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Molecular Cloning of a Novel 120-kDa TBP-Interacting Protein
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
229, 612–617 (1996)
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Molecular Cloning of a Novel 120-kDa TBP-Interacting Protein1 Shingo Yogosawa,*,2 Yasutaka Makino,*,2 Tatsushi Yoshida,* Toshihiko Kishimoto,* Masami Muramatsu,† and Taka-aki Tamura*,3 *Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba-263, Japan; and †Department of Biochemistry, Saitama Medical School, Moroyama, Iruma-gun, Saitama-350, Japan Received November 8, 1996 TATA-binding protein (TBP) is a central component for transcriptional regulation and is a target for various transcription regulators. Using histidine-tagged TBP as a ligand for affinity-purification of proteins bound to TBP, we purified a 120-kD protein, termed TBP-interacting protein 120 (TIP120), from rat liver nuclear extracts. The entire cDNA sequence of TIP120 contained an open reading frame encoding a novel polypeptide of 1230 amino acids. The recombinant TIP120 interacted directly with TBP under a physiological condition in vitro. Immunoprecipitation analysis indicated that TIP120 was associated with TBP in nuclear extracts. Interestingly, the N-terminal region of TIP120 exhibited sequence similarity to that of Drosophila TAF80, which was shown to bind directly to TBP. This novel TBP-binding protein is considered to participate in transcription regulation through the interaction with TBP. q 1996 Academic Press, Inc.
In eukaryotes, the TATA-binding protein (TBP) is an essential transcription factor for RNA polymerases I, II, and III, and thus appears to be required for all nuclear transcription (1). The general transcription factors, i. e., SL1, TFIID, TFIIIB, are composed of TBP and different sets of TBP-associated factors (TAFs) (2-4). TAFs in the TFIID complex have been shown to interact selectively with different classes of transcriptional regulators, and certain TAFs are required to mediate transcriptional enhancement by activators (5, 6). Studies on viral transactivators such as VP16, E1A, and Zta show that contact of those regulators with TBP is a critical step for activation (7-9). Several cellular transcription factors, e.g., p53 and c-Myc have a domain that binds to TBP in vitro (10, 11). Eventually, those proteins are thought to regulate transcription by direct or indirect interaction with TBP. According to the above context, a factor that can bind to TBP is assumed to fall into the category of a transcription regulator. Discovery of a new TBP-binding protein would thus expectedly lead to identification of a novel mechanism of transcription regulation. In this study, to search for new regulatory factors, we examined rat liver nuclear extracts for the presence of TBP-binding proteins by in vitro binding assay. We isolated a cDNA of a novel 120-kD TBP-interacting protein that exhibits significant homology to Drosophila TAF80 (12, 13). MATERIALS AND METHODS Purification and amino acid sequence determination of TIP120. HXmTBP was expressed in E. coli and purified to near homogeneity as previously described (14). Nuclear extracts were prepared from rat liver as described (20). The extracts were incubated with HXmTBP and Ni-agarose beads. After extensively washing, TIP120 was eluted with 3 M urea, resolved by SDS-PAGE, and then electrotransferred onto a polyvinylidene difluoride membrane. The
1 The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank databases with the following accession number: D87671. 2 S.Y. and Y.M. contributed equally to this work. 3 Corresponding author. Fax: (81)43-290-2824.
612 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Detection of TIPs in rat liver nuclear extracts. (A) TIPs as specifically bound to HXmTBP in vitro. TIPs were obtained from rat liver nuclear extracts with HXmTBP followed by 10% SDS-PAGE and silver staining. Reactions were carried out in the presence (/) or absence (0) of each protein sample. E. coli: E. coli proteins purified by Ni-agarose. NE: rat liver nuclear extract. Positions of HXmTBP and E. coli proteins are indicated by an arrow and brackets, respectively. (B) Detection of TIP120 by heat treatment. Nuclear extract was heated at 47, 52, and 577C for 20 min. TIPs present in the heated-extract were analyzed by 7.5% SDS–PAGE and silver stained. Dots denote background proteins.
