A Functional Sp1 Binding Site Is Essential for the Activity of the Adult Liver-Specific Human Insulin-Like Growth Factor II Promoter
Richard J. T. Rodenburg,
P. Elly Holthuizen and
John S. Sussenbach
Laboratory for Physiological Chemistry Graduate School of
Developmental Biology Utrecht University Utrecht, The
Netherlands
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ABSTRACT
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The human gene encoding insulin-like growth factor
II contains four promoters (P1P4) that are differentially activated
in various tissues during development. Expression of insulin-like
growth factor II in adult liver tissue is directed by P1, which is
activated by liver-enriched members of the CCAAT/enhancer binding
protein family of transcription factors. In the present report we show
that the region around -48 relative to the transcription start site
contains a high affinity Sp1 binding site. This was demonstrated by
electrophoretic mobility shift assays using nuclear extracts from Hep3B
hepatoma cells and with specific antibodies directed against Sp1.
Competition electrophoretic mobility shift assays revealed that the Sp1
binding site of P1 and a consensus Sp1 binding site bind Sp1 with
comparable efficiencies. Mutation of the Sp1 binding site results in an
85% decrease in P1 promoter activity in transient transfection assays
using two different cell lines, COS-7 and Hep3B. Investigation of P1
mutants in which the spacing of the Sp1 binding site and the
transcription start site was increased showed that the role of the Sp1
binding site in regulation of P1 is position dependent. Interestingly,
the Sp1-responsive element cannot be exchanged by a functional TATA
box. Activation of P1 by transactivators CCAAT/enhancer binding
protein-ß and hepatocyte nuclear factor-3ß is strongly impaired
after mutation of the Sp1 binding site. These results demonstrate that
the specific presence of a binding site for the ubiquitously expressed
transcription factor Sp1 is of eminent importance for
efficient activation of P1 by liver-enriched transactivators.
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INTRODUCTION
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Human insulin-like growth factor II (IGF-II) is a polypeptide of
67 amino acids that regulates both the proliferation and
differentiation of many different cell types (1). The production of
IGF-II in various tissues is extensively regulated. Transcriptional
regulation of the human IGF-II gene is controlled by four promoters
(P1P4) that are differentially activated (2, 3, 4). In the liver, which
is the main source for circulating IGF-II, the most active promoter in
the fetal stage of development is P3, whereas in the adult stage P1 is
exclusively active (2).
The adult liver-specific promoter P1 is a TATA-less promoter containing
70% G-C base pairs. Using sensitive reverse transcription-PCR
techniques, P1-derived messenger RNAs (mRNAs) have been detected in a
restricted number of tissues (5, 6). By far the highest levels of
P1-derived mRNAs are found in liver tissue after birth (2, 7). In fetal
liver, P1-derived mRNAs are hardly detectable, whereas hepatoma cell
lines usually produce low levels of P1-derived transcripts (8). The
expression pattern of P1 in liver cells at various stages of liver cell
development correlates very well with the expression levels of the
liver-enriched members of the CCAAT/enhancer binding protein (C/EBP)
family of bZIP transcription factors (8). The C/EBP transcription
factors stimulate P1 promoter activity by binding to an element that is
located approximately 100 bp in front of the transcription start site
of P1 (8). In addition, P1 contains at least two elements (denoted
PE1-1 and PE1-2) that are binding sites for ubiquitously expressed
proteins (9, 10). Promoters of genes that are activated in a
tissue-specific manner are often regulated by a combination of
tissue-specific and ubiquitous transcription factors. In a number of
these promoters a ubiquitous transcription factor facilitates or
enhances the action of one or more tissue-specific transcription
factors. A clear example is the albumin promoter, which is regulated by
a number of liver-enriched transcription factors, including hepatocyte
nuclear factor-1
(HNF-1
) and C/EBP
, and the ubiquitously
expressed transcription factor nuclear factor-Y, which cooperates with
both HNF-1
and C/EBP
in activation of the albumin promoter (11, 12). This illustrates the important role that ubiquitously expressed
transcription factors can play in regulation of tissue-specific gene
expression.
The objective of the present study was to determine the role that
promoter element PE1-1 plays in the regulation of IGF-II P1 activity in
the liver. Our results show that element PE1-1 is a binding site for
the ubiquitously expressed transcription factor Sp1. Mutational
analysis reveals that the presence of the Sp1 binding site in P1 is
required for efficient activation by liver-enriched transcription
factors, and that it cannot be replaced by a TATA box. These results
indicate that Sp1 plays a central role in the regulation of P1 promoter
activity.
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RESULTS
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IGF-II Promoter P1 Contains an Sp1 Binding Site
Promoter element PE1-1 is located at positions -38 to
-59 relative to the transcription start site of the liver-specific
IGF-II promoter P1. We previously showed that PE1-1 is bound by a
protein(s) that is expressed in both the hepatic cell line Hep3B and in
the nonhepatic cell line HeLa (9, 10). Electrophoretic mobility shift
analysis (EMSA) and deoxyribonuclease I (DNase I) footprinting
experiments revealed that PE1-1 binding proteins are present in
extracts derived from the cell lines 293 (adenovirus-transformed human
kidney cell line), PLC/PRF/5 (human hepatoma cell line), IN157 (human
rhabdomyosarcoma cell line), and COS-7 (simian virus 40-transformed
African green monkey kidney cell line) and in extracts from rat liver
and kidney (Fig. 1A
and data not shown). The center of the
PE1-1 footprint covers the nucleotide sequence 5'-CCC-CGC-CTC-3'.
