From the Department of Biochemistry and Molecular
Biology, Institute of Genetic Sciences, Yonsei University School of
Medicine, 134, ShinChon-Dong, SeoDaeMoon-Ku, Seoul, Korea 120-752 and
the § Department of Biochemistry and Molecular Biology,
Indiana University School of Medicine,
Indianapolis, Indiana 46202-5122
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ABSTRACT |
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The human alcohol dehydrogenase 5 gene (also
known as the formaldehyde dehydrogenase gene, ADH5/FDH) has
a GC-rich promoter with many sites at which transcription factors bind.
A minimal promoter extending from The regulated transcription of a typical eukaryotic gene is
governed by the combined action of multiple sequence-specific DNA-binding proteins (1, 2). The information provided by these proteins
is ultimately communicated to RNA polymerase II, resulting in a precise
transcription initiation frequency.
Sp1 is a well characterized sequence-specific DNA-binding protein that
plays a role in the transcription of many cellular and viral genes that
contain GC boxes (KRGGMGKRRY) in their promoters (3, 4). This includes
numerous housekeeping genes, with high G+C content in their promoters
(5). Additional human and rodent transcription factors (Sp2, Sp3, Sp4)
similar in structural and transcriptional properties to Sp1 have been
cloned, and form an Sp1 multigene family (6-9). Sp1, Sp3, and Sp4 have
a highly conserved zinc-finger DNA binding domain close to the C
terminus and contain glutamine- and serine/threonine-rich amino acid
stretches in the N-terminal region (10). Sp1, Sp3, and Sp4 can bind to the same recognition sequence (GC boxes) with identical affinity (6).
Sp1 and Sp4 generally act as transcriptional activators, while Sp3
generally acts as a repressor, and rarely as an activator (10-16). Sp2
has a DNA-binding specificity different from that of Sp1, Sp3, and Sp4.
The Sp1 multigene family is an important regulator of the cell cycle,
differentiation, and development (9, 15-17).
We previously cloned and characterized the
ADH5/FDH1
gene (18, 19), which encodes the human class III The very small DNA fragment extending to Here we test whether the members of the Sp1 multigene family recognize
the same cis-acting elements on the minimal promoter, and
examine the roles they play in transcriptional regulation. Analyses of
DNA-protein interactions in vitro and in vivo
demonstrate that the members of this multigene family compete for
binding, with different effects upon transcription. These data in part explain the ubiquitous expression of the gene and how different levels
of expression are achieved in different tissues. This may have
significant implications in understanding how many housekeeping genes
can be regulated (19, 22, 23).
Plasmid Constructs--
pGL luciferase, pCAT Control and pCAT
Basic were purchased from Promega (Madison, WI). pCAT Control contains
the SV40 promoter, enhancer, and CAT coding sequence. pCAT Basic
does not have an eukaryotic promoter and enhancer. pGL luciferase
contains a firefly luciferase gene driven by a cytomegalovirus (CMV)
promoter in a vector called pcDNA I (Invitrogen, San Diego, CA).
pCAT 5-2, which contains the ADH5/FDH minimal promoter
( Nuclear Extracts and Transcription Factors--
Nuclear extracts
from cultured HeLa cells were made according to Shapiro et
al. (29). HeLa cell nuclear extract and the purified transcription
factors Sp1 and AP2 were purchased from Promega. Antibodies to Sp1
(PEP2), Sp3, Sp4, and AP2 were purchased from Santa Cruz Biotechnology
Inc. (Santa Cruz, CA). Antibody to the Ku antigen was kindly provided
by Dr. Westly Reeves (University of North Carolina). Flagtag antibody
was purchased from Eastman Kodak Corp. Zinc-finger DNA binding domains
(ZFDs) of Sp3 (from amino acid 501 to amino acid 697) and Sp4 (from
amino acid 616 to amino acid 784) were prepared by subcloning
polymerase chain reaction products into pCITE vector (Novagen, Madison,
WI) or pT7-7 vector (obtained from Dr. Stan Tabor, Harvard Medical
School). Each DNA binding domain was tagged with Flag peptide (Kodak)
at the C terminus. Primers used to amplify ZFD-Sp3 are
CATATGGGGGACCAACAACATCAAGAAGGA (5' foward) and
GGGATCCTCACTTGTCATCGTCGTCCTTGTAGTCCTCCATTGTCTCATTTCCAGAAA (3'
foward). Primers used to amplify ZFD-Sp4 are
CATATGCCTGGCAAGAGGCTTCGAAGAGTT (5' forward) and
GGGATCCTCACTTGTCATCGTCGTCCTTGTAGTCGAATTCTTCCATGTTGGTTGAAC (3'
forward). The plasmids were transcribed and translated in vitro to produce functional zinc-finger DNA binding domains using the Single Tube System 2 (Novagen, Madison, WI). The resulting proteins
were used for electrophoretic mobility shift assays.
