Cloning of an Inr- and E-box-binding protein, TFII-I, that interacts physically and functionally with USF1
1997; Springer Nature; Volume: 16; Issue: 23 Linguagem: Inglês
10.1093/emboj/16.23.7091
ISSN1460-2075
Autores Tópico(s)RNA regulation and disease
ResumoArticle1 December 1997free access Cloning of an Inr- and E-box-binding protein, TFII-I, that interacts physically and functionally with USF1 Ananda L. Roy Ananda L. Roy Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Department of Pathology and Program in Immunology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA, 02111 USAA.L.Roy and H.Du contributed equally to this work Search for more papers by this author Hong Du Hong Du Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Division and Program of Human Genetics, Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati, Cincinnati, OH, 45229 USAA.L.Roy and H.Du contributed equally to this work Search for more papers by this author Polly D. Gregor Polly D. Gregor Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Memmorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY, 10021 USA Search for more papers by this author Carl D. Novina Carl D. Novina Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Department of Pathology and Program in Immunology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA, 02111 USA Search for more papers by this author Ernest Martinez Ernest Martinez Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Search for more papers by this author Robert G. Roeder Corresponding Author Robert G. Roeder Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Search for more papers by this author Ananda L. Roy Ananda L. Roy Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Department of Pathology and Program in Immunology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA, 02111 USAA.L.Roy and H.Du contributed equally to this work Search for more papers by this author Hong Du Hong Du Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Division and Program of Human Genetics, Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati, Cincinnati, OH, 45229 USAA.L.Roy and H.Du contributed equally to this work Search for more papers by this author Polly D. Gregor Polly D. Gregor Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Memmorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY, 10021 USA Search for more papers by this author Carl D. Novina Carl D. Novina Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Department of Pathology and Program in Immunology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA, 02111 USA Search for more papers by this author Ernest Martinez Ernest Martinez Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Search for more papers by this author Robert G. Roeder Corresponding Author Robert G. Roeder Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA Search for more papers by this author Author Information Ananda L. Roy1,2, Hong Du1,3, Polly D. Gregor1,4, Carl D. Novina1,2, Ernest Martinez1 and Robert G. Roeder 1 1Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021 USA 2Department of Pathology and Program in Immunology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA, 02111 USA 3Division and Program of Human Genetics, Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati, Cincinnati, OH, 45229 USA 4Memmorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY, 10021 USA The EMBO Journal (1997)16:7091-7104https://doi.org/10.1093/emboj/16.23.7091 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The transcription factor TFII-I has been shown to bind independently to two distinct promoter elements, a pyrimidine-rich initiator (Inr) and a recognition site (E-box) for upstream stimulatory factor 1 (USF1), and to stimulate USF1 binding to both of these sites. Here we describe the isolation of a cDNA encoding TFII-I and demonstrate that the corresponding 120 kDa polypeptide, when expressed ectopically, is capable of binding to both Inr and E-box elements. The primary structure of TFII-I reveals novel features that include six directly repeated 90 residue motifs that each possess a potential helix–loop/span–helix homology. These unique structural features suggest that TFII-I may have the capacity for multiple protein–protein and, potentially, multiple protein–DNA interactions. Consistent with this hypothesis and with previous in vitro studies, we further demonstrate that ectopic TFII-I and USF1 can act synergistically, and in some cases independently, to activate transcription in vivo through both Inr and the E-box elements of the adenovirus major late promoter. We also describe domains of USF1 that are necessary for its independent and synergistic activation functions. Introduction Transcription of eukaryotic protein-coding genes is