(Received for publication, September 26, 1995; and in revised form, January 25, 1996)
From the
Transcription enhancer factor-1 (TEF-1) has been implicated in transactivating a placental enhancer (CSEn) that regulates human chorionic somatomammotropin (hCS) gene activity. We demonstrated that TEF-1 represses hCS promoter activity in choriocarcinoma (BeWo) cells (Jiang, S. W., and Eberhardt, N. L.(1995) J. Biol. Chem. 270, 13609-13915), suggesting that TEF-1 interacts with basal transcription factors. Here we demonstrate that hTEF-1 overexpression inhibits minimal hCS promoters containing TATA and/or initiator elements, Rous sarcoma virus and thymidine kinase promoters in BeWo cells. Cotransfection of TEF-1 antisense oligonucleotides alleviated exogenous TEF-1-mediated repression and increased basal hCS promoter activity, indicating that endogenous TEF-1 exerts repressor activity. GST-TEF-1 fusion peptides fixed to glutathione-Sepharose beads retained in vitro-generated human TATA-binding protein, hTBP. The TEF-1 proline-rich domain was essential for TBP binding, but polypeptides also containing the zinc finger domain bound TBP with higher apparent affinity. TBP supershifted hTEF-GT-IIC DNA complexes, but TEF-1 inhibited in vitro binding of TBP to the TATA motif. Coexpression of TBP and TEF-1 in BeWo cells alleviated TEF-1-mediated transrepression, indicating that the TBP-TEF-1 interaction is functional in vivo. The data indicate that TEF-1 transrepression is mediated by direct interactions with TBP, possibly by inhibiting preinitiation complex formation.
Transcription enhancer factor-1 TEF-1 ()appears to be
a ubiquitous factor that has been implicated in directing the
expression of a wide variety of genes, including the SV40 early promoter (Davidson et al., 1988; Xiao et
al., 1991; Gruda et al., 1993; Hwang et al.,
1993), human papillomavirus-16 E6 and E7 oncogenes
(Ishiji et al., 1992), muscle-specific genes (Shimizu et
al., 1993; Kariya et al., 1993; Stewart et al.,
1994), and the human chorionic somatomammotropin (hCS) gene
enhancer (CSEn) function in placental cells (Walker et al.,
1990; Jacquemin et al., 1994; Jiang and Eberhardt, 1994).
The mechanism of TEF-1 action has not been elucidated but appears to be complex. TEF-1 binds to sequences related to the general ``enhancer core consensus,'' 5`-TGTGG(T/A)(T/A)(T/A)G-3` (Weiher et al., 1983; Kariya et al., 1993), and mutation of the binding sites for TEF-1 results in loss of transcriptional activation (Xiao et al., 1991). Nevertheless, when cells are cotransfected with TEF-1 expression vectors and promoter constructs containing the TEF-1 binding site, inhibition or squelching of promoter activity instead of activation is typically observed (Xiao et al., 1991; Ishiji et al., 1992; Hwang et al., 1993; Jiang and Eberhardt, 1995). This has led to the concept that TEF-1 requires a limiting transcription factor for its transactivation function. In the case of the placenta-specific enhancer CSEn, we recently provided evidence that a factor, designated CSEF-1, which is distinct from TEF-1 and is abundant in choriocarcinoma and COS cells, bound to the GT-IIC and Sph-I/Sph-II enhansons with binding specificity identical to that of TEF-1 (Jiang and Eberhardt, 1995). CSEn transactivation in COS and BeWo cells was correlated with the presence of CSEF-1, but not TEF-1, since COS cells contain very low levels, if any, of TEF-1. This raises the question of whether TEF-1 mediates transrepression but not transactivation in some cell types.