DNase I Footprinting Assays--
To examine DNA-protein
interactions in the minimal promoter region, pCAT AX plasmid (19) was
digested with HindIII and filled in with the Klenow fragment
in the presence of [ Electrophoretic Mobility Shift Assays (EMSAs)--
EMSAs were
carried out as described previously (19). Sequences of various
oligonucleotides are as follows (only the top strand is shown): site A
oligonucleotide (+40 bp to +60 bp): GATCCCCCCGGATGTCAGCCCCCCGCGCC; site
B oligonucleotide (+1 bp to +22 bp): GATCGCGCTCGCCACGCCCATGCCTC; site
C-1 oligonucleotide ( Transient Expression Assays in Mammalian Tissue Culture
Cells--
Alexander cells (human hepatoma), were grown on 10-cm
dishes in minimal essential medium (Life Technologies, Inc.)
supplemented with 5% fetal calf serum. When they reached 50-60%
confluence, the medium was replaced. Four hours later,
CaCl2-DNA coprecipitates (30) were added: 6 µg of pCAT
5-2, 3 µg of pCMVSp1, Sp3, or Sp4, and an appropriate amount of
pUC18 DNA to total 16 µg of DNA were added to each dish. The DNA was
allowed to remain on the cells for 4 h, after which the medium was
removed, medium containing 20% glycerol was added for 2 min, and fresh
growth medium was added. Cells were incubated for 48 h. After the
plates were gently washed four times with cold phosphate-buffered
saline, the cells were harvested and cell pellets were resuspended in 150 µl of lysis buffer (100 mM KPO4, pH 7.8, 1 mM dithiothreitol). Cells were broken by three cycles of
freezing and thawing.
We did not use reporter plasmids as internal controls because their
promoters contain binding sites for Sp1 and related transcription factors. Therefore, the introduction of Sp1, Sp3, and Sp4 expression vectors into cells would also affect the internal control. Instead, we
used a fixed amount of protein extract for each CAT assay. CAT assays
were conducted by incubating cell extracts containing 40 µg of
protein in 200 µl of a reaction mixture containing 0.25 M
Tris-HCl (pH 7.8), 50 mM acetyl CoA, 100 nCi of
[14C]chloramphenicol, 5 mM EDTA at 37 °C
for 4 h. The acetylation of chloramphenicol was analyzed by silica
gel thin-layer chromatography and quantitated with a Fuji Phosphoimage
Analyzer (Tokyo, Japan). CAT activity was expressed as relative CAT
activity compared with the control (pCAT 5-2 only), and was the
average of six independent experiments.
Transient Expression Assays in Schneider's Drosophila Line 2 Cells (SL2)--
SL2 cells were grown on 10-cm dishes in Schneider
cell culture medium (Life Technologies, Inc.) supplemented with 10%
fetal calf serum. When they reached 50-60% confluence, the cells were transfected. To investigate the role of Sp1, Sp3, and Sp4 on the minimal prompter, 12 µg of pCAT 5-2, 3 µg of pPac luciferase, varying amounts of Sp1, Sp2, and Sp3 expression vector (1-9 µg of
pPacSp1, pPacSp3, and pPacSp4) and appropriate amounts of pUC18 DNA to
fill the total DNA amount to 31 µg were transfected into the cells
using the CaCl2-DNA coprecipitation method (30). The pPac
vector contains the Drosophila actin promoter and was not affected by the addition of Sp1 and related factors. The DNA
precipitate was allowed to remain on the cells for 48 h, and then
the medium was removed. After the plates were gently washed four times
with cold phosphate-buffered saline, the cells were harvested and cell pellets were resuspended in 100 µl of 1× reporter lysis buffer (Promega, WI). The cellular extract (10 or 20 µl) was assayed for
luciferase activity (31) to normalize plate to plate variation in
transfection efficiency. Cell extracts representing 200,000 RLU of
luciferase activity were then used for CAT assays. CAT assays were
conducted by incubating cell extracts in 174 µl of reaction mixture
containing 0.25 M Tris-HCl (pH 7.8), 2.2 mM
acetyl CoA, 100 nCi of [14C]chloramphenicol, 5 mM EDTA at 37 °C for 8 h. The acetylation of
chloramphenicol was analyzed as described above. CAT activity was
expressed as relative CAT activity compared with the control and was
the average of three or five independent experiments. To investigate
the interaction among the Sp1 multigene family, cells were transfected
with 12 µg of pCAT 5-2, 1 µg of pPac luciferase, 1 µg of
pPacSp1, pPacSp3, or pPacSp4 (3-9 µg) and appropriate amounts of
pUC18 DNA to total 31 µg of DNA, using the CaCl2-DNA coprecipitation method as above. Extracts representing 67,000 RLU were
used for CAT assays.
Site-directed Mutagenesis of pCAT 5-2--
To investigate the
role of the cis-elements immediately flanking the
transcription start site, mutations were introduced into the Sp1
consensus sequences using a QuickChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA). pCAT 5-2 M1 (CC at Transient Expression Analysis of pCAT 5-2 Mutants--
To
investigate the importance of the core cis-elements in
transcriptional regulation, 12 µg each of pCAT 5-2, pCAT 5-2 M1, pCAT 5-2 M2, pCAT 5-2 M3, pCAT 5-2 M4, and pCAT 5-2 M5 were
separately cotransfected into Drosophila SL2 cells with a
mixture of the following plasmids: 1 µg of pPac luciferase, 1 µg of
pPacSp1, and appropriate amount of pUC18 (to total 31 µg of DNA).
