From the
Previous analyses of the uteroglobin promoter revealed seven
distinct regions, which contribute to its overall activity in
epithelial cells from endometrium and lung. Most significantly, a
mutation of the promoter sequence around 65 base pairs upstream of the
transcriptional start site severely impairs promoter activity. The
transcription factor acting through this sequence has not been
identified yet. Here, we report that members of the Sp transcription
factor family specifically recognize this non-classical GC box, in
addition to another functional motif located 230 base pairs upstream of
the transcriptional start site. We have characterized in detail the
interaction of recombinant Sp3 with both motifs by DNase I footprinting
and methylation protection using the wild-type uteroglobin promoter and
various linker scanning mutants as templates. Electrophoretic mobility
shift analyses show that Sp1 and Sp3 both bind with similar affinity to
these elements. We demonstrate that the DNA-binding proteins in the
endometrial cell line Ishikawa which recognize these motifs are also
Sp1 and Sp3. Gene transfer experiments into Drosophila Schneider cells that do not contain endogenous Sp factors revealed
that both DNA motifs respond to transiently expressed Sp1 and Sp3. Our
results show thus that the level of transcription from the uteroglobin
promoter is controlled by members of the Sp transcription factor family
through unusual Sp binding sites.
The level and pattern of expression of a gene is determined
mainly by the combinatorial action of transcription factors binding to
distinct promoter and enhancer elements. In the last few years, we have
started to define the DNA elements and transcription factors
responsible for the expression of the rabbit uteroglobin gene in
several ontogenetically unrelated epithelial tissues. DNase I
protection experiments
(1) , promoter deletion, and linker
scanning analyses
(2) revealed that at least seven distinct
promoter regions (I-VII; see Fig. 1) contribute to the
activity of the promoter in epithelial cells. Region VII is defined by
the presence of an estrogen-responsive element
(3) . Region VI
located 230 bp(
In the present study, we
have analyzed in greater detail the role of the Sp transcription factor
family for the activity of the uteroglobin promoter. DNase I and
dimethyl sulfate protection experiments as well as electrophoretic
mobility shift analyses using the wild-type uteroglobin promoter and
appropriate linker scanning mutants as templates revealed that, in
addition to the proximal CACCC box of region VI, the transcriptionally
essential region II of the uteroglobin promoter is specifically
recognized by Sp3 and Sp1. We show that the proteins in nuclear
extracts of the endometrial cell line Ishikawa which bind this motif
are also Sp1 and Sp3. In addition, we demonstrate that the element is
responsive to these two transcription factors in gene transfer
experiments. The strong correlation between DNA binding data in
vitro and our functional results suggests that (a) member(s) of
the Sp multigene family control the activity of the uteroglobin
promoter through binding to two non-GC box Sp binding sites.
Bacterial extracts containing
recombinant Sp3
(14) were preincubated for 10 min at 0 °C
(total volume of 20 µl) in a buffer containing 12.5 mM
HEPES, pH 7.5, 6.25 mM MgCl
Schneider cells
(26) were
maintained in Schneider medium supplemented with 10% fetal calf serum
at 25 °C. One day prior to transfection cells were plated onto 6-cm
plastic dishes at a density of 4.5
To
show also how Sp factors interact with the CACCC box, methylation
protection experiments were performed with promoter fragments
containing element VI. Seven guanine residues of the lower strand
(positions -223 to -226, -228, -230, and
-231) are protected completely from methylation by Sp3
(Fig. 3C, compare lane19 with
lane20). The guanine residue at position -232
of the upper strand is only partially protected. No guanine residue was
hypermethylated.
In the linker scanning mutant
LS-222/-229, the four protected guanine residues at
positions -223, -224, -226, and -228 are
replaced by non-guanine bases, which explains why Sp3 could not confer
DNase I protection to this promoter variant (see Fig. 2).
Moreover, the exchange of important contact sites for Sp factors in the
linker scanning mutants LS-64/-72 and
LS-222/-229 could also explain why promoter activity is
reduced in these two promoter variants.
Close inspection of CACCC boxes of various promoters and enhancers
revealed that six of the seven protected guanine residues are present
in the CACCC boxes of the tryptophan oxygenase gene, the tyrosine
aminotransferase gene, and the
Immediately upstream of the functional uteroglobin CACCC
box lies an estrogen-responsive element
(3) . Our gene transfer
experiments demonstrated that the replacement of the CACCC box does not
influence the inducibility of the uteroglobin promoter by estrogens,
indicating that no synergism occurs between the estrogen-responsive
element and the CACCC box. Thus, synergism of a CACCC box and an
adjacent hormone-responsive element appears to be not a general feature
of this combination of promoter elements.
