila Schneider studies, cells were
co-transfected with 1 µg of experimental promoter/luciferase construct, the indicated amount of expression plasmids, and 0.5 µg of
pRSV-
-gal. The total amount of DNA transfected was kept constant (2 µg) with the addition of pPacUO. SL2 cells were seeded at a density
of 2-3 × 106 cells/ml at the time of transfection. A
cytomegalovirus-based heterologous eukaryotic expression cassette for
Elf-1 (pcDNAI/neo-Elf-1) was kindly provided by J. M. Leiden
(University of Chicago, Chicago, IL).
80 °C for subsequent assay. The Bio-Rad protein assay was used
to determine protein concentration using bovine serum albumin as a
reference standard. Reverse transcription-PCR was used to characterize
RNA species produced from transfected DNA constructs to confirm that
episomal eNOS promoter/reporter constructs utilized comparable sites
for transcription initiation as the native promoter (data not shown).
Luciferase assays were carried out on the MonoLight 2010 luminometer
(Analytical Luminescence Laboratory, Ann Arbor, MI). Coenzyme A and ATP
were from Calbiochem (La Jolla, CA). D-Luciferin and
luminometer cuvettes were from Analytical Luminescence Laboratory.
Briefly, 25 µl of protein extract, 100 µl of
D-luciferin (1 mM), 100 µl of luciferase
assay buffer (30 nM Tricine (Calbiochem, La Jolla, CA), 3 mM ATP, 15 mM MgSO4, 10 mM DTT, 1 mM coenzyme A), and a pH of 7.8 were
used. Measurements of light units were integrated over a 10-s interval. Data in raw luciferase units (RLU) were normalized for the nonspecific background of mock-transfected cells, which represented 0.5% of most
experimental luciferase activities. Intra- and inter-assay coefficients
of variation averaged 7% and 9%, respectively.
-Galactosidase Assay--
CPRG (chlorophenol
red-
-D-galactopyranoside, Boehringer Mannheim) was used
as the chromogenic substrate for
-D-galactosidase measurements. 40 µl of cell extract was mixed with 160 µl of assay reagent containing 70 mM sodium phosphate buffer (pH 7.3),
9 mM MgCl2, 8 mM CPRG solution, and
0.1 M 2-mercaptoethanol. CPRG was made fresh for each assay
in 0.1 M sodium phosphate buffer (pH 7.3). Reaction
mixtures were incubated at 37 °C for 20 min and subsequently stopped
with 500 µl of 1 M Na2CO3.
Enzymatic activity was measured at 570 nm. As a index of the endogenous
-galactosidase activity, 40 µl of mock-transfected cell extract
was used and for BAEC averaged 1% of raw values. As a positive
control, 1 µl of
-galactosidase (Sigma, 50 units/ml) was added to
40 µl of mock-transfected cell extract.
456 to +133 (Table II).
Peripheral blood genomic DNA was obtained from 25 healthy, unrelated,
and ethnically diverse individuals using standard methodology after
informed consent. PCR reactions were carried out in 25-µl reaction
volumes with 25 ng of peripheral blood genomic DNA, 12.5 pmol of each
primer, 1.25 units of Taq polymerase (Life Technologies,
Inc.), 100 µM of each dNTP, 1 µCi of
[
-32P]dCTP (NEN Life Science Products), 0.75-1.0
mM MgCl2, 20 mM Tris-HCl, pH 8.4, and 50 mM KCl. A "hot start" was carried out with the addition of [
-32P]dCTP and dNTP after an initial
denaturation at 94 °C for 5 min and was followed by 30 cycles of
94 °C for 30 s, optimal annealing temperature for 30 s,
and a 72 °C extension for 30 s, followed by a single final
extension at 72 °C for 15 min. At the completion of thermocycling, 7 µl of the PCR product was mixed with 5 µl of loading buffer (95%
formamide, 10 mM NaOH, 0.25% bromphenol blue, 0.25%
xylene cyanol). The mixture was heated at 100 °C for 10 min and then
iced for 2 min prior to loading. A 0.5× MDE (AT Biochem, Malvern, PA),
0.6× TBE vertical electrophoresis gel was prepared according to the
manufacturer's instructions. 3 µl of each sample was loaded per
lane. A non-heat-denatured sample was also included to assist in the
identification of the double-stranded amplicon. Size-fractionation was
carried out using a S2 vertical electrophoresis unit (Life
Technologies, Inc.) under two distinct electrophoresis conditions: 1000 V, 15 mA, and 7 watts at 22 °C or 4 °C, for 8-10 h. Gels were
dried and subjected to autoradiography.
Primer sets used in SSCP analysis of the eNOS promoter region
80 °C. For EMSA
reactions, 3-10 µg of nuclear extract were incubated for 10 min on
ice in 20 µl of binding buffer (25 mM Tris-HCl (pH 8.0),
50 mM KCl, 6.25 mM MgCl2, 10%
glycerol, 50 µg/ml bovine serum albumin, 1 µg of poly(dI)-(dC) (Boehringer Mannheim, mean chain length = 5000 bp)). Adenine
5'-[
-32P]triphosphate ([
-32P]ATP;
6000 Ci/mmol) was from NEN Life Science Products. Two pmol of
double-stranded probes (Table III) were
labeled using T4 polynucleotide kinase and
[
-32P]ATP. Two fmol of labeled probe (2.5 × 104 dpm) was added to the binding reaction mix and
incubated at 22 °C for 50 min. Where recombinant Sp1 (Promega,
Madison, WI) or recombinant YY1 (Santa Cruz Biotechnology Inc., Santa
Cruz, CA) were used in EMSA, 0.03% Nonidet P-40 was added to the
binding reaction and 0.05% Nonidet P-40 was added to the acrylamide
gel. For "supershift" analyses, antibodies were added 30 min after probe addition and incubated an additional 20 min at 22 °C. Binding reactions were size-fractionated on a non-denaturing, 4% acrylamide gel (37:1, mass:mass,
acrylamide:N,N'-methylenebisacrylamide), run at 200 V at
4 °C for 2 h in 0.5× Tris-borate buffer. The gel was
subsequently dried and autoradiographed at
80 °C with intensifying screens. Monoclonal and polyclonal antibodies were from Santa Cruz
Biotechnology: Sp1 (PEP2), Sp2 (K-20), Sp3 (D-20), Sp4 (K-20), YY-1
(H-414), GATA-2 (CG2-96), Ets-1 (NH2 terminus, N-276),
Ets-1 (COOH terminus, C-20), Ets-2 (C-20), PU.1 (Spi-1, T-21), Erg-1 (C-17), Fli-1 (C-19), PEA3 (16), and Elk-1 (I-20). Anti-Elf-1 (rabbit
polyclonal), anti-MAZ (murine monoclonal), and anti-GATA-2 (rabbit
polyclonal) were generous gifts from J. M. Leiden (University of
Chicago, Chicago, IL), K. B. Marcu (State University of New York, Stony Brook, NY), and S. H. Orkin (Harvard Medical
School, Boston, MA), respectively.
Oligonucleotides used in EMSA analysis
RESULTS
-galactosidase
activity and protein concentrations. As shown in Fig.
1A, the construct pGL2
1193/+109 evidenced maximal functional eNOS promoter activity in
BAEC, and averaged 10 ± 1% (mean ± S.E.) of the activity
of the SV40 promoter/enhancer (n = 3, triplicate
determinations). Characterization of RNA species transcribed from
transfected DNA constructs with reverse transcription-PCR confirmed
that episomal eNOS promoter/reporter constructs utilized sites for
transcription initiation that were comparable to the chromatin
transcription unit (data not shown). The constructs pGL2
3500/+109
and pGL2
1900/+109, which contain further 5'-flanking sequences, had
moderately lower activities than pGL2
1193/+109 (~70% and ~80%,
respectively, of the activity of pGL2
1193/+109), suggesting the
presence of cis-regulatory DNA sequences exhibiting negative functional
effects between
3500 and
1193. Deletion of sequences from
1193 to
1001 produced a ~20% decrease in activity. Removal of sequences
spanning
1001 to
265 did not produce an appreciable drop in
functional promoter activity. When sequences from
265 to
14 were
deleted, a marked drop in activity was evident. Values for the
14/+109 construct essentially reflected those seen for the
promoterless luciferase plasmid, pGL2-Basic.
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Fig. 1.
Activity profiles of human eNOS
promoter/reporter luciferase 5'- to 3'-deletion constructs in BAEC
(A and B) and HUVEC
(C). To control for transfection efficiency,
cells were co-transfected with pRSV- -gal and relative luciferase
activity was normalized for protein and
-galactosidase values. The
data from these transient transfections are expressed as percent
luciferase activity relative to pGL2
1193/+109 and represent the
mean ± S.E. (three independent experiments, triplicate
determinations). The maximally active construct, pGL2
1193/+109,
displayed 10-20% of the activity an SV40 promoter/enhancer-directed
luciferase control vector, pGL2-control. Where error
bars are not evident, S.E. is below the figure
resolution.
265 and
14 in greater detail (Fig. 1B) (n = 3, triplicate determinations).
Constructs with 5'-end points at
265,
217,
185 and
151
demonstrated comparable activity that represented approximately
60-75% of the most active construct, pGL2
1193/+109. Deletion of
sequences between
151 and
133 produced an additional 33% drop in
activity and removal of the sequences from
133 to
92 abrogated
remaining functional promoter activity of the eNOS 5'-flanking region.
Taken together, these findings suggest that two positive regulatory
regions exist in the proximal core promoter of the human eNOS gene, the
first between
151 and
133, and the second between
133 and
92.
Comparison of the functional promoter activity of a series of human
eNOS promoter/reporter constructs in HUVEC (Fig. 1C)
revealed qualitatively similar functional activity response profiles.
This suggests that BAEC and HUVEC share in common a series of
functionally relevant trans-acting factors necessary for basal
expression of the eNOS promoter.
133 to be defined and allow transcriptional regulation
of eNOS to be analyzed while maintaining the native topological and
spatial relationships of the DNA helix. Twelve linker-scanning mutant
constructs were created in the context of the maximally active pGL2
1193/+109 promoter/luciferase construct and spanned a 120-bp region
from
164 to
45 relative to the start site of transcription. As
shown in Fig. 2A, four of the
eNOS promoter/reporter linker-scanning mutants demonstrated
significantly decreased functional activity in BAEC compared with
wild-type sequences (n = 4, triplicate determinations).
The
144/
135mut,
134/
125mut, and
124/
115mut linker-scanning
constructs displayed a significant 40-50% drop in functional promoter
activity relative to the wild-type construct (p < 0.0001). In addition, the 10-bp mutation spanning
104/
95 (
104/
95mut) resulted in a profound 85% drop in luciferase activity (p < 0.0001).
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Fig. 2.
Activity profiles of human eNOS
promoter/reporter luciferase linker-scanning mutations in BAEC.
A, linker-scanning mutants introduced a 10-bp substitution
(5-GCAGATCCGC-3'). B, comparison of promoter activity of PRD
I and II mutant pGL2 1193/+109 and pGL2
265/+109 constructs. To
control for transfection efficiency, cells were co-transfected with
pRSV-
-gal, and relative luciferase activity was normalized for
protein and
-galactosidase values. The data from these transient
transfections are expressed as percent luciferase activity relative to
pGL2
1193/+109 and represent the mean ± S.E. (three independent
experiments, triplicate determinations). The maximally active
construct, pGL2
1193/+109, displayed 10-20% of the activity of SV40
promoter/enhancer-directed luciferase control vector, pGL2-control.
Where error bars are not evident, S.E. is below
the figure resolution.
265/+109 construct. This construct contains a shorter eNOS
5'-flanking region. A comparison between the activity profile of the
shorter promoter linker mutants with the activity profile of the same
10-bp mutations in the context of a longer eNOS promoter region allowed
large domain interactions to be defined and/or mapped. Functional
reporter activities revealed no important differences compared with
linker-scanning mutations in the setting of the maximally active
1193/+109 construct. A synopsis of these data is presented in Fig.
2B and indicated that functional contributions from PRD I
and PRD II are not dependent upon additional sequences present between
1193 and
265 (n = 3, triplicate determinations). Although bacterial methylase recognition sites (GATC and CCWGG) are
found in the proximal eNOS promoter, studies performed with plasmid DNA
prepared in dam
and
dcm
bacterial strains indicated no functional
effects (data not shown). Taken together as a whole, deletion and
linker-scanning functional studies defined two regions of interest:
104 to
95 (designated as PRD I) and
144 to
115 (PRD II).
496 to +171 in 50 chromosomes (25 peripheral
blood genomic samples, four overlapping SSCP amplicons) revealed no
electrophoretic SSCP variants. This suggested that there were no common
allelic variants for human eNOS genomic regions internal to the SSCP
primers (
475 to +152) and that findings relevant to the current work
were broadly applicable (data not shown).
120/
91) representing residues
120/
91
in the human eNOS 5'-flanking region, and thereby spanning PRD I,
formed a series of protein-DNA complexes with BAEC nuclear extracts
(Fig. 3A, complexes
A, B, C, and D,
lane 2). Similar protein-DNA complexes were
observed with HUVEC nuclear extracts (Fig. 3C). With the
addition of 50-fold and 100-fold molar excess of unlabeled
120/
91
probe, all four protein-DNA complexes were effectively competed away
(Fig. 3C, lanes 4 and 5).
With the addition of 100-fold molar excess of an oligonucleotide containing a consensus Sp1 site, all four complexes (A,
B, C, and D) were competed away (Fig.
3A, lane 3). Unlabeled
oligonucleotides with a mutation in the Sp1 binding site located at
104 were ineffectual in competition studies, whereas mutating the
GATA site at
108/
105 had no effect on competition (Fig.
3A, lanes 4 and 5,
respectively). When protein-DNA complexes were incubated with a
monoclonal antibody directed against Sp1, a "supershift" of complex
A was evident (Fig. 3, B, lane 4, and
C, lane 6). Using monoclonal and
polyclonal antibodies directed against GATA-2, no supershift or shift
abrogation was observed. For instance, minor bands evident in Fig.
3A (lane 5) were not affected by
anti-GATA (data not shown). Incubating protein-DNA complexes with
polyclonal antibodies directed against Sp2 (data not shown) and Sp4
(Fig. 3B, lane 2) resulted in no supershift or
shift abrogation. However, exposing protein-DNA complexes to a
polyclonal antibody directed against Sp3 resulted in both supershift
and shift abrogation of complexes B and C (Fig. 3B,
lane 3). A specific protein-DNA complex formed
when the -120/
91 probe was utilized in an EMSA with recombinant Sp1
but not AP-2, comigrated with complex A, and was supershifted with an
antibody directed against human Sp1 (data not shown). Moreover,
competition studies performed with unlabeled
120/
91 probe and a
heterologous consensus Sp1 high affinity site from the early SV40
promoter indicated comparable effectiveness in competition studies
(data not shown).
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Fig. 3.
EMSAs of endothelial nuclear protein binding
to 120/
91 PRD I eNOS 5'-flanking region. A,
lane 1 represents probe alone and in
lane 2, 3 µg of BAEC nuclear extract was added.
Four specific protein-DNA complexes are evident: A,
B, C, and D. In lanes
3-5, 100-fold molar excess of Sp1 consensus,
120/
91mutSp1, and
120/
91mutGATA oligonucleotides were added to
the binding reaction as competitors. B, lane
1 represents
120/
91 probe incubated with 3 µg of BAEC
nuclear extract. In lanes 2-4, anti-Sp4, -Sp3,
and -Sp1 were added to the binding reaction, respectively. 2.5 × 104 dpm of labeled
120/
91 probe was used in each
binding reaction. C, lane 1 represents
probe alone; lanes 2 and 3 represent 5 and 3 µg, respectively of HUVEC nuclear extract incubated with probe.
In lanes 3 and 4, 50- and 100-fold
molar excess of unlabeled
120/
91 probe was added. An antibody
directed against Sp1 was added in lane 5.
140/
111
probe and BAEC nuclear extracts (Fig.
4A, lanes
2 and 3). Adding 100-fold molar excess of
unlabeled
140/
111 probe resulted in the disappearance of these
complexes, suggesting that they are specific (Fig. 4A,
lane 4). Competition was also observed with the
addition of 100-fold molar excess of unlabeled
155/
120 (Fig.
4B, lane 4). Sequence inspection of
PRD II regions indicated a variety of putative DNA cis-elements: Ets
(GGAA/T) (35), YY1 (5'-CCATT-3') (36, 37), low affinity Sp1
(5'-GGGAGG-3') (38), MAZ (GGGAGGG) (39, 40), p53 half-site
(5'-GGGCTTGTTC-3') (41), and paired domain PAX family members
(5'-GTTCC-3') (42). Competitor oligonucleotides containing consensus
Pax-2, Pax-8, or a variety of p53 binding sites had no effect on the
formation of nucleoprotein complexes (data not shown). Multiple
consensus p53 recognition sites were used given that neighboring DNA
sequences and the phosphorylation state of p53 influence binding site
selection. In contrast to these negative findings, unlabeled
oligonucleotides containing various Ets family member binding sites
were very effective in competition studies (Fig. 4A). For
example, the long terminal repeat sequence of the HIV-2 promoter has
been reported to bind Elf-1, an Ets family member (43), and as shown in
Fig. 4A (lane 5), demonstrated clear
competition. Other Ets binding site-containing oligonucleotides,
including the human T cell lymphotrophic virus type I long terminal
repeat (HTLV-I LTR) site (Ets-1) (44), the rat stromelysin 1 promoter
site (Ets-2) (45), and the polyomavirus PEA3 site (PEA3) (46) also
competed, though to a lesser extent (Fig. 4A,
lanes 6-8). Addition of monoclonal or polyclonal
antibodies directed against varied members of the Ets member family
(Ets-1, Ets-2, PU.1, Erg-1, Fli-1, PEA3, and Elk-1) failed to modify
nucleoprotein complexes formed with the
140/
111 probe. These
antibodies have been demonstrated to exhibit cross-reactivity across
species. However, our findings are complicated by the well described
difficulties inherent in characterizing which specific members of the
Ets family are implicated in the formation of protein-DNA complexes in
nuclear extracts and may be related, in part, to the contribution of
the autoinhibitory binding domain in Ets family members (47-49).
Exposure of complexes formed with the
140/
111 probe to a rabbit
polyclonal antibody directed against Elf-1 resulted in a supershift of
the fastest migrating complex (Fig. 4B, lane
5). These findings imply that the fast migrating complex
contains a protein that is antigenically-related to Elf-1. To confirm
that an Elf-1-like protein is present in the BAEC nuclear extracts, the
HIV-2 LTR oligonucleotide probe was labeled and incubated with BAEC
nuclear extract (Fig. 4E). This activator recognition
sequence is known to bind Elf-1 in T lymphocytes (50). Addition of
100-fold molar excess of
140/
111 and
155/
120 probes resulted in
competition (Fig. 4E, lanes 4 and
5) of the fast migrating protein-DNA complex and anti-Elf-1 resulted in a supershift of the fastest migrating complex (Fig. 4E, lane 7). These findings are taken
to indicate that Elf-1 is present in endothelial cell nuclear extracts
and that this Ets family member can participate in nucleoprotein
complex formation with PRD II.
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Fig. 4.
EMSAs of BAEC nuclear protein binding to
140/
111 PRD II eNOS 5'-flanking region. A,
lane 1 represents probe alone. Lanes
2 and 3 represent the addition of 3 and 10 µg
of BAEC nuclear extract, respectively. In lanes
4-8, 250-fold molar excess of various competitor
oligonucleotides were added to 3 µg of BAEC nuclear extract:
unlabeled
140/
111 (lane 4), HIV-2 CD3R
(lane 5), HTLV-I LTR (lane
6), STROM (lane 7), and PEA3
(lane 8) (2.5 × 104 dpm of
labeled probe). B, lane 1 represents
140/
111 probe alone and 10 µg of BAEC nuclear extract was added
in lane 2. 100-fold molar excess of unlabeled
140/
111 and
155/
120 was added in lanes 3 and 4. An antibody directed against Elf-1 was added in
lane 5. C, lane
1 represents probe alone and 10 µg of BAEC extract was
added in lane 2. 100-fold molar excess of cold
140/
111 was added in lane 3. An antibody
directed against MAZ was added in lane 4.
D, lane 2 represents probe alone and
10 µg of BAEC extract was added in lane 1.
100-fold molar excess of cold
140/
111 and YY1 oligonucleotides were
added in lanes 3 and 4, respectively.
E, EMSAs were performed with an HIV-2 CD3R LTR probe
containing an Elf-1 recognition sequence and BAEC nuclear extracts.
Lane 1 represents probe alone (2.5 × 104 dpm labeled probe), and 10 µg of BAEC nuclear extract
was added in lane 2. 100-fold molar excess of
unlabeled competitor DNA was added in lane 3 (HIV-2 CD3R), lane 4 (
155/
120),
lane 5 (
140/
111), and lane
6 (
120/
91). Anti-Elf-1 was added in lane
7. For all gels, arrows on the left
represent protein-DNA complexes and arrows on the
right represent supershifted complexes and/or abrogated
complexes.
140/
111 probe was exposed to a competitor oligonucleotide
containing the YY1 site found in the upstream conserved region of
Moloney murine leukemia virus (36) (Fig. 4D, lane
4). A clear reduction in one of the fastest migrating bands
is evident upon addition of this competitor, suggesting that YY1
participates in the formation of protein-DNA complexes formed upon PRD
II. Recombinant YY1 protein also formed protein-DNA complexes with the
140/
111 PRD II probe in EMSA (data not shown).
155 to
120 also formed a series of unique and specific protein-DNA
complexes (Fig. 5A,
lanes 2 and 3). Addition of 100-fold molar excess of cold
140/
111 resulted in competition for some, but
not all of the complexes (Fig. 5A, lane
4). This may suggest that the protein-DNA complexes that
were not effectively competed with the
140/
111 oligonucleotide,
represent interactions of trans-acting factors with portions of the
155/
120 probe that are not present on
140/
111. The slowest
migrating complex disappeared with the addition of anti-Sp1, whereas
the next two complexes failed to form upon addition of anti-Sp3 (Fig.
5B, lanes 2 and 3,
respectively). No change in complex pattern was observed with the
addition of anti-Sp4 or anti-Sp2 (data not shown). Upon the addition of
100-fold molar excess of cold
120/
91, which contains a high
affinity Sp1 cis-DNA sequence, clear competition of these slow
migrating complexes was evident (data not shown). In contrast, when
100-fold molar excess of cold
155/
120 was added as a cold competitor to the complexes formed with the
120/
91 probe, only slight competition was observed. Taken together, these data suggest the
presence of a low affinity Sp1 cis-DNA element on the
155/
120 probe, but not on the
140/
111 probe. When a polyclonal antibody directed against Elf-1 was added to the
155/
120 binding reaction, a
reduction was evident in the intensity of the fastest migrating specific complex and a supershift was seen (Fig. 5A,
lane 5).