Transfection and analysis of the reporter gene activities were carried
out as described above. CAT activity was expressed as relative CAT activity compared with the control and was the average of four independent experiments.
Sp1 Binding to the ADH5/FDH Minimal Promoter--
We demonstrated
that the fragment from
We characterized the minimal promoter by DNase I footprinting analysis
using various amounts of Sp1 (Fig. 2). As
shown in Fig. 2, Sp1 can bind to virtually all regions of the
ADH5/FDH minimal promoter, and has a very high affinity for
the core cis-elements (boxes B and C) immediately flanking
the transcription site. Sites B (+1 bp to +23 bp) and C ( Sp1, Sp3, and Sp4 Regulate ADH5/FDH Minimal Promoter Activity in
Mammalian Cells--
Because the members of the Sp1 multigene family
share the same binding consensus sequence (GC box), they may play
important roles in the transcriptional regulation by directly
interacting with the core cis-elements (Fig. 2,
sites B and C). Thus, we tested whether the members of the Sp1 multigene family can regulate the ADH5/FDH gene through the minimal promoter by cotransfecting
the minimal promoter-CAT fusion construct (pCAT 5-2) and the Sp1, Sp3,
Sp4, and rat Sp1 expression vectors into human Alexander cells. Sp1 did
not alter the promoter activity (Fig. 3),
probably because there was already enough endogenous Sp1 in the cells. However, rat Sp1 significantly activated transcription. Sp3 repressed the promoter activity by 40%. Surprisingly, Sp4, a known transcription activator (16, 30), also repressed transcription by 45%. The data
indicated that members of the Sp1 multigene family can regulate the
transcription of the ADH5/FDH gene by interacting with the minimal promoter.
Proteins Interacting with the Minimal Promoter in HeLa Cell Nuclear
Extract--
We carried out a series of EMSAs to determine which
transcription factors in a HeLa cell nuclear extract were interacting with the ADH5/FDH minimal promoter. We specifically tested
whether Sp1, Sp3, and Sp4 can indeed interact with the
cis-elements on the minimal promoter to give the transient
expression assay data described above (Fig. 3).
Sp1 bound strongly to probe A (+40 bp to +61 bp), and the band was
supershifted by the Sp1 antibody (Fig.
4A, lane
2 and 3). HeLa nuclear extract gave two main
retarded bands (labeled Sp1 and Ku), that could
be competed by excess cold probe (lanes 9-11). One complex could be supershifted by the antibody against Sp1 (band Sp1, lanes 4 and
5), indicating that Sp1 in HeLa nuclear extract can interact
with probe A. We suspected that the protein which produced the major
fast-moving complex that was observed in all gel mobility-shift assays
may be the Ku antigen, a DNA-binding subunit of the
DNA-dependent protein kinase complex. Indeed, the major
fast-moving complex (labeled Ku) was clearly supershifted by
the Ku antibody (Fig. 4E) (32, 33). Ku is known to bind to
the ends of DNA (34). We will not further discuss this band in the
present report.
Sp1 could also bind to probe B (+1 bp to +22 bp) (Fig. 4B).
A similar complex was detected in the HeLa extract, and most of this
band was supershifted by the Sp1 antibody (Fig. 4B).
Antibodies against Sp3 and Sp4 did not shift much of the Sp1-containing
band (Fig. 4B, lanes 6-9), although
some material was apparently shifted to the wells when larger amounts
of extract were tested. Following a much longer exposure of the gel,
very faint bands in lanes 7 and 9 appeared in a position similar to the supershifted Sp1-probe-antibody complex, which may be the Sp3- or Sp4-probe-antibody complex (data not
shown). Probe B could be bound more strongly by Sp1 than was probe A,
judging by the relative intensities of the Sp1 and Ku bands (Fig. 4,
B and E).
Based on DNase I footprint analysis with Sp1, we divided the upstream
footprinted region (
Probes B and C-1, the cis-elements immediately flanking the
transcription start site, bound most strongly to Sp1 (Fig.
4E). Despite the consensus AP2 sites noted above, EMSA did
not give any indication for AP2 binding; none of the retarded bands
formed by the various probes and HeLa extract was able to be
supershifted by the AP2 antibody (data not shown).
Zinc-finger DNA Binding Domains of Sp3 and Sp4 Can Bind
Specifically to the Core cis-Elements--
Because Sp3 and Sp4 may be
present in low levels in HeLa cells (10), and the size of the retarded
DNA-protein complex or DNA-protein-antibody complex overlap other
bands, we were not able in the experiments shown in Fig. 4 to clearly
demonstrate that Sp3 or Sp4 can bind to the minimal promoter. To
investigate the possible interaction between Sp3 and Sp4 and various
cis-elements, we prepared, by in vitro
transcription and translation, shorter versions of the transcription
factors that contain only the zinc-finger DNA binding domains tagged
with Flag peptide (named ZFD-Sp3 and ZFD-Sp4). ZFD-Sp3 and ZFD-Sp4 were
able to bind selectively to the probe B and C-1, the two core
cis-elements immediately flanking the transcription start
site, as demonstrated in the EMSA shown in Fig.