blotted proteins were subjected to trypsin digestion. Protein sequence determination was performed by a protein sequencer. cDNA cloning. On the basis of the determined peptide sequences, degenerated oligonucleotides were synthesized. To isolate cDNA clones for TIP120, we screened a rat liver cDNA library in lgt11 with the 32P-labeled oligonucleotide. Antibodies and immunoprecipitation. Histidine-tagged TIP120 was expressed in Sf9 cells and purified with Niagarose. The protein was further purified on Mono Q column and then used for in vitro binding assay. To generate antibody, the TIP120 was further separated on a preparative SDS-PAGE and electroeluted from the gel. The resulting protein was used to immunize rabbits. The antibody was affinity-purified using TIP120-immobilized beads. For immunoprecipitation, nuclear extracts were dialyzed against IP buffer (25 mM HEPES/KOH [pH7.9], 0.1 M KCl, 5 mM MgCl2 , 0.5 mM DTT, 0.1% NP-40, 10% glycerol) and incubated with antibody-coupled protein A-Sepharose. After extensively washing, the beads resuspended in the SDS loading buffer. Precipitated proteins were analyzed by Western blotting. Protein binding assay. 0.2 mM of TBP and TIP120 were incubated at 4 7C for 60 min in the IP buffer. Anti-TBP or -TIP120 antibody immobilized on protein A-Sepharose beads was added and washed extensively with IP buffer. The precipitated proteins were analyzed by SDS-PAGE and silver stained.
RESULTS AND DISCUSSION Detection of a 120-kD TBP-Interacting Protein in Rat Liver Nuclear Extracts We used histidine-tagged TBP (HXmTBP) to directly detect TBP-binding proteins in rat liver nuclear extracts. As previously reported, HXmTBP was produced in E. coli and purified to near homogeneity (14). After incubation of HXmTBP with nuclear extracts, polypeptides bound to the HXmTBP were purified with Ni-agarose and separated by SDS-PAGE. Many polypeptides with molecular masses ranging from ú200 to 40-kD were detected (Fig. 1A, 613
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lane 5), whereas only few polypeptides bound nonspecifically to the beads were observed (lanes 4 and 6), if we used nuclear extract or HXmTBP alone. We found E. coli proteins present in the HXmTBP preparation (Fig. 1A, lane 6). Although these proteins might serve as an adaptor for the polypeptides detected, we confirmed that no polypeptides bound to those contaminants (Fig. 1A, lanes 1-3), suggesting that almost all polypeptides in lane 5 are dependent on the presence of HXmTBP. We referred to the proteins specifically bound to TBP in vitro as TBP-interacting proteins (TIPs). These TIPs were recovered dependent on the Cterminal domain of TBP (data not shown). To reduce TIPs detected, we heated nuclear extracts (47 7C, 20 min) before the addition of HXmTBP. Most of TIPs seen in Fig. 1A disappeared (Fig. 1B, lane 1), whereas one major TIP of 120-kD and a few minor TIPs remained after the heat treatment. When the temperature was increased to 52 7C or 57 7C, 120-kD protein was predominantly observed (Fig. 1B). The protein, designated as TIP120, was thus suspected to interact, presumably directly, with HXmTBP. A question arose as how HXmTBP interacts with TIPs in vitro. An exchange reaction may occur between HXmTBP and endogenous TBP. Alternatively, HXmTBP might bind to the TBP moiety in native TBP-containing complexes, since TBP can self-associate in vitro and TFIID exists as a dimer in vivo (14-16). Molecular Cloning