(Received for publication, July 12, 1995)
From the
Sp4 is a human sequence-specific DNA binding protein with structural features similar to those described for the transcription factors Sp1 and Sp3. These three proteins contain two glutamine-rich regions and a highly conserved DNA binding domain composed of three zinc fingers. Consistently, Sp1, Sp3, and Sp4 do have the same DNA binding specificities. In this report, we have embarked on a detailed analysis of the transcriptional properties of Sp4 in direct comparison to Sp1 and Sp3. Cotransfection experiments into Drosophila SL2 cells lacking endogenous Sp factors demonstrate that Sp4 is an activator protein like Sp1. However, in contrast to Sp1, Sp4 is not able to act synergistically through adjacent binding sites. The transactivation function of Sp4 resides, like that of Sp1, in the N-terminal glutamine-rich region. Sp4 can function as a target for the Sp1 activation domains in a superactivation assay, suggesting that the activation domains of Sp1 and Sp4 are functionally related. Furthermore, we show that Sp4-mediated transcriptional activation can be repressed by Sp3. Taken together, our results demonstrate that the transcription factor Sp4 exhibits specific functional properties distinct from Sp1 and Sp3.
The properly timed and coordinated expression of eukaryotic genes requires the combinatorial action of multiple sequence-specific DNA binding proteins. These transcription factors recognize distinct promoter and enhancer elements, thereby acting positively or negatively on transcription. One of the first and best characterized mammalian transcription factors was Sp1 (1, 2) which binds to GC boxes and related motifs (3) present in many promoters. However, Sp1 is not the only protein binding to and acting through these DNA motifs. At least two other more recently cloned human proteins, designated Sp3 and Sp4, do bind with identical affinity to the same recognition sequence as Sp1 (4) . Note that Sp2, yet another factor homologous to Sp1, seems to have DNA binding specificities different from Sp1, Sp3, and Sp4(5) .
Sp1, Sp3, and Sp4 represent a family of GC box binding proteins with very similar structural features. In addition to the highly conserved DNA binding domain close to the C terminus, all three proteins contain two glutamine- and serine/threonine-rich amino acid stretches in the N-terminal part of the molecule. For Sp1, the glutamine-rich domains have been identified as transactivation domains(2, 6) . Two additional domains of Sp1 (C and D) located adjacent to the zinc finger region also influence the transcriptional activation function, one being weakly basic (C) and the other (D) showing no significant homology to known activation domains(6) . The D domain of Sp1 plays a key role in mediating the ability of Sp1 to activate transcription synergistically (7) .
The high degree of structural conservation between Sp1, Sp3, and Sp4 suggested that Sp3 and Sp4 do exert similar activation functions. A functional analysis of Sp3 using transfection experiments into mammalian cell lines and into Drosophila SL2 cells lacking endogenous Sp factors demonstrated, however, that Sp3 is not simply a functional equivalent of Sp1. Sp3 failed to activate Sp1-responsive promoter constructs. Instead, it repressed Sp1-mediated transcriptional activation(8, 9, 10) , suggesting that Sp3 is an inhibitory member of the Sp family. The intriguing finding that Sp1 and Sp3 can exert opposite transcriptional regulation prompted us to analyze thoroughly the transcriptional properties of the Sp4 protein.
We have performed cotransfection experiments into mammalian cells and into insect cells that lack endogenous Sp factors. Our studies demonstrate that Sp4 is an activator protein like Sp1. However, in contrast to Sp1, Sp4 is not able to act synergistically through adjacent binding sites. Moreover, Sp4-mediated activation is strongly enhanced (superactivated) in the presence of a non-DNA binding mutant of Sp1, suggesting that Sp1 can interact directly with Sp4. We show further that Sp4-mediated transcriptional activation is repressed by Sp3. Our results thus demonstrate that Sp4 exhibits a unique spectrum of functional properties distinct from those found for Sp1 and Sp3.
The reporter plasmid G5E1bSV is a derivative of the plasmid G5E1b (12) and was constructed as follows. The uteroglobin promoter in the plasmid pUG(-395)CATSV (13) was replaced by a 130-base pair PstI-BamHI fragment from G5E1b containing five Gal4 binding sites fused to the E1bTATA box.