5 (A and B,
lanes 2 and 5). The retarded bands
containing ZFD-Sp3 and ZFD-Sp4 were either supershifted or lost by the
addition of antibodies against Sp3 or Sp4 (Fig. 5, A and
B, lanes 3 and 6). Adding
the Flag antibody also resulted in the disappearance of the bands (Fig.
5, A and B, lanes 4 and
7). The binding of the proteins to probe B was stronger than
to probe C-1, which is in line with data showing that the GGGCGTGG
motif (probe B) has a slightly higher affinity toward Sp3 or Sp4 than
to Sp1 (6). EMSA revealed that ZFD-Sp3 and ZFD-Sp4 were not able to
bind to the cis-elements contained in probes A, C-2, and C-3
(data not shown).
Sp1, Sp3, and Sp4 Regulate ADH5/FDH Minimal Promoter Activity in
Drosophila SL2 Cells, Which Lack Endogenous Sp1--
Since Sp1 and
related factors are expressed in virtually all mammalian cells, and
such endogenous expression could affect the interpretation of
cotransfection experiments, we decided to analyze the effects of these
transcription factors in Drosophila SL2 cells, which are
known to lack them (35, 36). We introduced the minimal promoter-CAT
fusion plasmid pCAT 5-2 along with the Drosophila
expression vectors pPacSp1, pPacSp3, and pPacSp4 into Drosophila SL2 cells. pCAT 5-2 alone was not able to drive
transcription at all (Fig. 6,
lanes 1 and 11). This demonstrated the
lack of a critical transcription factor essential for the formation of the transcription initiation complex. Addition of the Sp1 expression vector, pPacSp1, drastically increased transcription in a
dose-dependent manner (Fig. 6, lanes
2-4); stimulation was 51-fold at 9 µg of pPacSp1. Sp3 and
Sp4 did not significantly stimulate transcription from the
ADH5/FDH promoter. We also introduced pCAT 5-2 (12 µg) with 9 µg each of pPacSp3 and Sp4 into Schneider cells to investigate the potential interaction between Sp3 and Sp4 on the minimal promoter. Sp3 and Sp4 gave only a very weak transcriptional activation, barely
detectable over the background (data not shown). Thus, Sp3 and Sp4
function very differently than Sp1 in the transcription process.
We further investigated whether transcriptional activation by Sp1 could
be repressed by either Sp3 or Sp4. pCAT 5-2 (12 µg), pPacSp1 (3 µg), and 3-9 µg of pPacSp3 or pPacSp4 were introduced into
Drosophila SL2 cells. Transcription activation by Sp1 could be clearly repressed by both Sp3 and Sp4 (Fig.
7, lanes 2-5). Sp3
and Sp4 may abort the formation of the transcriptional initiation complex by competing with Sp1 for the same core
cis-elements.
The Core cis-Elements Are Critical for Transcriptional Activation
by Sp1--
We tested the role of the core cis-elements in
transcriptional activation by Sp1, using site-directed mutagenesis. We
prepared five mutated minimal promoter constructs, with mutations
introduced at one or more Sp1 binding sites (pCAT 5-2 M1 at
Unexpectedly, mutating both sites B and C (in pCAT 5-2 M3 and M5) did
not knock out the transcription completely (Fig. 8B). The
residual transcription (22-30%) observed in pCAT 5-2 M3 and M5 might
be caused by some residual binding of Sp1 to the mutated elements, or
to cis-elements outside of this core. We investigated the
possibility that the newly mutated sequence may be recognized by
endogenous Drosophila transcription factors. We were not
able to observe any transcriptional activation with these mutated
constructs in the absence of Sp1, suggesting that Drosophila
transcription factors were not capable of stimulating these mutated
promoters (data not shown).
We investigated the regulation of transcription of the
ADH5/FDH minimal promoter by members of the Sp1 multigene
family member. DNase I footprinting analysis and EMSA showed that the
cis-elements on the minimal promoter differ in their binding
affinity toward Sp1 and related transcription factors; the affinities
vary by as much as 5-10-fold. Sp1 can preferentially bind to the core cis-elements immediately flanking the transcription start
site, sites B and C-1, extending from Analyses of the DNA-protein interactions on the minimal promoter, using
both purified proteins and HeLa cell nuclear extract, demonstrate that
Sp1 is the major transcription factor binding to the
cis-acting elements. Although virtually the entire promoter region can be footprinted by 0.5 fpu of Sp1, the core
cis-elements flanking the transcription start site have the
highest binding affinity toward Sp1 (5-10-fold higher than the other
sites; Figs. 2 and 4). Binding of Sp1 or related factors to this unique
arrangement of cis-acting elements may allow the nucleosome
surrounding the minimal promoter to enter into an "open" state by
actively displacing the histone (37-39). Since Sp3 and Sp4 were
reported to bind to the same cis-elements that are bound by
Sp1 (i.e. GC boxes), we examined the binding of Sp3 and Sp4
to this promoter. The zinc-finger DNA binding domains of both Sp3 or
Sp4 can bind selectively to the core cis-elements, but not
elsewhere in the minimal promoter (Figs. 4 and 5). Therefore it is very
likely that competition among Sp1 multigene family members and the
interaction among them are mainly occurring on the core
cis-elements in vivo.