of a cDNA Encoding TIP120 Amino acid (a.a.) sequences of two tryptic peptides derived from TIP120 were determined and used to design oligonucleotide probes. A rat liver cDNA library was screened and a 4.4kb cDNA containing a poly(A) tail was cloned. Northern analysis with this cDNA detected 4.5-kb mRNA (data not shown). The DNA sequence of this clone revealed an open reading frame of 1230 amino acids. Two peptide sequences obtained from the tryptic digests were found within the same open reading frame (Fig. 2A). The a.a. sequence of TIP120 predicted a protein with a calculated molecular mass of 135-kD, a value similar to that estimated by SDS-PAGE. We expressed TIP120 with a histidine tag at the N-terminus in Sf9 cells and purified it to near homogeneity. The recombinant TIP120 was resistant against heat treatment at 52 7C, consistent with the result of Fig. 1B (data not shown). Fig. 3A shows that antiTIP120 antibody reacted with the recombinant TIP120, with TIP120 purified with HXmTBP, and with endogenous TIP120 in the nuclear extract (lanes 1-5). Thus, we decided that the cloned 4.4-kb cDNA encoded full-length TIP120. TIP120 Interacts with TBP To investigate whether TIP120 interacts directly with TBP, a mixture of equimolar amounts (0.2 mM) of TBP and TIP120 was immunoprecipitated with the respective antibodies. AntiTBP antibody coimmunoprecipitated TIP120 together with TBP (Fig. 3B, lane 2). Similarly, anti-TIP120 antibody coimmunoprecipitated TBP together with TIP120 (Fig. 3B, lane 7), but control antibody did not (lane 1). Since both recombinant proteins contain a histidine tag, each antibody might crossreact with the tag. Anti-TBP and -TIP120 antibodies immunoprecipitated their respective proteins specifically (Fig. 3B, lanes 3 and 6), whereas anti-TIP120 and -TBP antibodies did not immunoprecipitated TBP and TIP120, respectively (lanes 4 and 5), suggesting no crossreactivity of either antibody with the tag. We concluded that TIP120 can bind to TBP. In a previous study, we estimated that the concentration of TBP was approximately 2-6 mM in rat liver nuclei (14). Taggart et al. (19) reported that the TBP concentration is ú 1 mM in the HeLa cell. Concentration of TIP120 in the nucleus was estimated to be 0.5 mM. Thus, the interaction between TIP120 and TBP can occur in nuclei. When nuclear extracts were immunoprecipitated with anti-TIP120 antibody, TBP was coimmunoprecipitated with TIP120 (Fig. 3C, lane 2). This association occurred even in a higher-salt condition (0.2 M KCl) (data not shown). No interaction between TBP and TIP120 was observed when a control 614
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FIG. 2. Structure of TIP120. (A) Nucleotide sequence of a cDNA encoding TIP120. The amino acid sequence is listed by the single-letter codes. Underlined amino acids represent peptide sequences derived from microsequence of the purified TIP120. (B) Schematic representation of TIP120 structure. The lower line indicates amino acid sequence positions. Several characteristic sequences such as charged (13-104 residues), acidic (267-344 residues), and leucinerich regions (600-779 and 1003-1149 residues) are indicated. The dTAF80 homology region is also shown. Two asterisks denote possible phosphorylation sites for casein kinase II (CKII). (C) Sequence similarity between TIP120 and dTAF80. Identical and homologous amino acid residues were indicated by vertical lines and double dots, respectively.
