Structure-Function Analysis of TFII-I

ROLES OF THE N-TERMINAL END, BASIC REGION, AND I-REPEATS*

Venugopalan CheriyathDagger and Ananda L. RoyDagger §||**

From the Dagger  Department of Pathology and Programs in § Immunology and  Genetics, || Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

Received for publication, September 13, 2000, and in revised form, December 4, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The transcription factor TFII-I can bind specifically to several DNA sequence elements and is implicated in both basal and activated transcription. There are four alternatively spliced isoforms of TFII-I, all characterized by the presence of six I-repeats, R1-R6, each containing a potential helix-loop-helix motif implicated in protein-protein interactions. These isoforms exhibit both homomeric and heteromeric interactions that lead to nuclear localization. In this study we mapped two distinct regions in TFII-I that affect its DNA binding. Deletion of either of these regions led to abrogation of DNA binding and transcriptional activation from both the Vbeta and c-fos promoters. The I-repeats, as expected, were capable of mediating homomeric interactions either individually or in combination. Unexpectedly, an additional homomeric interaction domain was found within the N-terminal end of TFII-I that includes a putative leucine zipper motif. These data suggest a model in which TFII-I undergoes regulated homomeric interaction mediated by both the N-terminal end and the I-repeats.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TFII-I is a unique transcription factor, because it can function both as a basal factor and as an activator (1-11). Consistent with these functional activities, it has been shown to bind a core promoter element, Inr, and various upstream elements apparently through distinct DNA binding domains (1, 4, 6-11). The primary structure of TFII-I is compatible with its multifunctional properties, consisting of six direct reiterated I-repeats, R1-R6, each containing a putative helix-loop-helix motif, HLH,1 but only one basic region preceding R2 (4, 9). The latter, by analogy with more conventional HLH proteins, was postulated to constitute a DNA binding domain (12, 13). The I-repeats, by virtue of having the potential HLH motifs, are conjectured to present protein-protein interaction surfaces (4, 9, 12). More recently, it has been shown that there are altogether four alternatively spliced isoforms of TFII-I (8). All isoforms contain the I-repeats and the basic region and exhibit both homomeric and heteromeric interactions with themselves that lead to their preferential nuclear localization (8). Although each isoform individually bound to DNA and activated transcription both from the Vbeta and c-fos promoters, a particular combination of isoforms differentially regulated the two promoters (8). We and others (14-16) have also shown that a variety of extracellular signals mediating through cell surface receptors, including growth factor receptors, lead to enhanced tyrosine phosphorylation and increased transcriptional activity of TFII-I. Given these unusual structural and functional features, it is important to determine the regions/domains in TFII-I that is/are responsible for its DNA binding properties and the role of the I-repeats in mediating protein-protein interactions.

Here we searched for potential DNA binding domains and regions responsible for homomeric interactions. The deletion of either the N-terminal 90 amino acids, including a putative leucine zipper but containing an intact basic region, or a basic region (containing an intact N terminus) led to a loss of binding to the Vbeta Inr and the c-fos upstream sites. Consistent with their lack of DNA binding activities, these mutants failed to transcriptionally activate either the Vbeta or the c-fos promoters in transient transfection assays. Interaction studies revealed that deletion of either the basic region or repeats R6 through R3 from the C-terminal end had no significant effects on homomeric interactions when compared with the wild type TFII-I. However, the N-terminal 90-amino acid-deleted mutant, containing all the repeats, failed to exhibit homomeric interactions with the wild type protein. Interestingly, the I-repeats R1 and R2, either in combination or individually, could also mediate homomeric interactions. So, although the repeats can mediate homomeric interactions, they alone are insufficient and additionally require the N-terminal 90 amino acids. Based on these results, we propose a model for regulated homomeric interactions of TFII-I involving both the N-terminal end and the I-repeats containing the HLH motifs.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture

COS7 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO), 50 units of penicillin/ml, and 50 µg of streptomycin (Life Technologies, Inc.). The cells were maintained at 37 °C in a 5% CO2 environment.