To investigate the roles of Sp1 family members in transcriptional
regulation, we carried out transient expression assays using the
minimal ADH5/FDH promoter-CAT fusion constructs. In human Alexander cells, which contain endogenous Sp1, rat Sp1 but not human
Sp1 stimulated transcription (Fig. 3), probably because there is
already enough endogenous Sp1. Sp3 inhibited transcription, as might be
expected given prior reports of its function as a repressor (10, 14).
Surprisingly, given its usual function as a transcriptional activator
(14), Sp4 also inhibited transcription (Fig. 3).
To better analyze the transcriptional roles of the Sp1 family members,
we carried out further assays in Drosophila SL2 cells, which
do not express endogenous Sp1 (35, 36). pCAT 5-2, a minimal promoter
construct, was not able to drive transcription at all in these SL2
cells (Fig. 6). Addition of Sp1 (by cotransfection) potently activated
transcription, more than 50-fold. This demonstrates that Sp1 is a
critical factor in transcriptional initiation at this promoter.
Mutations in the core cis-elements resulted in substantial
reductions of the transcription (Fig. 8), indicating that these
elements are critical for transcriptional initiation. Considering the
particular location of Sp1 binding sites relative to the transcription
start site and other reports on the interaction between Sp1 and TBP or
TAF110 (11, 26, 40-43), Sp1 may play a critical role in the formation
or recruitment of the transcription initiation complex onto the core
promoter (Fig. 9). Synergistic activation
by Sp1 is often made possible by having two or more Sp1 sites located
next to each other in a promoter (10, 44). However, the two core
cis-elements in the ADH5/FDH promoter did not
show synergism (Fig. 8).
34 base pairs (bp) to +61 bp
directs high levels of transcription in several different cells,
consistent with the ubiquitous expression of the gene. Nearly the
entire minimal promoter can be bound by Sp1. We analyzed the
transcriptional regulation of ADH5/FDH by members of the
Sp1 multigene family. Two core cis-elements (
22 bp to +22
bp) had the highest affinity for Sp1. Mutagenesis revealed that these
cis-elements are critical for transcriptional activation.
The zinc-finger domains of Sp3 and Sp4 also bind selectively to the
core cis-elements. In Drosophila SL2 cells,
which lack endogenous Sp1, the minimal promoter cannot drive
transcription. Introduction of Sp1 activated transcription over
50-fold, suggesting that Sp1 is critical in the initiation of
transcription. Neither Sp3 nor Sp4 was able to activate transcription in those cells, and transcriptional activation by Sp1 was repressed by
Sp3 or Sp4. These data suggest that Sp3 and Sp4 can repress transcription by competing with Sp1 for binding to the core
cis-elements. The content of Sp1, Sp3, and Sp4 in different
cells may be critical factors regulating transcription of the
ADH5/FDH gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-alcohol
dehydrogenase (ADH, EC 1.1.1.1) that is also the
NAD+/glutathione-dependent formaldehyde
dehydrogenase (FDH, EC 1.2.1.1) (20). This gene is believed to be
the ancestral ADH gene (21). It is important in the oxidation of
various alcohols and of formaldehyde (in the presence of glutathione)
(see Refs. 19 and 22-24, and references therein). ADH5/FDH
is ubiquitously expressed, although to different levels in different
cells (22, 23). The ADH5/FDH promoter is very different from
those of other ADH genes, which are expressed in different
tissue-specific patterns. It is very rich in GC base pairs (73% up to
200 bp; 82% in the minimal promoter) and does not contain either a
TATA or CCAAT box (18). It thus has the characteristics of a
housekeeping gene (25, 26). The regulation of housekeeping genes is
poorly understood.
34 bp of ADH5/FDH
is a strong promoter in all cells tested (19). Promoter elements from
38 bp to +22 bp, flanking the transcriptional start site, are
footprinted by all nuclear extracts tested, and nearly the entire
minimal promoter can be bound by Sp1 (Fig. 1) (19). This is an unusual
configuration of GC boxes flanking the transcription start site.
Although this may lead one to expect a simple promoter, the
ADH5 promoter is surprisingly complex and the regulation of this ubiquitously expressed gene is quite complicated (19).
EXPERIMENTAL PROCEDURES
34 bp to +61 bp) in front of the CAT coding region in
pCAT Basic, and pCAT AX (
64 bp to +61 bp) are described elsewhere
(19). pCMVSp1 and pPacSp1 were generous gifts from Dr. Robert Tjian
(27). pCMVSp3, pCMVSp4, pPacSp3, and pPacSp4 were kindly provided by
Dr. Guntram Suske and Dr. Luigi Lania (10, 13, 14). pRatSp1 and BTEB
was kindly provided by Dr. Fujii-Kuriyama (7). pCMVAP2 was provided by Dr. Trevor Williams (28). The pPac vector used to construct pPac
luciferase contains the Drosophila actin promoter, and was kindly provided by Dr. Carl Thummel (University of Utah).