immunoglobulin was used (Fig. 3C, lane 1). This result suggests that TIP120 is associated with TBP in nuclear extracts. Primary Structure of TIP120 Analysis of the a.a. sequence of TIP120 failed to detect any typical structural motifs usually found in transcription factors, but revealed some other characteristic structures. As shown in Fig. 2B, the N-terminal region of TIP120 (a.a. 13-104) was highly charged with 37% of the residues either acidic or basic. The TIP120-coding sequence contained two regions rich in leucine residues (a.a. 600-779, 22%; a.a. 1003-1149, 20%). These regions may be implicated in the interaction with other proteins through a leucine zipper-like structure. The N-terminal one-third was rich in acidic a.a. residues (a.a. 267-344; 36% being glutamic or asparatic acid residues). The acidic domain had some consecutive negatively charged regions, two of which (335-SDDE-338 and 340-SDDD-343) were identical to a consensus target site for casein kinase II, which is known to control the activity of transcription factors (17-19). 615
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FIG. 3. Characterization of the TIP120. (A) Western blot analysis of recombinant and endogenous TIP120. Recombinant TIP120 (lanes 1-3; 30 ng, 15 ng, or 7.5 ng), affinity-purified TIP120 (lane 4; 10 ng), and nuclear extract (lane 5; 10 mg) were analyzed by Western blotting using anti-TIP120 antibody. Arrow indicates the position of TIP120. (B) Interaction of TIP120 with TBP. The recombinant proteins (0.2 mM each) were incubated as indicated. The mixtures were immunoprecipitated with control IgG (lane 1), anti-TBP antibody (lanes 2, 3, and 5), and anti-TIP120 antibody (lanes 4, 6, and 7)-coupled protein A-Sepharose. The immunoprecipitates were resolved by 10% SDS-PAGE and stained with silver. An asterisk indicates the IgG heavy chain. (C) Association of TIP120 with TBP in nuclear extracts. The extracts were immunoprecipitated with control IgG or anti-TIP120 antibody-coupled protein A-Sepharose beads (lanes 1 and 2). The precipitated proteins and nuclear extracts (lane 3) were resolved by 10% SDS-PAGE and analyzed by Western blotting with anti-TBP antibody. Arrow indicates the position of TBP. An asterisk indicates the IgG heavy chain.
Comparison of the TIP120 a.a. sequence with known sequences in databases by means of the BLAST program without any gaps revealed that the TIP120 exhibited significant homology with the N-terminus of the Drosophila TFIID subunit p85, also called dTAF80 (Fig. 2C) (12, 13). These two proteins were 20% identical over a 95 a.a. overlap and 44% similar when conservative a.a. substitutions were taken into account. The N-terminal region of dTAF80 is essential for the interaction with TBP (13). The homologous sequence may be an evolutionary conserved interface for the interactions with TBP. These observations suggest that TIP120 is a novel TBP-binding protein and might regulate transcription through the interaction with TBP. ACKNOWLEDGMENTS We thank Dr. K. Kato for helpful discussions. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from The Japanese Ministry of Education, Science, Sports, and Culture.
REFERENCES 1. Hernandez, N. (1993) Genes Dev. 7, 1291–1308. 2. Comai, L., Zomerdijk, J. C. B. M., Beckmann, H., Zhou, S., Admon, A., and Tjian, R. (1995) Science 266, 1966– 1972. 3. Hoey, T., Weinzieri, R. O. J., Gill, G., Chen, J. L., Dynlacht, B. D., and Tjian, R. (1993) Cell 72, 247–260. 4. Taggart, A. K. P., Fisher, T. S., and Pugh, B. F. (1992) Cell 71, 1015–1028. 5. Chen, J. L., Attardi, L. D., Verrijzer, C. P., Yokomori, K., and Tjian, R. (1994) Cell 79, 93–105. 6. Chiang, C., and Roeder, R. G. (1995) Science 267, 531–536. 616
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7. Horikoshi, N., Maguire, K., Kraill, A., Maldonado, E., Reinberg, D., and Weinmann, R. (1991) Proc. Natl. Acad. Sci. USA 88, 5124–5128. 8. Lee, W. S., Kao, C., Bryant, G. O., Liu, X., and Berk, A. J. (1991) Cell 67, 365–376. 9. Lieberman, P. M., and Berk, A. J. (1991) Genes Dev. 5, 2441–2454. 10. Hateboer, G., Timmers, H. T. M., Rustgi, A. K., Billaud, M., Van’t Veer, L. J., and Bernard, R. (1993) Proc. Natl. Acad. Sci. USA 90, 8489–8493. 11. Seto, E., Usheva, A., Zambetti, G. P., Momand, J., Horikoshi, N., Weinmann, R., Levine, A. J., and Shenk, T. (1992) Proc. Natl. Acad. Sci. USA 89, 12028–12032. 