Despite the fact that P1 is very G-C rich, this is the only sequence in
the P1 promoter that resembles the consensus binding sequence for the
ubiquitous transcription factor Sp1, which consists of three nucleotide
triplets: 5'-CCC-CGC-CCC-3'. Each triplet is contacted by one of the
three zinc finger domains that make up the Sp1 DNA binding domain (13, 14). To determine whether Sp1 is responsible for the PE1-1 footprint, 1
bp in each of the three triplets was mutated, resulting in the sequence
5'-CCA-AGC-TTC-3' (mutated bases are
italicized). DNase I footprinting analysis using a P1
fragment containing the mutated PE1-1 element clearly showed that the 3
bp substitutions completely abolished binding to the mutated P1
fragment (Fig. 1B
). To further substantiate that PE1-1 is an Sp1
binding site, an EMSA experiment was performed with a 20-bp double
stranded (ds) oligonucleotide (positions -60 to -41) covering the
PE1-1 element. Incubation of this probe with Hep3B nuclear extract
resulted in the formation of a major and a minor protein DNA complex
(Fig. 2A
, lane 1). Very similar binding patterns were
observed for other Sp1 binding sites in EMSAs with crude nuclear
extracts (15). Subsequent addition of a specific Sp1 antibody gave rise
to a supershift of most of the major complex (Fig. 2A
, lane 2). A
control antibody directed against C/EBP
did not affect binding (Fig. 2A
, lane 3). To establish that PE1-1 is a genuine Sp1 binding site, a
competition EMSA was performed in which ds oligonucleotides containing
either PE1-1 or the consensus Sp1 binding site were added to EMSA
reaction mixtures containing a proximal P1 fragment (-139 to +1) as a
probe and Hep3B nuclear extract (this extract contains Sp1 and related
proteins, but no C/EBP
or C/EBPß). Incubation of P1 (-139 to +1)
with Hep3B extract led to the formation of a number of Sp1 and
Sp1-related proteins containing complexes (Fig. 2B
) that were not
formed when a P1 (-139 to +1) fragment with the three above-described
mutations in PE1-1 was used (Fig. 2B
, lane M). The major Sp1 complex
and the minor complexes containing Sp1-related proteins were
efficiently competed for by both element PE1-1 and the consensus Sp1
oligonucleotide (Fig. 2B
, left and middle
series), but not by increasing amounts of an oligonucleotide with
a C/EBP binding site (Fig. 2B
, right series). These
experiments demonstrate that PE1-1 behaves as a high affinity Sp1
binding site.

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Figure 1. Protection of Element PE1-1 by COS-7 Nuclear Proteins
in a DNase I Footprinting Experiment
A, Incubation of a labeled P1 fragment (-186/+52) with COS-7
nuclear extract or without extract, followed by partial degradation of
the probe with increasing amounts of DNase I. On the right,
the sequence of the upper strand of the protected region is indicated;
the putative Sp1 binding site is boxed. B, Result of a similar
experiment with a P1 fragment (-186/+52) carrying a mutated PE1-1
element. The mutated PE1-1 sequence is shown on the left.The GA tracks are mol wt markers obtained by piperidine cleavage
of the P1 fragments used as probes in the footprint reactions.
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Figure 2. EMSAs Showing Binding of Sp1 to Element PE1-1
A, P1 probe PE1-1 (-60/-41) was incubated with Hep3B nuclear extract
containing Sp1 (first lane). As indicated, reaction mixtures were
incubated with purified antibodies directed against Sp1 or C/EBP .
The major Sp1 complex and the supershifted Sp1 complex are indicated.
Note that unbound PE1-1 probe has run off the gel. B, Competition
experiment using 0.5 ng of P1 fragment (-139/+1) as a probe. The
following amounts of competitor DNA were added: PE1-1, 0, 1, 2.5, 5,
10, 15, and 25 ng; Sp1 consensus, 0, 1, 5, 10, 15, and 25 ng; C/EBP
binding site of P1, 1, 10, and 25 ng. Lane M shows the result of an
EMSA using a P1 fragment (-139/+1) carrying a mutated PE1-1 element as
a probe. The various probe/competitor mixtures were incubated with
equal amounts of Hep3B nuclear extract.
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Element PE1-1 Is Important for P1 Promoter Activity
To investigate the role of the PE1-1 Sp1 binding site in
regulation of P1 promoter activity, a mutant P1-luciferase reporter
gene construct was created in which the Sp1 binding site was replaced
by the mutant PE1-1 element bearing three point mutations, which was
shown not to bind Sp1 in EMSA experiments (Figs. 1B
and 2B
, lane M).