-32P]dCTP. The labeled DNA was
further digested with XbaI (a restriction site in the
polylinker), and the labeled restriction fragment was purified by
electrophoresis in a 4% non-denaturing polyacrylamide gel. For DNase I
footprinting assays from the opposite end of the fragment, the order of
digestion was reversed so that the XbaI site was labeled.
DNase I digestion and electrophoresis were as described previously
(19). Purified Sp1 transcription factor (0.1-2 fpu), probe (40,000 cpm), and poly(dI-dC) (1 µg) were used in each DNase I digestion reaction.
21 bp to: 3 bp): GATCACGCCCCGCCCCCCTCGCT; site
C-2 oligonucleotide (
38 bp to
22 bp): GAATTCATTGCAAGCCCCCCC; site
C-3 oligonucleotide (
29 bp to
15 bp): GAATTCCCCCCCCACGCCCC. Each
binding reaction was carried out in 20 µl, and contained 10 mM HEPES (pH 7.9), 60 mM KCl, 1 mM
dithiothreitol, 1 mM EDTA, 7% glycerol, and appropriate
nuclear extract or protein (0.1-2 footprint units of Sp1, 6-36 µg
of HeLa cell nuclear extract). Where indicated, excess unlabeled
competitor oligonucleotide (20-200-fold excess) and antibodies (1 µg
each) against Sp1, Sp3, Sp4, or Flag peptide was added to the binding mixture.
12/
13 bp to AA),
pCAT 5-2 M2 (CC at +13/+14 bp to AA), pCAT 5-2 M3 (CC at
12/
13
and at +13/+14 bp to AA), pCAT 5-2 M4 (CC at
12/
13 and
17/
18
bp to AA), and pCAT 5-2 M5 (CC at
12/
13,
17/
18, and +13/+14 bp
to AA) were prepared and used for transient expression assays in
Drosophila SL2 cells. Oligonucleotides used in mutagenesis
were as follows: M1: CCCCCCACGCCCCGAACCCCTCGCTAGGCG and
CGCCTAGCGAGGGGTTCGGGGCGTGGGGGG; M2:
GGCGCTCGCCACGAACATGCCTCCGTCGC and GCGACGGAGGCATGTTCGTGGCGAGCGCC; M3:
oligonucleotides used to prepare M1 and M2; M4:
AAGCCCCCCCCACGAACCGAACCCCTCGCT and AGCGAGGGGTTCGGTTCGTGGGGGGGGCTT; M5:
oligonucleotides used to prepare M2 and M4.
RESULTS
34 bp to +61 bp was able to promote
transcription in all tissue culture cell lines tested (19), and defined
it as an ADH5/FDH minimal promoter. The minimal promoter of
the human ADH5/FDH is GC-rich (82%) and contains several
consensus binding sequences for Sp1 and for AP2 protein (Fig.
1A; Refs. 18 and 19). Two Sp1
sites immediately flank the transcription start site. The region from
40 bp to +22 bp was footprinted by all nuclear extracts (19).
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Fig. 1.
The ADH5/FDH minimal
promoter. A, nucleotide sequence from 40 bp to +61 bp
(see Ref. 18). Potential binding sites by Sp1, AP2, NF-S, and ETS-1 are
marked by solid lines above or below the
sequence. B, summary of DNase I footprints. The
transcription start site is marked by an arrow. Footprints
are marked by filled ovals or round
rectangles on the depiction of the gene (top
solid line). Below is a tally of sites bound by
purified Sp1 or by nuclear extracts prepared from cells and tissues.
Differences in shading in the region from
90 bp to +61 bp indicate
the differences observed in the footprinting intensity.
4 bp to
27
bp), which immediately flank the transcription start site, had the
highest binding affinity for Sp1: they began to show footprints with
0.1 fpu (Fig. 2). Site A could be bound by Sp1 at 0.5 fpu (Fig.
2B). Site D (
39 bp to
64 bp) was upstream of the minimal
promoter, and was footprinted when Sp1 was above 0.25 fpu. Thus, even
though Sp1 could bind to the entire minimal promoter, the affinity of
the different cis-elements to Sp1 varied by as much as
5-10-fold. Considering their high affinity to Sp1 and locations
relative to the transcription start point, the core
cis-elements may be the most important
cis-elements regulating transcription.
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Fig. 2.
DNase I footprinting with increasing amounts
of Sp1. Sp1, ranging from 0.1 to 2 fpu, was added to the DNase I
footprinting reactions. A, antisense strand; B,
sense strand. G and G+A indicate Maxam and
Gilbert sequencing reactions of the DNA fragment. C, control
reaction without Sp1. The transcription start site is marked by an
arrow.
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[in a new window]
Fig. 3.
Effects of Sp1, Sp3, and Sp4 on transcription
in Alexander cells (of human liver origin). Six µg of pCAT 5-2
was cotransfected with 3 µg of various expression vectors encoding
mammalian transcription factors, all driven by the CMV promoter
(pCMVSp1, pCMVSp3, pCMVSp4, pCMVratSp1). pCAT Basic contained neither
promoter nor enhancer, and served as a negative control. Forty µg of
total cellular protein was used for each CAT assay. The reporter
activities are presented as relative CAT activity, the ratio of CAT
activity of each transfection compared with that of pCAT 5-2 without
cotransfected transcription factors, and are the average of six
independent assays. Bars represent standard
deviations.