12. Dynlacht, B. D., Weinzierl, R. O. J., Admon, A., and Tjian, R. (1993) Nature 363, 176–179. 13. Kokubo, T., Gong, D.-W., Yamashita, S., Takada, R., Roeder, R. G., Horikoshi, M., and Nakatani, Y. (1993) Mol. Cell. Biol. 13, 7859–7863. 14. Kato, K., Makino, Y., Kishimoto, T., Yamauchi, J., Kato, S., Muramatsu, M., and Tamura, T. (1994) Nucl. Acids Res. 22, 1179–1185. 15. Coleman, R. A., Taggart, A. K. P., Benjamin, L. R., and Pugh, B. F. (1995) J. Biol. Chem. 270, 13842–13849. 16. Taggart, A. K. P., and Pugh, B. F. (1996) Science 272, 1331–1333. 17. Kuenzel, E. A., Mulligan, J. A., Sommercorn, J., and Krebs, E. G. (1987) J. Biol. Chem. 262, 9136–9140. 18. Lu¨scher, B., Christenson, E., Litchfield, D. W., Krebs, E. G., and Eisenman, R. N. (1990) Nature 344, 517–522. 19. Berberich, S. J., and Cole, M. D. (1992) Genes Dev. 6, 166–176. 20. Tamura, T., Ohya, Y., Miura, M., Aoyama, A., Inoue, T., and Mikoshiba, K. (1989) Technique 1, 33–36.
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Molecular Cloning of a Novel 120-kDa TBP-Interacting Protein1 Shingo Yogosawa,*,2 Yasutaka Makino,*,2 Tatsushi Yoshida,* Toshihiko Kishimoto,* Masami Muramatsu,† and Taka-aki Tamura*,3 *Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba-263, Japan; and †Department of Biochemistry, Saitama Medical School, Moroyama, Iruma-gun, Saitama-350, Japan Received November 8, 1996 TATA-binding protein (TBP) is a central component for transcriptional regulation and is a target for various transcription regulators. Using histidine-tagged TBP as a ligand for affinity-purification of proteins bound to TBP, we purified a 120-kD protein, termed TBP-interacting protein 120 (TIP120), from rat liver nuclear extracts. The entire cDNA sequence of TIP120 contained an open reading frame encoding a novel polypeptide of 1230 amino acids. The recombinant TIP120 interacted directly with TBP under a physiological condition in vitro. Immunoprecipitation analysis indicated that TIP120 was associated with TBP in nuclear extracts. Interestingly, the N-terminal region of TIP120 exhibited sequence similarity to that of Drosophila TAF80, which was shown to bind directly to TBP. This novel TBP-binding protein is considered to participate in transcription regulation through the interaction with TBP. q 1996 Academic Press, Inc.
In eukaryotes, the TATA-binding protein (TBP) is an essential transcription factor for RNA polymerases I, II, and III, and thus appears to be required for all nuclear transcription (1). The general transcription factors, i. e., SL1, TFIID, TFIIIB, are composed of TBP and different sets of TBP-associated factors (TAFs) (2-4). TAFs in the TFIID complex have been shown to interact selectively with different classes of transcriptional regulators, and certain TAFs are required to mediate transcriptional enhancement by activators (5, 6). Studies on viral transactivators such as VP16, E1A, and Zta show that contact of those regulators with TBP is a critical step for activation (7-9). Several cellular transcription factors, e.g., p53 and c-Myc have a domain that binds to TBP in vitro (10, 11). Eventually, those proteins are thought to regulate transcription by direct or indirect interaction with TBP. According to the above context, a factor that can bind to TBP is assumed to fall into the category of a transcription regulator. Discovery of a new TBP-binding protein would thus expectedly lead to identification of a novel mechanism of transcription regulation. In this study, to search for new regulatory factors, we examined rat liver nuclear extracts for the presence of TBP-binding proteins by in vitro binding assay. We isolated a cDNA of a novel 120-kD TBP-interacting protein that exhibits significant homology to Drosophila TAF80 (12, 13). MATERIALS AND METHODS Purification and amino acid sequence determination of TIP120. HXmTBP was expressed in E. coli and purified to near homogeneity as previously described (14). Nuclear extracts were prepared from rat liver as described (20). The extracts were incubated with HXmTBP and Ni-agarose beads. After extensively washing, TIP120 was eluted with 3 M urea, resolved by SDS-PAGE, and then electrotransferred onto a polyvinylidene difluoride membrane. The
1 The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank databases with the following accession number: D87671. 2 S.Y. and Y.M. contributed equally to this work. 3 Corresponding author. Fax: (81)43-290-2824.