The promoter activities of wild-type P1 (SN-Luc) and mutant P1 (SNSp1
mut-Luc) reporter gene constructs were determined in a transient
transfection experiment using Hep3B and COS-7 cells. This showed that
mutation of the 3 bp in the PE1-1 element results in a reduction of P1
promoter activity of more than 85% in both cell lines (Fig. 3
, left panel). Obviously, the Sp1 binding
site containing element PE1-1 is an important cell type-independent
contributor to the promoter activity of P1, and without this element
the basal activity of P1 is remarkably reduced, albeit above
background.

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Figure 3. Comparison of the Wild-Type P1 Luciferase Construct
SN-Luc (-892/+52; Hatched Bars) with a P1 Luciferase
Construct Carrying a Mutated Element PE1-1 (SNSp1 mut-Luc;
Stippled Bars)
Left panel, Basal activities of the two P1 luciferase
constructs as observed in transient transfection experiments using
Hep3B cells and COS-7 cells. The promoter activity of the SN-Luc
construct was set to 100% for each cell line. The absolute luciferase
values obtained after transfection of SN-Luc were similar in both cell
lines, and 50- to 100-fold higher than the background luciferase
levels. Right panel, Transactivation of SN-Luc and SNSp1
mut-Luc by C/EBPß in a transient transfection experiment using Hep3B
cells. The basal activity of each construct was set at 1, and fold
induction by C/EBPß is indicated. Transfection results were corrected
for transfection efficiency using RSV-LacZ as an internal standard. The
SE in three independent experiments is indicated by
error bars. The luciferase activity measured after
transfection of Hep3B cells or COS-7 cells with the PE1-1 mutant was
only 23 times higher than that measured after transfection of cells
with a promoterless luciferase construct (data not shown).
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Element PE1-1 Is Indispensable for Efficient Activation of P1 by
C/EBPß
In a previous report, we showed that C/EBP transcription factors
interact with a binding site located around position -97 in P1,
leading to strong activation of P1 transcription (8, 9). Since the Sp1
binding site appears to play an important role in the regulation of P1
promoter activity as well, it was of interest to test the effect of
mutation of element PE1-1 on transactivation of P1 by C/EBP
transcription factors. This was investigated by cotransfecting Hep3B
cells with P1 constructs carrying a wild-type or mutated element PE1-1
(constructs SN-Luc and SNSp1 mut-Luc, respectively), and an expression
vector encoding C/EBPß. Whereas wild-type P1 was activated
approximately 14-fold by C/EBPß, the P1 mutant containing a disrupted
PE1-1 element, but intact C/EBP site, was activated only 2.5-fold (Fig. 3
, right panel). Apparently, C/EBPß activates P1 much more
efficiently when an intact Sp1 binding site is present.
Previously, C/EBPß and Sp1 have been shown to synergistically
activate the CYP2D5 promoter, which is due to cooperative binding of
C/EBPß and Sp1 to two adjacent binding sites (16). To investigate
whether cooperative binding of these factors also applies to IGF-II P1
transcription, an EMSA was performed using a P1 fragment (positions
-139 to +1) containing both the PE1-1 and the C/EBP binding sites.
This probe was incubated with increasing amounts of Hep3B extract (as a
source for Sp1) and recombinant C/EBPß (Hep3B cells have no
detectable endogenous C/EBP
or C/EBPß levels in EMSA assays). A
number of complexes were detected that represent binding of Sp1 to
element PE1-1 and binding of C/EBPß to the C/EBP binding site (Fig. 4A
). Only when large amounts of extract were added to
the EMSA reaction mixture, a slowly migrating complex representing
simultaneous binding of C/EBPß and Sp1 to the P1 fragment was
observed (Fig. 4A
, lane 2). Furthermore, when increasing amounts of
unlabeled P1 fragment were added, the amount of slowly migrating
complex rapidly declined, and only individual binding of C/EBPß and
Sp1 was observed. When EMSA experiments with the P1 fragment were
performed with a constant amount of Sp1 and increasing amounts of
C/EBP, the amounts of Sp1-containing complexes stayed constant,
independent of the amount of C/EBP added. Complexes representing
fragments that carry both Sp1 and C/EBPß were only observed at the
highest concentration of C/EBPß. These results support the idea that
Sp1 and C/EBP bind independently (Fig. 4A
). Also, the reciprocal
experiment in which EMSA experiments were performed with a constant
amount of C/EBP and increasing amounts of Sp1 demonstrated that binding
of C/EBP and Sp1 occurs independently (Fig. 4A
, right
lanes).