To address these issues, we reexamined the effects of overexpression of hTEF-1 on hCS promoter and enhancer function in choriocarcinoma cells (BeWo). Exogenous TEF-1 expression led to marked repression of basal hCS promoter activity without affecting relative enhancer-mediated stimulation of promoter activity. Interestingly, this repression appears to be independent of a GT-IIC binding site and was observed with several promoters, suggesting that basal transcriptional factors might be involved. Cotransfection of hTEF-1 antisense oligonucleotides not only reversed these effects, but also stimulated basal hCS promoter activity, indicating that endogenous TEF-1 was acting as a repressor in BeWo cells. Based on these studies we undertook an analysis of the ability of TEF-1 to bind to known components of the basal transcription apparatus. We found that TEF-1 bound to TATA-binding protein (TBP) and that the TEF-1 proline-rich and zinc finger domains were involved in this interaction. Since Hwang et al.(1993) demonstrated that these domains were required for both autoinhibitory and stimulatory responses, our data suggest that TEF-1-TBP interactions were important for TEF-1 function in vivo. Interestingly, TEF-1 inhibited the binding of TBP to the hCS proximal promoter region containing the TATA box, whereas TBP supershifted TEF-1-GT-IIC DNA complexes. Importantly, overexpression of TBP in BeWo cells alleviated the TEF-1-mediated inhibition of hCS promoter activity, indicating that TBP-TEF-1 interaction is functional in vivo. Our data are consistent with the hypothesis that TEF-1 transrepression may be the result of interference with the binding of TBP to the TATA element and that TEF-1-mediated transactivation requires additional factors. Alternatively, transactivation formally attributed to TEF-1 may be mediated by different factors. The latter possibility is strongly supported in the case of the placental enhancer, since transactivation of CSEn is highly correlated with the distinct GT-IIC-binding factor, CSEF-1 (Jiang and Eberhardt, 1995).
Figure 1:
TEF-1 overexpression inhibits
basal but not enhancer-mediated activity of hCSp.LUC and EnA_hCSp.LUC gene activity in BeWo cells. Promoter activities
were expressed as light units/µg of protein ± S.E.
10
. Panel A, cotransfection of increasing
amounts of CMVp.TEF (40-1080 ng) with hCSp.LUC (15 µg) resulted in progressive repression of basal hCS promoter activity (ANOVA, p < 0.0001) and total
CSEn-stimulated activity (ANOVA, p < 0.0001). Panel
B, relative enhancer-stimulated activity (fold stimulation) from
the EnA hCSp.LUC gene was not affected by increasing amounts
(40-1,080 ng) of cotransfected CMVp.TEF DNA (ANOVA, p < 0.2).
The hCS promoter is
dominantly controlled by Sp1, TATA box, and initiator elements in BeWo
cells (Jiang et al., 1995). We therefore analyzed CS promoter mutants to determine which of these sequences might be
involved in mediating TEF-1 transrepression. Substitution mutation of
the InrE binding site (sequences hCS -20/+1, 493CS_APA), deletion of the TATA element (493CS_
TATA) in the context of the otherwise intact hCS promoter, or deletion of sequences upstream of the TATA
box, including the Sp1 site that contributes to basal hCS promoter activity (36CS) did not alleviate the inhibitory
activity of TEF-1 (Fig. 2A). Similarly, TEF-1
overexpression inhibited the activities of the heterologous RSV and TK promoters (Fig. 2, B and C) in a manner indistinguishable from the hCS promoter. This repression was not due to the presence of excess
promoter DNA, since cotransfection of large amounts of DNAs containing
the CMV or TK promoters was without effect on hCS, RSV, or TK promoter activity (Fig. 2, A-C). These data further support the
concept that overexpression of TEF-1 interferes with a common, basal
transcription mechanism.
Figure 2:
TEF-1 overexpression inhibits truncated hCS promoter constructs lacking important control elements and
the TK and RSV promoters. Promoter activities were
expressed as light units/µg of protein ± S.E.
10
. Panel A, various amounts of CMVp.TEF (5-5,000 ng) were cotransfected with 15 µg
of the 493CS_
APAp.LUC (hatched bars, lacks
initiator element), 493CS_
TATAp.LUC (stippled
bars, lacks TATA element), and 36CSp.LUC (checkered
bars, lacks Sp1 and other upstream elements) genes. To control for
the potential effects of cotransfection of large amounts of promoter
DNA, 5 µg of the TKp. CAT or CMVp.
GAL gene
was cotransfected along with the mutated CS promoter
constructs. ANOVA analyses for TEF-1 inhibition of 493CS_
APAp.LUC, 493CS_
TATAp.LUC, and 36CSp.LUC promoter activity were all significant at the p < 0.0001 level. Panel B, cotransfection of RSVp.LUC (15 µg) and increasing amounts (50-5,000
ng) of CMVp.TEF or CMVp.