Plasmids

TFII-I has four alternatively spliced isoforms: alpha  (977 amino acids), beta  (978 amino acids), gamma  (998 amino acids), and Delta  (957 amino acids) (8). The construction of wild type expression plasmids of TFII-IDelta - and beta -isoforms (used in this study), pEBGII-I, pEBBGFPII-I, and p70 mutant plasmid pEBGp70 were detailed elsewhere (6, 8). TFII-I mutants used here were generated by a PCR-based strategy. In the first round, two PCR reactions were done. In the first reaction, a restriction enzyme-specific sequence 5' to the deleted region was used as a forward primer (primer 1) and a TFII-I-specific sequence, just downstream of the deleted sequence, was used as a reverse primer (primer 2). In the second reaction, a TFII-I-specific sequence, just upstream of the deleted sequence, was used as a forward primer (primer 3) and a restriction enzyme-specific sequence 3' to the deleted sequence was used as a reverse primer (primer 4). The products of each reaction (0.25 µg) were mixed, and the second round of PCR was performed using the primers 1 and 4. The final PCR product was gel-purified and digested with restriction enzymes, the recognition sequences of which were used as the primers 1 and 4. The restriction-digested PCR product was ligated into TFII-I-expressing plasmid, pEBGII-I or pEBBGFPII-I. The specific sequences of these primers and the corresponding restriction enzymes used are detailed as follows:

Delta N20 (Deletion of Amino Acids 1-20)-- Reaction 1: primer 1, 5'-ATTCCCCTCTAGAAATAATTTTG-3'; primer 2, 5'-CATGAGGAATGTCACCATATGGCTGCCGCG-3'. Reaction 2: primer 3, 5'-ATGGTGGTGACATTCCTCATGTC-3'; primer 4, 5'-GGAGAGATGCATAAAAT- GAAATCT-3'. The final product was gel-isolated, digested with XbaI and NsiI, and then ligated with pEBGII-I.

Delta N90 (Deletion of Amino Acids 1-90)-- Reaction 1: primer 1, 5'-GTGGATCCACCATGGGCAG-3'; primer 2, 5'-CATGAGGAATGTCACCATATGGCTGCCGCG-3'. Reaction 2: primer 3, 5'-ATAGGATCCATGCATAAAATGAA-3'; primer 4, 5'-GGGTTTACGTAGATCAGTGATG-3'. The purified PCR product was digested with BamHI and SnaB1, and then ligated with pEBGII-I.

Delta linker (Deletion of Amino Acids 232-252)-- Reaction 1: primer 1, 5'-AATGAGCTACCGCAGCCACCAGTCCCG-3'; primer 2, 5'-TTGAATGTTATATTGATAATAATC-3'. Reaction 2: primer 3, 5'-CAATATAACATTCAAGATGATGATTATTCT-3'; primer 4, 5'-GGGTTTACGTAGATCAGTGATG-3'. The final product was gel-isolated, digested with Acc65I and SnaB1, and then ligated with pEBGII-I.

Delta BR (Amino Acids 301-306)-- Reaction 1: primer 1, 5'-AATGAGCTACCGCAGCCACCAGTCCCG-3'; primer 2, 5'-GAGTTCAACTTCGAGAAATGGAAT-3'. Reaction 2: primer 3, 5'-GAAGTTGAACTCAGCATTGGCGGG-3'; primer 4, 5'-TTGAATGTTATATTGATAATAATC-3'. The PCR product was digested with Acc65I and SnaB1 and ligated with pEBGII-I.

p46 (Amino Acids 1-428)-- The region between amino acids (1) was PCR-amplified by using the following primers: primer 1, 5'-GTGGATCCACCATGGGCAG-3'; primer 2, 5'-AGGCGGCCGCCTTTCTTAATTT-3'. The final product was digested with BamHI and NotI and ligated to pEBG vector.

R1 (Amino Acids 104-176)-- primer 1, 5'-GTGGATCCATTGAAACACTCAGAAAA-3'; primer 2, 5'-GGGCGGCCGCTAAAAAAGGTCTCTT-3'. The PCR-amplified product was gel-extracted, digested with BamHI and NotI, and ligated to pEBG.

R2 (Amino Acids 309-397)-- primer 1, 5'-GTGGATCCTTCGAGAAATGGAATGCT-3'; primer 2, 5'-GGGCGGCCGCAAGCTCATCTTTC-3'. The PCR-amplified product was gel-isolated, digested with BamHI and NotI, and ligated with pEBG vector.

All the mutant constructs were confirmed by DNA sequencing.

Eukaryotic Expression and Purification of Wild Type and Mutant TFII-I

COS7 cells were transfected with 7.5 µg of expression plasmids of either wild type TFII-IDelta -isoform (pEBGII-I) or its various mutants (pEBGDelta N90II-I, pEBGDelta BR), and the recombinant proteins were isolated as described previously (6, 8).

Western Blot Analysis

The protein samples were separated by 10% SDS-PAGE, transferred onto nitrocellulose membrane by semi-dry blotting method, and processed as described previously (8). The primary antibodies were used as follows: anti-TFII-I, 1:2500; anti-GST (Sigma), 1:3000; and anti-GFP (CLONTECH, Palo Alto, CA) 1:300. Either the secondary anti-rabbit (Zymed Laboratories Inc., South San Francisco, CA; 1:8000 dilution) or anti-mouse (Roche Molecular Biochemicals, Indianapolis, IN; 1:4000 dilution) horseradish peroxidase-linked antibodies were incubated in TBS containing 0.05% Tween-20. All Western blots were developed by enhanced chemiluminescence (Renaissance, PerkinElmer Life Sciences).