Sp4 expression vectors for Drosophila melanogaster Schneider cells (SL2 cells) were
generated as follows. The plasmid pPacSp4 was obtained by cloning a
3-kb HindIII-NotI fragment from pBS-Sp4 into the
single BamHI site of pPac via decameric BamHI
linkers. Sp4 expression plasmids containing the Ubx leader sequence
(pPac773Sp4 and pPac747Sp4) were generated by replacing the Sp1 cDNA in
pPacSp1 (6) by the 2.7-kb SmaI or the 2.6-kb blunted SauI-XhoI fragment, respectively, from the pBS-Sp4
plasmid A8O (4) via 8-mer and 12-mer XhoI linkers. The
expression plasmids for Sp1 (pPacSp1) and fingerless Sp3
(pPacSp3ZnD = pPacSp3-DBD in (8) ) were described
previously(6, 8) . The expression plasmid for the
fingerless Sp1 mutant (pPacSp1
ZnD), in which the 165 C-terminal
codons of Sp1 were removed, was obtained by replacing the Sp4 insert of
pPac747Sp4 by a 1.6-kb BamHI fragment of pPacSp1 leading to
pPacSp1
ZnD. The plasmid for the expression of the DNA binding
domain of Sp3 (pPacSp3DBD) was generated by replacing the NdeI-XbaI insert of an expression plasmid for
dTAFII110 (pPacG4-110, kindly provided by R. Tjian) with a 0.8-kb NdeI-XbaI fragment obtained from
pET-3c/A3O(4) .
SL2 cells (17) were maintained in
Schneider medium supplemented with 10% fetal calf serum at 25 °C. 1
day prior to transfection, cells were plated onto 6-cm plastic dishes
at a density of 4.3 10
cells per plate. Cells were
transfected by the calcium phosphate method described by DiNocera and
Dawid(18) . Every plate received up to 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.
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.(19) . Protein concentrations in the CAT
assays 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 acetylated and non-acetylated forms of
[C]chloramphenicol was performed with an
automated Imaging Scanner (United Technologies Packard). The ratio of
acetylated to total chloramphenicol was displayed as percentage of
conversion. The
-galactosidase assays were performed according to
Hall et al.(20) . The values were used to normalize
the CAT conversion data for plate to plate variations in transfection
efficiency.
The sequences of the oligonucleotides were as follows: GT box binding site, 5`-AGCTTCCGTTGGGGTGTGGCTTCACGTCGA-3` and 3`-TCGAAGGCAACCCCACACCGAAGTGCAGCT-5`; Gal4 binding site, 5`-GCTTAGCGGAGTACTGTCCTCCGATCCC-3` and 3`-CGAATCGCCTCATGACAGGAGGCTAGGG-3`; unspecific oligonucleotide, 5`-CAGCGACTAACATCGATCGC-3` and 3`-GTCGCTGATTGTAGCTAGCG-5`.
Figure 1: Transient expression of Sp1 and Sp4 proteins in SL2 cells. Gel retardation assays were performed with crude nuclear extracts from SL2 cells. Cells were transfected with 8 µg of pPac vector (lanes 1-3), 8 µg of pPacSp4 (lanes 4-6), or 8 µg of pPacSp1 (lanes 7-9). In lane 10, a bacterial extract containing Sp4 was used as control(4) . All reactions contained 0.1 ng of labeled GT oligonucleotide. In lanes 2, 5, and 8, a 50-fold molar excess of a nonspecific oligonucleotide (U) and in lanes 3, 6, and 9, a 50-fold molar excess of unlabeled GT oligonucleotide (S) was included in the binding reaction. Arrows indicate the free oligonucleotide and specifically retarded protein-DNA complexes, respectively.
To test the putative transcriptional activity of Sp4 in direct comparison with Sp1, we cotransfected expression vectors for Sp4 and Sp1 together with BCAT-1 as test promoter construct (see Fig. 2A). BCAT-1 contains a single Sp1 binding site from the HIV promoter fused to the E1b TATA box and the CAT gene. This plasmid has been used to characterize activation domains of Sp1 in SL2 cells(7) . A constant amount of the reporter plasmid BCAT-1 was transfected into Schneider cells along with 4 µg of the Sp4 expression plasmid or various amounts of Sp1 expression plasmid. Under these conditions, Sp4 activated the test promoter 5-6-fold. Essentially the same degree of activation was obtained with 20 ng of the Sp1 expression plasmid (Fig. 2A). Electrophoretic mobility shift analysis experiments with nuclear extracts prepared from these two plates revealed that equal amounts of Sp1 and Sp4 protein engender roughly equal activation of BCAT-1 (Fig. 2B). Thus, it appears that Sp4 can mediate transcriptional activation of BCAT-1 in SL2 cells to the same extent as Sp1.
Figure 2: Activation properties of Sp4 in SL2 cells in comparison to Sp1. A, 8 µg of the reporter plasmids BCAT-1 or BCAT-2, respectively, were transfected into SL2 cells along with variable amounts of pPacSp1 (2, 10, 20, 100, and 200 ng) or 4 µg of pPacSp4 as indicated. The cells were subsequently lysed, and CAT activities were determined as described under ``Experimental Procedures.'' B, gel retardation assays with crude nuclear extracts from SL2 cells transfected with 20 ng of pPacSp1 (lanes 2-4), 4 µg of pPacSp4 (lanes 5-7), or 4 µg of vector (pPac) (lane 1). All reactions contained 0.1 ng of labeled GT oligonucleotide. In lanes 2 and 7, a 20-fold molar excess of unlabeled GT oligonucleotide (S) and in lanes 3 and 6, a 20-fold molar excess of a nonspecific oligonucleotide (U) was included in the binding reaction. Arrows indicate the free oligonucleotide and specifically retarded protein-DNA complexes, respectively.
Figure 3: Activation properties of Gal4-Sp fusion proteins in the mammalian cell line Ishikawa. A, schematic representation of the expression constructs Gal4, Gal4-Sp1, Gal4-Sp3, and Gal4-Sp4. The hatched boxes indicate the glutamine-rich domains designated A and B. B, transient expression of Gal4-Sp fusion proteins in Ishikawa cells. Gel retardation assays were performed with crude nuclear extracts from Ishikawa cells transfected with 8 µg of expression plasmids for Gal4-Sp1 (lanes 2-4), Gal4-Sp3 (lane 5), Gal4-Sp4 (lane 6), or mock DNA (pUC8 plasmid) (lane 1). All reactions contained 0.2 ng of labeled Gal4 oligonucleotide and 2.4 µg of protein extract. In lanes 3 and 4, a 100-fold molar excess of unlabeled Gal4 oligonucleotide (S) or nonspecific oligonucleotide (U) was included in the binding reaction. C, transactivation of G5E1bSV. Ishikawa cells were transfected with 2 µg of G5E1bSV along with 2 µg of expression plasmids for Gal4, Gal4-Sp1, Gal4-Sp3, or Gal4-Sp4 as indicated. The cells were subsequently lysed and assayed for CAT activities. The CAT values are expressed relative to the CAT activity obtained with the Gal4 expression plasmid, which has been given the arbitrary value of 1. The mean value and the standard deviation of at least three transfections are displayed.
Next, we performed cotransfection experiments with G5E1bSV as reporter plasmid. G5E1bSV is a derivative of the plasmid G5E1b(12) . It contains five Gal4 binding sites fused to the E1b TATA box, the CAT gene, and the SV40 enhancer. These experiments revealed that the N-terminal region of Sp4 can stimulate transcription efficiently in Ishikawa cells (Fig. 3C). Essentially the same degree of activation was obtained with the corresponding domains of Sp1 but not with those of Sp3. This result suggests that the glutamine-rich domains of Sp4 possess the potential for transcriptional activation like those present in Sp1. The corresponding N-terminal region of the Sp3 protein, however, appears to be inactive under these conditions.
To test further the functional relationship between Sp1 and Sp4, we asked whether Sp4 could function as a target for the Sp1 activation domains in a superactivation assay. We performed a series of gene transfer experiments into SL2 cells with the Sp4 and Sp1 expression constructs in the absence and presence of an expression construct for an Sp1 deletion mutant lacking the DNA binding domain. As reporter constructs, we used again BCAT-1 and BCAT-2. The results of these experiments are summarized in Fig. 4. Consistent with previous results obtained with the SV40 early promoter, which contains six Sp1 binding sites(7, 27) , fingerless Sp1 enhanced Sp1-mediated activation of BCAT-2 (two Sp1 binding sites) up to 10-fold (Fig. 4B). Surprisingly, fingerless Sp1 was not able to enhance Sp1-mediated transcriptional activation from a promoter that contains only one Sp binding site (Fig. 4C). Essentially the same extent of superactivation by fingerless Sp1 on BCAT-2 as reporter construct was achieved with Sp4 as activator (Fig. 4D). This result demonstrates that the N-terminal part of Sp1 can interact functionally not only with Sp1 but also with Sp4. Very likely, the functional interaction between fingerless Sp1 and Sp4 reflects specific protein-protein interactions involving some portions of the Sp1 and Sp4 molecules closely linked to their activation domains (see ``Discussion'').
Figure 4:
Superactivation of the transcription
factor Sp4 by a DNA binding-deficient mutant of Sp1. A,
schematic illustration of the activator plasmids for Sp1 and Sp4 and
the fingerless mutant of Sp1 (Sp1ZnD). The hatched boxes and the black bars indicate the glutamine-rich domains
and the zinc fingers, respectively. B and C, 8 µg
of BCAT-2 (B) or BCAT-1 (C) were transfected with
increasing amounts of pPacSp1 in the absence (open circles)
and presence (solid triangles) of 1 µg of an expression
plasmid for a fingerless Sp1 mutant (Sp1
ZnD). D, 8 µg
of BCAT-2 were transfected with different amounts of pPacSp4 (20, 200,
and 2000 ng) in the absence (
) and presence (
) of 1 µg
of an expression plasmid for a fingerless Sp1 mutant (Sp1
ZnD). E, schematic representation of the model for the
superactivation of Sp1 and Sp4 by a fingerless mutant of Sp1. At least
two binding sites for Sp1 are necessary for the enhancement of the Sp1
activity by the superactivator Sp1
ZnD.