GAL DNA (5 µg)
indicated that TEF-1 inhibited RSVp.LUC promoter activity
(ANOVA, p < 0.0005). Panel C, cotransfection of TKp.LUC (15 µg) and increasing amounts (50-5,000 ng)
of CMVp.TEF or CMVp.
GAL DNA (5 µg) indicated
that TEF-1 inhibited TKp.LUC promoter activity (ANOVA, p < 0.0113).
Figure 3:
TEF-1 represses hCSp.LUC mRNA
levels in BeWo cells. After transfection with hCSp.LUC (15
µg) and either 5 µg of CMVp.TEF or CMVp.GAL DNA, BeWo cell RNA was isolated and resolved on a 2% agarose gel. Upper panel, to control for sample loading and RNA quality, 28
S and 18 S RNA were revealed by UV shadowing. Lower panel, the hCSp.LUC transcripts were detected using
P-labeled luciferase cDNA
probe.
Figure 4: TEF-1 antisense oligonucleotide relieves transcriptional inhibition mediated by TEF-1 in BeWo cells. Data expressed as light units/µg of protein ± S.E. were derived from triplicate transfections. Panel A, the hCSp.LUC (15 µg) and CMVp.TEF (5 µg) DNAs and varying concentrations of antisense (diamonds) or sense (squares) TEF-1 oligonucleotides were introduced into BeWo cells. Panel B, the hCSp.LUC (15 µg) and varying concentrations of antisense (diamonds) or sense (squares) TEF-1 oligonucleotides were introduced into BeWo cells.
Figure 5:
TEF-1 interacts specifically with TBP but
not TFIIB. Panel A, expression of GST-TEF fusion protein in E. coli. GST-TEF fusion protein was expressed in HB101 and
purified using glutathione-Sepharose 4B beads. Aliquots (30 ng) of
thrombin-cleaved, intact GST-TEF fusion protein and GST peptide were
resolved by SDS-PAGE and visualized by silver staining. Panel
B, S-labeled hTBP, hTFIIB, and hTR
from in
vitro translation. No
S-labeled product was observed
from control reactions lacking plasmid DNA (TNT). Panel
C, recovery of
S-labeled proteins after binding to
GST-TEF-coated glutathione-Sepharose 4B
beads.
We next analyzed gel shift experiments with TEF and the GT-IIC enhanson in the presence or absence of TBP. When purified TBP was added to gel shift assays using different amounts of in vitro-generated TEF-1, a weak but visible supershift was observed (Fig. 6). Since neither GST protein nor TBP binds to the GT-IIC probe, the supershift represents the TBP-TEF-GT-IIC complex. This result confirms the TEF-TBP interaction and indicates that this protein-protein interaction does not affect the ability of TEF-1 to bind DNA.
Figure 6: TBP binds to TEF-GT-IIC complex resulting in supershifted complexes. Gel shift assays were performed with increasing amounts of GST-TEF and an oligonucleotide containing the GT-IIC enhanson. The addition of 2 footprinting units (fpu) of hTBP to the binding reaction resulted in a supershifted complex. No protein-DNA complex was observed when 500 ng of GST peptide or 2 fpu of TBP was incubated with the GT-IIC-containing oligonucleotide. The molar ratio of TBP to GST-TEF-1 was estimated to be about 2.1, assuming that the TEF-1 and TBP were 100% active, GST-TEF-1 was 50% pure (Fig. 5A), and using Promega quality control data on the recombinant TBP (95% pure as estimated from Coomassie-stained SDS-PAGE gels and 20 ng of protein/fpu).
Figure 7: TEF-1 inhibits the binding of TBP to DNA containing the TATA motif. Purified hTBP (4 footprinting units, fpu) and oligonucleotides containing the AdML-TATA box motif were used in gel shift assays in the presence or absence of varying amounts (100-300 ng) of in vitro-generated GST-TEF. Inhibition of TBP binding to the TATA motif was due to TEF-1, since GST peptide (500 ng) had no inhibitory effect. The molar ratios of TBP to GST-TEF-1 at 100, 200, and 300 ng input of GST-TEF-1 were estimated to be about 3.3, 1.7, and 1.1, respectively, as detailed in the legend to Fig. 6.