Electrophoretic Mobility Shift Analysis

The EMSA reactions in Fig. 1 was performed with either an Inr probe derived from the Vbeta promoter (17) or with a probe derived from upstream sequences of the c-fos promoter (10). The EMSA was performed as described previously (8).

GST Pull-down Assay

Whole cell extracts (200 µg) from COS7 cells, cotransfected with GFP-tagged wild type TFII-I (either Delta - or beta -isoforms) and either GST-tagged wild type TFII-I or the GST-tagged mutants, were subjected to GST pull-down assay as described (8). The precipitated proteins were separated by 10% SDS-PAGE, Western-blotted, and probed with an anti-GFP antibody (CLONTECH). The blots were stripped to remove the anti-GFP immune complex (17) and reprobed with anti-GST antibody (Sigma).

Transient Transfection and Indirect Immunofluorescence

COS7 cells were transfected with either the GST construct alone or the GST-tagged wild type TFII-I or its mutants as described (8). After 30-h post-transfection, cells were fixed with 4% paraformaldehyde and prepared for indirect immunofluorescence (8). For immunodetection, monoclonal anti-GST antibody (Sigma) was used as a primary antibody at a dilution of 1:4000, and Alexa 594 goat anti-mouse IgG (H+L) (Molecular Probes) as a secondary antibody at a dilution of 1:30,000. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) dye (Sigma). Immunofluorescence was detected using a fluorescent microscope (Nikon, E400) with a 100× objective.

Reporter Assays

Transient transfection and luciferase reporter assays were done as described (6, 8). Each experiment was done in triplicate and repeated twice.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Multiple Interdependent DNA Binding Regions in TFII-I-- Based on its multiple DNA sequence element, recognition properties, and indirect competition experiments, it was postulated that TFII-I might have more than one distinct DNA binding domains/regions (1). To gain insight into its DNA binding regions/domains, we generated several mutants of TFII-I and tested their ability to bind to the Vbeta -derived Inr and the c-fos-derived upstream sequence elements. Of particular interest to us were two mutants. The first mutant (Delta N90) was created such that the entire N-terminal end (first 90 amino acids) before I-repeat 1, including the putative leucine zipper, was deleted. The second mutant (Delta BR) was created such that the basic region (amino acids 301-306) preceding I-repeat 2 was deleted, because, by analogy with known basic-helix-loop-helix (bHLH) proteins, this could constitute a bona fide DNA binding domain (11, 12). EMSA was performed with the wild type and mutant TFII-I proteins on probes containing either Vbeta -derived Inr sequences or c-fos-derived upstream sequences overlapping the serum response element (Fig. 1). For this and all subsequent experiments, unless otherwise stated, the Delta -isoform was used (Ref. 8, see also "Experimental Procedures"). Compared with the wild type protein (WT), neither the Delta N90 nor the Delta BR gave any significant shifted complex on any of the probes (nearly 6-fold reduction in DNA binding between the wild type and either of the mutants). The amount and integrity of various proteins used for EMSA were monitored by Western blot assay (data not shown). Although the amount of Delta N90 used for EMSA was slightly less than the wild type TFII-I, increasing it even 2-fold did not produce any appreciable DNA binding (data not shown). That the lack of appreciable DNA binding capability of these mutants is due to specific and not general mutation in TFII-I is proven by the fact that the p70 mutant exhibits greater than wild type levels of DNA binding activity (6). Hence, we conclude that both the N-terminal 90 amino acids and the basic region are required for DNA binding and, although they may constitute separate DNA binding regions/domains, neither one alone is sufficient to mediate DNA binding.



View larger version (58K):
[in this window]
[in a new window]
 
Fig. 1.   DNA binding activity of wild type and mutant TFII-I EMSA with Vbeta Inr (lanes 1-4) and c-fos (lanes 5-8). Lanes 1 and 5, vector only (probe); lanes 2 and 6, wild type TFII-I (WT, 82 ng); lanes 3 and 7, Delta BR (84 ng); lanes 4 and 8, Delta N90 (68 ng).