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Fig. 4.
EMSA with purified Sp1 and HeLa nuclear
extract. One footprint unit of Sp1 or 6 to 36 µg of HeLa nuclear
extract was used. A, probe A (+40 bp to +60 bp).
B, probe B (+1 bp to +22 bp). C, probe C-1 ( 22
bp to
3 bp). D, probes C-2 (
38 bp to
22 bp) and C-3
(
29 bp to
15 bp). E, supershifts with antibody to Ku.
Sp1-Ab band, Sp1-probe-
Sp1 Ab complex; Sp1
band, Sp1-probe complex; Ku band, Ku-probe complex;
Ku-Ab band, Ku-probe-
Ku Ab complex; X band,
C-1 probe-unknown protein complex; X1, X2 bands,
probe-unknown protein complex enhanced by addition of Ku
antibody.
37 bp to
2 bp: footprints C + C' in
Fig. 2A, and C in Fig. 2B) into three
partially overlapping probes, C-1, C-2, and C-3. Sp1 (and the Ku
antigen) were the major proteins interacting with these probes (Fig. 4,
C and D). Probes C-1 (
22 bp to
3 bp) and C-3
(
29 bp to
15 bp) bound strongly to Sp1, while probe C-2 (
38 bp to
21 bp) bound more weakly. We suspect that the presence of two closely
positioned Sp1 binding elements enhanced the interaction between Sp1
and probe C-1.
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Fig. 5.
Binding of Sp3 and Sp4 to the core
cis-elements. A, EMSA with probe B (+1 bp
to +22 bp) and ZFD-Sp3 and ZFD-Sp4. B, EMSA with probe C-1
( 22 bp to
3 bp) and ZFD-Sp3 and ZFD-Sp4. In vitro
synthesized ZFDs of Sp3 or Sp4, tagged with Flag peptide, were used in
the binding reactions. In the control lanes (lane
1 of A and B), rabbit reticulocyte
extract with the in vitro synthesized product of the
lacZ gene was used instead. Where noted, 1 µg of
antibodies against Sp3, Sp4, or Flag peptide was added
(lanes 3, 4, 6, and
7). C, control (lane 1);
ZFD-Ab, supershifted probe-ZFD-antibody complex;
NS, nonspecific probe-protein complex; ZFD-Sp3,
ZFD-Sp3-probe complex; ZFD-Sp4, ZFD-Sp4-probe complex.
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Fig. 6.
Effects of Sp1, Sp3, and Sp4 on transcription
in Drosophila SL2 cells. Twelve µg of pCAT 5-2, 3 µg of pPac luciferase, and varying amounts of pPacSp1, pPacSp3, and
pPacSp4 (1-9 µg) were cotransfected into Drosophila SL2
cells. Cell extracts representing 200,000 RLU of luciferase (from the
internal control pPac luciferase) were used for CAT activity assays.
Data are presented as relative CAT activity compared with the control
pCAT 5-2 without cotransfected expression vector, and are the average
of three independent assays. Bars represent standard
deviations.
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Fig. 7.
Transcription activation by Sp1 can be
repressed by Sp3 or Sp4 in Drosophila SL2 cells. Cells
were transfected with 12 µg of pCAT 5-2, 1 µg of pPac luciferase,
3 µg of pPacSp1, and varying amounts of pPacSp3 and pPacSp4 (3-9
µg). Cell extracts representing 67,000 RLU of luciferase activity
were used for CAT assays. Data are presented as relative CAT activity
compared with the control (12 µg of pCAT 5-2 + 1 µg of pPacSp1),
and are the average of five independent assays. Bars
represent standard deviations.
12/
13,
GGGGCGGG
GGTTCGGG; pCAT 5-2 M2 at +13/+14,
TGGGCGTGG
TGTTCGTGG; pCAT 5-2 M3 with both
previous mutations, at
12/
13 and +13/+14; pCAT 5-2 M4 at
17/
18, and
12/
13; pCAT 5-2 M5 at
17/
18,
13/
12, and
+13/+14) (Fig. 8A). pCAT 5-2
M1 and pCAT 5-2 M2 constructs showed reduction in transcription by
53% and 78%, respectively (Fig. 8B). Site B (+9 to +16 bp)
seems to play a more important role in transcription than site C-1
(
17 to
10 bp). In the pCAT 5-2 M3 and M5 constructs, a significant
reduction (greater than 70%) in transcription resulted by having two
to three mutations introduced at the core Sp1 binding sites. Comparing
the activity of M4 with M1, there is little effect of the second
mutation in C-3, given a mutation in C-1. This is also seen comparing
M3 and M5. Thus, in the context of a promoter with a mutation in C-1, site C-3 has little or no effect.
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Fig. 8.
The two Sp1 binding core
cis-elements are the critical center in the transcriptional
activation by Sp1. A, five mutant versions of pCAT 5-2
and the sites of mutations introduced by site-directed mutagenesis.
B, CAT assay results of pCAT 5-2 wt and mutant versions.
