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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human gene encoding insulin-like growth factor II contains four promoters (P1–P4) 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (P1–P4) 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{alpha} (HNF-1{alpha}) and C/EBP{alpha}, and the ubiquitously expressed transcription factor nuclear factor-Y, which cooperates with both HNF-1{alpha} and C/EBP{alpha} 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo 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. 1BGo). 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. 2AGo, 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. 2AGo, lane 2). A control antibody directed against C/EBP{alpha} did not affect binding (Fig. 2AGo, 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{alpha} 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. 2BGo) that were not formed when a P1 (-139 to +1) fragment with the three above-described mutations in PE1-1 was used (Fig. 2BGo, 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. 2BGo, left and middle series), but not by increasing amounts of an oligonucleotide with a C/EBP binding site (Fig. 2BGo, right series). These experiments demonstrate that PE1-1 behaves as a high affinity Sp1 binding site.



View larger version (80K):
[in this window]
[in a new window]
 
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.

 


View larger version (25K):
[in this window]
[in a new window]
 
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{alpha}. 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.

 
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. 1BGo and 2BGo, 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. 3Go, 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.



View larger version (28K):
[in this window]
[in a new window]
 
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 2–3 times higher than that measured after transfection of cells with a promoterless luciferase construct (data not shown).

 
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. 3Go, 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{alpha} 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. 4AGo). 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. 4AGo, 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. 4AGo). 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. 4AGo, right lanes).



View larger version (56K):
[in this window]
[in a new window]
 
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 4–7, 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 9–12, competition with increasing amounts of unlabeled C/EBPß oligonucleotide (5-, 10-, 20-, and 50-fold, respectively) is performed.

 
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. 4BGo). 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. 4BGo, 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. 4BGo, 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. 4BGo, 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. 4BGo, lanes 4–7), 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 9–12). As demonstrated in Fig. 4BGo, 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. 5AGo). 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. 5BGo). These results indicate that the C/EBP binding site functions as a normal enhancer element acting relatively independent of its position in promoter P1.



View larger version (27K):
[in this window]
[in a new window]
 
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.

 
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. 6AGo). 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. 6Go, 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. 7AGo). 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. 7BGo). 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.



View larger version (25K):
[in this window]
[in a new window]
 
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).

 


View larger version (22K):
[in this window]
[in a new window]
 
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.

 
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. 8AGo). 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. 8BGo). 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. 8CGo). 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.



View larger version (20K):
[in this window]
[in a new window]
 
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.

 
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{alpha} (Fig. 9Go). The basal activities of both P1 constructs, which are only 15% that of wild-type P1 (Fig. 3AGo), were set at 1. HNF-3ß activated the wild-type P1 5-fold, whereas C/EBP{alpha} 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{alpha}, C/EBPß, and HNF-3ß requires a functional Sp1 binding site.



View larger version (28K):
[in this window]
[in a new window]
 