Our
methylation protection experiments with recombinant Sp3 and Sp1 (data
not shown) revealed protection of guanines on both strands of element
II. This finding is consistent with the observation that Sp1 does not
bind efficiently to single-stranded DNA
(32) . Contacts with
guanine residues on both strands have been described also for the Sp1
binding sites present in the HTLV-III retrovirus
(32) and in a
parvovirus promoter
(33) . These binding sites also differ from
the classical GC boxes found for instance in the SV40 promoter
region
(34) . With respect to the flexibility for deviations from
the consensus Sp binding site
(35) , the previous work of Thiesen
and Bach
(36) is illuminating. Using a target detection assay,
they determined putative DNA binding sites for Sp1 and rescued 11
strong binding sites. One of the selected binding sites (S16:
GGGGCAGGGC) differs only in one position compared with the core
sequence of element II (AGGGCAGGGC). Thus, our results further
demonstrate the high variability of the recognition sequence for the
members of the Sp transcription factor family. The variability of Sp
binding sites may explain why promoters that do not contain GC boxes
are stimulated by Sp1
(37) . Indeed, as far as we know, there
exists no strong promoter whose activity is independent of Sp1 in
transfection experiments.
The control of
uteroglobin gene expression is even more complex. The level of
expression in endometrium and lung is regulated by various steroid
hormones
(41) . The enhancer region, which is responsible for the
induction of transcription of the gene in the endometrium by
progesterone, is located 2.4 kilobases upstream of the transcriptional
start site
(42) . So far, we do not know how this enhancer region
communicates with the promoter and whether the described Sp binding
sites are directly involved in this interaction. However, Sp1 seems to
be active in mammalian cells only in response to a remote enhancer
(43). Thus, it is possible that element II and/or VI of the uteroglobin
promoter are directly involved in promoter/enhancer interactions.
Further investigations will be necessary to unravel the functional
interplay between the uteroglobin enhancer and promoter and to
determine the specific roles of Sp1 and Sp3 in this interaction.
The values represent mean values of two independent transfection
experiments and are expressed relative to the wild type, which have
been given the arbitrary value of 100. The values were normalized for
We thank W. Lorenz for technical assistance. Dr. R.
Tjian kindly provided us with Sp1 cDNA clones. We gratefully
acknowledge Drs. A. Baniahmad, M. Kalff-Suske, and J. Klug for
critically reading the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
upstream of the transcription
start site contains two so-called CACCC boxes found in several other
promoters and enhancers, including the tyrosine aminotransferase gene
(4, 5), the tryptophan oxygenase gene
(6, 7) , the
-globin gene
(8, 9) , and the SV40
enhancer
(10, 11, 12) . Region V spans at least
60 nucleotides between -208 and -148 and probably contains
several DNA elements that have not been characterized further. Regions
III and IV contain degenerated octamer motifs, which are bound by Oct I
in vitro. The strongest phenotypes are displayed by mutations
affecting regions I and II, suggesting that these two regions determine
mainly the activity of the uteroglobin promoter. A linker scanning
mutation in region II (LS-64/-72; see Fig. 1) reduces
the promoter activity 20-fold (Ref. 2, ). However, the
transcription factor acting through this sequence has not been
identified yet. Region I contains a noncanonical TATA box motif (TACA
box), which is bound specifically by two factors, the TATA core factor
TCF and the TATA palindrome factor TPF
(13) . Both are different
from the TATA box-binding protein TBP.
Figure 1:
The rabbit uteroglobin promoter
structure and nucleotide sequences of elements II and VI in the
wild-type promoter and the corresponding linker scanning promoter
mutants LS-64/-72, LS-68/-75,
LS-232/-240, and LS-222/-229. Seven regions
(I-VII) mapped by DNase I footprinting (1), deletion,
and linker scanning analyses (2) contribute to the overall activity of
the promoter. The two CACCC sequences present in region VI are depicted
by solidlines. TACA, TACA box (noncanonical
TATA element; Ref. 13); OCT, octamer factor binding sites;
ERE, estrogen-responsive element.
Using an oligonucleotide
containing region VI of the uteroglobin promoter, we have cloned
previously two transcription factors (Sp3 and Sp4), which specifically
bind to this element
(14) . Both proteins are paralogs of the
human transcription factor Sp1. Sp1, Sp3, and Sp4 have similar
structural features. Most significantly, the DNA binding domains of all
three proteins are highly conserved and recognize the GC box and a
CACCC motif present in region VI of the uteroglobin promoter (14, 15).