Twelve µg each of pCAT 5-2 w/t, pCAT 5-2 M1, pCAT 5-2 M2, pCAT
5-2 M3, pCAT 5-2 M4, and pCAT 5-2 M5 were separately cotransfected
with pPac luciferase (1 µg) and pPacSp1 (1 µg). Cell extracts
representing 67,000 RLU were used for the CAT assays. Data are
presented as relative CAT activity (% conversion) compared with the
control (12 µg of pCAT 5-2 wt + 1 µg pPacSp1) and are the average
of four independent assays. Bars represent standard
deviations.
DISCUSSION
22 bp to +20 bp. Sp1 is a
strong transcriptional activator of this promoter, acting through these sites. Sp3 and Sp4 do not significantly activate this promoter, and
compete with Sp1 for the key sites, leading to a reduction in
transcription. These data provide the first demonstration that Sp4 can
act as a repressor.
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Fig. 9.
A hypothetical model of the transcriptional
regulation by the Sp1 multigene family at the ADH5/FDH
minimal promoter. B and C-1 are core cis-elements
immediately flanking transcription start point (Tsp, marked
by an arrow). TBP, TATA-binding protein. TAF110,
TBP-associated factor 110. Double arrows indicate
the interaction between transcription factors (Sp1, Sp3, Sp4) and TBP
or TAF110. Arrows under transcription indicate
the degree of transcriptional activation.
We investigated whether Sp3 or Sp4 could activate transcription in the absence of Sp1, using SL2 cells. There was little or no transcription, even in the presence of the highest amount of cotransfected expression vectors (Fig. 6). This suggests that Sp3 and Sp4 lack the ability to interact with TBP or TAF110 in this promoter context. We also tested whether transcriptional activation by Sp1 can be affected by Sp3 or Sp4. Transcriptional activation by Sp1 was repressed by Sp3 or Sp4 (Fig. 7). The transcriptional repression by Sp3 or Sp4 is surprisingly similar to the results obtained with the mutant constructs where one of the two core cis-elements is destroyed by site-directed mutagenesis. Because Sp3 or Sp4 cannot themselves activate transcription on this particular promoter, the occupation of one of the two sites by Sp3 or Sp4 and the other by Sp1 is like the situation where only one site is occupied by Sp1 due to the mutation introduced. By having one site occupied by Sp3 (or Sp4), or by having one Sp1 binding site destroyed, a significant drop in transcription can result. The drop might be more dramatic if higher levels of Sp3 or Sp4 were induced by the expression vectors; others have reported that the levels achieved are not high (10). In contrast, even at 20 ng of added DNA, pPac-Sp1 expresses Sp1 sufficiently to activate transcription strongly (10).
These data suggest that Sp3 and Sp4 can act as repressors by competing with Sp1 for binding to the core cis-elements and preventing the formation of the transcription initiation complex. Sp4 had not previously been shown to repress transcription. Based on these findings, we propose a hypothetical model on the transcriptional regulation at the ADH5/FDH minimal promoter (Fig. 9). The two core cis-elements are the critical center for transcriptional initiation and activation by Sp1. These elements can be preferentially bound by Sp1, Sp3, and Sp4. If only Sp1 occupies these elements, it can interact with TBP or TAF110 and can promote strong transcription. By having two core binding sites for Sp1, transcription activation by Sp1 is ensured and strong transcription is made possible. If the core sites are occupied by Sp3 or Sp4 alone, the interaction between the transcription factor with one or more components of general transcription machinery is either absent or weak, which can result in little or no transcriptional activation. If one site is occupied by Sp1 and the other by Sp3 or Sp4, the transcription activation will be relatively low compared with the situation where two sites are occupied by Sp1 only.
Therefore, the cellular content of Sp1, Sp3, and Sp4 and their
interactions on the minimal promoter may be critical factors influencing transcription of the ADH5/FDH gene in various
human tissues (22, 23). This model may also be applicable to many housekeeping genes with GC boxes located either in the proximal promoter or around the transcription start site, as is the case in the
ADH5/FDH gene.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Robert Tjian for the generous gift of pCMVSp1 and pPacSp1. We thank Dr. Suske and Dr. Lania for pCMVSp3, pCMVSp4, pPacSp3, and pPacSp4. Our thanks also go to Dr. Fujii-Kuriyama for rat Sp1. Antibody to Ku antigen was kindly provided by Dr. Westly Reeves. We appreciate the vigorous discussion and careful reading of the manuscript by the members of the Department of Biochemistry and Molecular Biology at the Yonsei University School of Medicine.
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FOOTNOTES |
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* This work was supported mainly by a 1996 Dr. Kim Myung Sun Memorial Foundation grant (to M.-W. H.) and Grant AA06460 from the National Institute on Alcohol Abuse and Alcoholism (to H. J. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 82-2-361-5187; Fax: 82-2-312-5041; E-mail: mwhur{at}shinbiro.co.kr.
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ABBREVIATIONS |
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The abbreviations used are: ADH5/FDH, alcohol dehydrogenase 5/formaldehyde dehydrogenase; Ab, antibody; bp, base pair(s); CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; fpu, footprinting unit(s); RLU, relative light unit(s); SL2, Drosophila Schneider line 2 cells; ZFD, zinc-finger DNA binding domain..
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REFERENCES |
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