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{alpha}, respectively. The basal activities of either P1 construct was set at 1, and fold activation by the various liver-enriched transcription factors is indicated.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}, 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{alpha} 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{alpha}, and C/EBPß.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 [{gamma}-32P]ATP and T4-kinase, and purified over a Sephadex G-50 column. Specific antibodies directed against Sp1 or C/EBP{alpha} 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 [{gamma}-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 60–80% 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{alpha}, 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34[Medline]
  2. de Pagter-Holthuizen P, Jansen M, van Schaik FMA, van der Kammen R, Oosterwijk C, Van den Brande JL, Sussenbach JS 1987 The human insulin-like growth factor II gene contains two development specific promoters. FEBS Lett 214:259–264[CrossRef][Medline]
  3. de Pagter-Holthuizen P, Jansen M, van der Kammen R, van Schaik FMA, Sussenbach JS 1988 Differential expression of the human insulin-like growth factor II gene. Characterization of the IGF-II mRNAs and a mRNA encoding a putative IGF-II associated protein. Biochim Biophys Acta 950:282–295[Medline]
  4. Holthuizen P, van der Lee FM, Ikejiri K, Yamamoto M, Sussenbach JS 1990 Identification and initial characterization of a fourth leader exon and promoter of the human IGF-II gene. Biochim Biophys Acta 1087:341–343[Medline]
  5. Jin IH, Sinha G, Yballe C, Vu TH, Hoffman AR 1995 The human insulin-like growth factor II promoter P1 is not restricted to liver: evidence for expression of P1 in other tissues and for a homologous promoter in baboon liver. Horm Metab Res 27:447–449[Medline]
  6. Vu TH, Hoffman AR 1994 Promoter specific imprinting of the human insulin-like growth factor II gene. Nature 371:714–717[CrossRef][Medline]
  7. Jansen M, de Pagter-Holthuizen P, Van den Brande JL, Sussenbach JS 1987 Somatomedin gene structure and expression. In: Christiansen C, Riis BJ (eds) Highlights on Endocrinology. Proceedings of The First European Congress on Endocrinology, Copenhagen, pp 217–224
  8. Rodenburg RJT, Teertstra W, Holthuizen PE, Sussenbach JS 1995 Postnatal liver specific expression of human insulin-like growth factor II is highly stimulated by the transcriptional activators liver enriched activating protein and CCAAT/enhancer binding protein. Mol Endocrinol 9:424–434[Abstract]
  9. van Dijk MA, Rodenburg RJT, Holthuizen P, Sussenbach JS 1992 The liver specific promoter of the human insulin-like growth factor II gene is activated by CCAAT/enhancer binding protein (C/EBP). Nucleic Acids Res 20:3099–3104[Abstract]
  10. van Dijk MA, van Schaik FMA, Bootsma HJ, Holthuizen P, Sussenbach JS 1991 Initial characterization of the four promoters of the human insulin-like growth factor II gene. Mol Cell Endocrinol 81:81–94[CrossRef][Medline]
  11. Tronche F, Rollier A, Sourdive D, Cereghini S, Yaniv M 1991 NFY or a related CCAAT binding factor can be replaced by other transcriptional activators for co-operation with HNF-1 in driving the rat albumin promoter in vivo. J Mol Biol 221:31–43[CrossRef][Medline]
  12. Milos PM, Zaret KS 1992 A ubiquitous factor is required for C/EBP related proteins to form stable transcription complexes on an albumin promoter segment in vitro. Genes Dev 6:991–1004[Abstract]
  13. Kuwahara J, Yonezawa A, Futamura M, Sugiura Y 1993 Binding of transcription factor Sp1 to GC box DNA revealed by footprinting analysis: different contact of three zinc finger and sequence recognition mode. Biochemistry 32:5994–6001[Medline]
  14. Kadonaga JT, Carner KR, Masiarz FR, Tjian R 1987 Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51:1079–1090[Medline]
  15. Hagen G, Möller S, Beato M, Suske G 1994 Sp1 mediated transcriptional activation is repressed by Sp3. EMBO J 13:3843–3851[Abstract]
  16. Lee YH, Yano M, Liu SY, Matsunaga E, Johnson PF, Gonzalez FJ 1994 A novel cis acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBPß and an Sp1 factor. Mol Cell Biol 14:1383–1394[Abstract]
  17. Zawel L, Reinberg D 1995 Common themes in assembly and function of eukaryotic transcription complexes. Annu Rev Biochem 64:533–561[CrossRef][Medline]
  18. Talianidis I, Tambakaki A, Toursounova J, Zannis VI 1995 Complex interactions between SP1 bound to multiple distal regulatory sites and HNF-4 bound to the proximal promoter lead to transcriptional activation of liver specific human apoCIII gene. Biochemistry 34:10298–10309[Medline]
  19. Dusing MR, Wiginton DA 1994 Sp1 is essential for both enhancer mediated and basal activation of the TATA less human adenosine deaminase promoter. Nucleic Acids Res 22:669–677[Abstract]
  20. Blake MC, Jambou RC, Swick AG, Kahn JW, Azizkhan JC 1990 Transcriptional initiation is controlled by upstream GC box interactions in TATAA less promoter. Mol Cell Biol 10:6632–6641[Medline]
  21. Lu J, Lee W, Jiang C, Keller EB 1994 Start site selection by Sp1 in the TATA less human Ha-ras promoter. J Biol Chem 269:5391–5402[Abstract/Free Full Text]
  22. Pugh BF, Tjian R 1991 Transcription from a TATA less promoter requires a multisubunit TFIID complex. Genes Dev 5:1935–1945[Abstract]
  23. Pugh BF, Tjian R 1990 Mechanism of transcriptional activation by Sp1: evidence for coactivators. Cell 61:1187–1197[Medline]
  24. Kaufmann J, Smale ST 1994 Direct recognition of initiator elements by a component of the transcription factor IID complex. Genes Dev 8:821–829[Abstract]
  25. Dynlacht BD, Hoey T, Tjian R 1991 Isolation of coactivators associated with the TATA binding protein that mediate transcriptional activation. Cell 66:563–576[Medline]
  26. Adams CC, Workman JL 1995 Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative. Mol Cell Biol 15:1405–1421[Abstract]
  27. Das G, Hinkley CS, Herr W 1995 Basal promoter elements as a selective determinant of transcriptional activator function. Nature 374:657–660[CrossRef][Medline]
  28. Karlseder J, Rotheneder H, Wintersberger E Interaction of Sp 1 with the growth- and cell cycle-regulated transcription factor E2F. Mol Cell Biol 16:1659–1667
  29. Kang SH, Brown DA, Kitajima I, Xu H, Heidereich O, Gryaznov S, Nerenberg M 1996 Binding and functional effects of transcriptional factors Sp1 on the murine interleukin-6 promoter. J Biol Chem 271:7330–7335[Abstract/Free Full Text]
  30. Leggett RW, Armstrong SA, Barry D, Mueller CR 1995 Sp1 is phosphorylated and its DNA binding activity down regulated upon terminal differentiation of the liver. J Biol Chem 270:25879–25884[Abstract/Free Full Text]
  31. De Wet JR, Wood KV, Deluca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737[Medline]
  32. Pruijn GJ, van Driel W, van Miltenburg RT, van der Vliet PC 1987 Promoter and enhancer elements containing a conserved motif are recognized by nuclear factor III, a protein stimulating adenovirus DNA replication. EMBO J 6:3771–3778[Abstract]
  33. Maxam AM, Gilbert W 1980 Sequencing end labeled DNA with base specific chemical cleavages. Methods Enzymol 65:499–560[Medline]
  34. Knowles BB, Howe CC, Aden DP 1980 Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209:497–499[Medline]
  35. Gluzman Y 1981 SV40 transformed simian cells support the replication of early SV40 mutants. Cell 23:175–182[Medline]
  36. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning–A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY