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Molecular Cloning and Characterization of CAPER, a Novel Coactivator of Activating Protein-1 and Estrogen Receptors

2002; Elsevier BV; Volume: 277; Issue: 2 Linguagem: Inglês

10.1074/jbc.m110417200

ISSN

1083-351X

Autores

Dong-Ju Jung, Soon-Young Na, Doe Sun Na, Jae Woon Lee,

Tópico(s)

Chromatin Remodeling and Cancer

Resumo

Transcriptional coactivators either bridge transcription factors and the components of the basal transcription apparatus and/or remodel the chromatin structures. We isolated a novel nuclear protein based on its interaction with the recently described general coactivator activating signal cointegrator-2 (ASC-2). This protein CAPER (for coactivator of activating protein-1 (AP-1) and estrogen receptors (ERs)) selectively bound, among the many transcription factors we tested, the AP-1 component c-Jun and the estradiol-bound ligand binding domains of ERα and ERβ. Interestingly, CAPER exhibited a cryptic autonomous transactivation function that becomes activated only in the presence of estradiol-bound ER. In cotransfections, CAPER stimulated transactivation by ERα, ERβ, and AP-1. Thus, CAPER may represent a more selective transcriptional coactivator molecule that plays a pivotal role for the function of AP-1 and ERs in vivo in conjunction with the general coactivator ASC-2. Transcriptional coactivators either bridge transcription factors and the components of the basal transcription apparatus and/or remodel the chromatin structures. We isolated a novel nuclear protein based on its interaction with the recently described general coactivator activating signal cointegrator-2 (ASC-2). This protein CAPER (for coactivator of activating protein-1 (AP-1) and estrogen receptors (ERs)) selectively bound, among the many transcription factors we tested, the AP-1 component c-Jun and the estradiol-bound ligand binding domains of ERα and ERβ. Interestingly, CAPER exhibited a cryptic autonomous transactivation function that becomes activated only in the presence of estradiol-bound ER. In cotransfections, CAPER stimulated transactivation by ERα, ERβ, and AP-1. Thus, CAPER may represent a more selective transcriptional coactivator molecule that plays a pivotal role for the function of AP-1 and ERs in vivo in conjunction with the general coactivator ASC-2. activating protein-1 basic region-leucine zipper activating function 2 steroid receptor coactivator-1 CREB-binding protein (where CREB is cAMP-response element-binding protein) activating signal cointegrator-2 retinoic acid receptor thyroid hormone receptor estrogen receptor peroxisome proliferator-activated receptor γ PPARγ coactivator coactivator of AP-1 and ERs glutathione S-transferase RNA recognition motif ribonucleoprotein heterogeneous nuclear RNP coactivator activator estradiol The activation protein-1 (AP-1)1 transcription factors are immediate early response genes involved in a diverse set of transcriptional regulatory processes (for a review see Ref. 1Shaulian E. Karin M. Oncogene. 2001; 20: 2390-2400Google Scholar). The AP-1 complex consists of a heterodimer of a Fos family member and a Jun family member. This complex binds the consensus DNA sequence (TGAGTCA) (termed AP-1 sites) found in a variety of promoters. The Fos family contains four proteins (c-Fos, Fos-B, Fra-1, and Fra-2), whereas the Jun family is composed of three proteins (c-Jun, Jun-B, and Jun-D). Fos and Jun are members of the basic region-leucine zipper (bZIP) family of sequence-specific dimeric DNA-binding proteins (1Shaulian E. Karin M. Oncogene. 2001; 20: 2390-2400Google Scholar). The C-terminal half of the bZIP domain is amphipathic, containing a heptad repeat of leucines that is critical for the dimerization of bZIP proteins, whereas the N-terminal half of the long bipartite helix is the basic region that is responsible for the sequence-specific DNA binding. The nuclear receptor superfamily is a group of ligand-dependent transcriptional regulatory proteins that function by binding to specific DNA sequences named hormone-response elements in the promoters of target genes (reviewed in Ref. 2Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Google Scholar). The superfamily includes receptors for a variety of small hydrophobic ligands such as steroids, triiodothyronine, and retinoids as well as a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors. The C terminus of the ligand binding domain of these proteins harbors an essential ligand-dependent transactivation function, activation function 2 (AF2), whereas the N terminus of many nuclear receptors often includes AF1. Genetic studies implicated that