initiated by interactions of RNA polymerase II and general initiation factors at common core promoter elements, and is regulated by various gene-specific activators that act through adjacent or distal regulatory elements (Roeder 1991, 1996; Conaway and Conaway, 1993; Zawel and Reinberg, 1995; Orphanides et al., 1996). Therefore, communication between the activation and basal (core promoter) components of eukaryotic transcription is critical for appropriate gene expression. In metazoans, the most common core promoter elements, which can act independently or in concert to determine the transcription start site, are the TATA box near position −30 and a pyrimidine-rich initiator (Inr) element (consensus YYA+1NT/AYY) located near the start site (Breathnach and Chambon, 1981; Smale and Baltimore, 1989; Javahery et al., 1994). With respect to core promoter functions, the minimal factor requirement and corresponding pre-initiation complex (PIC) assembly pathway are best understood for TATA-directed basal transcription, in which case TATA recognition by the TATA-binding protein (TBP) component of TFIID is sufficient to nucleate the assembly of other general initiation factors and RNA polymerase II into a functional complex (Roeder, 1996). The pyrimidine-rich Inr-directed basal transcription is more complicated and less well understood, but requires several factors, including both the TBP-associated factor (TAF) subunits of TFIID and other novel factors, that are not required for TATA-directed transcription (Martinez et al., 1994; Roeder, 1996, Smale, 1997). Factors which have been demonstrated or inferred to recognize the Inr and to nucleate PIC assembly include the TAF components of TFIID, RNA polymerase II and novel Inr-binding proteins (reviewed in Smale, 1997). Consistent with the latter possibility, several factors have been reported to bind at or near Inr elements (Novina and Roy, 1996; Smale, 1997) and, in some cases, shown to facilitate core promoter functions in vitro (Roy et al., 1991, 1993a; Seto et al., 1991; Usheva and Shenk, 1994). This multiplicity of Inr-binding proteins could reflect diversity in core promoter elements, especially in view of the loose consensus for such elements (Kaufman et al., 1996). Alternatively, as suggested (Wiley et al., 1992; Kaufman and Smale, 1994), these observations could also reflect juxtaposition or overlap of binding sites for various regulatory factors and Inr sites that could be recognized by a universal (but still unidentified) factor. In any case, what is needed to settle the issue unequivocally is identification and characterization of the protein factors directly involved in Inr function. TFII-I was identified originally as a factor that could bind to Inr elements and stimulate transcription from the potent TATA- and Inr-containing adenovirus major late (AdML) promoter in a system reconstituted with partially purified components (Roy et al., 1991, 1993a). Somewhat surprisingly, TFII-I was also found to bind to a distinct upstream element (E-box) on the AdML promoter that originally was identified as a recognition site for the transcriptional activator USF, a member of the basic helix–loop–helix-leucine zipper (bHLH-LZ) family of proteins (reviewed in Murre and Baltimore, 1992) that activates the AdML promoter both in vitro and in vivo (Pognonec and Roeder, 1991; Du et al., 1993; Luo and Sawadogo, 1996). Similarly, USF1 was also shown to bind not only to the E-box but also to the Inr (Roy et al., 1991; Du et al., 1993). Consistent with these observations, as well as synergistic interactions at both Inr and E-box elements, ectopic expression of USF1 was found to enhance expression of TATA-containing promoters either through an adjacent Inr element (AdML promoter) or through upstream E-boxes (E1b promoter) (Du et al., 1993). Although it is not yet clear whether USF1 is unique with respect to its apparent dual function through two distinct promoter elements, and whether these functions might be linked in some promoters, these observations suggested novel mechanisms of gene regulation and the possible involvement of TFII-I as a co-regulator that can integrate regulatory responses of USF1 to the basal machinery. The involvement of such co-regulators may also help explain the differential functions of distinct bHLH-LZ proteins through common E-box elements in different promoters (Weintraub et al., 1994; Molkentin et al., 1995). As part of our investigation of these questions, we now report the purification of native TFII-I and the cloning of a cognate cDNA whose ectopically expressed product, like its native counterpart, exhibits specific Inr- and E-box-dependent binding. Consistent with our model, the ectopically expressed TFII-I markedly enhances both USF1 binding in vitro