(Received for publication, February 7, 1996)
From the
Sp3 is a member of the Sp family of transcription factors, and it binds to the GC box with an affinity and specificity comparable with that of Sp1. Previous studies have shown that Sp3 repressed Sp1-mediated transcriptional activation, suggesting that Sp3 is an inhibitory member of the Sp family. The experiments described here demonstrate that Sp3 contains a portable repression domain that can function independently from the zinc finger DNA-binding domain. We found that the amino-terminal region of Sp3 tethered to a promoter DNA by connecting to a heterologous DNA-binding protein domain represses transcriptional activation by different positive regulators. Moreover, we determined that Sp3 targeted to a promoter-proximal RNA sequence acts as a transcriptional repressor. Taken together, our results suggest that Sp3 functions as a repressor by protein-protein interaction with components of the general transcription complex.
Originally identified as a cellular transcription factor required for SV40 gene expression, Sp1 stimulates transcription by binding to a GC box present in a wide variety of cellular and viral promoters(1) . Recently, three Sp1-related genes (Sp2, Sp3, and Sp4) have been cloned based on their homology with the Sp1 DNA-binding domain(2, 3) . The DNA-binding domains of the Sp3 and Sp4 proteins are highly conserved, and they recognize GC boxes with specificity and affinity closely similar to that of Sp1. Contrary to these factors, Sp2 seems to have different DNA binding specificities(3) . It has been shown that both Sp1 and Sp3 proteins are ubiquitously expressed at a high level in many mammalian cell lines, whereas Sp4 expression appears to be restricted to certain cell types of the brain(2, 4, 5) . Functional analysis of Sp3 and Sp4 in direct comparison with Sp1, using transfection experiments into mammalian cell lines and Drosophila SL2 cells lacking endogenous Sp factors, demonstrated that Sp4, like Sp1, is a transcriptional activator of Sp1-responsive promoters, whereas Sp3 represses Sp1-mediated transcription(4, 5, 6, 7) . These results suggest that Sp3 is an inhibitory member of the Sp family.
The intriguing finding that Sp3 represses Sp1-mediated transcription prompted us to embark on an analysis of the transcriptional properties of Sp3. We performed in vivo transfections, in which the non-finger region of Sp3 repressor and a defined activating domain were both targeted to a promoter. We found that Sp3 contains a portable repressor activity, which can function independently from the zinc finger DNA-binding domain. In addition we demonstrated that the Sp3 amino terminus region is a transcriptional repressor of several activators, and the repression is not influenced by the arrangement of basal promoter elements. Finally, we determined that the Sp3 repressor is functional when targeted to a promoter-proximal RNA sequence. Our results indicate that Sp3 acts as a transcriptional silencing of RNA polymerase II promoters either when tethered to DNA by fusion to a DNA-binding protein domain or when targeted to a promoter-proximal RNA sequence.
Figure 1: A, schematic representation of the T7G5-TATA and T7G5-I reporter plasmids containing the E1b TATA or the AdMLP Inr sequences as core promoter element, respectively. B, reporter plasmids T7G5-TATA (2 µg, open bars) and T7G5-I (2 µg, solid bars) were cotransfected into HeLa cells with the GAL4 derivatives (1 µg) as indicated. The transcriptional activity of each GAL4 derivative relative to the sample without activator is diagrammed at the right. The values are representative of four independent duplicated experiments; vertical lines indicate the standard deviations.
To determine the Sp3-mediated repression of the activation function of each GAL4 derivative, the reporter plasmids described in Fig. 1A were co-transfected into HeLa cells with the indicated GAL4 fusion proteins in the presence of an increasing amount of the Tet-Sp3 expression vector. Co-expression of Tet-Sp3 protein was found to repress in a dose-dependent manner GAL4 chimeric protein-mediated activation of both the TATA and Inr-containing promoters (Fig. 2). The specificity of Sp3-mediated repression was demonstrated by the results reported in Fig. 2B, showing that neither the Tet-Sp3 fusion protein affected the activation function of a reporter lacking the tetO sequences nor did the pCMV-Sp3DBD, encoding the fingerless portion of Sp3 lacking the DNA-binding domain(6) , influence significantly promoter activity.
Figure 2: Sp3 represses transcription in the presence of defined activators. On the top a schematic representation of the chimeric protein Tet-Sp3 is shown. A, reporter plasmids T7G5-TATA (2 µg, open bars) and T7G5-I (2 µg, solid bars) were cotransfected into HeLa cells together with 1 µg of the indicated GAL4-activator plasmid in the presence of increasing amounts (1, 5, and 10 µg) of Tet-Sp3 expression vector in a total of 20 µg adjusted with the parental plasmid. CAT activities relative to the samples without repressor (taken as 100%) are shown. The values are representative of four independent duplicated experiments; vertical lines indicate the standard deviations. B, repression specifically depends upon DNA binding. T7G5-TATA (open bars) and G5E1b (striped bars) reporter plasmids (2 µg each) were transactivated with 1 µg of GAL4-VP16 expression plasmid. Coexpression of CMV-Sp3DBD (10 µg) or Tet-Sp3 (10 µg), respectively, did not affect transactivation.