transcription coregulators (or cofactors) with no specific DNA binding activity are essential components of transcriptional regulation, which ultimately led us to identify a series of coregulatory proteins (for reviews, see Refs. 3McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Google Scholarand 4Lee J.W. Lee Y.C. Na S.Y. Jung D.J. Lee S.K. Cell. Mol. Life Sci. 2001; 58: 289-297Google Scholar). They appear to function by either remodeling chromatin structures and/or acting as adapter molecules between transcription factors and the components of the basal transcriptional apparatus. These proteins include the steroid receptor coactivator-1 (SRC-1) family, CREB-binding protein (CBP)/p300, activating signal cointegrator-2 (ASC-2), and many others (3McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Google Scholar, 4Lee J.W. Lee Y.C. Na S.Y. Jung D.J. Lee S.K. Cell. Mol. Life Sci. 2001; 58: 289-297Google Scholar). SRC-1 and its homologue ACTR, along with CBP and p300, were recently shown to contain histone acetyltransferase activities and associate with yet another histone acetyltransferase protein P/CAF (3McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Google Scholar, 4Lee J.W. Lee Y.C. Na S.Y. Jung D.J. Lee S.K. Cell. Mol. Life Sci. 2001; 58: 289-297Google Scholar). Interestingly, unliganded retinoic acid receptor (RAR) and thyroid hormone receptor (TR) bind to their target genes and repress transcription. “The silencing mediators of RAR and TR” and “nuclear receptor corepressors” are known to mediate this repression (3McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Google Scholar, 4Lee J.W. Lee Y.C. Na S.Y. Jung D.J. Lee S.K. Cell. Mol. Life Sci. 2001; 58: 289-297Google Scholar). Interestingly, the silencing mediators of RAR and TR and nuclear receptor corepressors appear to interact with the estrogen receptor (ER) and the progesterone receptor only in the presence of their respective antagonists (5Xu J. Nawaz Z. Tsai S.Y. Tsai M.J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12195-12199Google Scholar, 6Jackson T.A. Richer J.K. Bain D.L. Takimoto G.S. Tung L. Horwitz K.B. Mol. Endocrinol. 1997; 11: 693-705Google Scholar, 7Smith C.L. Nawaz Z. O'Malley B.W. Mol. Endocrinol. 1997; 11: 657-666Google Scholar, 8Lavinsky R.M. Jepsen K. Heinzel T. Torchia J. Mullen T.M. Schiff R. Del-Rio A.L. Ricote M. Ngo S. Gemsch J. Hilsenbeck S.G. Osborne C.K. Glass C.K. Rosenfeld M.G. Rose D.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2920-2925Google Scholar). These proteins harbor transferable repression domains that associate with various histone deacetylases. These results are consistent with the notion that acetylation of histones destabilizes nucleosomes and relieves transcriptional repression by allowing transcription factors access to recognition elements, whereas deacetylation of the histones stabilizes the repressed state (3McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Google Scholar, 4Lee J.W. Lee Y.C. Na S.Y. Jung D.J. Lee S.K. Cell. Mol. Life Sci. 2001; 58: 289-297Google Scholar). It is important to note that many of these coregulatory proteins have shown a very broad spectrum of action with many different nuclear receptors and transcription factors, including AP-1 (3McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Google Scholar, 4Lee J.W. Lee Y.C. Na S.Y. Jung D.J. Lee S.K. Cell. Mol. Life Sci. 2001; 58: 289-297Google Scholar). More recently, however, a series of more target-selective coactivators have been isolated. These include peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1 (PGC-1) (9Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Google Scholar), the human homologue of the yeast DNA repair and TFIIH regulator MMS19 (10Wu X. Li H. Chen J.D. J. Biol. Chem. 2001; 276: 23962-23968Google Scholar) and p68 RNA helicase (11Endoh H. Maruyama K. Masuhiro Y. Kobayashi Y. Goto M. Tai H. Yanagisawa J. Metzger D. Hashimoto S. Kato S. Mol. Cell. Biol. 1999; 19: 5363-5372Google Scholar), both of which were shown to be AF1-specific coactivators of ERα, and prothymosin α, which selectively enhances ER activity by interfering with “the repressor of ER” activity (12Martini P.G. Delage-Mourroux R. Kraichely D.M. Katzenellenbogen B.S. Mol. Cell. Biol. 2000; 20: 6224-6232Google Scholar, 13Montano M.M. Ekena K. Delage-Mourroux R. Chang W. Martini P. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6947-6952Google Scholar). In addition, ARA160 was recently reported as the first androgen receptor N-terminal-associated coactivator in human prostate cancer cells (14Hsiao P.W. Chang C. J. Biol. Chem. 1999; 274: 22373-22379Google Scholar), and a neuronal-specific corepressor “neuronal interacting factor X 1” was shown to repress transactivation by a subset of nuclear receptors (15Greiner E.F. Kirfel J. Greschik H. Huang D. Becker P. Kapfhammer J.P. Schule R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7160-7165Google Scholar). Interestingly, Smad3 specifically represses transcriptional activation mediated by androgen receptor in prostate cancer cells (16Hayes S.A. Zarnegar M. Sharma M. Yang F. Peehl D.M. ten Dijke P. Sun Z. Cancer Res. 2001; 61: 2112-2118Google Scholar) while acting as a coactivator specific for ligand-induced transactivation of vitamin D receptor by forming a complex with a member of the SRC-1 family in the nucleus (17Yanagisawa J. Yanagi Y. Masuhiro Y. Suzawa M. Watanabe M. Kashiwagi K. Toriyabe T. Kawabata M. Miyazono K. Kato S. Science. 1999; 283: 1317-1321Google Scholar). ARA70 has also been shown to stimulate selectively PPARγ and androgen receptor (18Heinlein C.A. Ting H.J. Yeh S. Chang C. J. Biol. Chem. 1999; 274: 16147-16152Google Scholar). Finally, JAB1, interacting with c-Jun and JunD but not with JunB or v-Jun, is known to selectively potentiate transactivation by c-Jun or JunD (19Claret F.X. Hibi M. Dhut S. Toda T. Karin M. Nature. 1996; 383: 453-457Google Scholar). In this report, we describe the molecular cloning and characterization of a nuclear protein, which was originally isolated as a novel autoantigen from a patient with liver cirrhosis who progressed to hepatocarcinoma (20Imai H. Chan E.K. Kiyosawa K. Fu X.D. Tan E.M. J. Clin. Invest. 1993; 92: 2419-2426Google Scholar). Our results show that this protein functions as a specific transcriptional coactivator of AP-1, ERα and ERβ. HCC1.4 and HCC1.3 (20Imai H. Chan E.K. Kiyosawa K. Fu X.D. Tan E.M. J. Clin. Invest. 1993; 92: 2419-2426Google Scholar) were kind gifts from Dr. Eng M. Tan at University of California, San Diego. Polymerase chain reaction fragments encoding CAPER (for coactivator of AP-1 and ERs), CAPER-N, -MI, -MII, -C, -ΔC1, -ΔC2, -ΔN1, -ΔN2, -MII-1, -MII-2, -ASC2-4Δ1, -ASC2-4Δ2, and -ASC2-4Δ3 were constructed intoEcoRI and XhoI restriction sites of the LexA fusion vector pEG202PL, the B42 fusion vector pJG4-5, the mammalian two-hybrid vector pCMX/Gal4 and pCMX/VP16, the mammalian expression/in vitro translation vector pcDNA3, and the glutathione S-transferase (GST) fusion vector pGEX4T-1. Similarly, PCR fragments encoding ERα-ABC, -ABCD, -EF, -D, and -DE were cloned into EcoRI and XhoI restriction sites of pJG4-5. Gal4 fusions to ERα and ERβ, LexA fusions to ERαΔAF2, ASC2-4, ASC2-4a, ASC2-4b, and ASC2-4LR, B42 fusions to ERα, ERβ, c-Jun, c-Fos, JunΔ1, JunΔ2, JunΔ3, FosΔ1, FosΔ2, and FosΔ3, GST fusions to ERα and ERβ, mammalian expression/in vitro translation vector for ERα, ASC-2, ASC2-4, c-Jun, and c-Fos, the reporter constructs ERE-Luc, AP1-Luc, Gal4-Luc, and LexA-β-gal, and the transfection indicator construct pRSV-β-gal were as described (21Lee S.-K. Choi H.S. Song M.R. Lee M.O. Lee J.W. Mol. Endocrinol. 1998; 12: 1184-1192Google Scholar, 22Lee S.-K. Kim H.J. Na S.-Y. Kim T.S. Choi H.-S. Im S.Y. Lee J.W. J. Biol. Chem. 1998; 273: 16651-16654Google Scholar). Finally, PCR fragment encoding the full-length ERα was subcloned into the HindIII andXhoI restriction sites of the yeast expression vector p425-Gal1 (23Mumberg D. Muller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Google Scholar). The LexA-ASC2-4 (24Lee S.-K. Na S.Y. Jung S.Y. Choi J.E. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Google Scholar) was used as a bait to screen a mouse liver cDNA library in pJG4-5 to identify ASC-2-interacting proteins, and the screening was executed essentially as described previously (25Lee J.W. Choi H.S. Gyuris J. Brent R. Moore D.D. Mol. Endocrinol. 1995; 9: 243-254Google Scholar). The yeast β-galactosidase assay was done as described (25Lee J.W. Choi H.S. Gyuris J. Brent R. Moore D.D. Mol. Endocrinol. 1995; 9: 243-254Google Scholar). For each experiment, at least three independently derived colonies expressing chimeric proteins were tested. The GST fusions or GST alone was expressed in Escherichia coli, bound to glutathione-Sepharose 4B beads (Amersham Biosciences), and incubated with labeled proteins expressed by in vitro translation by using the TNT-coupled transcription-translation system, with conditions as described by the manufacturer (Promega, Madison, WI). Specifically bound proteins were eluted from beads with 40 mm reduced glutathione in 50 mm Tris (pH 8.0) and analyzed by SDS-PAGE and autoradiography as described (26Lee J.W. Ryan F. Swaffield J.C. Johnston S.A. Moore D.D. Nature. 