Contrary to these factors, Sp2, another Sp1-related protein, seems to
have different DNA binding specificities
(16) . Functional
analysis of Sp3 and Sp4 in direct comparison with Sp1 revealed that
Sp4, like Sp1, is a transcriptional activator, whereas Sp3 represses
Sp1-mediated transcription
(15) .
Plasmid Constructions
Vectors for Sp1 and Sp3
expression in Drosophila Schneider cells (pPacSp1 and pPacSp3)
have been described previously
(15, 17) . The
-galactosidase expression plasmid p97b was constructed by P.
DiNocera and generously supplied by L. Lania. The plasmids
(EII)
-CAT, (EII*)
-CAT, and
(EVI)
-CAT are derivatives of BCAT-2
(17) and were
generated as follows. First, we constructed the reporter plasmid
BCAT-0, which lacks the two Sp1 binding sites of the HTLV promoter but
still contains the E1B TATA box fused to the CAT gene. For this, BCAT-2
was cut with HindIII and XbaI, filled in with Klenow
polymerase, and religated. The oligonucleotides OEII, OEII*, and OEVI
(Fig. 4A) were concatemerized via their HindIII
and SalI ends and cut with HindIII. The resulting
dimeric oligonucleotides were then cloned into the XhoI site
of BCAT-0, resulting in (EII)
-CAT, (EII*)
-CAT,
and (EVI)
-CAT. The construction of all other plasmids,
including the wild-type uteroglobin promoter construct
pUG(-395)-CATSV as well as the appropriate linker scanning
mutants fused to the CAT gene and the bacterial expression plasmids for
Sp1 and Sp3, has been described
(2, 14, 15) .
Figure 4:
Electrophoretic mobility shift analysis
with uteroglobin promoter elements II and recombinant Sp1 and Sp3.
A, oligonucleotides used for the EMSA of this and the
following figure. Sequences present in the wild-type uteroglobin
promoter are written in uppercase letters. The Sp binding
sites in the oligonucleotides OEII, OEVI, and OGC are
underlined. B, the oligonucleotide OEII shown in
panel A was end-labeled and incubated with bacterially
expressed Sp1 (lane1), Sp3 (lane2), or a mixture of both proteins (Sp1/Sp3; lanes
3-15). Competitions were performed with x-fold
molar excess of cold oligonucleotides OGC, OEVI, OEII, and OEII* as
indicated.
Bacterial Extracts, Nuclear Extracts, and Electrophoretic
Mobility Shift Analysis (EMSA)
Bacterial extracts containing
recombinant Sp1 and Sp3 have been prepared according to Kadonaga et
al..
(18) . Nuclear extracts from Ishikawa cells were
prepared from one 10-cm plate according to Andrews and
Faller
(19) . Gel retardation assays were essentially performed
as described
(20, 21) with oligonucleotides containing
the EII, EII*, and EVI motifs as binding sites, respectively.
Annealing, labeling, and purification of oligonucleotides has been
performed as described by Klug et al.(13) . The
sequences of the oligonucleotides are shown in Fig. 4A.
For supershift assays, 1 µl of the appropriate antiserum was added
to the binding reaction 10 min prior to the loading of the gel.
DNase I Protection Experiments
Fragments used for
DNase I protection experiments were isolated from pUG(-395)CATSV
and the appropriate linker scanning derivatives
(2) . The
plasmids were cut with either XbaI (for the footprint on
element II) or XhoI (for the footprint on element VI) and
end-labeled. The subsequent restriction digest with XhoI and
XbaI, respectively, released a 425-bp DNA fragment containing
the uteroglobin promoter sequence from -395 to +14, which
was purified further by polyacrylamide gel electrophoresis. The probe
(30,000 cpm) was mixed with 10 µg of bacterial extract in a buffer
containing 12.5 mM HEPES, pH 7.5, 6.25 mM
MgCl, 9% glycerol, 5 µM ZnSO
, 50
mM KCl, and 40 µg/ml poly(dI-dC), and incubated for 30 min
at 4 °C. Then, 2.5 µl of DNase I (5 ng or 10 ng) in 50
mM MgCl
and 10 mM CaCl
was
added and incubated for 30 s at 20 °C. The reaction was stopped by
addition of 100 µl 100 mM Tris-HCl, pH 7.6, 20 mM
EDTA, 0.5% SDS, 100 mM NaCl, 100 µg/ml proteinase K, and
20 µg of glycogen. After phenol extraction and ethanol
precipitation, DNA fragments were resolved on 6.5% denaturing
polyacrylamide gels. Sequencing ladders were prepared according to
standard procedures
(22) .
Dimethyl Sulfate Protection Experiments
The
following templates were used for methylation protection experiments.