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Fig. 5.
EMSAs of BAEC nuclear protein binding to
155/
120 PRD II eNOS 5'-flanking region. A,
lane 1 represents probe alone and 10 µg of BAEC
nuclear extract was added in lane 2. 100-fold
molar excess of unlabeled
155/
120 and
140/
111 probes were added
in lanes 3 and 4, respectively
(2.5 × 104 dpm of labeled probe). An antibody
directed against Elf-1 was added in lane 5.
B, lane 1 represents probe alone and
10 µg of BAEC nuclear extract was added in lane
2. Antibodies were introduced in lanes
3-5: anti-Sp1 (lane 3), anti-Sp3
(lane 4), and anti-MAZ (lane
5). For all gels, arrows on the left
represent protein-DNA complexes and arrows on the
right represent supershifted complexes and/or abrogated
complexes.
191,
146,
99,
75,
62, and
47. An
important facet of MAZ function is the participation of partner
proteins. For instance, enhancer activity of the CD4 gene is critically
dependent upon MAZ and an Ets consensus site that binds Elf-1 (51, 52).
Functional interactions between MAZ and Sp1 occur with a number of
genes, including the adenovirus major late and the TATA-less serotonin 1a receptor promoters (33). Because proteins shown to functionally interact with Ets and/or Sp1 family members in other promoters became
candidate participants for nucleoprotein complex formation upon PRD II,
studies assessed MAZ binding. A monoclonal antibody directed against
MAZ resulted in shift abrogation of complexes formed upon the
155/
120 probe, especially one of the prominent fastest migrating
ones (Fig. 5B, lane 5). Consistent
with protein-protein interactions involving MAZ with Sp1 and/or Sp3, a
clear reduction in the slower migrating protein-DNA complexes was also
observed following the addition of MAZ antibody. Protein-DNA complexes formed by the
140/
111 probe also demonstrated a clear reduction with the addition of MAZ antibody (Fig. 4C, lane
4). In summary, studies of endothelial cell nuclear extracts
and double-stranded oligonucleotide probes representing PRD I and PRD
II functional domains demonstrate nucleoprotein complexes composed of
Sp1, variants of Sp3, Ets-1, Elf-1, MAZ, and YY1. The majority of
protein-DNA complexes seen at this EMSA resolution have been accounted for.
104/
95 and
146/
141)
resulted in a reduction of eNOS promoter/reporter activity (Fig.
2A). Similarly, mutating the region encompassing an Ets
recognition sequence (
129/
126) also resulted in a reduction of
promoter activity (Fig. 2A). Based upon this background, a
model can be proposed wherein Sp1, Sp3, and Ets family members are
essential for in vivo eNOS promoter function. To evaluate
this hypothesis, a series of transient transfection experiments were
performed in cells that lack constitutive Sp1, Sp3, and Ets activities,
namely the Drosophila Schneider cell line (for each
experimental series, n = 3; triplicate determinations) (31, 32). Ets-1 is a well characterized member of the Ets family of
transcription factors that is known to be robustly expressed in
endothelial cells (53, 54). In the absence of Sp1, Sp3, or Ets-1, the
pGL2
1193/+109 reporter construct exhibited trivial functional
activity, being essentially equivalent to mock-transfected cells. As
shown in Fig. 6A,
cotransfection of increasing amounts of Sp1 expression cassette,
over the range 1-250 ng/plate, resulted in a
concentration-dependent increase in functional promoter
activity (n = 3, triplicate determinations). 250 ng of Sp1 expression plasmid resulted in a maximal 140-fold increase.
Increasing amounts of expression cassette encoding full-length Sp3,
over the range 1-250 ng/plate, also stimulated luciferase activity in
a concentration-dependent fashion, with a maximal 35-fold
increase (Fig. 6A). Co-expression of Sp3 with half-maximal
amounts of Sp1 (15-40 ng) demonstrated cooperative positive functional
interaction with the eNOS promoter (Fig. 6A). Transfecting
increasing amounts of Ets-1 expression plasmid alone failed to modify
functional eNOS promoter activity over the range 1-250 ng/dish (Fig.
6B). However, co-expression of Ets-1 with half-maximal
amounts of Sp1 resulted in a cooperative positive functional
interaction with the eNOS promoter (Fig. 6B) (n = 3, triplicate determinations). Although threshold
amounts of Ets-1, Sp3, and Sp1 expression cassettes (5 ng) alone had
minimal effects on the pGL2
1193/+109 luciferase reporter construct
(Fig. 6C) (n = 3, triplicate
determinations), the combined addition of threshold amounts of Ets-1,
Sp3, and Sp1 activated the eNOS promoter in a cooperative fashion. For
example, the addition of 5 ng of Ets-1 expression plasmid resulted in a
5-fold increase in functional promoter activity compared with the
combined addition of Sp1 and Sp3 alone. These results highlight the
cooperative and complex nature of Ets-1, Sp3 and Sp1 interactions in
functional eNOS promoter activity and underscore their essential
contributions to nucleoprotein complex formation for the eNOS promoter.
To demonstrate that these factors were acting through cis-DNA sequences
found in PRD I and II, these factors were co-transfected with three linker-scanning mutant constructs. Co-transfection of the PRD I
linker-scanning mutant construct with threshold amounts of Sp1, Sp3,
and Ets-1 demonstrated an approximate 80% decrease in activity relative to the activity of the wild-type eNOS construct (Fig. 6C). When activator sequences between
144 and
135 in PRD
II were mutated an approximate 50% decrease in functional promoter activity was observed confirming the important contribution of 5'-regions of PRD II. When sequences corresponding to the Ets recognition site in PRD II were mutated, an approximate 70% decrease in functional promoter activity was observed (Fig. 6C).
These results identify PRD I and II as critical activator recognition sequences for eNOS promoter function. Mutating the Sp1/Sp3 sites in PRD
I and II and mutating the Ets site in PRD II resulted in dramatic
decreases in functional promoter activity both in endothelial cells and
the Drosophila Schneider heterologous expression system.
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Fig. 6.
Sp1, Sp3, and Ets-1 transactivate human eNOS
promoter/reporter luciferase constructs in Drosophila
Schneider cells. A, assay of promoter activity of
pGL2 1193/+109 promoter/reporter luciferase construct upon
co-transfection with increasing amounts of pPacUSp1 (5-250 ng,
left panel), pPacUSp3 (5-250 ng,
middle panel), and pPacUSp3 (5-250 ng) with
half-maximal amounts of pPacUSp1 (40 ng) (right
panel). B, assay of promoter activity of pGL2
1193/+109 promoter/reporter luciferase construct upon co-transfection
with increasing amounts of pPacUSp1 (5-250 ng, left
panel), pPacUEts-1 (5-250 ng, middle
panel), and pPacUEts-1 (5-250 ng) with half-maximal amounts
of pPacUSp1 (40 ng) (right panel). C, assay of
promoter activity of wild-type and linker-scanning mutant pGL2
1193/+109 promoter/reporter luciferase constructs following
co-transfection with threshold amounts of pPacUSp1 (5 ng), pPacUSp3 (5 ng), and/or pPacUEts-1 (5 ng). Shown are representative experiments
(triplicate determinations), each performed four times. Data are
expressed as -fold increase in luciferase activity ± S.E.
relative to pGL2
1193/+109.
1193/+109
eNOS promoter/reporter luciferase construct (Fig.
7A) (n = 3, triplicate determinations). Addition of 100 ng of MAZ expression
construct to threshold amounts of Ets-1, Sp3, and Sp1 resulted in an
approximate 90% decrease in functional promoter activity relative to
the addition of threshold amounts of Ets-1/Sp3/Sp1 alone (Fig.