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Figure 4. EMSA of a Proximal P1 Fragment (-139 to +1)
Showing Independent Binding of Sp1 and C/EBPß
A, The left panel shows a competition experiment in
which increasing amounts of unlabeled P1 fragment (compC+H; 5-, 10-,
and 25-fold excesses, respectively) were added to the labeled probe and
incubated with a constant amount of recombinant C/EBPß (C) and Hep3B
nuclear extract (H). In the middle panel, the probe was
incubated with a fixed amount of Hep3B nuclear extract and increasing
amounts of C/EBPß. In the right panel, the amount of
recombinant C/EBPß was kept constant, and increasing amounts of Hep3B
nuclear extract were added. Complexes formed with C/EBPß and Sp1 as
well as the ternary complex containing both C/EBPß and Sp1 are
indicated. B, The proximal P1 fragment (-139 to +1) was incubated with
Hep3B extract (Sp1 binding only; lane 1), recombinant C/EBPß (lane
2), and the combination of Sp1 and C/EBPß (lanes 3, 8, and 13). In
lanes 47, the constant amounts of recombinant C/EBPß and Hep3B
nuclear extract were competed with increasing amounts of unlabeled Sp1
oligonucleotide (5-, 10-, 20-, and 50-fold, respectively). In lanes
912, competition with increasing amounts of unlabeled C/EBPß
oligonucleotide (5-, 10-, 20-, and 50-fold, respectively) is
performed.
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The independent binding of C/EBPß and Sp1 to P1 was further
demonstrated in an experiment in which the proximal P1 fragment (-139
to +1) was incubated with the combination of Sp1 (present in Hep3B
extract) and recombinant C/EBPß. Complex formation was performed in
the presence of increasing amounts of oligonucleotides with binding
sites for Sp1 or C/EBPß (Fig. 4B
). Incubation of the probe with the
Hep3B extract leads to formation of a major band representing the
Sp1-containing complex and a few minor bands of Sp1-related protein-DNA
complexes (Fig. 4B
, lane 1). Incubation of the probe with C/EBPß
leads to the formation of a complex that migrates faster than the
Sp1-containing complexes and represents C/EBP binding (Fig. 4B
, lane
2). When the probe is incubated with Sp1 and C/EBPß simultaneously,
each factor binds with the same efficiency as if the factors are
incubated separately with the probe (Fig. 4B
, lane 3). This experiment
demonstrates that the binding of Sp1 is not dependent on the amounts of
C/EBPß, and conversely, the binding of C/EBPß is not affected by
the amount of Sp1. When the probe is incubated with the combination of
Sp1 and C/EBPß (Fig. 4B
, lanes 47), we studied the effect of the
addition of a 5- to 50-fold excess of an unlabeled Sp1 oligonucleotide
or a C/EBPß oligonucleotide, respectively, on complex formation
(lanes 912). As demonstrated in Fig. 4B
, modulation of the amount of
C/EBP or Sp1 available to the fragment did not affect the efficiency of
binding of the other transcription factor. In conclusion, these
experiments show that binding of Sp1 and that of C/EBPß to P1
fragment -139 to +1 occur independently of each other and that no
physical association or cooperativity of C/EBPß and Sp1 can be
detected.
Positional Effects of the C/EBP and Sp1 Binding Sites
To gain more insight into the mechanism underlying Sp1-dependent
transactivation of P1 by C/EBPß, it was investigated whether this
phenomenon is dependent on the relative positions of the binding sites
of Sp1 and C/EBPß in P1. A set of P1 mutant reporter gene constructs
was constructed in which the C/EBP binding site was positioned 5 bp
(half a helical turn), 10 bp (full helical turn), or 32 bp (three
helical turns) closer to the PE1-1 element in the promoter, or 4 bp
(half a helical turn) further away from the PE1-1 element (Fig. 5A
). The Sp1 binding site in all of these constructs was
present at the same position as in the wild-type P1. Hep3B cells were
transfected with these constructs either with or without addition of
C/EBPß expression vector. The basal promoter activities of these
spacer mutants were similar to the basal activity of wild-type P1 (data
not shown) and were set at 1 in each lane. The effect of the altered
positioning of the C/EBP binding site on C/EBPß transactivation of P1
was very moderate, as the mutants were activated 8- to 12-fold by
C/EBPß, whereas wild-type P1 was activated 14-fold (Fig. 5B
). These
results indicate that the C/EBP binding site functions as a normal
enhancer element acting relatively independent of its position in
promoter P1.

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Figure 5. Position-Dependent Effect of the C/EBP-Binding Site
in P1 on Transactivation of P1 by C/EBPß
A, Schematic representation of the wild-type P1 construct SN-Luc
(-892/+52) and P1 mutants derived from SN-Luc containing a C/EBP
binding site (stippled box) at different positions in
P1. In all constructs, element PE1-1 (hatched box) is
located around position -48. Numbers indicate the distance of the
middle of the binding site from the transcription start site (tss). B,
Transactivation by C/EBPß of the wild-type and mutant P1 constructs
represented in A in Hep3B cells. The basal activity of each construct
was set at 1, and fold activation by C/EBPß is indicated.