1995; 374: 91-94Google Scholar). CV-1 cells were grown in 24-well plates with medium supplemented with 10% charcoal-stripped serum. After 24 h of incubation, cells were transfected with 100 ng of β-galactosidase expression vector pRSV-β-gal and 100 ng of an indicated reporter gene, along with c-Fos, ASC-2, ERα, CAPER, CAPER-MII-2, and Gal4 fusions to ERα and ERβ as well as various CAPER fragments. Total amounts of expression vectors were kept constant by adding decreasing amounts of the CDM8 expression vector to transfections. Twelve hours later, cells were washed and refed with Dulbecco's modified Eagle's medium containing 10% charcoal-stripped fetal bovine serum. After 12 h, cells were left unstimulated or stimulated with 0.1 μm ligand. Cells were harvested 24 h later, and luciferase activity was assayed as described (26Lee J.W. Ryan F. Swaffield J.C. Johnston S.A. Moore D.D. Nature. 1995; 374: 91-94Google Scholar), and the results were normalized to the β-galactosidase expression. To search for interacting proteins with the recently described transcriptional coactivator ASC-2 (24Lee S.-K. Na S.Y. Jung S.Y. Choi J.E. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Google Scholar, 27Lee S.-K. Anzick S.L. Choi J.E. Bubendorf L. Guan X.Y. Jung Y.K. Kallioniemi O.P. Kononen J. Trent J.M. Azorsa D. Jhun B.H. Cheong J.H. Lee Y.C. Meltzer P.S. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Google Scholar, 28Lee S.-K. Jung S.Y. Kim Y.S. Na S.Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Google Scholar), we screened a yeast two-hybrid-based mouse liver cDNA library using ASC2-4 (i.e. the ASC-2 residues 1172–1729) (24Lee S.-K. Na S.Y. Jung S.Y. Choi J.E. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Google Scholar) as a bait. A few independent cDNAs encoded a protein similar to human proteins HCC1.3 and HCC1.4 (20Imai H. Chan E.K. Kiyosawa K. Fu X.D. Tan E.M. J. Clin. Invest. 1993; 92: 2419-2426Google Scholar). These proteins were described previously as novel nuclear autoantigens identified with antibodies from human hepatocarcinoma. These proteins are identical except the presence of additional six amino acids in HCC1.4. The isolated mouse clones retained the internal six amino acids, like HCC1.4, and had only two amino acid changes from the human proteins (results not shown). HCC1.3 and HCC1.4 were indistinguishable in their binding and transcriptional coactivation properties (results not shown), and thus we focused only on HCC1.3 for the rest of the studies presented here. Based on their functional properties (this paper) and ubiquitous expression pattern (20Imai H. Chan E.K. Kiyosawa K. Fu X.D. Tan E.M. J. Clin. Invest. 1993; 92: 2419-2426Google Scholar), we renamed these proteins CAPER (for coactivator ofAP-1 and ERs). Despite the lack of direct sequence homology, it is interesting to note that CAPER and PGC-1, the recently defined transcription coactivator of PPARγ (9Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Google Scholar), has a few conserved features in common (Fig.1A). These include a cryptic autonomous transactivation domain (Fig. 5B), a region rich in Ser-Arg pairs (so called SR domains) (reviewed in Ref. 29Tacke R. Manley J.L. Curr. Opin. Cell Biol. 1999; 11: 358-362Google Scholar), and an RNA recognition motif (RRM) (30Shamoo Y. Abdul-Manan N. Williams K.R. Nucleic Acids Res. 1995; 23: 725-728Google Scholar) with homology to corresponding domains found in hnRNP proteins and SR factors (Fig. 1B). The RRM, consisting of two highly conserved peptide motifs, ribonucleoprotein (RNP)-1 and RNP-2, confers both RNA and single-stranded DNA binding activity (30Shamoo Y. Abdul-Manan N. Williams K.R. Nucleic Acids Res. 1995; 23: 725-728Google Scholar). The hnRNP family of proteins includes members that are involved in all aspects of RNA metabolism (reviewed in Ref. 31Krecic A.M. Swanson M.S. Curr. Opin. Cell Biol. 1999; 11: 363-371Google Scholar), and some of them have been shown to have transcriptional activity through association with single-stranded DNA enhancer sequences in the promoter region of the genes they regulate (32Eggert M. Michel J. Schneider S. Bornfleth H. Baniahmad A. Fackelmayer F.O. Schmidt S. Renkawitz R. J. Biol. Chem. 1997; 272: 28471-28478Google Scholar, 33Dempsey L.A. Hanakahi L.A. Maizels N. J. Biol. Chem. 1998; 273: 29224-29229Google Scholar, 34Du Q. Melnikova I.N. Gardner P.D. J. Biol. Chem. 1998; 273: 19877-19883Google Scholar). The association of SR domains and RRMs is typical of the classical SR splicing factors that play a key role in both constitutive splicing and in the regulation of alternative splicing in vivo (29Tacke R. Manley J.L. Curr. Opin. Cell Biol. 1999; 11: 358-362Google Scholar). Interestingly, PGC-1 