Transcriptional Properties of the Mutants-- We next tested the transcriptional properties of the mutants as compared with the wild type protein on both the Vbeta and c-fos promoters. For this assay, we chose an additional mutant (p70) in which the C-terminal 222 amino acids, containing an activation domain, are deleted (6). Although the wild type TFII-I stimulated the Vbeta promoter 3-fold, none of the mutants gave any significant transcriptional stimulation and the transcriptional activity of Delta BR and p70 mutants were similar to or below the basal levels (Fig. 2A). Delta N90 failed to give any significant stimulation. Like the Vbeta promoter, the wild type and the Delta BR and p70 mutants behave similarly to the c-fos promoter (Fig. 2B). Although the transcriptional activities of Delta BR and p70 mutants were similar to or below the basal levels, the Delta N90 mutant failed to produce any significant transcriptional activation (Fig. 2B). From these experiments we conclude that the DNA binding domains and the C-terminal activation domain of TFII-I are necessary for its transcriptional activation either as a basal factor or as an activator. Control Western blot analysis from these experiments (Fig. 2, lower panels) showed that the expression levels and integrity of the mutants were comparable to that of the wild type and thus, the differences in the functional activities were not due to alteration in protein levels.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2.   Transcription function of TFII-I mutants. A, the top panel shows the transactivation potentials of individual and combinations of wild type and TFII-I mutants on the basal Vbeta promoter. COS7 cells were transiently cotransfected with 600 ng of Vbeta and 400 ng of either pEBG vector alone (vector) or pEBG plasmids expressing TFII-I mutants, Delta N90, Delta BR, and p70. To normalize the transfection efficiency, 35 ng of plasmid encoding renilla luciferase was also included in all transfections. The result is an average of two independent experiments done in triplicate. The bottom panel shows control Western blot analysis of lysates from a representative transfection assay. B, the top panel shows the transactivation potentials of wild type and mutant TFII-I on the c-fos promoter. COS7 cells were cotransfected with 600 ng of the c-fos promoter either with 400 ng of empty vector plasmid alone (top panel, vector) or with 400 ng of plasmid encoding wild type TFII-I (WT) or mutant TFII-I (Delta N90, Delta BR, and p70). The result shown is an average of two independent experiments done in triplicate. The bottom panel shows Western blot analyses of lysates from transfection assays either in the absence or in the presence of hEGF.

Nuclear Localization of the Wild Type and Mutant Forms of TFII-I-- It could be argued that the lack of transcriptional activity of the TFII-I mutants is due to a lack of their proper nuclear localization. This is particularly relevant for the Delta BR mutant, because a classical nuclear localization signal (NLS) is rich in basic amino acids (18) and thus, the basic region might constitute an NLS or contribute to the proper localization of TFII-I. However, like the wild type TFII-I, all of the mutants (Delta N90, Delta BR, and p70) preferentially localized to the nucleus when ectopically expressed in COS7 cells (Fig. 3). Therefore, none of the mutations impaired the nuclear localization signal of TFII-I, and the lack of the transcriptional function is indeed due to a lack of either the DNA binding or the transcriptional activation domains.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Nuclear localization of wild type and mutant TFII-I. Localization of the TFII-I mutants was compared with the wild type by expressing them in COS7 cells as GST fusion proteins. COS7 cells were transfected with 600 ng of expression plasmid encoding either GST alone (A, B, and C), or wild type TFII-IDelta (D, E, and F), or Delta N90 (G, H, and I), or Delta BR (J, K, and L), or p70 (M, N, and O). 30 h post-transfection, cells were fixed with 4% paraformaldehyde and the ectopically expressed proteins were visualized by indirect immunofluorescence with monoclonal anti-GST antibody and Alexa 594 goat anti-mouse IgG secondary antibody (A, D, G, J, and M). Nuclei were stained with DAPI (B, E, H, K, and N). Superimposition (Merge) of Alexa 594 and DAPI images is shown in the bottom panels (C, F, I, L, and O). Images were obtained by using a Nikon E400 fluorescence microscope with a 100× objective.