and, most importantly, the in vivo function of ectopic USF1 through both Inr and E-box elements in the AdML promoter. Taken together, these results indicate that TFII-I may serve as a novel co-regulator for USF1 in addition to, or in conjunction with, its potential role as an Inr-binding basal transcription factor. Results Purification of TFII-I Using an electrophoretic mobility shift assay (EMSA) to monitor site-specific binding to the AdML promoter Inr element (Roy et al., 1991), TFII-I was purified according to the scheme shown in Figure 1A and detailed in Materials and methods. The TFII-I activity eluted predominantly with a 120 kDa polypeptide at the dsDNA cellulose step (Figure 1B, lane 1) and exclusively with this polypeptide at the final HPLC (SP-5PW) step (Figure 1B, lane 2 and data not shown). Figure 1.Purification of TFII-I. (A) Purification scheme of native TFII-I. (B) Silver-stained dsDNA cellulose (lane 1) and HPLC-purified (lane 2) native TFII-I subsequent to SDS–PAGE. Download figure Download PowerPoint Primary structure of TFII-I The purified material from the SP-5PW HPLC column was resolved by SDS–PAGE, transferred to nitrocellulose and subjected to microsequencing. The 120 kDa polypeptide yielded four peptide sequences, indicated by underlining in Figure 2A, three of which were used to design primers for screening a Namalwa (B cell)-derived cDNA library (Scheidereit et al., 1988). Extensive screening yielded a cDNA clone with a 957 amino acid open reading frame (ORF) that was unique (GenBank database) and contained all four peptide sequences derived from microsequencing (Figure 2A). Most strikingly, analysis of the amino acid sequence (Figure 2B) revealed six direct repeats (R1–R6), each 90 amino acids long, suggesting that TFII-I probably arose via gene duplication. The internal or core repeats, R2–R5, are more closely related to each other than to either of the flanking repeats (R1 and R6). The remarkable sequence conservation amongst R2–R5 is highlighted by a region (underlined) that is nearly identical amongst these repeats. Several other interesting structural features also are apparent. First, the presence of a hydrophobic zipper-like region at the N-terminal portion of the protein (indicated by bold amino acids, Figure 2A) suggests a protein interaction domain, although the functional significance of this zipper-like region is not known at present. Moreover, unlike the conventional basic leucine zipper DNA-binding proteins, this region is not flanked by a conserved basic region that could be involved in DNA binding (Ferre-D'Amare et al., 1993). Second, the region within the N-terminal 90 amino acids, and before the beginning of R1, includes two clusters of four acidic amino acids. A third acidic cluster is also apparent between R1 and R2 (indicated by + signs, Figure 2A). Although the functional significance of these acidic clusters is uncertain at present, they are reminiscent of acidic activation domains present in eukaryotic transcriptional activator proteins (Triezenberg, 1995). Importantly, all of these special structural features in TFII-I lie outside of or between the direct repeats in the 'linker' regions. Figure 2.Amino acid sequence of TFII-I. (A) Primary structure of TFII-I protein indicating the four peptides (underlined) derived from microsequencing. A leucine zipper-like region is indicated by bold amino acids (VLLV). Acidic amino acid regions are indicated by overhead + signs. The putative basic region preceding repeat 2 (R2) is overlined and indicated as BR. A peptide comprised of amino acids 301–321, which included the BR, was employed to generate the anti-peptide antibody. A consensus MAPK site (PRSP) is apparent at amino acids 631–634. Src autophosphorylation sites (EDXDY) are at positions 244–248 and 273–277. Finally, a putative SH3 recognition helix is present at positions 290–297. (B) Arrangement of six direct repeats in TFII-I, starting from position 102 and extending to position 906 with the internal (core) repeats (R2–R5) showing a closer sequence relationship to each other than to the flanking repeats (R1 and R6). In turn, the flanking repeats are more closely related to each other than to the internal repeats. The most highly conserved amino acids are indicated at the bottom. The amino acids in bold represent identity in all six repeats. Amino acids that are conserved in at least five of the repeats are also indicated. The most conserved region within these repeats is indicated by the solid line and is termed the 'I-repeat'. (C) The putative helix–loop/span–helix homology in TFII-I compared with the helix–loop–helix proteins USF and c-MYC. For the sake of simplicity, only the HLH homology in R2 is shown. Other repeats also have similar homology. There is a greater