Element II, upper and lower strand, was a 150-bp
XhoI-SspI fragment obtained from pUG(-395)CATSV
and LS-68/-75, respectively
(2) . Element
VI, upper strand, was a 250-bp XbaI-BglII fragment
obtained from LS-142/-146
(2) . Element VI,
lower strand, was a 260-bp XbaI-SspI fragment
obtained from pUG(-395)CATSV.
, 9% glycerol, 5
µM ZnSO
, 50 mM KCl, 50 ng/µl
poly(dI-dC), and 50 ng/µl bovine serum albumin. The appropriate
end-labeled fragments (200,000 cpm) were added and incubated for 10
min. The methylation reaction was started by adding 2 µl of a 2%
dimethyl sulfate solution. After incubation for 3 min at 20 °C, the
reaction was stopped by adding 2 µl of 60 mM
-mercaptoethanol. Free and bound DNA were separated on 4%
polyacrylamide gels and blotted onto DE81 ion exchange paper. After
elution (2 h at 65 °C in 200 µl 10 mM Tris/Cl, pH 8.0,
1 mM EDTA, 1.5 M NaCl), the DNA fragments were
ethanol-precipitated and treated with piperidine essentially as
described
(23) . Finally, the reactions were analyzed on 7%
denaturing sequencing gels along with sequencing reactions (G+A
and C+T) obtained from the same DNA fragments.
Cell Culture and Transfections
Ishikawa cells were
grown as monolayers in minimum essential medium supplemented with 10%
fetal calf serum (routinely stripped from hormones by charcoal
treatment), L-glutamate, and antibiotics. For the hormone
response experiments, the cells were transferred to Dulbecco's
minimal essential medium without phenol red (Life Technologies, Inc.)
and the DEAE-dextran method was used for transfection
(24) .
Every plate (9 cm) received 8 µg of reporter plasmid, 1 µg of
RSVgal, and 200 ng of human estrogen receptor expression plasmid
HEGO
(25) . Hormonal response was tested 20 h post-transfection
by addition of the synthetic estrogen diethylstilbestrol
(10
M).
10
cells/plate.
Cells were transfected by the calcium phosphate method described by
DiNocera and Dawid
(27) . Every plate received 14 µg of DNA
including 4 µg of the
-galactosidase expression plasmid p97b
as internal reference. Variable amounts of expression plasmids were
compensated with the plasmid pPac. 24 h after addition of DNA, the
medium was changed, and 24 h later the cells were washed twice with
phosphate-buffered saline and harvested.
CAT and
For CAT assays,
cells were suspended in 250 mM Tris/Cl, pH 7.8, and lysed by
three rounds of freezing and thawing. CAT assays were carried out
according to Gorman et al.,
(28) . Protein
concentrations in the CAT assay and reaction times were adjusted to
bring the extent of CAT conversion into a range that is linear with the
CAT enzyme concentration. CAT conversion was assayed by thin layer
chromatography and quantitation of the acetylated and non-acetylated
forms of [-Galactosidase Assays
C]chloramphenicol performed with an
automated Imaging Scanner (United Technologies Packard). The ratio of
acetylated to total chloramphenicol is displayed as percentage of
conversion. The
-galactosidase assays were performed according to
Hall et al..
(29) . The values were used to normalize
the CAT conversion data for plate to plate variations in transfection
efficiency.
The Uteroglobin Promoter Does Contain Two Binding Sites
for Members of the Sp Transcription Factor Family
Region VI of
the uteroglobin promoter contains two adjacent CACCC motifs
(Fig. 1). Previously, we have shown that an oligonucleotide
containing the proximal CACCC box is bound specifically by Sp1, Sp3,
and Sp4
(14, 15) . To see whether the distal CACCC motif
is also recognized by members of the Sp family, we performed DNase I
protection experiments with recombinant Sp3 using the wild-type
uteroglobin promoter (WT) and the appropriate linker scanning
mutants LS-232/-240 and LS-222/-229
(Fig. 1) as templates. In LS-232/-240 and
LS-222/-229 either the distal or the proximal CACCC
sequence is replaced by a BglII linker sequence
(Fig. 1). A footprint over element VI was obtained with the
wild-type uteroglobin promoter (WT in Fig. 2A;
compare lane8 with lanes9 and
10) and the linker scanning mutant LS-232/-240
containing the mutated distal CACCC motif (Fig. 2A,
compare lane5 with lanes6 and
7). With the promoter variant in which the proximal CACCC
motif is mutated (LS-222/-229), no protection from DNase I
cleavage is visible (Fig. 2A, compare lane11 with lanes12 and 13).