was recently shown to mediate mRNA splicing (35Monsalve M. Wu Z. Adelmant G. Puigserver P. Fan M. Spiegelman B.M. Mol. Cell. 2000; 6: 307-316Google Scholar), suggesting the presence of a novel class of proteins that may coordinate the coupled events of transcription and mRNA processing in vivo(reviewed in Ref. 36Hirose Y. Manley J.L. Genes Dev. 2000; 14: 1415-1429Google Scholar). Thus, it will be interesting to examine whether CAPER and the recently defined “PGC-1-related coactivator,” a serum-inducible coactivator of nuclear respiratory factor 1-dependent transcription in mammalian cells (37Andersson U. Scarpulla R.C. Mol. Cell. Biol. 2001; 21: 3738-3749Google Scholar), are also involved with mRNA processing. When LexA fusion to ASC2-4 was coexpressed in yeast with the amphiphatic acidic transactivation domain B42 (25Lee J.W. Choi H.S. Gyuris J. Brent R. Moore D.D. Mol. Endocrinol. 1995; 9: 243-254Google Scholar) fused to various CAPER constructs (Fig.2A), only the full-length CAPER and CAPER-C enhanced the LexA-ASC2-4-directed transactivation (Fig. 2B). These results suggest that the interaction interface with ASC-2 may involve the C-terminal region of CAPER (i.e. CAPER-C in Fig. 2A). Consistent with these yeast results, GST fusions to the full-length CAPER and CAPER-C but not GST alone interacted with in vitro translated ASC2-4 (Fig.2C). The CAPER-interacting domain was further mapped to the N-terminal region of ASC2-4 (i.e. ASC2-4Δ3 consisting of the ASC-2 residues 1172–1273) in yeast, as shown in Fig.2D.Figure 5The autonomous transactivation domain ofCAPER. A, CV-1 cells were transfected withlacZ expression vector (100 ng), the increasing amount of expression vectors for Gal4 alone or Gal4 fusions to the full-length CAPER and various CAPER fragments, and a reporter geneGal4-Luc (100 ng), as indicated. Open andsolid boxes indicate 50 and 100 ng of each Gal4 construct, respectively. Normalized luciferase expressions from triplicate samples were calculated relative to the lacZ expressions. The experiments were repeated at least three times, and the representative results were expressed as fold activation (n-fold) over the value obtained with Gal4 alone, with the error bars as indicated. B, expression vectors for LexA/CAPER, LexA/CAPER-MII, and ERα were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described (24Lee S.-K. Na S.Y. Jung S.Y. Choi J.E. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Google Scholar). Open, hatched, and solid boxesindicate the absence of ERα, the presence of ERα, and ERα plus 100 nm of E2, respectively. NormalizedlacZ expressions from triplicate samples were calculated relative to the result with LexA alone. The data are representative of at least two similar experiments, and the error bars are as indicated.View Large Image Figure ViewerDownload (PPT)Figure 2CAPER as an ASC-2-interactor. A, a series of 10 fragments of CAPER are as shown. B, the indicated B42- and LexA-ASC2-4-encoding plasmids were transformed into a yeast strain containing an appropriate lacZreporter gene, as described (24Lee S.-K. Na S.Y. Jung S.Y. Choi J.E. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Google Scholar). Normalized lacZexpressions from triplicate samples were calculated relative to the result with B42 alone. The data are representative of at least two similar experiments. C, ASC2-4 was labeled with [35S]methionine by in vitro translation and incubated with glutathione beads containing GST alone and GST fusions to the full-length CAPER and CAPER-C, as indicated. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-polyacrylamide gel electrophoresis. Approximately 20% of the total reaction mixture was loaded as input. D, B42 fusion to CAPER-C was coexpressed with indicated LexA fusions to different ASC-2 fragments in a yeast strain containing an appropriate lacZ reporter gene, as described (21Lee S.-K. Choi H.S. Song M.R. Lee M.O. Lee J.W. Mol. Endocrinol. 1998; 12: 1184-1192Google Scholar). The receptor-interacting LXXLL motif (38Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Google Scholar, 39Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Google Scholar) of ASC2-4 is as indicated. Each LexA fusion was transcriptionally inert in yeast (results not shown). +++, strongly blue colonies after 2 days of incubation; ++, light blue colonies after 2 days of incubation; +, light blue colonies after more than 2 days of incubation; −, white colonies.View Large Image Figure ViewerDownload (PPT) To explore the possibility of CAPER as a transcriptional coregulator, we examined bindings of CAPER with a series of different transcription factors in yeast. These