Regions Necessary for Homomerization-- TFII-I has four alternatively spliced isoforms that can undergo both homo- and heteromerization with each other (8). Furthermore, either homo- or heteromerization of isoforms facilitates nuclear translocation. Given the fact that Delta N90, Delta BR, and p70 mutants readily translocate to the nucleus, we anticipated that all of them would exhibit homo- and perhaps heteromerization capabilities. Furthermore, because of the presence of a putative leucine zipper (LZ) toward the N-terminal end (amino acids 23-44) of TFII-I that is conserved in all human isoforms and in mouse TFII-I (19, 20), we reasoned that it might be involved in either homo- and/or heteromerization. To test this idea and to further identify other potential interaction domains, we constructed a series of deletion mutants (as GST fusion proteins) and analyzed their homo- and heteromerization potentials. All the mutants are schematically shown in Fig. 4. In Delta N20, the first 20 amino acids from the N-terminal end, including the first acidic cluster, are removed, but the putative LZ remains intact (4). Delta N90 is described above. In Delta linker, part of the linker region between R1 and R2 (amino acids 232-252) is removed, but the LZ remains intact. This mutant is particularly interesting, because this deletion creates a shorter version of TFII-I that is naturally absent in humans but present in mice (20). Therefore, we wanted to test whether such a mutant/isoform can have homomeric and heteromeric interactions. The p46 is a deletion mutant that contains only the first 428 amino acids from the N-terminal end (containing only repeats 1 and 2), and when expressed, migrates as a 46-kDa protein. The p70 mutant, containing the first four repeats, has been described before (Ref. 6, see also Fig. 4).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   Schematic diagram of TFII-I mutants. The schematics of wild type and various deletion constructs of the TFII-IDelta -isoform. The open boxes represent the I-repeats (denoted as R1-R6), and the closed box denoted LZ represents the putative leucine zipper motif between amino acids 23 and 44. The functional nuclear localization sequence (amino acids 278-284) and the basic region (amino acids 301-306) are marked as NLS and BR, respectively.

Either the GFP-tagged Delta - (Fig. 5A) or the beta -isoforms (Fig. 5B) were used as baits to determine homo- and heteromeric interactions in GST pull-down assays. These proteins were ectopically co-expressed in COS cells with either the GST-tagged wild type Delta - or beta -isoforms or with various mutants derived from the Delta -isoform. GST protein (GST) was used as a negative control (shown only with the Delta -isoform, Fig. 5A, lane 1). The GST pull-down precipitates were analyzed by Western blot first by an anti-GFP antibody (top panels) and, after stripping, by an anti-GST antibody (middle panels). The bottom panel shows the quantity of GFP-tagged TFII-I proteins expressed in whole extracts. Except for Delta N90 (lane 3), all other mutants interact with the Delta -isoform (Fig. 5A, top panel). The lack of interaction of the Delta -isoform with Delta N90 is not due to lack of protein expression, because the GFP-tagged Delta -isoform is expressed comparably in all lanes (bottom panel). This is contrary to our expectations, because Delta N90 readily translocates to the nucleus (Fig. 3). The slightly diminished amounts of GFP-TFII-I in the last two lanes directly reflect the lower amount of GST-tagged p70 and Delta BR (lanes 7 and 8) pulled down under our assay conditions and do not constitute a true quantitative difference with the other mutants. Also, the amount of GFP-TFII-I expression in these two lanes was less than the other lanes (see bottom panel). The slightly higher mobility of the Delta N20 mutant (lane 2) is due to its hyperphosphorylation (data not shown). In contrast to the Delta -isoform, the beta -isoform clearly showed interactions with Delta N90 and all other mutants (Fig. 5B, top panel). Once again, the expression of the GFP-beta -isoform was comparable in all lanes (bottom panel). Thus, the first 90 amino acids appear to be important for homomerization of the Delta -isoform but not for its heteromerization with the beta -isoform. Moreover, the first 428 amino acids (as in p46) contain information that is sufficient to mediate both homo- and heteromerization. The Delta linker mutation had no significant effects in interaction with either isoforms suggesting that this form of TFII-I, although only naturally present in mice, can have both homo- and heteromeric interactions.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5.   The N-terminal 90 amino acids of TFII-I are required for homomeric interactions. A, either GST alone (lane 1) or GST-tagged wild type TFII-I (lane 4) or its mutants: Delta N20 (lane 2), Delta N90 (lane 3), Delta linker (lane 5), p46 (lane 6), p70 (lane 7), and Delta BR (lane 8) were used as baits for GST pull-down assays. COS7 cells were cotransfected with 7.5 µg each of expression plasmids encoding the GST baits and GFP-tagged wild type TFII-IDelta (top panel, lanes 1-8). 36 h post-transfection, whole cell lysates were prepared and 200 µg of lysate was subjected to GST pull-down assay. Blots were first probed with anti-GFP antibody (upper panel) and then stripped and reprobed with anti-GST antibody (middle panel). Western blot analysis shows comparable expression of GFP-tagged TFII-I in various lanes. B, the N-terminal 90 amino acids are dispensable for heteromeric interactions with beta -isoform of TFII-I. As in A, COS7 cells were cotransfected with 7.5 µg each of expression plasmids encoding either GST-tagged wild type TFII-IDelta (lane 3) or its various mutants (lanes 1, 2, and 4-6) with the GFP-tagged wild type TFII-Ibeta -isoform (lanes 1-6) and processed as in A. Western blot analysis shows comparable expression of GFP-tagged TFII-Ibeta in various lanes.