These results show that region VI of the uteroglobin promoter contains
only a single Sp binding site and that only the proximal CACCC box is
part of the Sp recognition sequence.
Figure 2:
DNase I protection of the uteroglobin
promoter by recombinant Sp3. Reactions contained 10 µg of bacterial
protein with (+) or without (-) Sp3 and no (-), 5, or
10 ng DNase I, respectively, as indicated. LanesG/A contain Maxam-Gilbert sequencing ladders prepared from the
wild-type uteroglobin promoter fragment. A, DNase I protection
experiments using the wild-type uteroglobin promoter (lanes2 and 8-10) and the linker scanning
promoter mutants LS-232/-240 (lanes1 and
5-7) and LS-222/-229 (lanes3 and 11-13) labeled approximately 400 bp
upstream of the transcriptional start site. The sequences on the
right show the Sp3 binding region of the wild-type promoter
(WT) and the corresponding linker scanning mutants
LS-232/-240 and LS-222/-229. The mutations
present in the linker scanning mutants are indicated by lowercase
letters. The proximal CACCC motif region is boxed. The
bar (topright) indicates the second
protected region around -65. B, DNase I protection
experiment with the wild-type uteroglobin promoter (WT,
lanes1 and 4-6) and the linker
scanning promoter mutant LS-64/-72 (lanes2 and 7-9) labeled close to the 3`-end of the
promoter. The sequences on the right show the Sp3 binding
region of the wild-type promoter (WT) and the corresponding
sequence of the linker scanning mutant LS-64/-72. The
mutations in the linker scanning mutant are indicated by lowercase
letters.
In the upper part of the gel
shown in Fig. 2A another protection from DNase I
cleavage (around -65) is visible. This region was protected also
when footprinting experiments were performed with nuclear extracts from
Ishikawa and HeLa cells
(1) . To further delineate the sequence
protected by Sp3, we labeled the uteroglobin promoter close to the
transcriptional start point and performed footprinting experiments with
the wild-type promoter and a linker scanning mutant
(LS-64/-72, see Fig. 1), which reduced the promoter
activity almost to background levels (at least 20-fold) in gene
transfer experiments (2). A footprint over element II was obtained with
the wild-type uteroglobin promoter but not with the
LS-64/-72 mutant (Fig. 2B, compare lane4 with lanes5 and 6 and
lane7 with lanes8 and
9). This result suggests that the absolute requirement of
region II for the activity of the uteroglobin promoter might be due to
an unusual Sp binding site.
Guanine Contacts of Sp3 with the Non-classical GC Box of
Element II and the CACCC Box Binding Site
In order to obtain a
more detailed picture of the interaction between Sp3 and the unusual
binding site of element II, we performed dimethyl sulfate methylation
protection experiments (Fig. 3). Sp3-DNA complexes were allowed
to form at 4 °C and subjected subsequently to partial methylation
by dimethyl sulfate. Bound and free DNA were separated through native
polyacrylamide gels and analyzed on denaturing sequencing gels.
Figure 3:
Methylation protection pattern of the
uteroglobin promoter obtained with recombinant Sp3. Appropriate
end-labeled uteroglobin promoter fragments were incubated with
recombinant Sp3. After methylation, bound DNA (lanesB) was separated from free DNA (lanesF) by native polyacrylamide electrophoresis, eluted, and
applied to denaturing polyacrylamide gels. LanesC contain non-methylated free DNA, lanes A+G and
C+T Maxam-Gilbert sequencing ladders. Specific guanine
residues protected from dimethyl sulfate (DMS) methylation by
Sp3 are depicted by large (completely protected residues) and
small (partially protected residues) triangles. A and B, DMS methylation protection in region II (lower
(A) and upper (B) strand) obtained with the wild-type
promoter (lanes 1-4 and 9-12) and the
linker scanning mutant LS-68/-75 (lanes5-8 and 13-17). C and
D, DMS methylation protection in region VI (lanes
18-25; lower (C) and upper (D) strand).
E, summary of the protection pattern of guanine residues in
elements II and VI.