included p53, the NFκB component p50, the activating protein-1 components c-Jun and c-Fos, serum response factor, retinoid X receptor α, RARα, TRα and -β, PPARα, liver X receptor α and β, farnesoid X receptor, glucocorticoid receptor, and ERα and -β. Among these factors, only c-Jun, ERα, and ERβ appeared to interact significantly with CAPER (see below). Transactivation mediated by LexA fusion to CAPER was stimulated by B42 fusions to the full-length c-Jun and JunΔ3 but not JunΔ1, JunΔ2, c-Fos, FosΔ1, FosΔ2, and FosΔ3 in yeast (Fig. 3, Aand B). In addition, B42-JunΔ3 stimulated transactivation by LexA fusion to CAPER-MII fragment but not -N, -MI, and -C fragments (Fig. 3C). It is noteworthy that the transcriptionally inert full-length CAPER contains a cryptic autonomous transactivation function ascribed to the MII region (i.e. compare the basal activities of LexA fusions to the full-length CAPER and CAPER-MII in Fig. 3C). Corroborating these yeast results, GST fusion to CAPER-MII interacted with in vitro translated c-Jun but not c-Fos (Fig. 3D). Thus, the MII subregion of CAPER appears to interact specifically with the C-terminal region of c-Jun (i.e. JunΔ3). Transactivation directed by LexA fusions to the full-length CAPER and CAPE-MII but not CAPER-N, -MI, and -C was stimulated by B42 fusion to ERα and ERβ in an E2-dependent manner (Fig.4A). Interestingly, these E2-dependent interactions were retained with a mutant ERα that lacks the AF2 core region (i.e.ERαΔAF2). This AF2-independent interaction of CAPER with ERα is consistent with the lack of LXXLL motif in CAPER, which was recently shown to be a binding interface for many AF2-dependent coactivators (38Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Google Scholar, 39Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Google Scholar). In contrast to the wild type ERα, however, ERαΔAF2 also bound CAPER in the presence of partial antagonist tamoxifen. The yeast results further suggested that the interaction interfaces involve the EF domains of ERα (Fig.4B) and the MII-2 fragment of CAPER (i.e. the CAPER residues 356–400) (Fig. 4C). This prediction was also confirmed in the in vitro GST pull-down assays, in which radiolabeled CAPER or CAPER-MII specifically interacted with GST fusion to ERα only in the presence of E2 (Fig. 4D). Similarly, CAPER-MII also interacted with GST fusion to the full-length ERβ only in the presence of E2 but not tamoxifen. Overall, these results clearly indicated that CAPER binds the AP-1 component c-Jun and both ERα and β.Figure 4CAPER as ER interactant. A andC, the indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described (24Lee S.-K. Na S.Y. Jung S.Y. Choi J.E. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Google Scholar). 100 nm E2 or tamoxifen was used where indicated. F indicates the full-length CAPER, whereas N, MI, MII, and C are as shown in Fig. 2A. All the ER fragments used were fusions to B42 (C). Normalized lacZexpressions from triplicate samples were calculated relative to the result with B42 alone. The data are representative of at least two similar experiments. B, the full-length ERα as well as five ERα fragments are as indicated, in which A–F denotes functional modules of nuclear receptors (2Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Google Scholar). C and E represent the DNA- and ligand-binding domains, respectively. D, the full-length CAPER and CAPER-MII were labeled with [35S]methionine byin vitro translation and incubated with glutathione beads containing GST alone and GST-ERα and ERβ, as indicated. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-polyacrylamide gel electrophoresis. 100 nm ligand was used where indicated. Approximately 20% of the total reaction mixture were loaded as input.View Large Image Figure ViewerDownload (PPT) Many transcription coactivators are known to exhibit transcriptional activities when forced to bind DNA (2Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Google Scholar, 3McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Google Scholar, 4Lee J.W. Lee Y.C. Na S.Y. Jung D.J. Lee S.K. Cell. Mol. Life Sci. 2001; 58: 289-297Google Scholar). Interestingly, Gal4 fusions to the full-length CAPER or CAPER-ΔC1, -ΔC2, -N, -ΔN1, -ΔN2, and -C fragments directed transcriptional activities lower than that mediated by Gal4 alone in CV-1 cells (Fig.5A). However, a cryptic activation function was unraveled with the MII fragment, which was further localized to the MII-1 fragment (i.e. the CAPER residues 291–355). These results also suggested the presence of independent transcriptional repression function, both at the N- and C-terminal regions of CAPER. However, it