7B) (n = 3, triplicate determinations). This
suggested that MAZ has a negative effect on the cooperative interaction
among Ets-1, Sp3, and Sp1. MAZ had a similar effect on Sp1 alone (Fig.
7C) (n = 3, triplicate determinations). An approximate 95% drop in activity was observed when 100 ng of MAZ expression construct was added to a half-maximal dose of Sp1, compared
with Sp1 alone. Various linker-scanning mutant constructs were used to
define the requirements for PRD I and PRD II cis-elements (Fig.
7D) (n = 3, triplicate determinations). A
lower amount of MAZ (5 ng) resulted in a 60% reduction of wild-type
functional promoter activity relative to Ets-1/Sp3/Sp1 alone. Although
base-line activity was obviously lower, MAZ was still able to repress
promoter/reporter activity directed by PRD I (
104/
95) or PRD II
(
144/
135) mutants. In contrast, MAZ failed to inhibit functional
activity when
134/
125 regions of PRD II were mutated (
134/
125
mut). This suggested that the Ets site located in PRD II is necessary
for MAZ to exhibit its maximal repressive effect. In summary, these
results suggest (i) that MAZ exhibits a negative effect on eNOS
promoter activity in Drosophila Schneider cells and (ii)
that this repressor activity is especially dependent on the Ets site in
PRD II.
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Fig. 7.
Effect of MAZ on eNOS promoter activity in
Drosophila Schneider cells. A,
promoter activity of pGL2 1193/+109 construct following
co-transfection with increasing amounts of pPacUMAZ (5-250 ng).
B, MAZ inhibits pGL2
1193/+109 promoter/reporter
luciferase construct activity in the presence of threshold amounts of
pPacUSp1 (5 ng), pPacUSp3 (5 ng), and pPacUEts-1 (5 ng). C,
MAZ (pPacUMAZ, 100 ng) inhibits pGL2
1193/+109 promoter/reporter
luciferase construct activity in the presence of half-maximal amounts
of pPacUSp1 (15 ng). D, effect of MAZ (pPacUMAZ, 5 ng) on
promoter activity of wild-type and linker-scanning mutant pGL2
1193/+109 promoter/reporter luciferase constructs following
co-transfection with threshold amounts of pPacUSp1 (5 ng), pPacUSp3 (5 ng), and pPacUEts-1 (5 ng). Shown are representative experiments
(triplicate determinations), each performed three times. Data are
expressed as -fold increase in luciferase activity ± S.E.
relative to pGL2
1193/+109.
NH2) was no longer capable of
trans-activating the promoter by itself over the range 1-250 ng/plate
(compare with Sp3 in Fig. 6A). Sp3
NH2, however, dramatically enhanced the cooperative activity exhibited by
threshold amounts of Ets-1, Sp3, and Sp1, at both 5 or 100 ng of
Sp3
NH2 (Fig. 8B) (n = 3, triplicate determinations). On the other hand, when varied amounts of
Sp3
NH2 were co-transfected with half-maximal amounts of
Sp1 expression construct (15-40 ng), a biphasic effect on Sp1-mediated
activation of the eNOS promoter was observed (Fig. 8C)
(n = 3, triplicate determinations). A low amount of
Sp3
NH2 (1 ng) exhibited a repressive effect on
Sp1-mediated activation, whereas cooperativity was demonstrated between
Sp1 and higher amounts of Sp3
NH2 (250 ng). Low amounts
of Sp3
NH2 was also able to repress Sp3-mediated
activation (100 ng) of the eNOS promoter in Drosophila
Schneider cells (data not shown).
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Fig. 8.
Effect of NH2-deleted Sp3
(Sp3 NH2) on eNOS promoter activity
in Drosophila Schneider cells. A,
promoter activity of pGL2
1193/+109 construct following
co-transfection with increasing amounts of pPacUSp3(
Nterm) (1-250
ng). B, Sp3
NH2 (pPacUSp3(
Nterm), 5 and 100 ng) augments pGL2
1193/+109 promoter/reporter luciferase construct
activity in the presence of threshold amounts of pPacUSp1 (5 ng),
pPacUSp3 (5 ng), and pPacUEts-1 (5 ng). C, effect of low (1 ng) and high (100 ng) concentrations of Sp3
NH2
(pPacUSp3(
Nterm)) on Sp1-induced (pPacUSp1, 15 ng) activation of eNOS
promoter activity. Shown are representative experiments (triplicate
determinations), each performed three times. Data are expressed as
-fold increase in luciferase activity ± S.E. relative to pGL2
1193/+109.
1193/+109 eNOS promoter/reporter construct was
co-transfected with threshold amounts of Ets-1/Sp3/Sp1 expression
plasmids (Fig. 9C) (n = 3, triplicate
determinations). Conversely, cytomegalovirus-directed expression of
Elf-1 protein in BAEC had a stimulatory effect on the
1193/+109 eNOS
promoter/reporter construct (Fig. 9D). This augmentation of
eNOS promoter activity occurred over a range of added Elf-1
heterologous eukaryotic expression cassette (100 ng to 1 µg). Elf-1
enhanced eNOS promoter activity 3.2-13.6-fold above expression vector
alone (n = 4, triplicate determinations, 1 µg).
Moreover, Elf-1-induced activation of functional promoter activity in
BAEC was significantly blunted when Elf-1 was co-expressed with the
134/
125mut linker-scanning construct compared with the wild-type
1193/+109 eNOS promoter/reporter construct (data not shown).
View larger version (24K):
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Fig. 9.
Effect of Elf-1 on eNOS promoter activity in
Drosophila Schneider cells and BAEC.
A, promoter activity of pGL2 1193/+109 construct in
Drosophila Schneider cells following co-transfection with
increasing amounts of pPacUElf-1 (15-250 ng). B, Elf-1
inhibits pGL2
1193/+109 promoter/reporter luciferase construct
activity in Drosophila Schneider cells in the presence of
half-maximal amounts of pPacUSp1 (40 ng). C, effect of Elf-1
(pPacUElf-1, 100 ng) on pGL2
1193/+109 promoter/reporter luciferase
construct activity in Drosophila Schneider cells in the
presence of threshold amounts of pPacUSp1 (5 ng), pPacUSp3 (5 ng), and
pPacUEts-1 (5 ng). D, Elf-1 augments pGL2
1193/+109
promoter/reporter luciferase construct activity in BAEC
(pcDNAI/neo-Elf-1, 1 µg). Shown in A, B,
and C are representative experiments (triplicate
determinations), each performed three times. Data are expressed as
-fold increase in luciferase activity ± S.E. relative to pGL2
1193/+109. Shown in D are results from four experiments
(triplicate determinations) expressed as -fold increase in luciferase
activity ± S.E. relative to empty expression vector alone.
DISCUSSION
104/
95) and PRD II
(
144/
115). Analysis of trans-factor binding and functional
expression studies revealed a surprising degree of cooperativity and
complexity in PRD I and PRD II structure and function. Through analysis
of nucleoprotein complexes in endothelial cells and functional domain
studies in Drosophila Schneider cells and endothelial cells,
we demonstrate positive and negative protein-protein cooperativity
involving Sp1, variants of Sp3, Ets-1, Elf-1, MAZ, and YY1. PRD I and
II function is conserved across species, given that the activity of
human eNOS promoter/reporter constructs exhibited similar trends in
activity in BAEC and HUVEC (Fig. 1). As well, there is a high degree of
relatedness in human and bovine genomic DNA sequences for PRD I and PRD
II (56), and no common allelic variants in these genomic regions for
human eNOS were detected (
475 to +152).