The
results obtained with both strands of element II are shown in
Fig. 3
(A and B). In the presence of recombinant
Sp3, four guanine residues (at positions -58, -60,
-61, and -62) of the lower strand (compare lanes3 and 4 in Fig. 3A) and two
residues of the upper strand (positions -63 and -68;
compare lanes9 and 10 in
Fig. 3B) of element II showed specific and almost
complete protection from methylation. In addition, three guanine
residues of the lower (positions -65 to -67) and one of the
upper (position -69) strand are partially protected. In the
linker scanning mutant LS-68/-75, guanine residues at
positions -72, -73 (lower strand), and -76 (upper
strand) are substituted by adenine, thymidine, or cytidine residues. A
promoter fragment containing these mutations showed essentially the
same protection pattern and the same extent of protection from
methylation as the wild-type promoter sequence (Fig. 3, A and B, compare lane4 with lane8 and lane10 with lane16), indicating that no interactions occur between Sp3
and the guanine-rich sequence immediately upstream of the protected
area. A promoter fragment that contains the linker scanning mutation of
LS-64/-72 (Fig. 1) did not bind to Sp3; therefore, no
protection from methylation could be observed (data not shown). In
LS-64/-72, the strong protected G residue at position
-68 of the upper strand and the two partially protected G
residues at positions -65 and -67 of the lower strand are
replaced by thymidine or adenine residues, respectively (see
Fig. 1
), indicating that at least one of these three guanine
residues is required for the interaction of Sp3 with element II.
Electrophoretic Mobility Shift Analyses (EMSA) with
Recombinant Sp1 and Sp3
To examine further the interaction
between Sp factors and element II of the uteroglobin promoter and to
determine the relative binding affinity of this site, we performed EMSA
experiments with bacterially expressed Sp1 and Sp3 and oligonucleotides
containing the wild-type sequence of element II (OEII oligonucleotide)
and the sequence of the corresponding linker scanning mutant
LS-64/-72 (OEII* oligonucleotide). As reference probes, we
used oligonucleotides containing a classical GC motif (OGC
oligonucleotide) or the proximal CACCC box of element VI (OEVI
oligonucleotide) (Fig. 4A). The element II-containing
oligonucleotide was labeled and incubated with recombinant Sp1 and Sp3
fragments. A predominant complex was generated with the 263 C-terminal
amino acids encoding Sp3 fragment, whereas full-length Sp1
(14) produced two specific complexes (Fig. 4B).
The two Sp1-containing complexes have been observed previously. Both
are very likely degradation products of the complete Sp1
protein
(14) . All three complexes were competed equally with
increasing amounts of unlabeled oligonucleotides containing the EII,
EVI, and GC motif showing that Sp1 and Sp3 do have the same binding
specificity for all three motifs. In contrast, the EII*
oligonucleotide, which contains the corresponding sequence of the
linker scanning mutant LS-64/-72, did not at all compete
the labeled oligonucleotide (Fig. 4B, compare lane3 with lane15). Obviously, the GC
box-containing oligonucleotide (OGC) competed significantly better than
the EVI and the EII oligonucleotides. Quantitative evaluations of the
band shift data show also that the affinity of the element
II-containing oligonucleotide for Sp1 and Sp3 is approximately 2-fold
lower compared with the CACCC box-containing oligonucleotide.
Sp1 and Sp3 Are the Nuclear Proteins Binding to Element
II of the Uteroglobin Promoter
Next, we asked whether the
nuclear proteins binding to element II are indeed Sp1 and Sp3 or
whether other additional transcription factors are able to recognize
this element. To address this question, we performed EMSA experiments
with crude nuclear extracts from Ishikawa cells. As labeled DNA probe,
we used the EII oligonucleotide. In high resolution gels, two slow
migrating and two fast migrating complexes were observed
(Fig. 5A). Essentially, the same migration pattern is
observed with the element VI-containing oligonucleotide
(Fig. 5B). All four bands were specifically competed
with oligonucleotides containing elements II or VI, but not with the
EII* oligonucleotide (Fig. 5, A and B). In the
presence of a Sp1 antiserum, the slowest migrating complex disappeared.
When the anti-Sp3 serum was present in the binding reaction, the three
other complexes were shifted but the slowest migration complex was
unaltered. If antisera against Sp1 and Sp3 were present in the binding
reaction, all four bands disappeared. This experiment demonstrates that
other factors distinct from Sp1 and Sp3, which may bind to element II
(and VI), are not present in the nuclear extract from Ishikawa cells,
suggesting that Sp1 and/or Sp3 are responsible for the transcriptional
activity of both promoter elements.
Figure 5:
Nuclear proteins binding to uteroglobin
promoter elements II and VI are Sp1 and Sp3. A, sequences of
the oligonucleotides OEII, OEII*, and OEVI are shown in Fig.
4A. The oligonucleotide OEII was labeled and incubated with
nuclear extracts from Ishikawa cells. One microliter of protein
A-Sepharose-purified sera against Sp1 (Sp1), Sp3
(
Sp3), or a mixture of both (
Sp1/3) were
included in the binding reactions as indicated at the top.
Specific complexes for Sp1 and Sp3 are indicated on the left.