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To analyze whether the Sp1 binding site displays position-dependent
effects on P1 promoter activity, a number of additional P1 mutants were
tested containing the Sp1 element PE1-1 at different positions in P1
(Fig. 6A
). When the original Sp1 site at position -48
was mutated, the corresponding reporter construct SNSp1 mut-Luc showed
little promoter activity. Insertion of an Sp1 element to SNSp1 mut-Luc
at position -928 (construct SNins880-Luc) did not enhance basal or
activated promoter activities (Fig. 6
, B and C). This indicates that
the PE1-1 element is not functional at such a long distance from the
transcription start site. Even when the PE1-1 element was inserted at
position -94 (SNins46-Luc), the basal activity was still only 2-fold
higher compared with the activity of SNSp1 mut-Luc. As this construct
lacks a functional C/EBP binding site, transactivation by C/EBPß was
not tested. When element PE1-1 was located at position -56
(SNins8-Luc), promoter activity was approximately 20% reduced compared
with that of wild-type P1, and activation by C/EBPß was only slightly
impaired. Interestingly, the negative effect of shifting or mutating
the Sp1 binding site on basal transcription seems to correlate with the
negative effect on transactivation by C/EBPß, suggesting that basal
and activated transcriptions are in a similar manner dependent on Sp1.
These results clearly show that the function of the PE1-1 element is
dependent on its position in P1, which is different from that of a
normal enhancer element. The properties of PE1-1 as an enhancer element
were further investigated by placing a second PE1-1 element in front of
both a full-length P1-containing 892-bp promoter sequence (PE1-1SN-Luc)
and a truncated P1 of only 57 bp (PE1-1NhN-Luc; Fig. 7A
). In this manner, the additional PE1-1 element was
positioned around positions -928 or -94, respectively. The promoter
activities of these two constructs in Hep3B cells were compared with
the promoter activities of their normal counterparts. For both
constructs the additional Sp1 site did not influence promoter activity
(Fig. 7B
). This shows that the addition of a second PE1-1 element has
no effect on promoter activity in either full-length or truncated P1,
indicating that element PE1-1 as such lacks intrinsic enhancer
activity.

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Figure 6. Position-Dependent Effect of Element PE1-1 on Basal
P1 Promoter Activity and Transactivation by C/EBPß
A, Schematic representation of wild-type P1 construct SN-Luc
(-892/+52) and mutated P1 constructs. The distances of the middle of
element PE1-1 (hatched box) and the C/EBP binding site
(stippled box) from the transcription start site (tss)
in the various constructs are indicated. B, Basal activities of the P1
constructs shown in A upon transfection in Hep3B cells. The promoter
activities (in arbitrary light units) were corrected for transfection
efficiency using RSV-LacZ as an internal standard. C, Transactivation
of the mutant P1 constructs shown in A by C/EBPß. The basal
activities of the various constructs (shown in B) were set at 1, fold
activation was measured after cotransfection of Hep3B cells with the
different P1 luciferase constructs, and a C/EBPß expression vector is
indicated. As mutant SNins46-Luc lacks a C/EBP binding site, activation
of this construct by C/EBPß was not determined (nd).
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Figure 7. Element PE1-1 Lacks Intrinsic Enhancer Activity
A, Schematic representation of wild-type P1 construct SN-Luc
(-892/+52), truncated P1 construct NhN-Luc (-57/+52), and mutant P1
constructs containing an additional element PE1-1 at two different
positions. In construct PE1-1SN Luc, the second PE1-1 element is
located 928 bp in front of the transcription start site (tss), whereas
in construct PE1-1NhN-Luc, the distance of the second PE1-1 element to
the tss is 94 bp. The original PE1-1 element is located at position
-48 in all four constructs. B, Promoter activities measured after
transfection of Hep3B cells with the constructs shown in A. The
promoter activities of the various constructs are shown in arbitrary
light units, which were normalized using RSV-LacZ as an internal
standard.
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Specificity of the Effects of Sp1 and C/EBPß on P1 Promoter
Activity
The crucial role that is played by element PE1-1 in the regulation
of P1 is very similar to that of the TATA box in the regulation of many
other promoters of RNA polymerase II-transcribed genes. The TATA box is
bound by the TATA binding protein, after which the basal transcription
machinery is recruited, giving rise to a transcriptionally active
promoter (17). As P1 lacks a TATA box, it was tested whether
introduction of a TATA box at position -25 in P1 restores the promoter
activity of a mutant P1 lacking a functional Sp1 binding site (Fig. 8A
). However, introduction of a TATA box in the absence
of element PE1-1(construct SNTATA-Luc) results in only a 2-fold higher
basal activity compared with that of a P1 construct containing a
mutated Sp1 binding site (Fig. 8B
). Likewise, SNTATA-Luc was activated
only 2-fold by C/EBPß, again comparable to the level of C/EBP
activation of the P1 carrying a mutated PE1-1 element (Fig. 8C
). These
observations show that a TATA box at position -25 is not functionally
equivalent to the PE1-1 element with respect to the regulation of basal
and activated P1 promoter activity. From these experiments it is
concluded that a TATA box cannot functionally replace the Sp1 binding
site in P1.