identity to the USF sequence (indicated by solid lines) than to the c-MYC sequence. (D) Northern blot analysis on poly(A)+ RNA isolated from HeLa and Namalwa (Nam) shows a predominant 4.7 kb TFII-I RNA (left panel). It is about three times more abundant in Namalwa cells than in HeLa cells. Northern analysis on a multiple tissue blot shows that although the TFII-I RNA is ubiquitously expressed, the levels vary significantly in various tissues (right panel). Moreover, these tissues contain a second (4.2 kb) RNA whose exact relationship to the 4.7 kb RNA is not clear yet. Download figure Download PowerPoint Careful analysis of the primary amino acid structure of TFII-I demonstrated a putative HLH-like domain (Figure 2C) within each of the repeats. However, there appears to be only one putative basic region (BR, between amino acids 301 and 321) that, by analogy to known basic-HLH domain proteins, could constitute a DNA-binding domain. In contrast to the conventional HLH domains in which the loop ranges from six to 20 amino acids (reviewed in Ferre-D'Amare et al., 1993), but more like the long loop region in AP-4 (Hu et al., 1990), the loop region in TFII-I is ∼70 amino acids. This fact, and the presence of multiple putative HLH motifs, makes TFII-I a unique transcription factor, potentially capable of interacting with a variety of HLH regulators (Roy et al., 1993b; Roy and Roeder, 1994). The presence of multiple HLH-like motifs also raises the possibility that in addition to forming intermolecular heteromeric interactions with other classical HLH proteins, TFII-I may dimerize intramolecularly and thereby display different configurations (e.g. two distinct DNA-binding domains) depending on the particular combination of intramolecular interactions. Finally, we tested the expression pattern of TFII-I in various tissue types. Northern blot analysis in HeLa- and Namalwa-derived poly(A)+ RNA revealed that TFII-I is expressed as a single 4.7 kb message under stringent hybridization conditions (Figure 2D, left panel). Furthermore, as expected, a multiple tissue Northern blot analysis also showed that TFII-I is widely expressed (consistent with Western blot analyses, data not shown), although the extent of expression varied among different tissues (Figure 2D, right panel). Curiously, in these primary tissue types, in addition to the 4.7 kb TFII-I RNA, a shorter RNA at 4.2 kb was also visible. The structure of this RNA is unclear at present. Expression of a recombinant TFII-I that is competent in DNA binding For further functional tests, the cDNA encoding TFII-I was expressed via a bacterial expression vector that adds a hexa-histidine tag to the N-terminus of the protein, and recombinant protein was purified from crude bacterial lysate on a Ni2+-agarose column. A Western blot analysis with antibody raised against the putative DNA-binding domain (basic region, see above) of TFII-I (Figure 3A) showed a dominant 120 kDa band and several degradation products in the bacterially expressed recombinant TFII-I (TFII-IR, lane 1) in comparison with a single 120 kDa band (arrow) in native purified TFII-I (TFII-IN, lane 2). The anti-TFII-I antibody also recognized 120 kDa/TFII-I in various nuclear extracts (data not shown, and Manzano-Winkler et al., 1996). Most importantly, as revealed by an EMSA with an oligonucleotide probe (MLI1) containing Inr1, the recombinant TFII-I showed site-specific binding to AdML initiator elements (Figure 3B); the observed complex (lane 1) was shown to be specific by virtue of competition with intact MLI1- and MLI2-containing oligonucleotides (lane 2 and data not shown), but not with a mutant MLI2 oligonucleotide (lane 3). Furthermore, the binding of recombinant TFII-I to the Inr site was not competed by an E-box-containing oligonucleotide (lane 4). Finally, an EMSA with an oligonucleotide probe (ML-U) containing the AdML E-box demonstrated specific and direct binding of recombinant TFII-I to this element; the observed complex (lane 5) was competed by an oligonucleotide containing a wild-type E-box (lane 6), but not by an oligonucleotide containing a mutant E-box (lane 7) and only weakly by an Inr-containing oligonucleotide (lane 8). However, this binding could be inhibited specifically by an anti-TFII-I antibody (lanes 9–11, Figure 3B). Therefore, the Inr- and E-box-binding properties described here for the recombinant 120 kDa protein mirror those described for the native TFII-I (Roy et al., 1991). Figure 3.Analysis of recombinant TFII-I expressed in bacteria. (A) Western blot analysis of recombinant TFII-I (TFII-IR, lane 1) and the HPLC-purified native TFII-I (TFII-IN, lane 2). The arrow shows the 120 kDa polypeptide. (B) Specific binding of recombinant TFII-I to AdML Inr and E-box elements. Binding was monitored