In the competition experiments, an x-fold molar excess of
oligonucleotides OEII (lanes 5-8), OEVI (lanes
9-12), and OEII* (lane13) was included in
the binding reactions as indicated. B, the oligonucleotides
OEII* (lane1), OEII (lanes 2-4), and
OEVI (lanes 5-16) were end-labeled and used for EMSA
experiments with nuclear extracts from Ishikawa cells. Competitions
with cold oligonucleotides OEII (lanes 3 and
11-15) and OEVI (lanes 6-10) were
performed with 10-50-fold molar excess, respectively. In the
competition experiment with OEII* (lane16), a
100-fold molar excess of the oligonucleotide was used. Complexes
specific for Sp1 and Sp3 are indicated on the
left.
Element II of the Uteroglobin Promoter Mediates
Activation by Sp1 and Repression by Sp3 in SL2 Cells
To further
substantiate the conclusion that members of the Sp family control the
level of transcription from the uteroglobin promoter, we asked whether
Sp1 and Sp3 are able to act as transcriptional regulators through
binding to element II. We performed gene transfer experiments into the
Drosophilamelanogaster Schneider cell line (SL2
cells) that lacks endogenous Sp factors
(30, 15) . The
CAT reporter constructs designed for these experiments contain the
oligonucleotides OEII and OEII* (Fig. 4A) as dimers
upstream of the E1B TATA box (Fig. 6A). For direct
comparison, we also used (EVI)-CAT and BCAT-2 as reporter
plasmids. In (EVI)
-CAT two copies of the proximal CACCC
motif-containing oligonucleotide (OEVI in Fig. 4A) is
fused to the E1B TATA box. BCAT-2 contains two Sp1 binding sites from
the HTLV-III promoter in a tandem array fused to the E1B TATA box. This
construct has been used previously to characterize transcriptional
properties of Sp1 and Sp3
(15, 17) .
Figure 6:
Functional analysis of promoter elements
II and VI in SL2 cells. A, schematic representation of the
reporter plasmids (EII)-CAT, (EII*)
-CAT,
(EVI)
-CAT, and BCAT-2. B, 8 µg of the reporter
plasmids (EII)
-CAT, (EVI)
-CAT,
(EII*)
-CAT, and BCAT-2 were transfected in SL2 cells along
with 50 or 500 ng of pPacSp1 as indicated. The cells were subsequently
lysed and CAT activities determined as described under
``Experimental Procedures.'' The values represent the average
of two independent determinations. C, Sp3 represses
Sp1-mediated transactivation through uteroglobin promoter element II.
Eight micrograms of (EII)
-CAT was transfected along with
various amounts of pPacSp1 (2, 20, and 200 ng) and pPacSp3 (2, 20, 200,
and 2000 ng) as indicated. The cells were subsequently lysed and
assayed for CAT activity.
A constant amount
of the reporter constructs (EII)-CAT,
(EII*)
-CAT, (EVI)
-CAT, and BCAT-2 was
transfected into SL2 cells along with 50 or 500 ng of the expression
plasmid pPacSp1
(30) . The three reporter constructs
(EII)
-CAT, (EVI)
-CAT, and BCAT-2 were activated
by Sp1 (Fig. 6B). In contrast, the activity of the
construct containing the linker scanning mutation of
LS-64/-72 ((EII*)
-CAT) was not at all
influenced by Sp1. Sp3 did not activate any of the promoter constructs
(Fig. 6C and data not shown), although Sp3 is
efficiently expressed in transfected SL2 cells
(15) . However,
when we cotransfected (EII)
-CAT with 20 ng of Sp1
expression plasmid and an excess of the Sp3 expression plasmid,
Sp1-mediated activation was strongly repressed by Sp3
(Fig. 6C). Similar results were obtained previously with
BCAT-2. Sp3 repressed Sp1-mediated activation of BCAT-2 due to the
competition of both factors for their common binding sites
(15) .
Thus, our results obtained with the element II-containing reporter
construct demonstrate that Sp1 and Sp3 also bind to element II in
vivo, thereby acting as transcriptional regulators.
Elements II and VI of the Uteroglobin Promoter Mediate
Basal Activity but Not Estrogen Inducibility
The
estrogen-responsive element of the uteroglobin gene is located
immediately upstream of the CACCC motifs of region VI (Fig. 1).