612 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Detection of TIPs in rat liver nuclear extracts. (A) TIPs as specifically bound to HXmTBP in vitro. TIPs were obtained from rat liver nuclear extracts with HXmTBP followed by 10% SDS-PAGE and silver staining. Reactions were carried out in the presence (/) or absence (0) of each protein sample. E. coli: E. coli proteins purified by Ni-agarose. NE: rat liver nuclear extract. Positions of HXmTBP and E. coli proteins are indicated by an arrow and brackets, respectively. (B) Detection of TIP120 by heat treatment. Nuclear extract was heated at 47, 52, and 577C for 20 min. TIPs present in the heated-extract were analyzed by 7.5% SDS–PAGE and silver stained. Dots denote background proteins.
blotted proteins were subjected to trypsin digestion. Protein sequence determination was performed by a protein sequencer. cDNA cloning. On the basis of the determined peptide sequences, degenerated oligonucleotides were synthesized. To isolate cDNA clones for TIP120, we screened a rat liver cDNA library in lgt11 with the 32P-labeled oligonucleotide. Antibodies and immunoprecipitation. Histidine-tagged TIP120 was expressed in Sf9 cells and purified with Niagarose. The protein was further purified on Mono Q column and then used for in vitro binding assay. To generate antibody, the TIP120 was further separated on a preparative SDS-PAGE and electroeluted from the gel. The resulting protein was used to immunize rabbits. The antibody was affinity-purified using TIP120-immobilized beads. For immunoprecipitation, nuclear extracts were dialyzed against IP buffer (25 mM HEPES/KOH [pH7.9], 0.1 M KCl, 5 mM MgCl2 , 0.5 mM DTT, 0.1% NP-40, 10% glycerol) and incubated with antibody-coupled protein A-Sepharose. After extensively washing, the beads resuspended in the SDS loading buffer. Precipitated proteins were analyzed by Western blotting. Protein binding assay. 0.2 mM of TBP and TIP120 were incubated at 4 7C for 60 min in the IP buffer. Anti-TBP or -TIP120 antibody immobilized on protein A-Sepharose beads was added and washed extensively with IP buffer. The precipitated proteins were analyzed by SDS-PAGE and silver stained.