Figure 8: Schematic diagram indicating the localization of the P-rich, STY-rich, and zinc finger domains in TEF-1 required for transactivation and transrepression in HeLa cells (Hwang et al., 1993). The lower panel indicates the GST-TEF truncation polypeptides used to map the TBP binding domain. The ability of TBP to bind to the truncated polypeptides is indicated as derived from the data in Fig. 9C.
Figure 9:
Maximal TBP binding by truncated TEF-1
polypeptides is exhibited by those containing the P-rich and zinc
finger domains. Panel A, digestion of various pGEX-4T-3
plasmids containing the various truncated TEF-1 cDNA inserts. The
inserts were liberated with NotI/EcoRI except for
GST-TEF, which was obtained by NotI/BamHI digestion. The DNA standards are HindIII-digested
and HaeIII-digested
X
DNA. Panel B, E. coli expression of truncated forms
of TEF-1. GST-TEF fusion proteins purified from HB101 were resolved by
10% SDS-PAGE and stained with Coomassie Brilliant Blue (indicated by asterisks). Protein standards and a control HB101 bacterial
lysate are shown at the extreme left side of the panel. The purified GST peptide is shown in the lane marked pGEX-4T-3. Panel C,
S-labeled hTBP was incubated with glutathione-Sepharose 4B
beads coated with truncated and wild-type GST-TEF polypeptides. Input
S-labeled hTBP is shown in the extreme left lane.
Proteins retained on beads were recovered and resolved by 10%
SDS-PAGE.
Figure 10:
Overexpression of TBP inhibits
TEF-1-mediated transrepression of hCS promoter activity in
BeWo cells. The hCS promoter activities were expressed as
light units/µg protein ± S.E. 10
.
Fifteen µg of hCSp.LUC was cotransfected with various
amounts of CMVp.TEF (0, 0.2, 1.0, and 5 µg) along with 0.0 (open bars), 0.2 (striped bars), or 1.0 µg (solid bars) of the TBP expression vector hIID-CX.
The data for each condition represent the results from three
independent transfections. ANOVA analyses for TEF-1 inhibition of hCSp.LUC activity in the absence and presence of 0.2 µg of
the hIID-CX expression plasmid were significant at the p < 0.0002 and p < 0.0034 levels, respectively. ANOVA
analysis was not significant for the group transfected with 1.0 µg
of the hIID-CX expression plasmid (p < 0.24).
Significance levels within the three groups for post hoc Bonferonni t tests are
indicated.
TEF-1 is proposed to transactivate a variety of eukaryotic enhancers and promoters, including the SV40 (Davidson, 1988; Xiao et al., 1991; Hwang et al., 1993) and hCS (Walker et al., 1990; Jacquemin et al., 1994; Jiang and Eberhardt, 1994, 1995) enhancers, muscle-specific genes (Shimizu et al., 1993; Kariya et al., 1993; Stewart et al., 1994), human papillomavirus-16 E6 and E7 oncogenes (Ishiji et al., 1992), and mouse early developmental genes (Melin et al., 1993). Since overexpression of TEF-1 in various cells results in transcriptional squelching (Xiao et al., 1991; Ishiji et al., 1992; Hwang et al., 1993; Jiang and Eberhardt, 1995), it has been proposed that other limiting transcription factors along with TEF-1 are required for transactivation.
Recently we found that another factor, CSEF-1,
possibly unrelated to TEF-1, is correlated with CSEn transactivation in
choriocarcinoma cells (BeWo). In these cells TEF-1 overexpression
failed to activate the hCS enhancer and repressed hCS promoter activity in the absence of the enhancer (Jiang and
Eberhardt, 1995). These results mirror those found with the myosin
heavy chain gene, in which two distinct factors, a ubiquitous
mouse TEF-1 homolog and an unrelated muscle-specific factor, bind to
the GT-IIC-related element, and the ubiquitous TEF-1 homolog failed to
transactivate myosin heavy chain
gene expression in mouse
skeletal muscle cells (Shimizu et al., 1993). Also, Stewart et al.(1993) have shown that GAL4 chimeras containing a novel
isoform of chicken TEF-1 which has 13 additional COOH-terminal amino
acids can transactivate GAL4-dependent reporter genes, whereas chimeras
corresponding to the ubiquitous hTEF-1 isoform only exhibit squelching
activity. These studies raise the possibility that the dominant
function of the ubiquitous form of TEF-1 is a repressor.