This is reminiscent of the situation found in the tyrosine
aminotransferase gene
(4) and the tryptophan oxygenase gene
(6) where the CACCC boxes are also close to hormone-responsive
elements. Moreover, the hormone inducibility of the tryptophan
oxygenase gene is dependent on the integrity of the CACCC
box
(6) . Consequently, we raised the question whether the
enhancement of the uteroglobin promoter activity by estrogens
(3) is also dependent on the integrity of the adjacent CACCC
sequences. Since binding of Sp factors is common to element VI and
element II, we also tested the linker scanning mutant
LS-64/-72 that destroys the latter element (Fig. 1).
Constructs containing the CAT gene driven by the uteroglobin wild-type
promoter or the promoter linker scanning mutants
LS-232/-240, LS-222/-229, and
LS-64/-72 were transfected along with an expression vector
for the human estrogen receptor into Ishikawa cells. The result of
these experiments is summarized in . The mutations in
LS-222/-229 and LS-64/-72 but not those in
LS-232/-240 reduced significantly the basal activity of the
uteroglobin promoter, but the inducibility by diethylstilbestrol, a
synthetic estrogen, is in no case affected. The integrity of element II
and the proximal CACCC box of region VI is thus necessary to retain the
wild-type promoter activity. However, mutations in these regions do not
interfere with the inducibility of the uteroglobin promoter by
estrogens.
The Proximal but Not the Distal CACCC Motif of
Uteroglobin Promoter Element VI Interacts Specifically with Sp
Factors
Our footprinting analyses revealed that the proximal but
not the distal CACCC box of the uteroglobin promoter region VI is bound
by Sp factors. This finding is corroborated by the methylation
protection experiments, which showed that additional guanine residues
flanking the GGGTG core sequence are protected from methylation, which
are not present in the distal CACCC sequence. These in vitro DNA binding data correlate with our functional results.
Substitution of the proximal CACCC box reduced the promoter activity
more than 3-fold, whereas a mutation of the distal CACCC box did not
impair promoter activity significantly. Thus, the CACCC core sequence
alone is not sufficient to indicate a functional Sp binding site.
-globin gene, as well as in the
SV40 enhancer. It is known that the SV40 and the
-globin CACCC
sequences are able to bind Sp1
(11, 31) . Thus, it is
very likely that the CACCC boxes found in the tyrosine aminotransferase
gene and the tryptophan oxygenase gene are recognized also by members
of the Sp factor family. The CACCC motifs present in the tyrosine
aminotransferase gene and in the tryptophan oxygenase gene are in close
proximity to glucocorticoid-responsive elements. The
glucocorticoid-responsive element and the CACCC box of the tyrosine
aminotransferase enhancer act synergistically. Consequently, the
glucocorticoid induction of the tryptophan oxygenase gene is abolished
when the CACCC box is deleted (6). For these systems, it remains to be
clarified which member of the Sp transcription family binding to the
CACCC box mediates the synergistic effect with the glucocorticoid
receptor.
Element II of the Uteroglobin Promoter Contains an
Unusual Sp Binding Site
A second Sp binding site in the
uteroglobin promoter has been detected in element II around -64
relative to the transcriptional start site. Compared with the CACCC box
binding site or a classical GC box, the affinity of the Sp recognition
sequence in element II is weaker. Nevertheless, this site appears to be
more important for the uteroglobin promoter strength than the CACCC
box-containing binding site. Very likely, its location adjacent to the
TATA box is optimal for the activation properties of Sp1.
Functional Relevance of the Sp Binding Sites for the
Expression of the Uteroglobin Gene
Expression of the uteroglobin
gene is restricted to epithelial tissues of various organs including
the endometrium, the oviduct, the male genital tract, and the lung
(Ref. 38, and references therein). Consistently, the activity of the
uteroglobin promoter appears to be stronger in cell lines derived from
endometrium and lung as compared with fibroblast cell lines following
DNA-mediated transfection
(2) . Both Sp1 and Sp3 are present in
all the cell lines that we have tested and in all the tissues examined
in situ.()
Moreover, the relative
amounts of the two proteins vary only moderately between various cell
lines (15). Thus, the Sp binding sites of the uteroglobin promoter
appear to be of a general rather than of a cell type-specific nature.
So far, we have been unable to delineate clearly the elements of the
uteroglobin gene that mediate its cell type specificity. It appears
that the combinatorial action of various transcription factors rather
than a single transcription factor determines the cell type-specific
expression of the uteroglobin gene in the uterus. Lung Clara cell
expression has been analyzed for the rat homologue of rabbit
uteroglobin. Different members of the HNF-3 family of transcription
factors seem to contribute, at least in part, to its cell type-specific
expression in lung
(39, 40) .
Table:
Effects of uteroglobin promoter linker scanning
mutations in the absence and presence of diethylstilbestrol (DES)
-galactosidase activity for variations in transfection efficiency.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.