104 to
95
(Fig. 10). Protein-DNA complexes that
formed on an oligonucleotide spanning PRD I (
120/
91) contained Sp1
and multiple variants of Sp3. EMSA and functional studies also
suggested the presence of a low affinity Sp1 site (5'-CCTCCC-3') at
positions
146 to
141 in PRD II. An oligonucleotide spanning this
region of PRD II was also recognized by Sp3 variants.
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Fig. 10.
Schemata representing the proximal promoter
of the human eNOS gene. PRD I and II are indicated. Functional
cis-regulatory DNA elements are shown. The positions of three EMSA
oligonucleotides ( 155/
120,
140/
111, and
120/
91) are shown
as solid black lines.
Numbering is with respect to the start site of transcription
(arrow).
Sp3 is a bifunctional protein that can either activate or repress Sp1-responsive elements in a promoter-dependent context. Stimulation of transcription by Sp3 has been demonstrated for a number of native promoters, sometimes exhibiting synergy with Sp1 (57). This contrasts with other promoters, such as the HIV-1 LTR, wherein Sp3 acts as an inhibitor of Sp1-mediated activation (58). When Sp1 and Sp3 were expressed concomitantly in cells, Sp3 inhibited Sp1-mediated activation of the HIV-1 LTR (32). This repression was dependent on the DNA binding domain of Sp3. A mutant of Sp3, lacking the COOH-terminal DNA binding domain, did not repress Sp1-dependent transcription suggesting that the inhibitory effect of Sp3 may be a consequence of competition with Sp1 for their common DNA recognition sites. Work from others suggested a unique repressor domain in Sp3 immediately upstream of the DNA-binding domain (57).
Newer insight has evolved with the realization that Sp3 mRNA
encodes at least three transcription factors, two of which arise from
alternate translation initiation sites (55). Studies have revealed
three prominent proteins of 115, 80, and 78 kDa that are abundantly
expressed in a broad number of mammalian cell types. Each of these
variants are recognized by antisera prepared against the Sp3 protein
(55). The shorter Sp3 isoforms (78 and 80 kDa) co-migrate as a fast
protein-DNA complex, and the 115-kDa Sp3 isoform migrates as a slower
protein-DNA complex. EMSA results in endothelial cells with PRD I and
PRD II probes are consistent with this formulation. Kennet et
al. (55) concluded that the internally initiated Sp3 isoforms
function as potent inhibitors of Sp-mediated transcription, whereas the
full-length protein is an activator of transcription. In view of these
recent findings, the current studies sought to define the relative
functional contributions of the varied Sp3 isoforms. We report here
that promoter regions of the eNOS are cooperatively activated by Sp1
combined with full-length Sp3, both in the presence and absence of
Ets-1. Sp3NH2 failed to modulate eNOS promoter activity
in Drosophila Schneider cells by itself but potentiated the
stimulatory effects of the combined addition of Ets-1, Sp1, and
full-length Sp3. This enhancement was observed over a wide range of
Sp3
NH2 expression. A biphasic effect was seen when
Sp3
NH2 was co-expressed with Sp1 alone. Curiously,
repression was observed in the setting of limiting amounts of the
Sp3
NH2 mutant. Part of this complexity may be related to
the nature of the binding sites in PRD I and II. Others have suggested
that multimerization of Sp1 with short and long forms of Sp3 may exert
positive or negative effects on transcription depending on the number
of Sp1/Sp3 binding sites in the promoter (55). Limiting concentrations
of Sp3
NH2 protein may inhibit eNOS promoter
transactivation by Sp1 through disruption of intermolecular Sp1
interactions involving both PRD I and II. With the addition of
increasing amounts of Sp3
NH2 protein, this inhibition
may be overridden by the formation of transcriptionally active
multimeric complexes that involve interactions between different
domains of Sp1 and Sp3
NH2. A similar mechanism may be
operative when Sp3
NH2 is added to threshold amounts of
Ets-1, Sp1, and full-length Sp3 proteins. Here, highly co-ordinated
interactions created through protein-protein and protein-DNA
interactions creates multimeric nucleoprotein complexes that robustly
activate the eNOS promoter in a manner that is not dependent upon a
single or more limited repertoire of intermolecular Sp1 interactions.
It is clear that concomitant expression of variants of Sp3 along with Sp1 in endothelial cells adds a level of complexity to understanding the functional role of eNOS PRD I and PRD II. In Drosophila Schneider cells, Sp1-mediated stimulation of eNOS transcription is, for the most part, enhanced by Sp3 rather than abrogated. These findings may have relevance to changes in eNOS transcription that attend the varied unique exogenous stimuli to which the vascular endothelium can respond to, especially since the ratio of Sp1 and Sp3 molecules in a cell can vary during differentiation. A representative example is the involvement of Sp3 in Sp1-dependent activation of p21Cip1/WAF1 expression upon keratinocyte differentiation (59). In these cells it is Sp3 that accounts for the induction of expression, not Sp1. Cellular activation mechanisms in vascular endothelium may exert distinct effects on Sp1 and Sp3 structure and function, especially since post-translational modification of Sp1 family members is known to exert functional effects on promoter activity (60).
Studies in endothelial and Drosophila Schneider cells indicate a critical role for PRD II in efficient transactivation of the eNOS promoter. Although PRD I is necessary for eNOS activation it is not sufficient, given that mutating 5'- or 3'-regions of PRD II interferes with efficient eNOS transactivation. An interesting architectural feature of PRD II is the presence of many putative cis-DNA elements (Fig. 10). Especially intriguing was an Ets binding site. Ets proteins bind to the invariant core motif (GGAA/T). The Ets DNA binding domain, which covers approximately 85 amino acids, has no structural homology to other known DNA-binding motifs such as zinc finger, helix-turn-helix, or leucine zippers but is sufficient for specific DNA interaction (61). The contribution of Ets family members to gene regulation in vascular endothelium is a newer story. Vascular endothelium is known to constitutively express Ets-1, Ets-2, and Erg-1 protein (53, 62). Angiogenesis and cytokine activation represent important changes in endothelial phenotype that are associated with alterations in Ets family member expression and function (53, 63).
Given EMSA results (Figs. 4 and 5) suggesting involvement of Ets family members in nucleoprotein formation upon PRD II, we determined whether Ets-1, a major Ets family member in endothelial cells, participated in activation of the eNOS promoter. Results suggested that Ets-1 can cooperate with Sp1 and/or Sp3 in a cooperative fashion in cells known not to constitutively express Ets proteins, namely Drosophila Schneider cells (Fig. 6). This is well illustrated in studies that evaluated interactions between threshold amounts of Ets-1, full-length Sp3, and Sp1. Removing any one of these factors abrogated their activation potential. Mutating any of the activator recognition sites found in PRD I and II also resulted in a marked decrease in functional promoter activity (Fig. 6C). These results demonstrated the functionally important contribution of the Ets-1 transcription factor in activation of the eNOS promoter. The contribution of Ets proteins in transcriptional regulation often involves participation in protein-protein interactions with other nuclear factors, especially Sp1 family members, so the interdependence on other transcription factors was not unexpected. For example, the LTR of human HTLV-I contains a Ets-1-responsive element that is dependent on the integrity of an adjacent Sp1 cis-DNA element for synergistic activation of HTLV-I transcription (64). Similarly, the P4 promoter of parvovirus minute virus is synergistically transactivated via neighboring Ets-1 and low affinity Sp1 sites (65). In this regard, we propose that low and high affinity Sp1/Sp3 protein-DNA interactions participate with Ets-1 in activating the proximal eNOS promoter through PRD I and PRD II. This is the most plausible interpretation of the findings reported in the current work. It should be acknowledged that conclusive evidence of Ets-1 binding to PRD II is not provided in the present work, given the acknowledged difficulties with Ets family member antisera and cross-competition among Ets binding sites. Moreover, an autoinhibitory domain of Ets-1 can inhibit important protein-protein or protein-DNA interactions in EMSA studies. This has led to the realization that the Ets-1 protein is negatively regulated through conformational changes involving intramolecular interactions (47). Specifically, an inhibitory module allosterically modulates the DNA binding activity of Ets-1. Activation of Ets-1 requires a conformational change, and this autoinhibition of Ets-1 can be relieved by either protein partner(s) or post-translational modifications (47). This concept is relevant to a model discussed below.