Taken together the data from transfection experiments demonstrate that Sp3 suppresses transcription when allowed to bind next to the activator, and the extent of repression appears to be independent of the type of activator domain and of the presence of a specific core promoter element.
To clarify this point we chose to attempt regulation of HIV-1 promoter activity by designing a chimeric Tat negative trans-dominant mutant fused to the non-finger region of Sp3. We used the previously described pSVTat22/37, in which cysteines 22 and 37 have been substituted with glycine and serine, respectively(8) . It has been shown that these point mutations in the cysteine-rich region abolished the transacting effect of the protein(17) . The non-finger region of Sp3 (aa 1-527) was fused to the COOH terminus of Tat22/37 resulting in the pSVTat22/37-Sp3 expression vector. As reporter, the G5-83HIV-CAT plasmid, which contains five GAL4 DNA-binding sites located at position -83 of HIV-1 LTR(6) , was used. Using a similar reporter it has been shown (18) that the GAL4-VP16 fusion protein can potently activate expression from an HIV-1 LTR bearing multiple GAL4 DNA-binding sites. The G5-83HIV-CAT plasmid was transfected into HeLa cells together with the GAL4-VP16 effector plasmid in the presence of increasing amounts of pSVTat22/37 or pSVTat22/37-Sp3 effectors (see Fig. 3A). Accordingly with reported results the GAL4-VP16-mediated trans-activation of G5-83HIV-CAT reporter was very high(18) , and the presence of Tat22/37 did not change the promoter activity. Conversely, a dose-dependent repression of GAL4-VP16-mediated activation was observed in the presence of the Tat22/37-Sp3 fusion protein (Fig. 3A).
Figure 3:
Sp3 represses activated and basal HIV LTR
promoter activity when tethered to TAR. A, GAL4-VP16-mediated
activation of the HIV promoter is repressed by Sp3 targeted to TAR.
HeLa cells were transfected with G5-83HIV reporter (1 µg) in
the presence of GAL4-VP16 effector plasmid (1 µg) together with
increasing amounts of pSVTat22/37 or pSVTat22/37-Sp3 as indicated in a
total of 20 µg adjusted with the parental pSV expression vector.
The bars show the relative CAT activities, with CAT activity
obtained in the presence of GAL4-VP16 alone taken as 100%. B,
Sp3 represses basal HIV LTR promoter activity. HeLa cells were
transfected with the reporter G-83HIV (5 µg) alone (empty
bars) or in the presence of increasing amounts (5 and 10 µg)
of pSVTat22/37 (dashed bars) or increasing amounts (1, 5, and
10 µg) of pSVTat22/37-Sp3 (black bars) effectors as
indicated. C, similar transfections were performed using as
reporter the G5-83HIVTAR in which the TAR element was
destroyed by deleting TAR sequences downstream of position +25.
All CAT assays were determined as described in the text, after
normalization for the internal control
-galactosidase activity.
The values are representative of four independent duplicated
experiments; vertical lines indicate the standard
deviations.
To further substantiate the notion that
Sp3 targeted to nascent RNA is able to repress HIV-1 LTR promoter
activity we analyzed the Sp3-mediated repression of the LTR basal
activity. Dose-dependent repression of the HIV LTR basal activity was
mediated by Tat22/37-Sp3 fusion protein, whereas no significant effect
was observed in the presence of the Tat22/37 mutant (Fig. 3B). Moreover, Tat22/37-Sp3-mediated repression
was dependent on the presence of TAR element (Fig. 3C),
since the activity of the reporter plasmid G5-83HIV-TAR in
which the TAR sequences have been deleted was not affected by the
presence of the Tat22/37-Sp3 fusion protein. These data clearly
indicated that the Sp3 repressor domain can efficiently repress
transcription from nascent RNA target and demonstrated that
Sp3-mediated repression does not require a stable interaction with the
promoter DNA.
Several models can be envisaged to explain how Sp3 might repress transcription. For example, Sp3 may function by inactivating or squelching a protein that normally activates polymerase II expression (14, 15, 19) . This possibility seems very unlikely because Sp3-mediated repression is strictly dependent on binding in cis to the promoter or binding to a promoter-proximal RNA target. A number of repressors appear to use quenching as their mechanism. For example, the Drosophila Kruppel protein displays the ability to quench some activators but not others(20) . However, the quenching mechanism does not account for the ability of Sp3 to repress different types of activators, and the repression is not influenced by the presence of a specific core promoter element. Therefore, it appears that Sp3-mediated repression may act directly on the general transcription machinery.
The observation that Sp3 repressor function does not necessarily require an interaction with a DNA target sequence appears inconsistent with a repression model involving the formation on the DNA template of multiprotein complexes composed of Sp3 and other factors. Instead, it appears more likely that the RNA-bound Sp3 may interact directly with a component of the general transcriptional machinery and prevent an isomerization or disassembly step. Alternatively, Sp3 could load a putative ``corepressor(s)'' into the general transcription complex, which may then modify the initiation complex so that the rate of transcription is repressed. However, it remains to be demonstrated whether the mechanism of Sp3-mediated repression is the same when it is targeted to nascent RNA as it is when it is bound to promoter DNA.