The present studies expand our previous observation that TEF-1 represses hCS promoter activity independently of the presence of GT-IIC motifs (Jiang and Eberhardt, 1995). Consistent with the concept that TEF-1 acts at the level of transcription, steady-state levels of luciferase transcripts were diminished in BeWo cells cotransfected with the hCSp.LUC and CMVp.TEF-1 genes (Fig. 3). Repression was not restricted to the hCS promoter, since it occurs with the heterologous TK and RSV promoters (Fig. 2, B and C), which have not been previously known to respond to TEF-1. Furthermore, TEF-1 inhibits transcription from the minimal hCS promoter ( Fig. 2A), which contains a TATA box and an initiator element (Jiang et al., 1995). Equivalent repression was observed with constructs lacking either the TATA or initiator element (Fig. 2A). Thus TEF-1-mediated repression may involve interactions with basal transcriptional factors that can function with both TATA and initiator elements. The TEF-1-mediated repression could be overcome by cotransfecting antisense oligonucleotides (Fig. 4). More importantly, the data demonstrate that endogenous levels of TEF-1 negatively regulate hCS promoter activity in BeWo cells, since antisense oligonucleotides increased hCS promoter activity in the absence of cotransfected CMVp.TEF-1 (Fig. 4). This finding suggests that TEF-1 does not function as a transactivator in these cells.
Previous studies
suggested that TEF-1 might interact with TBP. First, in studies
primarily directed at SV40 T antigen-TEF-1 interactions, it
was observed that TBP could bind to TEF-1 (Gruda et al.,
1993). Second, Brou et al.(1993) had shown that the activity
of GAL-TEF-1 chimeras appeared to be mediated by at least two distinct
classes of TFIID complexes. Using GST-TEF-1 pull-down assays we
confirmed the observation of Gruda et al.(1993), demonstrating
that TEF-1 specifically interacts with hTBP, but not hTFIIB or as a
control the human TR receptor (Fig. 5C).
Interestingly, TBP was able to form supershifted complexes with TEF-1
and DNA containing the GT-IIC enhanson (Fig. 6); however, TEF-1
inhibited the ability of TBP to bind to the TATA element (Fig. 7), suggesting that TEF-1 binding to TBP might inhibit
preinitiation complex formation. We localized an essential region for
binding TBP to the proline-rich sequences located between amino acids
143 and 204 (Fig. 9C). In addition, polypeptides
containing the zinc finger domain bound TBP with higher affinity (Fig. 9C). Thus two of the three TEF-1 domains shown to
be required for its transactivation and transrepression in HeLa cells
(Hwang et al., 1993) are required for optimal TBP binding. Our
studies indicate that the domains containing the proline-rich and zinc
finger sequences link TEF-1 to the basal transcriptional machinery
through its ability to bind TBP.
On both the TATA-containing and non-TATA-containing promoters, TBP as part of the TFIID complex is the first component to enter the preinitiation complex. The association of TBP with the promoter may be rate-limiting (Chatterjee and Struhl, 1995; Klages and Strubin, 1995). Accordingly, it is not surprising that a large number of viral and cellular transcriptional factors have been found to interact with TBP (Table 2). These factors represent various classes of DNA-binding and non-DNA-binding proteins, and their interaction with TBP produces a variety of responses. For example, NC1, NC2, and DBF4 (Meisterernst et al., 1991) and Dr1 (Inostroza et al., 1992) do not affect the binding of TBP with DNA, but they do prevent the association of TBP with TFIIA and/or TFIIB. TFIIA increases the stability of the TBP-TATA complex (Yokomori et al., 1994) and in conjunction with TBP may be essential for committing a complex to transcription (Auble and Hahn, 1993). A yeast transcription factor, ADI, disrupts the TBP-TATA complex in an ATP-dependent manner, but this effect is blocked by prior association of TFIIA (Auble and Hahn, 1993). The HBV protein X-TBP interaction is also inhibited by nonhydrolyzable ATP analogs, suggesting that protein interactions with TBP which are ATP-dependent may be common. The Drosophila p230 subunit of TFIID inhibits the TBP TATA binding activity and represses transcription (Kokubo et al., 1994), activities that are shared by TEF-1.