With the exception of GABP, Ets family members bind the core motif
as a monomer. Approximately 10 bp of DNA sequence containing the Ets
core motif determines which Ets family member will bind and hence
specificity. Sequence comparison of PRD II of the eNOS gene at
132/
122 (5'-ACAGGAACAA-3') with previously identified Ets binding
sites revealed near identity with the HIV-2 LTR enhancer (5'-ACAGGAACAG-3'), which is known to bind Elf-1 (50)
(Table IV). Elf-1 is a transcription
factor previously implicated in the inducible activation of genes in
mature T cells. For example, Elf-1 participates in the inducible
regulation of CD4, granulocyte-macrophage colony-stimulating factor,
and interleukin-2 receptor
chain (IL-2R
) following T cell
activation (51, 66). Elf-1 plays a role in the developmental regulation
of the terminal transferase gene in early lymphocyte development (67).
Elf-1 is also required for inducible T cell trophic viruses including
HIV-2 and HTLV-1 (50, 68). Elf-1 is highly expressed in B cells, where
it participates in the regulation of a variety of genes, including IgH,
lyn, and lck (69). As a rule, the Ets family of
transcription factors are widely distributed in varied tissues and cell
types. Therefore, the cell-restricted expression of Elf-1 contrasts
with the expression of most Ets family members. Only a few Ets family
members demonstrate a cell type-specific expression pattern (61). For
example, PU.1 is B cell- and macrophage-restricted and ESE-1 is
primarily found in epithelial cells (70). Elf-1 protein expression was
thought to be relatively restricted to lymphoid and myeloid cells (66). The present work demonstrates that Elf-1 is constitutively expressed in
vascular endothelium and can bind to PRD II and consensus HIV-2 LTR
sequences. Elf-1 was not previously appreciated as playing a role in
the control of gene expression in vascular endothelium. It is of
interest that novel Elf-1-like proteins continue to be cloned and
characterized. Recently NERF, Elf-2, and MEF were identified (69, 71).
These proteins contain domains that are structurally similar to the Ets
binding domain of Elf-1 (69, 71). Therefore, we cannot exclude the
possibility that a protein antigenically related to Elf-1 exists in
endothelial cells.
|
An intriguing facet of Elf-1 biology is that the consensus motif for
optimal Elf-1 binding (5'-A(A/t)(C/a)CCGGAAGT(a/g/c)-3') determined by
a binding site selection method does not conform to known functional
Elf-1 sites (Table IV) (43). Conversion of a naturally occurring, low
affinity Elf-1 site in the IL-2R promoter to an optimal site
resulted in a decrease in the ability of Elf-1 to induce transcription
(43). Therefore, high affinity Elf-1 sites may lack sufficient
biological specificity even though Elf-1 is more discriminatory than
Ets-1 in binding site selection (72). The absence of naturally
occurring high affinity Elf-1 sites in Elf-1-regulated genes may
represent a mechanism to achieve greater inducibility and may emphasize
the importance of accessory proteins in modulating Elf-1 binding and
action. One exception to this generalization has been recently
provided. Elf-1 plays an essential role in the trans-activation of the
T cell receptor-
subunit, a constitutively expressed T cell-specific
gene, through a site that is the best match with the optimal Elf-1
consensus of any known mammalian Elf-1 binding site (73). This suggests that the requirement for post-translational modulation of Elf-1 or
other inducible trans-factors in Elf-1-mediated trans-activation may
decrease as the affinity of the Elf-1 site increases. In the current
work we report that (i) the Elf-1 binding site in PRD II does not
conform to the optimal Elf-1 consensus sequence, (ii) Elf-1 protein
minimally enhanced eNOS promoter activity in Drosophila Schneider cells by itself, (iii) Elf-1 was a potent inhibitor of
Sp1-mediated transactivation in Drosophila Schneider cells, (iv) Elf-1 did not repress eNOS promoter activity in the presence of
Ets-1 in Drosophila Schneider cells, (v) PRD II
nucleoprotein complexes in BAEC contain Elf-1, and (vi) augmented Elf-1
expression in BAEC enhanced eNOS promoter activity and this increase
was dependent upon the Ets binding site in PRD II.
In general, Elf-1 serves as a transcriptional activator for many
inducible T cell genes. These promoters often contain adjacent or
overlapping binding sites for the Elf-1 and NF-B/NFAT families of
transcription factors. For example, Elf-1 participates in the inducible
expression of the HIV-2 LTR via Ets binding sites and a neighboring
NF-
B/NFAT binding site (50). Elf-1 functions cooperatively with the
NF-
B family of transcription factors to activate transcription of
the HIV-2 LTR during T cell activation. Both proteins are necessary for
this activation. Overexpression of Elf-1 alone fails to activate the
HIV-2 LTR. Also, a dominant-negative mutant of NF-
B p50 that binds
DNA but fails to interact with Ets proteins inhibits the synergistic
activation of the HIV-2 enhancer by NF-
B (p50 + p65) and Ets family
members (74). As a further example, cell type-specific expression of
the IL-2R
promoter in T cells also involves, in part, synergistic
interactions between Elf-1, HMG-I(Y), and NF-
B family proteins (75).
The current studies demonstrated that Elf-1 exerts a repressive effect on Sp1-mediated promoter activation in Drosophila Schneider
cells but enhanced activity in endothelial cells. Further studies will be necessary to understand why the contribution of Elf-1 to
transcriptional regulation of the eNOS gene differs between
Drosophila Schneider cells and endothelial cells. Clearly
some members of the Ets family of transcription factors exhibit strong
transcriptional repressor activity, such as ERF (76). It may be
possible that Elf-1 requires a co-activator in order to demonstrate
activation potential. Also, activation of mitogen-activated protein
kinase signal transduction pathways results in changes in the activity
of many ETS domain transcription factors (77). Therefore, a further
possibility may be a requirement for post-translational modification of
Elf-1 for it to function as an activator, much like BOB kinase activity is required for BOB co-activator to functionally activate Oct1 or Oct2
in B and T cells (78). It is plausible that such a pathway(s) may not
be functional in Drosophila and Elf-1 protein may be in a
conformation that sterically inhibits its capabilities of functioning as an activator. Finally, it is known that Elf-1 forms complexes in T
cells with the underphosphorylated form of the retinoblastoma protein
(Rb) both in vitro and in vivo. Overexpression of
unphosphorylated Rb inhibits Elf-1-dependent
transcriptional activation in T cells (79). After T cell activation,
phosphorylation of Rb leads to the release of transcriptionally
competent Elf-1 (79). This coordinated regulation of Elf-1 may be
deficient in Drosophila Schneider cells.
EMSA analyses with PRD II oligonucleotides demonstrated binding of
recombinant YY1 and the existence of YY1 in PRD II nucleoprotein complexes (Fig. 4D), likely at position 121 to
117 (Fig.