RESULTS AND DISCUSSION Detection of a 120-kD TBP-Interacting Protein in Rat Liver Nuclear Extracts We used histidine-tagged TBP (HXmTBP) to directly detect TBP-binding proteins in rat liver nuclear extracts. As previously reported, HXmTBP was produced in E. coli and purified to near homogeneity (14). After incubation of HXmTBP with nuclear extracts, polypeptides bound to the HXmTBP were purified with Ni-agarose and separated by SDS-PAGE. Many polypeptides with molecular masses ranging from ú200 to 40-kD were detected (Fig. 1A, 613
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lane 5), whereas only few polypeptides bound nonspecifically to the beads were observed (lanes 4 and 6), if we used nuclear extract or HXmTBP alone. We found E. coli proteins present in the HXmTBP preparation (Fig. 1A, lane 6). Although these proteins might serve as an adaptor for the polypeptides detected, we confirmed that no polypeptides bound to those contaminants (Fig. 1A, lanes 1-3), suggesting that almost all polypeptides in lane 5 are dependent on the presence of HXmTBP. We referred to the proteins specifically bound to TBP in vitro as TBP-interacting proteins (TIPs). These TIPs were recovered dependent on the Cterminal domain of TBP (data not shown). To reduce TIPs detected, we heated nuclear extracts (47 7C, 20 min) before the addition of HXmTBP. Most of TIPs seen in Fig. 1A disappeared (Fig. 1B, lane 1), whereas one major TIP of 120-kD and a few minor TIPs remained after the heat treatment. When the temperature was increased to 52 7C or 57 7C, 120-kD protein was predominantly observed (Fig. 1B). The protein, designated as TIP120, was thus suspected to interact, presumably directly, with HXmTBP. A question arose as how HXmTBP interacts with TIPs in vitro. An exchange reaction may occur between HXmTBP and endogenous TBP. Alternatively, HXmTBP might bind to the TBP moiety in native TBP-containing complexes, since TBP can self-associate in vitro and TFIID exists as a dimer in vivo (14-16). Molecular Cloning of a cDNA Encoding TIP120 Amino acid (a.a.) sequences of two tryptic peptides derived from TIP120 were determined and used to design oligonucleotide probes. A rat liver cDNA library was screened and a 4.4kb cDNA containing a poly(A) tail was cloned. Northern analysis with this cDNA detected 4.5-kb mRNA (data not shown). The DNA sequence of this clone revealed an open reading frame of 1230 amino acids. Two peptide sequences obtained from the tryptic digests were found within the same open reading frame (Fig. 2A). The a.a. sequence of TIP120 predicted a protein with a calculated molecular mass of 135-kD, a value similar to that estimated by SDS-PAGE. We expressed TIP120 with a histidine tag at the N-terminus in Sf9 cells and purified it to near homogeneity. The recombinant TIP120 was resistant against heat treatment at 52 7C, consistent with the result of Fig. 1B (data not shown). Fig. 3A shows that antiTIP120 antibody reacted with the recombinant TIP120, with TIP120 purified with HXmTBP, and with endogenous TIP120 in the nuclear extract (lanes 1-5). Thus, we decided that the cloned 4.4-kb cDNA encoded full-length TIP120. TIP120 Interacts with TBP To investigate whether TIP120 interacts directly with TBP, a mixture of equimolar amounts (0.2 mM) of TBP and TIP120 was immunoprecipitated with the respective antibodies. AntiTBP antibody coimmunoprecipitated TIP120 together with TBP (Fig. 3B, lane 2). Similarly, anti-TIP120 antibody coimmunoprecipitated TBP together with TIP120 (Fig. 3B, lane 7), but control antibody did not (lane 1). Since both recombinant proteins contain a histidine tag, each antibody might crossreact with the tag. Anti-TBP and -TIP120 antibodies immunoprecipitated their respective proteins specifically (Fig. 3B, lanes 3 and 6), whereas anti-TIP120 and -TBP antibodies did not immunoprecipitated TBP and TIP120, respectively (lanes 4 and 5), suggesting no crossreactivity of either antibody with the tag. We concluded that TIP120 can bind to TBP. In a previous study, we estimated that the concentration of TBP was approximately 2-6 mM in rat liver nuclei (14). Taggart et al. (19) reported that the TBP concentration is ú 1 mM in the HeLa cell. Concentration of TIP120 in the nucleus was estimated to be 0.5 mM. Thus, the interaction between TIP120 and TBP can occur in nuclei. When nuclear extracts were immunoprecipitated with anti-TIP120 antibody, TBP was coimmunoprecipitated with TIP120 (Fig. 3C, lane 2). This association occurred even in a higher-salt condition (0.2 M KCl) (data not shown). No interaction between TBP and TIP120 was observed when a control 614
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FIG. 2. Structure of TIP120. (A) Nucleotide sequence of a cDNA encoding TIP120. The amino acid sequence is listed by the single-letter codes. Underlined amino acids represent peptide sequences derived from microsequence of the purified TIP120. (B) Schematic representation of TIP120 structure. The lower line indicates amino acid sequence positions. Several characteristic sequences such as charged (13-104 residues), acidic (267-344 residues), and leucinerich regions (600-779 and 1003-1149 residues) are indicated. The dTAF80 homology region is also shown. Two asterisks denote possible phosphorylation sites for casein kinase II (CKII). (C) Sequence similarity between TIP120 and dTAF80. Identical and homologous amino acid residues were indicated by vertical lines and double dots, respectively.