Importantly, the TEF-1-mediated
transrepression of the hCS promoter is alleviated by the
cotransfection of increasing amounts of TBP expression plasmids (Fig. 10), indicating that TBP and TEF-1 functionally interact in vivo. This result provides strong support for the concept
that the in vitro TBP-TEF-1 interactions observed in these
studies represents a physiologically relevant interaction and suggests,
but does not prove, that TEF-1 can interact with holo-TFIID. It is
noteworthy that when overexpressed in eukaryotic cells, like TEF-1,
many of the TBP-binding factors (Table 2) cause strong squelching
effects on heterologous promoters. Tax1 transactivates several viral
and cellular promoters and enhancers through specific DNA elements.
Tax1 interacts specifically with TBP and exerts strong squelching
effects on transcription (Caron et al., 1993). Like the
TBP-mediated inhibition of TEF-1 repression (Fig. 10),
overexpression of TBP was able to stimulate the transactivation of
GAL4-Tax1 chimeras and partially alleviates Tax1-mediated squelching
(Caron et al., 1993). Also, c-Fos- and FosB-mediated
inhibition of transcription can be partially relieved by overexpression
of TBP (Metz et al., 1994a, 1994b). These studies suggest that
TBP levels may be limiting, in which case squelching mechanisms
involving additional factors are considered unlikely to explain the
transcription inhibition. However, although TBP overexpression
increased p65/NFB-mediated transactivation, neither TBP nor TFIIB
overexpression was able to alleviate p65/NF
B-mediated squelching
(Schmitz et al., 1995), suggesting that additional proteins
were required for transactivation in this case. Also, in BeWo cells,
overexpression of TBP itself results in reduced promoter activity,
suggesting that other limiting factors may be involved in mediating hCS promoter function.
Metz et al. (1994b) have provided evidence for the existence of a TBP binding motif that is shared by VP16, E1A, and c-Fos. We therefore compared the essential proline-rich TEF-1 domain and a number of other transcription factors that were known to bind to TBP and whose TBP binding domain had been narrowly mapped. Alignment of the sequences around the TBP binding motif, (F/L)V(F/L)D, indicates that TEF-1 shares some homology with these TBP-binding proteins (Fig. 11). Interestingly, TEF-1, p53, and VP16 show a region of homology just downstream of the putative TBP binding motif in a proline-rich domain, suggesting that these factors are more closely related, and the proline-rich region might constitute part of a conserved TBP binding domain.
Figure 11: Comparison of identified TBP binding domains among transcription factors, whose interaction with TBP is essential for transcriptional activity. The protein sequences (Swissprotein data base) were initially aligned with the program Pileup (Wisconsin Sequence Analysis Package), and additional gaps were introduced manually to obtain maximal alignment. Underlined sequences in p230 have been shown to be important for TBP binding (Kokubo et al., 1994), and the arrows identify sites that have been demonstrated to be important for TBP binding in c-Fos (Metz et al., 1994b). Uppercase letters indicate homology between two sequences; uppercase bold letters indicate homology among three or more sequences.
Although TEF-1 blocks the formation of TBP-TATA complexes, this does not exclude the possibility that TEF-1 can serve as a transactivator in certain cell types. In such cells, additional cofactors may prevent TEF-1 inhibition of TBP-TATA complex formation in a manner similar to TFIIA inhibition of ADI-mediated disruption of the TBP-TATA complex (Auble and Hahn, 1993). However, the data do support the concept that TEF-1 may be an exclusive repressor in some cell types. This conclusion is strengthened by our recent findings that a 30-kDa GT-IIC-binding factor, apparently unrelated to TEF-1, is correlated with hCS enhancer activity in COS-1 cells, which express very low levels of TEF-1 (Jiang and Eberhardt, 1995). Accordingly, it is possible that TEF-1 provides a counterregulatory stimulus to the actions of other factors that mediate transactivation through the GT-IIC enhanson.