10). YY1 is a ubiquitously expressed 65-68-kDa GLI-Kruppel-related protein that contains four C2H2-type zinc
fingers at the COOH terminus (80) and is a multifunctional
transcriptional regulator. Depending on promoter and cellular context,
it can activate or repress transcription. YY1-binding proteins so far
identified include Sp1 (81), the oncoprotein c-Myc, cyclophilin A,
FK506-binding protein, p300, ATF/CREB, and the mammalian homologue of
RPD3 (82). Given this complexity, future studies will be needed that
address the functional contributions of YY1 to eNOS promoter structure and function in episomal and chromatin-based assays. Our current working model is that YY1 will activate transcription given that its
consensus binding site is an activator site located in 3'-regions of
PRD II. It is of interest that YY1 has been shown to physically interact with Sp1 (81) and tightly clustered YY1 and Ets binding sites
have been functionally characterized in other genes, such as the
cytochrome c oxidase subunit VIIc (NRF-2/NERF-2) or human P19 parvovirus (GABP
) promoter (83, 84). In the case of the eNOS
promoter, the YY1 and Ets binding sites are separated by 4 nt in PRD
II. It is presumed that the four zinc fingers of YY1 interact with 12 nt of the bottom strand, with the core motif positioned at the center
(83). This may indicate that the binding of one factor would sterically
hinder the binding of the other. One mechanism of YY1 repression
involves preventing the binding of activator proteins via overlapping
binding sites. For example, overlapping binding sites have been
observed for YY1 and NF-
B in the serum amyloid A1 gene promoter. YY1
can also enhance the binding of an activator to an adjacent binding
site by inducing DNA binding, thereby facilitating the interaction of
an activator with the basal transcriptional machinery (85). Perhaps in
the absence of YY1, Elf-1 is unable to interact with the basal
transcriptional machinery and thus requires the presence of YY1 to
facilitate this interaction. It is of interest that the eNOS gene does
not evidence a canonical TATAA element (86), given that YY1 has also
been implicated in the formation of preinitiation transcription complexes independent of TATA-binding protein (87).
The current work demonstrated that MAZ (myc-associated zinc
finger protein) participates in protein-DNA and protein-protein interactions in PRD II regulatory regions of the eNOS promoter. In
Drosophila Schneider cells, MAZ exhibits a negative effect on eNOS promoter activity, and this repressor activity is especially dependent on the Ets site in PRD II. MAZ has received increasing attention for its protean roles in gene regulation: transcription initiation, interference, and termination (39). MAZ is especially important in TATA-less promoters (33). Particularly well studied examples of MAZ involvement in transcriptional initiation include c-myc, the adenovirus major late promoter, the serotonin 1a
receptor, and CD4 (33, 39, 52). An important facet of MAZ function is
the participation of partner proteins. MAZ polypeptide contains multiple functional domains in addition to the six structurally important zinc fingers (His2Cys2) (39).
Functional interactions between MAZ and Sp1 occur in a number of genes,
including c-myc (33, 40). In the case of CD4, enhancer
activity during development is critically dependent upon MAZ and an Ets
consensus site that binds Elf-1 (51, 52). The functional contributions
of MAZ are complex considering that not only is MAZ necessary for
efficient initiation and transcriptional elongation of c-myc
P2 promoter transcripts, through the ME1a1 site in the P2 promoter, but
MAZ and ME1a1-like binding sites are also involved in the
transcriptional pausing/attenuation of the c-myc gene, and
also the human complement C2 gene (39). In certain respects the human
eNOS promoter evidences sequence similarity with the human
c-myc promoter: tandem CT elements in the P1 promoter and
the single functional CT (ME1a1) element of the P2 promoter are
reminiscent of the numerous CT elements in the eNOS promoter. Although
multiple potential MAZ-binding CT elements were identified in the eNOS
promoter at 191,
146,
99,
75,
62, and
47, the evidence in
the current work highlighted that the repressive effect of MAZ on eNOS
promoter activity in Drosophila Schneider cells is likely
mediated via PRD II. The complexity that has emerged from studies
assessing the contributions of MAZ to regulation of c-myc
expression suggests that further nuances of MAZ and eNOS expression
will evolve, perhaps also involving both regulation of transcriptional
initiation and pausing.
In summary, these studies demonstrate an unexpected complexity in the
regulation of eNOS gene expression. Sp1, variants of Sp3, Ets-1, and
Elf-1 play important roles in the activation of eNOS transcription in
endothelial cells (Fig. 10). Cooperativity between these trans-acting
factors is likely to require multiple protein-protein and protein-DNA
interactions (88, 89). These trans-acting factors may functionally
cooperate to present to the basal transcriptional machinery a
biochemical interface that is highly efficient in transcription
initiation. In the current work, coexpression of Sp1, Sp3, and Ets-1 in
Drosophila Schneider cells enhanced transcription of eNOS
promoter/reporter constructs compared with each factor alone. Mutating
activator recognition sites for these factors, or removal of any of
these factors, abolishes this cooperativity. This suggests that
activation of the eNOS promoter is dependent upon protein-protein
interactions between these factors as well as interactions between the
trans-factors and their corresponding cis-elements in PRD I and PRD II.
Sp1 is known to recruit and physically interact with itself and varied trans-acting factors: Sp3, AP-1, GATA members, NF-B, and Ets-1, among others. Based upon this background, a model can be proposed. Following an initial binding of Sp1 to a high affinity element in PRD
I, other Sp1 molecules and variant Sp3 proteins are recruited through
binding or tethering and interact with low affinity elements in PRD II.
DNA binding domains of Sp1 family members have been reported to unwind
DNA as well as bend DNA (90) upon binding. These DNA deformations may
be important in determining overall binding affinities as well as
influencing binding site preferences for neighboring sites, but are not
by themselves sufficient for transactivation (90). These changes may
also enhance the transactivation potential of Sp1 family members. Ets
factors also interact with other proteins to form either multisubunit
complexes or ternary complexes that are stable only in the presence of
DNA. Therefore, an initial recruitment of Sp1 may also facilitate the
binding of Ets-1. Domain interactions between Sp1 and Elf-1 proteins
has not yet been described, nor has an interaction between Elf-1 and Ets-1 proteins. We further suspect that post-translational
modifications of Elf-1 figure prominently in determining the functional
contributions of Elf-1 to eNOS promoter function in endothelial cells,
likely by modulating binding site affinity. The prior findings that MAZ is capable of inducing a 72° bend in the DNA helix and that YY1 has
also been shown to induce bending of DNA (80) indicate that protein-DNA
and protein-protein interactions on the eNOS promoter may be modulated
by complex architectural features. In this regard, future detailed
biochemical analysis of protein-DNA and protein-protein interactions on
the eNOS promoter will be needed to substantiate this model further. It
will be also be necessary to determine what alterations occur in the
complex eNOS promoter structure and function in conditions known to be
associated with biologically important alterations in eNOS mRNA
expression. Especially relevant to health and disease are alterations
in fluid shear stress and atherosclerosis.
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FOOTNOTES |
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* 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.
Recipient of a University of Toronto Connaught scholarship.
§ Recipient of a Career Investigator award from the Heart and Stroke Foundation of Canada. Supported by a grant from the Medical Research Council of Canada (Group Grant 13298). To whom correspondence should be addressed: Rm. 7358, Medical Sciences Bldg., University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-2441; Fax: 416-978-8765; E-mail: p.marsden{at}utoronto.ca.
The abbreviations used are:
eNOS, endothelial
nitric-oxide synthase; bp, base pair(s); kb, kilobase(s); nt, nucleotide(s); PCR, polymerase chain reaction; cDNA, complementary
DNA; cRNA, complementary RNA; RACE, rapid amplification of cDNA
ends; EMSA, electrophoretic mobility shift assay; RLU, raw luciferase
units; PRD, positive regulatory domain; DTT, dithiothreitol; HIV, human
immunodeficiency virus; IL-2R, interleukin-2 receptor
; HTLV, human T cell lymphotrophic virus type I; LTR, long terminal repeat; UTR, untranslated region; BAEC, bovine aortic endothelial cell; HUVEC, human umbilical vein endothelial cell; SSCP, single-stranded
conformation polymorphism; CPRG, chlorophenol
red-
-D-galactopyranoside.
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REFERENCES |
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