by EMSA with an AdML probe (MLI1) containing Inr1 (lanes 1–4) and with an AdML probe (ML-U) containing an E-box (lanes 5–11). Oligonucleotide competitors added at 50-fold molar excess contained: wild-type Inr2 sequences (MLI2), lanes 2 and 8; mutated Inr2 sequences (MLI2m), lane 3; wild-type E-box sequences (ML-U), lanes 4 and 6; and mutated E-box sequences (ML-Um), lane 7. Anti-TFII-I serum (α-I) and pre-immune serum (α-pI) were added in lanes 10 and 11, respectively. (C) Stimulatory effect of recombinant TFII-I on USF1 binding to the AdML Inr1-containing probe (MLI1). The binding of variable amounts of recombinant USF1 was monitored by EMSA in the absence (lanes 1–3) and presence (lanes 4–6) of a fixed amount of recombinant TFII-I, which was also analyzed in the absence of USF1 (lane 7). (D) Interactions of in vitro translated USF1 and TFII-I in the absence of DNA binding. Intact TFII-I and both wild-type USF1 and a USF1 mutant lacking the leucine zipper (USFΔLZ) were co-translated in rabbit reticulocyte lysates in the presence of [35S]methionine, both individually and in the combinations indicated above the lanes. Individual translation reactions (lanes 1–3, 5–7 and 8), as well as a mixture of independently translated TFII-I and USF1 (lane 4), were subjected to immunoprecipitation with anti-USF1 antibody (lanes 1–7) or with anti-TFII-I antibody (lane 8). Immunoprecipitations were subjected to SDS–PAGE and autoradiography. Direct analyses of translation reactions revealed that approximately equal amounts of radiolabeled TFII-I and USF1 were synthesized when the corresponding vectors were expressed independently or together (data not shown) Download figure Download PowerPoint Having established intrinsic DNA-binding properties of recombinant TFII-I, we next tested its ability to interact with USF1 both on DNA (Figure 3C) and in solution (Figure 3D). As shown in Figure 3C, and consistent with previous studies of native TFII-I (Roy et al., 1991), recombinant TFII-I significantly stimulated the binding of recombinant USF1 to the AdML-derived Inr element; recombinant TFII-I, like native TFII-I (Roy et al., 1991), also stimulated USF1 binding to the E-box (data not shown). However, contrary to expectations of heterodimer formation, we were unable to observe a stable heterodimeric complex consisting of both TFII-I and USF1 under these conditions. This may reflect either an instability of the heterodimeric complex under the electrophoretic conditions employed or a role for TFII-I in increasing the stability of USF1 on Inr and E-box elements via transient interactions. In order to test whether TFII-I and USF1 can interact stably under different conditions, we performed co-immunoprecipitation studies subsequent to ectopic expression and radiolabeling of both proteins in a rabbit reticulocyte lysate. Under these conditions, roughly equivalent amounts of USF1 and TFII-I were synthesized, as shown by direct analysis of radiolabeled proteins (data not shown). In the immunoprecipitation analysis (Figure 3D), USF1 (lane 1) but not TFII-I (lane 2) was immunoprecipitated by an anti-USF antibody when these proteins were expressed separately. In contrast, TFII-I and USF1 were co-immunoprecipitated by anti-USF1 antibody when both proteins were co-translated (lane 3). TFII-I was not co-immunoprecipitated by anti-USF1 antibody when the two proteins were post-translationally mixed (lane 4). As a further control, co-immunoprecipitation was performed following expression of a mutant USF1 protein that lacked the LZ domain (lane 5). In this case, anti-USF1 antibody failed to co-immunoprecipitate TFII-I even when both proteins were co-translated (lane 7). That TFII-I is translated efficiently under these conditions was also demonstrated by immunoprecipitation of TFII-I by an anti-TFII-I antibody (lane 8). Taken together, these data demonstrate that stable interactions between USF1 and TFII-I do occur when the proteins are allowed to fold together. Thus, the inability to detect a stable heteromeric complex on DNA may reflect either the failure of the independently synthesized proteins to interact stably, possibly because of improper folding, or the dissociation of TFII-I from the complex under the electrophoretic conditions. Although initial experiments have failed to detect formation of such a complex with co-translated USF1 and TFII-I (data not shown), this may reflect insufficient levels of synthesis in the in vitro system. Although the recombinant TFII-I behaved similarly to native TFII-I with respect to DNA-binding specificity and USF1 interactions, it did not show the in vitro transcription activity observed earlier (Roy et al., 1991, 1993a) for native TFII-I preparations (data not shown). Thus, while confirming the DNA-binding specificity