immunoglobulin was used (Fig. 3C, lane 1). This result suggests that TIP120 is associated with TBP in nuclear extracts. Primary Structure of TIP120 Analysis of the a.a. sequence of TIP120 failed to detect any typical structural motifs usually found in transcription factors, but revealed some other characteristic structures. As shown in Fig. 2B, the N-terminal region of TIP120 (a.a. 13-104) was highly charged with 37% of the residues either acidic or basic. The TIP120-coding sequence contained two regions rich in leucine residues (a.a. 600-779, 22%; a.a. 1003-1149, 20%). These regions may be implicated in the interaction with other proteins through a leucine zipper-like structure. The N-terminal one-third was rich in acidic a.a. residues (a.a. 267-344; 36% being glutamic or asparatic acid residues). The acidic domain had some consecutive negatively charged regions, two of which (335-SDDE-338 and 340-SDDD-343) were identical to a consensus target site for casein kinase II, which is known to control the activity of transcription factors (17-19). 615
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FIG. 3. Characterization of the TIP120. (A) Western blot analysis of recombinant and endogenous TIP120. Recombinant TIP120 (lanes 1-3; 30 ng, 15 ng, or 7.5 ng), affinity-purified TIP120 (lane 4; 10 ng), and nuclear extract (lane 5; 10 mg) were analyzed by Western blotting using anti-TIP120 antibody. Arrow indicates the position of TIP120. (B) Interaction of TIP120 with TBP. The recombinant proteins (0.2 mM each) were incubated as indicated. The mixtures were immunoprecipitated with control IgG (lane 1), anti-TBP antibody (lanes 2, 3, and 5), and anti-TIP120 antibody (lanes 4, 6, and 7)-coupled protein A-Sepharose. The immunoprecipitates were resolved by 10% SDS-PAGE and stained with silver. An asterisk indicates the IgG heavy chain. (C) Association of TIP120 with TBP in nuclear extracts. The extracts were immunoprecipitated with control IgG or anti-TIP120 antibody-coupled protein A-Sepharose beads (lanes 1 and 2). The precipitated proteins and nuclear extracts (lane 3) were resolved by 10% SDS-PAGE and analyzed by Western blotting with anti-TBP antibody. Arrow indicates the position of TBP. An asterisk indicates the IgG heavy chain.
Comparison of the TIP120 a.a. sequence with known sequences in databases by means of the BLAST program without any gaps revealed that the TIP120 exhibited significant homology with the N-terminus of the Drosophila TFIID subunit p85, also called dTAF80 (Fig. 2C) (12, 13). These two proteins were 20% identical over a 95 a.a. overlap and 44% similar when conservative a.a. substitutions were taken into account. The N-terminal region of dTAF80 is essential for the interaction with TBP (13). The homologous sequence may be an evolutionary conserved interface for the interactions with TBP. These observations suggest that TIP120 is a novel TBP-binding protein and might regulate transcription through the interaction with TBP. ACKNOWLEDGMENTS We thank Dr. K. Kato for helpful discussions. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from The Japanese Ministry of Education, Science, Sports, and Culture.
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