is not currently clear whether these regions have intrinsic repressive activities or simply mask the activation function within the MII-1 region. Surprisingly, the transcriptionally inert full-length CAPER fused to the DNA-binding protein LexA, upon coexpression of ERα in the presence of E2, became fully active in yeast (Fig. 5B). Consistent with the direct involvement of the activation function within the MII region in this E2/ERα-dependent activation of CAPER, transactivation mediated by LexA-CAPER-MII was further stimulated by E2/ERα. Similar results were also obtained with Gal4 fusions to CAPER and CAPER-MII in CV-1 cells (results not shown). These results are analogous to the recent report (40Puigserver P. Adelmant G. Wu Z. Fan M. Xu J. O'Malley B. Spiegelman B.M. Science. 1999; 286: 1368-1371Google Scholar), in which transcriptionally inactive PGC-1 was stimulated upon coexpression of activated PPARγ. They further demonstrated that the docking of PGC-1 to PPARγ stimulates an apparent conformational change in PGC-1 that permits binding of SRC-1 and CBP/p300, resulting in a large increase in transcriptional activity (40Puigserver P. Adelmant G. Wu Z. Fan M. Xu J. O'Malley B. Spiegelman B.M. Science. 1999; 286: 1368-1371Google Scholar). Because CAPER was isolated based on its interaction with ASC-2, which also serves as an excellent coactivator of ERs (27Lee S.-K. Anzick S.L. Choi J.E. Bubendorf L. Guan X.Y. Jung Y.K. Kallioniemi O.P. Kononen J. Trent J.M. Azorsa D. Jhun B.H. Cheong J.H. Lee Y.C. Meltzer P.S. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Google Scholar), activated-ERα may also cause a conformational change with CAPER, resulting in better bindings with ASC-2 or other coactivator molecules. This possibility is currently under investigation. The functional significance of the interactions of CAPER with c-Jun and ERs was tested in cotransfections. CAPER potently stimulated transactivation by AP-1 when tested with AP1-luciferase reporter construct (Fig. 6A). Interestingly, ASC-2 showed a relatively week synergy with CAPER only in the presence of 200 ng of ASC-2 expression vector (compare the transactivation levels of 50 ng of CAPER, 200 ng of ASC-2, and both in Fig. 6A). Similar results were also obtained with Gal4 fusions to c-Jun and c-Fos (results not shown). With both ERE-luciferase and Gal4-luciferase reporter constructs, coexpression of CAPER in CV-1 cells stimulated the E2-dependent level of transcriptional activities without significantly affecting the basal activities (Fig. 6B). In contrast, CAPER had no coactivation function with other transcription factors that did not show significant interactions with CAPER. These included TR, RAR, p53, and serum response factor (results not shown). Interestingly, CAPER-MII-2, which contains the ERα-interacting region (Fig.4C), exhibited a dominant negative phenotype with the ERα transactivation (Fig. 6C), suggesting the possible importance of the direct ERα-CAPER interactions in E2-mediated transactivation. Overall, these results strongly suggest that CAPER is a bona fide transcriptional coactivator molecule of AP-1 and ERs. In this report, we have shown that CAPER is a transcriptional coactivator molecule whose function, in contrast to multifunctional integrator molecules like CBP/p300, SRC-1, and ASC-2 (3McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Google Scholar, 4Lee J.W. Lee Y.C. Na S.Y. Jung D.J. Lee S.K. Cell. Mol. Life Sci. 2001; 58: 289-297Google Scholar), is rather selective to AP-1, ERα, and ERβ. As summarized in Fig.6D, CAPER contains distinct binding sites for c-Jun (Fig. 3) and ERs (Fig. 4) and significantly enhances their transactivation potential in cotransfections (Fig. 6, A and B). In particular, a fragment of CAPER that contains a binding site for ERα (i.e. CAPER-MII-2) acted as a potent dominant negative mutant for E2-dependent transactivation by ERα (Fig. 6C), suggesting the possible importance of the direct interactions between ERα and CAPER. It is noted that CAPER was originally isolated as a molecule that specifically interacts with transcriptional integrator ASC-2 (24Lee S.-K. Na S.Y. Jung S.Y. Choi J.E. Jhun B.H. Cheong J. Meltzer P.S. Lee Y.C. Lee J.W. Mol. Endocrinol. 2000; 14: 915-925Google Scholar, 27Lee S.-K. Anzick S.L. Choi J.E. Bubendorf L. Guan X.Y. Jung Y.K. Kallioniemi O.P. Kononen J. Trent J.M. Azorsa D. Jhun B.H. Cheong J.H. Lee Y.C. Meltzer P.S. Lee J.W. J. Biol. Chem. 1999; 274: 34283-34293Google Scholar, 28Lee S.-K. Jung S.Y. Kim Y.S. Na S.Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Google Scholar), and we further localized the ASC-2-binding site to the C-terminal region of CAPER (Fig. 2). Under our experimental conditions, however, we were not able to demonstrate clearly the synergistic activation function of CAPER and ASC-2, although some synergy was observed with the AP-1 transactivation in the presence of a higher dose of ASC-2 expression vector (Fig.6A). Further work is warranted to fully resolve this issue. CAPER contains an autonomous transactivation domain that was localized to the CAPER residues 291–355 (i.e. CAPER-MII-1) (Fig.5A). Strikingly, the full-length CAPER was transcriptionally inactive, unless coexpressed with E2-bound ERα (Fig.5B). The CAPER residues 291–400 (i.e. CAPER-MII) that also contains the neighboring ERα-binding site behaved similarly. These results strongly suggest that CAPER undergoes a conformational change upon binding activated ERα. Similar results were recently reported with PGC-1 (9Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Google Scholar), in which the docking of PGC-1 to PPARγ stimulated an apparent conformational change in PGC-1 that permits binding of SRC-1 and CBP/p300, resulting in a large increase in transcriptional activity (40Puigserver P. Adelmant G. Wu Z. Fan M. Xu J. O'Malley B. Spiegelman B.M. Science. 1999; 286: 1368-1371Google Scholar). The similarity between CAPER and PGC-1 also includes the presence of SR domains and RRMs (Fig. 1). The association of SR domains and RRMs is typical of the classical SR splicing factors that play a key role in both constitutive splicing and in the regulation of alternative splicing in vivo (29Tacke R. Manley J.L. Curr. Opin. Cell Biol. 1999; 11: 358-362Google Scholar). Indeed, PGC-1 was shown recently to mediate mRNA splicing (35Monsalve M. Wu Z. Adelmant G. Puigserver P. Fan M. Spiegelman B.M. Mol. Cell. 2000; 6: 307-316Google Scholar). PGC-1-related coactivator, a serum-inducible coactivator of nuclear respiratory factor 1-dependent transcription in mammalian cells, was also isolated as an RRM-containing coactivator molecule (37Andersson U. Scarpulla R.C. Mol. Cell. Biol. 2001; 21: 3738-3749Google Scholar). While this manuscript was in preparation, Iwasaki et al. (41Iwasaki T. Chin W.W. Ko L. J. Biol. Chem. 2001; 276: 33375-33383Google Scholar) reported another RRM-containing coactivator activator (CoAA) as a novel ASC-2-interacting protein. Interestingly, CoAA, in contrast to CAPER, showed rather broad target specificity, stimulating transactivation mediated by multiple hormone-response elements (41Iwasaki T. Chin W.W. Ko L. J. Biol. Chem. 2001; 276: 33375-33383Google Scholar). These results suggest an interesting possibility that ASC-2 may act as a platform to recruit various RRM-containing coactivator molecules such as CoAA and CAPER. Notably, p68 RNA helicase was recently isolated as a transcriptional coactivator specific for the AF1 of ERα (11Endoh H. Maruyama K. Masuhiro Y. Kobayashi Y. Goto M. Tai H. Yanagisawa J. Metzger D. Hashimoto S. Kato S. Mol. Cell. Biol. 1999; 19: 5363-5372Google Scholar), whereas RNA helicase A was found to mediate association of CREB-binding protein with RNA polymerase II (42Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Google Scholar). In addition, a novel transcriptional coactivator p52 interacted not only with transcriptional activators and general transcription factors to enhance activated transcription but also with the essential splicing factor ASF/SF2 both in vitro and in vivo to modulate ASF/SF2-mediated pre-mRNA splicing (43Ge H. Si Y. Wolffe A.P. Mol. Cell. 1998; 2: 751-759Google Scholar). It is important to note that post-transcriptional mRNA processings such as 5′-capping, splicing, and polyadenylation can take place cotranscriptionally in vivo (36Hirose Y. Manley J.L. Genes Dev. 2000; 14: 1415-1429Google Scholar). Thus, these proteins and CAPER may also act as adapter molecules to coordinate various pre-mRNA processings and transcriptional initiation of class II genes, in addition to functioning as transcriptional coactivators. Consistent with this idea, CAPER was originally found colocalized with splicing factors in nuclear speckles (20Imai H. Chan E.K. Kiyosawa K. Fu X.D. Tan E.M. J. Clin. Invest. 1993; 92: 2419-2426Google Scholar) like PGC-1 (35Monsalve M. Wu Z. Adelmant G. Puigserver P. Fan M. Spiegelman B.M. Mol. Cell. 2000; 6: 307-316Google Scholar). In conclusion, we have shown that CAPER is a novel transcriptional coactivator specific to ERs and AP-1. Studies of CAPER, along with PGC-1 and other related molecules, may provide an important insight into the coupling mechanisms of different mRNA processings in vivo. We thank Dr. Eng M. Tan for plasmids.

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