The Role of the I-repeats 1 and 2-- The lack of homomeric interaction of Delta N90, despite its nuclear localization, can be reconciled if Delta N90 interacts with itself but not with the wild type Delta -isoform. We further hypothesized that such potential interactions between Delta N90 could be mediated by the I-repeats. To test whether the two molecules of Delta N90 interact with each other and whether the first two I-repeats (as in p46) were necessary and sufficient to mediate such interactions, we employed GFP-tagged Delta N90 as bait. GFP-Delta N90 was ectopically coexpressed with GST-tagged Delta N90, GST-tagged R1, or GST-tagged R2, and GST-pull-down assay was performed. Western blot analysis with an anti-GFP antibody showed that GFP-Delta N90 was brought down by GST-Delta N90, suggesting that Delta N90 interacts with itself (Fig. 6, top panel, lane 1). In addition, both R1 and R2 were also capable of interacting with Delta N90, although to a much lesser extent than the Delta N90 with itself (Fig. 6, top panel, lanes 2 and 3). The blot was stripped and reprobed with an anti-GST antibody to show comparable amounts of GST-Delta N90 (lane 1, second panel), GST-R1 and GST-R2 (lanes 2 and 3, third panel) were precipitated in the pull-down assay. The bottom panel is a Western blot showing comparable amounts of GFP-Delta N90 expression in each extract. We emphasize that the interactions of R1 and R2 with Delta N90 are weak and it is likely that both R1 and R2 are necessary for a robust and physiological interaction. It is also possible that these repeats fail to reach the nucleus (note that they lack the NLS). Together, these results indicate that, although the I-repeats can mediate homomeric interactions, in the context of the full-length TFII-I, the N-terminal 90 amino acids are also required for such interactions.



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 6.   I-repeats can mediate protein-protein interactions. The expression plasmids coding for GST-Delta N90, I-repeat 1 (GST-R1) or I-repeat 2 (GST-R2) were cotransfected with GFP-Delta N90 in COS7 cells. 36 h post-transfection, lysates were prepared and 300 µg of lysate was subjected to GST pull-down assay. The precipitates were separated by 10% SDS-PAGE and Western-blotted. The blot was cut into two halves and processed simultaneously. The top part (70-230 kDa) of the blot was first probed with anti-GFP antibody to visualize GFP-Delta N90 (top panel), stripped, and reprobed with anti-GST antibody to visualize GST-Delta N90 (second panel). The bottom half (70-20 kDa) of the blot was only probed with anti-GST antibody to visualize either GST-R1 or GST-R2 (third panel). The expression of Delta N90-GFP was checked by Western blot analysis of each lysates (50 µg) with anti-GFP antibody (bottom panel).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Since the initial discovery of TFII-I as an Initiator binding protein (1), this transcription factor has remained intriguing. Although additional functions ranging from roles in signal transduction to a potential role in genetic disorders have been attributed to this complex molecule (reviewed in Ref. 8), some of the more fundamental information regarding its DNA binding ability and protein-protein interaction potentials have remained largely unknown. Given its broad potential biological role, we believe that it is important to determine these fundamental properties of TFII-I. In an attempt to understand the DNA binding and protein-protein interaction capabilities of TFII-I, we have begun its structure-function analysis and generated selective mutants and analyzed them in various functional assays.

One of the most interesting features of the TFII-I sequence is the presence of six highly conserved 90-residue I-repeats, each of which contains a potential helix-loop-helix (HLH)-like domain that is implicated in homo- and heterodimerization of conventional HLH proteins (reviewed in Ref. 12). However, unlike the conventional HLH proteins, in which a basic region that directly mediates DNA contact precedes the HLH regions, most of the repeats in TFII-I lack a basic region preceding them. The only notable exception is a basic region just before I-repeat 2 (BR, amino acids 301-306). Given this feature, and the fact that the anti-TFII-I antibody raised against the BR ablates the DNA binding ability of TFII-I (5-7), we surmised that the BR could either directly or indirectly affect the DNA binding of TFII-I. Consistent with this notion, deletion of the BR results in a DNA binding-deficient mutant that failed to bind to both a consensus Inr sequence and an upstream activation site. However, it should be noted that, although the TFII-I recognition element from the c-fos promoter lies upstream of the transcription start site, the sequence matches a consensus Inr element (9). Thus, the BR may recognize only this sequence albeit in a context-dependent fashion. If so, then there might be other DNA recognition surfaces present in TFII-I that could bind diverse DNA elements, such as an E-box (1, 4). Although identification of the BR as a DNA binding domain was important, it was somewhat expected. More surprising was the lack of significant DNA binding property of the Delta N90 mutant. The region that appears to be responsible for such a phenotype includes a putative leucine zipper, and it is very likely that this leucine zipper mediates a homo- or heteromeric interaction required for DNA binding (12, 13). However, homo- or heteromerization per se may not be sufficient for DNA binding, because the Delta N90 mutant homomerizes with itself (see below). Thus, the N-terminal region may directly contact DNA or indirectly contribute to DNA binding by altering the native structure, and removal of this region will expose a negative inhibitory domain that covers the DNA binding domain.