Figure 5:
Sp4-mediated transcriptional activation
is repressed by Sp3. 8 µg of BCAT-2 were transfected along with 4
µg of pPacSp4 (+) and increasing amounts of pPacSp3 (+,
20 ng; ++, 200 ng; and +++, 2000 ng),
pPacSp3ZnD (++, 200 ng; and +++, 2000 ng)
or pPacSp3DBD (++, 200 ng; and +++, 2000 ng)
as indicated. The structure of the Sp4, Sp3, Sp3
ZnD, and Sp3DBD
proteins is illustrated schematically. The cells were subsequently
lysed and assayed for CAT activities.
Under conditions where equal
amounts of Sp4 and Sp1 protein were detectable in SL2 nuclear extracts,
the extent of activation of a reporter construct containing a single Sp
binding site was similar. However, significant differences became
apparent with a reporter construct containing two binding sites. In
contrast to Sp1, Sp4 was not able to activate this construct
synergistically. What might be the molecular basis for this
observation? Since DNA binding studies failed to detect any evidence of
Sp1 binding cooperatively to two adjacent sites(7) , ()DNA binding is not the key to explain these differences
between Sp1 and Sp4.
Probably, the synergistic effect of Sp1 occurs at steps following DNA binding by generating more effective activation surfaces(7) . The regions of the Sp1 molecule, which are necessary for synergistic activation, have been mapped extensively(7) . Three domains, the glutamine-rich activation domains A and B and the most C-terminal region of Sp1 (domain D), are essential for the ability of Sp1 to activate transcription synergistically from two adjacent sites. Thus, differences in either of these domains may account for the failure of Sp4 to activate transcription synergistically. Sequence comparison of the D-domain of Sp1 with the corresponding domain of Sp4 revealed no significant homologies within this region. The absence of a functionally active domain D in Sp4 may thus account for the lack of synergistic activation. This interpretation is supported by our gene transfer experiments into mammalian cells using Gal4-Sp expression vectors. These constructs do not contain the most C-terminal domain of Sp1 (domain D). Consistently, the N-terminal region of Sp4 containing two glutamine-rich domains exhibits activation properties similar to the N-terminal region of the Sp1 molecule lacking the D domain.
The similarity of the glutamine-rich domains of Sp1 with those of Sp4 prompted us to consider a possible functional relationship between Sp1 and Sp4. We found that the N terminus of Sp1 is indeed able to superactivate Sp4-mediated transcriptional activation, suggesting that the non-DNA binding form of Sp1 directly interacts with Sp4. The implication of this finding is that the glutamine-rich domains of Sp4 and those of Sp1 are functionally related to each other. It should be noted that superactivation does not appear to be a general phenomenon of glutamine-rich activation domains but rather a factor-specific property. For instance, the Drosophila antennapedia and bicoid transcription factors cannot be superactivated by Sp1(27, 28) , suggesting that the glutamine-rich domains of these factors are functionally unrelated to those of Sp1 and Sp4.
The only protein besides Sp4 that has been shown to function as a target for Sp1 activation domains in a superactivation assay is the Drosophila TATA-box binding protein associated factor 110 (dTAFII110)(28) . Since Sp1 binds and requires dTAFII110 for activation in vitro(29) , it has been suggested that dTAFII110 may function as a coactivator by serving as a site of protein-protein contacts between Sp1 and the TFIID complex.
Recently, one of the two glutamine-rich domains of Sp1 (region B) has been mapped in more detail(30) . Certain bulky hydrophobic residues rather than the glutamine residues within this region are responsible for dTAFII110 interaction and transcriptional activation. Close inspection of the homologous region of Sp4 revealed a very similar glutamine-rich hydrophobic patch in Sp4 (Fig. 6), suggesting that the homologous glutamine-rich domains of Sp1 and Sp4 share functional equivalence. So far, we were not able to demonstrate a functional interaction between Sp4 and dTAFII110 in a superactivation assay. However, this negative result may be due to the low expression level of the Sp4 constructs in SL2 cells. Other experimental approaches, for instance a two-hybrid assay in yeast, could help to clarify this point.
Figure 6: Comparison of glutamine-rich regions present in Sp1 and Sp4. The Sp1 activation domain B (amino acids 450-473) has been shown to interact with dTAFII110(30) . Residues in Sp1B that are sensitive to mutations are indicated by asterisks. The glutamine-rich domains of Sp1B (amino acids 450-473), Sp4B (amino acids 462-486), Sp1A (amino acids 165-188), and Sp4A (amino acids 164-187) share a similar array of glutamine residues and large hydrophobic residues (circled).