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Figure 8. Specific Role of Sp1 in Basal and
C/EBPß-Activated P1 Promoter Activity
A, Schematic representation of wild-type P1 construct SN-Luc
(-892/+52), the P1 Sp1 mutant (SNSp1 mut-Luc), and the construct
lacking an Sp1 binding site in which a TATA box was introduced
(SNTATA-Luc). The positions of the various elements in P1 with respect
to the transcription start site (tss) region are indicated. All
constructs contain a C/EBP binding site around position -97. B, Basal
activities of the promoter constructs depicted in A upon transfection
of Hep3B cells. The promoter activities (in arbitrary light units) were
corrected for transfection efficiency using RSV-LacZ as an internal
standard. C, Transactivation of the P1 constructs shown in A by
C/EBPß. The basal activities of the various constructs (shown in B)
were set at 1, fold activation was measured after cotransfection of
Hep3B cells with the different P1 luciferase constructs, and a C/EBPß
expression vector.
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Finally, it was investigated whether other liver-specific activators of
P1 transcription are also dependent on the presence of the Sp1 binding
site in P1. Hep3B cells were cotransfected with wild-type P1 (SN-Luc)
or a P1 Sp1 mutant (SNSp1 mut-Luc) reporter gene construct and
expression vectors encoding HNF-3ß, C/EBPß, and C/EBP
(Fig. 9
).
The basal activities of both P1 constructs, which are only 15% that of
wild-type P1 (Fig. 3A
), were set at 1. HNF-3ß
activated the wild-type P1 5-fold, whereas C/EBP
gave rise to a 7-
to 8-fold activation of P1. C/EBPß was the strongest activator of P1
activity, as it activated P1 more than 14-fold. By contrast, all three
liver-enriched transcription factors had a very modest effect on the P1
Sp1 mutant. This experiment shows that activated transcription of P1 by
the liver-enriched transcription factors C/EBP
, C/EBPß, and
HNF-3ß requires a functional Sp1 binding site.

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Figure 9. Transactivation of Wild-Type P1 (Hatched
Bars) and the P1 Sp1 Mutant (Stippled Bars) by
Liver-Enriched Transcription Factors
Hep3B cells were cotransfected with the P1 luciferase constructs and
with expression vectors encoding HNF-3ß, C/EBPß, and C/EBP ,
respectively. The basal activities of either P1 construct was set at 1,
and fold activation by the various liver-enriched transcription factors
is indicated.
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DISCUSSION
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In this report we show that element PE1-1, which is located at
positions -38 to -59 of the IGF-II promoter P1, is functionally
identical to an Sp1 binding site. This conclusion is based on the
following observations. PE1-1 binding activity was observed in extracts
from several mammalian cell lines, indicating that it is ubiquitously
expressed. In EMSA experiments the major PE1-1 binding activity is
recognized by Sp1 antibodies and in EMSA competition experiments
element PE1-1 behaves indistinguishably from an oligonucleotide
carrying a consensus Sp1 binding site. Finally, mutation of PE1-1 at
three crucial positions in the putative Sp1 site completely abolishes
Sp1 binding.
Comparison of the expression of wild-type and Sp1 mutant
P1-luciferase vectors in Hep3B cells shows that the basal P1 promoter
activity is reduced to only 15% of the wild-type activity when three
point mutations are introduced into the Sp1 binding site. A functional
Sp1 binding site is also required for activation of P1 by the
liver-enriched transcription factors C/EBP
, C/EBPß, and HNF-3ß.
Furthermore, the position of the Sp1 site relative to that of the
transcription start site is also important, since the effect of the Sp1
binding site on both basal and activated transcription drastically
decreases when the Sp1 binding site is transferred from its original
position at -48 to regions further upstream in the promoter. This
indicates that the Sp1 binding site does not function as a
position-independent enhancer element. Taken together, these data show
that Sp1 binding is important for both basal and inducible IGF-II
expression from promoter P1.
Binding sites for Sp1 have previously been identified in other
promoters that are active in liver tissue, e.g. the
apolipoprotein CIII promoter and the promoter of the CYP2D5 cytochrome
P450 gene are highly dependent on functional Sp1 binding sites
(16, 17, 18). Two other examples in which Sp1 is involved in basal
transcription are the human adenosine deaminase promoter (19) and the
hamster dehydrofolate reductase promoter (20). Of the six Sp1 sites
that are present in the TATA-less human H-ras promoter, only
the most proximal Sp1 binding site located around position -45 is
required for full transient expression. Introduction of unrelated DNA
between this Sp1 site and the transcription start site resulted in
reduced promoter activity (21). Based on these results, it was
suggested that Sp1 is required for proper positioning of the basal
transcription machinery on the H-ras promoter. A major
difference between most Sp1-containing promoters and the human IGF-II
P1 is that these promoters contain multiple GC boxes to which Sp1 can
bind, whereas P1 is regulated by a single Sp1 binding site.
Basal transcription of TATA-less promoters containing multiple Sp1
binding sites (one of which is located around position -45) requires a
multisubunit TFIID complex (22, 23). DNase I footprinting experiments
using a TATA-less promoter containing Sp1 binding sites have shown that
binding of Sp1 stabilizes interaction of TFIID with the transcription
start site region (24). This mechanism deviates from that of
TATA-containing promoters where the TATA box directly binds TFIID (at
position -25) (25).
Activation of transcription involves an interplay between activators
bound to the cis-acting regulatory elements and factors
bound to the basal elements near the initiation of transcription site.
Binding of such factors does not necessitate interaction between the
transcription factors, but does require sequence-specific binding of
the factors to the elements (26). Changing the arrangement of the basal
elements can alter the transcriptional activation response (27). This
was shown for the murine TK promoter that carries only a single Sp1
motif and an E2F binding site. Mutational inactivation of the elements
or changing the distance between the elements almost completely
abolished promoter activity (28). Likewise, Sp1 was shown to play an
important role in basal and induced expression of the murine IL-6 gene
(29).
Recently, it was shown that the activity of Sp1 protein is decreased to
some extent during liver development, which is due to
development-dependent posttranslational modifications (30). However, as
the Sp1 expression level increases during liver development,
differentiated liver tissue probably contains sufficient levels of
active Sp1 protein to regulate Sp1-dependent promoters (16).
Based on the results presented in this report, we propose that Sp1
plays a central role in the regulation of P1 activity. In the absence
of a functional PE1-1 element, Sp1 does not bind to P1, and therefore,
the basal transcription machinery is inefficiently recruited. As a
consequence, basal P1 promoter activity is very low. In addition,
specific activators of P1 are unable to regulate P1 activity in the
absence of basal transcription factors. For example, C/EBP
and
C/EBPß, which activate transcription by interacting with the basal
transcription factors TFIID and TFIIB (16), have only a very small
effect on a P1 mutant carrying a disrupted Sp1 binding site. When a
functional Sp1 binding site is present in P1 around position -48, the
interaction between TFIID and P1 is stabilized by Sp1. This results in
a strong increase in the basal level of P1 activity, which can be
further stimulated by the liver-enriched transactivators HNF-3ß,
C/EBP
, and C/EBPß.
 |
MATERIALS AND METHODS
|
---|
Oligonucleotides and Plasmids
The following oligonucleotides were synthesized on a
Pharmacia/LKB Gene Assembler Plus: 9424,
5'-CCGGCCGTCCACCCGCCTGCCCAGCCTTGG-3'; 9427,
5'-CCCTGTTCCTGAAGCTCTGAGCTCACCCCTTCCCC-3'; 9471,
5'-AGC-TTCTGGAGGCGGGGCTAGCTG-3'; 9472,
5'-GATCCAGC-TAGCCCCGCCTCCAGA-3'; 9476,
5'-CCCAGCTAGCCAA-GCTTCCAGAGTGGGGGCCAAGGCTGGG-3'; 9477,
5'-GG-GGGCTAGCGCTGTGACATCTCTGGGTGGAGG-3';
9478,5'-GGGGGCTAGCGACATCTCTGGGTGGAGGGGGGC-3';
9480, 5'-AATGCTAGCTGGATGTGGTCATGGGGAAGGGGT-GAGCTC-3'; 9586,
5'-TAGGCTATAAAAGGGGTGGACGGC-C-3'; 9587,
5'-GGCCGTCCACCCCTTTTATAGCC-3'; 9590, 5'-TATGGCCCCCACTCTGGAAGCTTGG-3';
9591, 5'-CTAG-CCAAGCTTCCAGAGTGGGGGCCA-3'; Sp1 cs upper,
5'-ATTCGATCGGGGCGGGGCGAGC-3'; Sp1 cs lower,
5'-GC-TCGCCCCGCCCCGATCGAAT-3'; and M13rev,
5'-AGCG-GATAACAATTTCACACAGGA-3'.
The IGF-II P1 luciferase construct pSN-Luc contains a
SmaI-NcoI fragment of P1, consisting of 892 bp of
promoter sequence and 52 bp of exon 1 sequence, fused to the firefly
luciferase gene (31). The truncated pNhN-Luc contains P1 sequences from
the NheI restriction site at position -57 to the
NcoI site at +52. To generate P1 mutants, a subclone was
produced by cloning a P1 fragment from the SmaI site at
position -892 up to the transcription start site at position +1 into
the SmaI site of pUC18, resulting in the construct pSNpUC18.
Several P1 mutants were created by PCR using pSNpUC18 as a template and
various primers. A P1 construct bearing a mutated PE1-1 element (pSNSp1
mut-Luc) was obtained by PCR using upstream primer M13rev and
downstream primer 9476. The PCR product was digested with
SmaI and NheI and cloned into the SmaI
and NheI sites of pSN-Luc. Mutants pSND5-Luc in which 5 bp
between PE1-1 and the C/EBP binding site are deleted, pSND10-Luc (10 bp
deletion), and pSND32-Luc (32 bp deletion) were created in a similar
manner, using downstream primers 9477, 9478, and 9480, respectively. By
filling in the NheI site at -57 of pSN-Luc using Klenow DNA
polymerase, 4 bp were inserted in between element PE1-1 and the C/EBP
binding site, yielding construct pSNins4-Luc. To create a P1 construct
lacking an Sp1 binding site and containing a TATA box, two ds
oligonucleotides were generated. Ds oligonucleotide 9586/9587 contains
a TATA box that is derived from the adenovirus major late promoter, and
contains ends that are compatible with NdeI and
NaeI restriction sites. Ds oligonucleotide 9590/9591
contains a mutated PE1-1 element and NheI-NdeI
compatible ends. The two ds oligonucleotides were purified over a 6%
19:1 acrylamide gel, fused at their NdeI ends, and cloned
into the NheI-NaeI sites of pSN-Luc, thereby
replacing P1 sequences from -57 to -4 containing element PE1-1. The
resulting construct pSNTATA-Luc contains a mutated element PE1-1 and a
TATA box around position -25. Construct pPE1-1SN-Luc was obtained by
cloning ds oligonucleotide 9471/9472 containing element PE1-1 into the
HindIII and BamHI sites of pSN-Luc. In a similar
manner, clones pSNins880-Luc and pPE1-1NhN-Luc were created by cloning
9471/9472 into pSNSp1 mut-Luc and pNhN-Luc, respectively. By deleting a
BamHI-NheI fragment from pSNins880-Luc, mutant
pSNins46-Luc was created. Mutant pSNins8-Luc was obtained by cloning an
8-bp SalI linker with the sequence 5'-CGTCGACG-3' into the
NaeI site at position -4 of P1 in pSN Luc. All plasmids
were checked by DNA base pair sequence analysis.
EMSA and DNase I Footprinting
A probe covering the proximal P1 region was produced by PCR
using primers 9424 and 9427 on the template pSN-Luc, resulting in a
140-bp fragment containing P1 sequences from positions -139 to +1. Ds
oligonucleotides containing element PE1-1 and the consensus Sp1 site
were produced by annealing oligonucleotides 9471 and 9472 or Sp1 cs
upper and Sp1 cs lower, respectively, and subsequently purified over
gel. Probes were labeled using [
-32P]ATP and
T4-kinase, and purified over a Sephadex G-50 column. Specific
antibodies directed against Sp1 or C/EBP
were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Preparation of nuclear extracts
and C/EBPß-containing extracts, and EMSA binding conditions have been
described previously (32). Briefly, binding reactions were performed by
mixing DNA probes, competitor DNA, and binding buffer, after which
protein extract was added. Reactions were incubated for 1 h on
ice; for supershift experiments, binding reactions were incubated for
an additional 10 min with 0.1 mg polyclonal antibody. Bound and free
probe were separated on a 5% 37.5:1 polyacrylamide gel containing
0.5 x TBE (10 x TBE = 0.89 M Tris-HCl,
0.89 M boric acid, and 0.02 M EDTA). Gels were
dried and exposed to x-ray films using intensifying screens. For DNase
I footprinting experiments, XhoI-NcoI fragments
(-186/+52) were isolated from pSN-Luc and from pSNSp1 mut-Luc. These
fragments were labeled by filling in the NcoI site using
[
-32P]deoxy-CTP and Klenow DNA polymerase and
subsequently purified over a 5% 37.5:1 acrylamide gel. The probes were
incubated with a saturating amount of COS-7 extract (empirically
determined by EMSA) under standard EMSA binding conditions for 1 h
on ice, after which the samples were treated with varying amounts of
DNase I for 1 min at room temperature. Reactions were stopped by adding
5 mg salmon sperm DNA and 0.5% SDS, phenol extracted, precipitated,
and analyzed on a denaturing 19:1 polyacrylamide sequencing gel. As a
mol wt marker, a Maxam and Gilbert sequencing reaction was performed on
the same probes (33).
Transient Transfections
Transient transfections of Hep3B human hepatoma cells (34) and
COS-7 African green monkey kidney cells (35) were performed by a
modified Ca3(PO4)2 procedure using
Bes buffered saline as described previously (36). Briefly, cells were
grown in 25-cm2 flasks to 6080% confluence and
transfected with 2 µg luciferase construct and 0.25 µg pRSV-LacZ
(RSV, Rous sarcoma virus). For cotransfections, 0.5 µg pCMV-LAP
(encoding the major gene product of the C/EBPß gene), pCMV-HNF-3ß,
pCMV-C/EBP
, or empty CMV vector was added. The amount of DNA per
transfection was kept constant by the addition of herring sperm DNA up
to 10 µg. Sixteen hours after transfection, Hep3B cells were shocked
with medium containing 10% dimethylsulfoxide, and both cell lines
received fresh medium. Cells were harvested 40 h after
transfection, after which luciferase and ß-galactosidase assays were
performed as previously described (36). All transfection experiments
were repeated at least three times, and luciferase values were
corrected for transfection efficiency using the ß-galactosidase
activity obtained with pRSV-LacZ as an internal standard.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. P. Elly Holthuizen, Laboratory for Physiological Chemistry, Graduate School of Developmental Biology, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
This work was supported by a grant from the Netherlands Organization
for the Advancement of Pure Research.
Received for publication March 18, 1996.
Revision received November 4, 1996.
Accepted for publication November 11, 1996.
 |
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