Given the lack of DNA binding ability of the Delta BR and Delta N90 mutants, it is not surprising that they fail to activate the TFII-I-dependent reporters. Together with the p70 mutant, which exhibits DNA binding but lacks an activation domain (6), it is also not surprising that the Delta BR mutant that lacks DNA binding activity did not exhibit any significant transcriptional activity. Hence, proper transcriptional function of TFII-I requires both its DNA binding capabilities and its transcriptional activation domain. In this regard, it behaves like a classical transcription factor with an N-terminal DNA binding domain and a separable C-terminal activation domain.

Although the nuclear translocation of the mutants was normal, the lack of homomeric interactions of the Delta N90 mutant with the wild type TFII-IDelta -isoform was puzzling at first glance. This is largely because we have previously shown that homomeric or heteromeric interactions lead to preferential nuclear occupancy (8). We argued that, although Delta N90 failed to interact with the wild type Delta -isoform, it could interact with itself leading to nuclear localization. Interaction studies revealed that indeed Delta N90 interacts with itself. These observations raise a very interesting question: What is the role of the N-terminal end? The N-terminal end of the protein contains the leucine zipper motif (4) that is also present in all the isoforms of human and mouse TFII-I (8, 19, 20). Moreover, several TFII-I-related proteins contain this motif approximately in the same position (21-26).2 Based on our observations, we propose that the Delta -isoform remains in a "closed" conformation until the LZ motif becomes available to interact with other partners: either another molecule of Delta  or another molecule of beta  (Fig. 7). In contrast to the Delta -isoform, the beta -isoform can assume a constitutively open conformation. Although secondary interactions can happen through the I-repeats in the absence of the LZ motif, Delta N90, without the LZ cannot induce conformational changes in the wild type Delta  (closed conformation). However, Delta N90 can interact readily with beta  (open conformation). Thus, we conjecture that the regulated availability of the leucine zipper, perhaps by phosphorylation or other signal-induced events, might control the extent of complex formation and consequently, nuclear function of TFII-I. Such a mechanism of regulated zipper interaction via phosphorylation has been proposed for the Ikappa B kinase complex formation (reviewed in Ref. 27).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7.   Model for regulated homomeric interaction. a, the Delta -isoform remains in a "closed" conformation. The N-terminal LZ is denoted by four parallel lines, and the repeats are depicted as black boxes. Only the first two repeats are shown. b, signal-induced conformational changes may lead to availability of the leucine zipper and subsequent coiled-coiled homomeric interactions. Subsequent secondary interactions can occur via the direct repeats. c, homomeric interactions involving the beta -isoform may not primarily require the LZ but may require the direct repeats. The additional 20 amino acids between the repeats may introduce a "kink" and make the conformation constitutively "open." d, Delta N90, lacking the LZ, does not interact with the Delta -isoform because the LZ is required to induce conformation changes in the closed Delta -isoform. e, interactions of Delta N90 with the constitutively open beta -isoform do not require LZ. f, lack of LZ also makes the direct repeats constitutively available for interactions in Delta N90. For the sake of simplicity, we have depicted the interactions as dimeric.



    ACKNOWLEDGEMENTS

We are grateful to rotating student Mireia Guerau for her contribution. We thank Roy laboratory members for their input, valuable suggestions, and reading of the manuscript. We also thank Mike Byrne for DNA sequencing that was done at the Tufts University School of Medicine.


    FOOTNOTES

* This work was supported by Grant RPG-98-104-01-TBE from the American Cancer Society and Grant AI45150 from the National Institutes of Health (to A. L. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Dept. of Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6715; Fax: 617-636-2990; E-mail: ananda.roy@tufts.edu.

Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008411200

2 I. Tusie-Luna and A. L. Roy, submitted for publication.


    ABBREVIATIONS

The abbreviations used are: HLH, helix-loop-helix motif; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; GFP, green fluorescence protein; EMSA, electrophoretic mobility shift assay; DAPI, 4,6-diamidino-2-phenylindole; WT, wild type; NLS, nuclear localization signal.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Roy, A. L., Meisterernst, M., Pognonec, P., and Roeder, R. G. (1991) Nature 354, 245-248[CrossRef][Medline] [Order article via Infotrieve]
2. Roy, A. L., Malik, S., Meisterernst, M., and Roeder, R. G. (1993) Nature 365, 355-359[CrossRef][Medline] [Order article via Infotrieve]
3. Roy, A. L., Carruthers, C., Gutjar, T., and Roeder, R. G. (1993) Nature 365, 359-362[CrossRef][Medline] [Order article via Infotrieve]
4. Roy, A. L., Du, H., Gregor, P. D., Novina, C. D., Martinez, E., and Roeder, R. G. (1997) EMBO J. 16, 7091-7104[Abstract/Free Full Text]
5. Manzano-Winkler, B., Novina, C. D., and Roy, A. L. (1996) J. Biol. Chem. 271, 12076-12081[Abstract/Free Full Text]
6. Cheriyath, V., Novina, C. D., and Roy, A. L. (1998) Mol. Cell. Biol. 18, 4444-4454[Abstract/Free Full Text]
7. Wu, Y., and Patterson, C. (1999) J. Biol. Chem. 274, 3207-3214[Abstract/Free Full Text]
8. Cheriyath, V., and Roy, A. L. (2000) J. Biol. Chem. 275, 26300-26308[Abstract/Free Full Text]
9. Grueneberg, D. A., Henry, R. W., Brauer, A., Novina, C. D., Cheriyath, V., Roy, A. L., and Gilman, M. (1997) Genes Dev. 11, 2482-2493[Abstract/Free Full Text]
10. Kim, D.-W., Cheriyath, V., Roy, A. L., and Cochran, B. H. (1998) Mol. Cell. Biol. 18, 3310-3320[Abstract/Free Full Text]
11. Mobley, C. M., and Sealy, L. (2000) J. Virol. 74, 6511-6519[Abstract/Free Full Text]
12. Ferre-D'Amare, A. R., Prendergast, G. C., Ziff, E. B., and Burley, S. K. (1993) Nature 363, 38-45[CrossRef][Medline] [Order article via Infotrieve]
13. Ferre-D'Amare, A. R., Pognonec, P., Roeder, R. G., and Burley, S. K. (1994) EMBO J. 13, 180-189[Abstract]
14. Novina, C. D., Cheriyath, V., and Roy, A. L. (1998) J. Biol. Chem. 273, 33443-33448[Abstract/Free Full Text]
15. Novina, C. D., Kumar, S., Bajpai, U., Cheriyath, V., Zang, K., Pillai, S., Wortis, H. H., and Roy, A. L. (1999) Mol. Cell. Biol. 19, 5014-5024[Abstract/Free Full Text]
16. Yang, W., and Desiderio, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 604-609[Abstract/Free Full Text]
17. Novina, C. D., Cheriyath, V., Denis, M. C., and Roy, A. L. (1997) Methods 12, 254-263[CrossRef][Medline] [Order article via Infotrieve]
18. Vandromme, M., Gauthier-Rouviere, C., Lamb, N., and Fernandez, A. (1996) Trends Biochem. Sci. 21, 59-64[CrossRef][Medline] [Order article via Infotrieve]
19. Perez Juardo, L. A., Wang, Y.-K., Peoples, R., Coloma, A., Cruces, J., and Francke, U. (1998) Hum. Mol. Genet. 7, 325-334[Abstract/Free Full Text]
20. Wang, Y. K., Perez-Juardo, L. A., and Francke, U. (1998) Genomics 48, 163-170[CrossRef][Medline] [Order article via Infotrieve]
21. O'Mahoney, J. V., Guven, K. L., Lin, J., Joya, J. E., Robinson, C. S., Wade, R. P., and Hardeman, E. C. (1998) Mol. Cell. Biol. 18, 6641-6652[Abstract/Free Full Text]
22. Osborne, L. R., Campbell, T., Daradich, A., Scherer, S. W., and Tsui, L.-C. (1999) Genomics 57, 279-284[CrossRef][Medline] [Order article via Infotrieve]
23. Franke, Y., Peoples, R. J., and Francke, U. (1999) Cytogenet. Cell. Genet. 86, 296-304[CrossRef][Medline] [Order article via Infotrieve]
24. Tassabehji, M., Carette, M., Wilmot, C., Donnai, D., Read, A. P., and Metcalfe, K. (1999) Eur. J. Hum. Genet. 7, 737-747[Medline] [Order article via Infotrieve]
25. Yan, X., Zhao, X., Qian, M., Guo, N., Gong, X., and Zhu, X. (2000) Biochem. J. 345, 749-757[CrossRef][Medline] [Order article via Infotrieve]
26. Bayarsaihan, D., and Ruddle, F. H. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7342-7347[Abstract/Free Full Text]
27. Zandi, E., and Karin, M. (1999) Mol. Cell. Biol. 19, 4547-4551[Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.