of the cDNA-encoded protein, these results also raise the possibility that the bacterially expressed TFII-I is improperly folded and/or lacking post-translational modifications that play a critical role in effecting the transcription function, but not the intrinsic DNA-binding activity. Another possibility is that although the 120 kDa polypeptide is competent in DNA binding, the transcriptional activity, as seen with the partially purified TFII-I, may reflect additional polypeptides associated with the 120 kDa polypeptide. Independent and synergistic functions of TFII-I and USF1 via the AdML Inr element in transfected cells We reasoned that whether the inactivity of recombinant TFII-I in an in vitro transcription assay results from a lack of post-translational modifications or a lack of associated polypeptides, an analysis of TFII-I function in eukaryotic cell lines by transient transfection assays might circumvent these problems and reveal associated transcription functions. Furthermore, these assays would also enable us to test whether the synergism between TFII-I and USF1, as seen at the DNA-binding level, is manifested at the transcriptional level in a physiological situation. In order to study activation via AdML Inr sites, we used a reporter plasmid (MLICAT) containing the AdML core promoter (−45 to +65) fused to the CAT gene (Du et al., 1993). In addition to the TATA element, the core promoter contains initiator elements at positions −3 to +9 (Inr 1) and +45 to +57 (Inr 2) (Roy et al., 1991). HeLa cells were co-transfected with MLICAT and either an empty vector (pCX) or vectors expressing TFII-I (pCX-II-I) and/or the human USF1 (pCX-USF1) (Figure 4A). The reporter was activated significantly (up to 18-fold) by ectopic USF1 in a dose-dependent manner (lanes 1–4), but only marginally (1- to 1.5-fold) by ectopic TFII-I expression (lanes 5–7 versus lane 1). In contrast, at an intermediate level of USF1 expression (5 μg of pCX-USF1) that gave only a 3-fold increase in reporter activity, co-expression of TFII-I resulted in markedly enhanced levels of activity that were up to 73-fold above the control values (lanes 9–11 versus lane 1). At the highest level of activity (lane 10), the overall activity was 25-fold greater than that expected (on the basis of additivity) from the independent expression of comparable levels of USF1 (lane 3) and TFII-I (lane 6). Greater than additive levels of activity were also observed at higher and lower levels of TFII-I co-expression with USF1 (lane 9 versus lanes 3 and 5, and lane 11 versus lanes 3 and 8). Hence, the effects of ectopic USF1 and TFII-I are clearly synergistic. Figure 4.Independent and synergistic effects of ectopic USF1 and TFII-I expression on transcriptional activation through Inr elements in transfected cells. (A) Activation from the AdML core promoter. HeLa cells were co-transfected with the wild-type MLICAT reporter and with variable amounts of pCX-USF1 and pCX-II-I expression vectors, both alone and in combination, as indicated at the top of the figure. Transfection conditions were as described in Du et al. (1993). (B) Activation from AdML core promoters containing intact versus mutated Inr1 and Inr2 elements. HeLa cells were co-transfected with wild-type or Inr-mutated MLICAT reporters and with the indicated combinations of the pCX-USF1 expression vector (5 μg), the pCX-II-I expression vector (5 μg) or the control pCX vector (5 or 10 μg, to bring the amount of total transfected DNA to 10 μg). Transfection conditions were as described by Chen and Okayama (1987) and resulted in slightly higher transfection efficiency than the method used in Figure (A) and in Figures 5,6,7. In both (A) and (B), the relative CAT activities were normalized to the level of activity observed with the control pCX vector alone (lane 1) and are indicated above the figure. Download figure Download PowerPoint To test whether the transcriptional synergism between USF1 and TFII-I was Inr dependent, reporter plasmids containing either wild-type or mutant Inr1 and Inr2 core promoters were co-transfected with TFII-I or TFII-I plus USF1 (Figure 4B). Under the higher efficiency co-transfection conditions of this analysis (see legend to Figure 4B), the independent levels of activation by TFII-I and USF1 were slightly higher. Thus, the reporter was activated 17-fold by USF1 alone (lane 2), 5-fold by TFII-I alone (lane 3) and 33-fold by comparable concentrations of both together (lane 4). Although the greater effects of independently expressed USF1 and TFII-I resulted in a level of synergism lower than that observed in the analysis of Figure 4A, this did permit an analysis of the effect of Inr mutations on both independent and synergistic effec
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