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Endocrinology 147(1):377–388 Copyright © 2006 by The Endocrine Society doi: 10.1210/en.2005-0450
Isolation and Characterization of a Transcriptional Cofactor and Its Novel Isoform that Bind the Deoxyribonucleic Acid-Binding Domain of Peroxisome Proliferator-Activated Receptor-␥ Takuya Tomaru, Teturou Satoh, Satoshi Yoshino, Takahiro Ishizuka, Koshi Hashimoto, Tsuyoshi Monden, Masanobu Yamada, and Masatomo Mori Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan in transfection assays and short interfering RNA targeting PDIP1 mRNA significantly reduced transactivation by PPAR␥. No potent intrinsic activation domain was identified in either PDIP1 isoforms in mammalian one-hybrid assays, and mutation of all LXXLL motifs did not affect enhancement of PPAR␥-mediated transactivation. PDIP1␣ and - similarly augmented transactivation by PPAR␣, PPAR␦, thyroid hormone receptor (TR)-␣1, TR1, and retinoid X receptor-␣. PDIP1␣ also enhanced estrogen receptor␣- and androgen receptor-mediated transactivation, whereas PDIP1 did not. PDIP1␣ showed receptor-specific synergism with activation function-2-interacting coactivators in PPAR␥- and TR1-mediated transactivation. Together, PDIP1 might function as a transcriptional cofactor for a broad range of nuclear receptors, possibly in collaboration with specific activation function-2 interacting coactivators. (Endocrinology 147: 377–388, 2006)
Using the DNA-binding domain (DBD) and hinge region of human peroxisome proliferator-activated receptor (PPAR)-␥ as bait in yeast two-hybrid screen, we isolated partial cDNA identical with that of the C terminal of KIAA1769. KIAA1769 encodes a 2080-amino acid protein (molecular mass, 231 kDa) that was recently identified to interact with PPAR␣ and termed PPAR␣-interacting cofactor 285 (here referred to as PPAR␥-DBD-interacting protein 1 (PDIP1)-␣). PDIP1 mRNA was expressed in 3T3-L1 adipocytes and THP-1 macrophages. We also identified the expression of the N terminal extended form of PDIP1␣ (referred to as PDIP1) consisting of 2649 amino acids (295 kDa) in human cultured cell lines by RT-PCR, and 5ⴕ rapid amplification of cDNA ends. Ribonuclease protection assay revealed that PDIP1 mRNA was expressed more abundantly than PDIP1␣ mRNA. The C-terminal region of PDIP1 directly binds DBD of PPAR␥, and multiple LXXLL motifs in PDIP1 were not required for the interaction. PDIP1␣ and - similarly enhanced PPAR␥-mediated transactivation
P
EROXISOME PROLIFERATOR-activated receptor (PPAR)␥ belongs to the nuclear hormone receptor (NR) superfamily and is involved in a variety of biological processes such as insulin sensitization, adipogenesis, atherosclerosis, inflammation, and carcinogenesis (1–3). PPAR␥ binds to the PPAR response element (PPRE) as a heterodimer with the retinoid X receptor (RXR) and activates the transcription of target genes in a ligand-dependent manner (1–3). Recent cumulating evidence established that DNA-bound NRs cooperatively function with
multiple cofactor proteins that capture NRs in a protein-protein interaction fashion (4, 5). Cofactors interacting in a ligand-dependent manner with the carboxyl-terminal activation function (AF)-2 of PPAR␥ include PPAR-binding protein (PBP)/thyroid hormone receptor-associated protein (TRAP) 220/vitamin D receptor-associated protein (DRIP) 205 (6 – 8), steroid receptor coactivator (SRC)-1 (9), and cAMP response element-binding protein-binding protein (CBP)/p300 (10). In addition to the AF2-interacting cofactors, the PPAR␥-interacting coactivator (PGC)-1␣ that binds the DNA-binding domain (DBD) and part of the hinge region of PPAR␥ has been isolated (11) and its molecular and physiological functions intensively analyzed (12–15). PGC-2 was also identified as an amino (N)-terminal AF1-interacting coactivator of PPAR␥ (16). In addition to AF2 and AF1, the DBD of NRs has recently been shown to function as a direct interaction interface for other transcription factors (11, 17–19). Using the yeast twohybrid system, we recently identified Tat-binding protein-1, a subunit of 26S proteasome, as a DBD-interacting coactivator specifically functioning with thyroid hormone receptor (TR) (18). Because the expression of PGC-1␣, a key coactivator of PPAR␥, is restricted in tissues such as adipose tissues, skeletal muscles, and liver (20), we speculated that additional tissue-specific cofactors binding to the DBD/ hinge region of PPAR␥ might exist to exert the pleiotropic function of this NR on the body (1–3).
First Published Online October 20, 2005 Abbreviations: aa, Amino acids; AF, activation function; aP2, fatty acid-binding protein; AR, androgen receptor; CBP, cAMP response element-binding protein-binding protein; CD36, scavenger receptor; DBD, DNA-binding domain; DBD-H, DBD and part of the hinge region; ER, estrogen receptor; EST, expression sequence tag; GST, glutathioneS-transferase; h, human; LBD, ligand-binding domain; Luc, luciferase; N-CoR, nuclear receptor corepressor; NR, nuclear hormone receptor; P/CAF, p300/CBP-associated factor; PDIP1, PPAR␥-DBD-interacting protein 1; PGC, PPAR␥-interacting coactivator; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; PRIC, PPAR␣-interacting cofactor; RACE, rapid amplification of cDNA end; RNase, ribonuclease; RNB, RNase B; RXR, retinoid X receptor; si, short interfering; SRC, steroid receptor coactivator; TR, thyroid hormone receptor; TRAP, thyroid hormone receptor-associated protein; Tro, troglitazone; UAS-TK, upstream activating sequence-thymidine kinase. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.
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Using the DBD/hinge region of human PPAR␥ as bait in a yeast two-hybrid screen, we isolated a 2681-bp partial cDNA fragment apparently identical with that of a human expression sequence tag (EST), KIAA1769, whose gene product was recently identified to interact with PPAR␣ and is known as PPAR␣-interacting cofactor (PRIC) 285 (21). In addition, we isolated an N-terminal extended isoform of PRIC285 generated by alternative splicing. We characterized the interaction domains between PRIC285 and PPAR␥ and examined whether functional differences between two PRIC isoforms exist in a variety of nuclear receptor-mediated transactivations. Materials and Methods Plasmids Expression vectors for human (h)PPAR␥, PPAR␣, TR␣1, TR1, RXR␣, estrogen receptor (ER)-␣ and androgen receptor (AR) were previously described (18). Firefly luciferase reporter plasmids, thyroid response element (TRE)-luciferase (Luc), PPRE/RXR-Luc, estrogen response element (ERE)-Luc and mouse mammary tumor virus-Luc were described previously. The full-length cDNA of KIAA1769 in pBluescript II SK⫹ was provided by Kazusa DNA Research Institute (Chiba, Japan) and subcloned into pSVSPORT1 (Gibco BRL, Gaithersburg, MD). Expression vectors for SRC-1, CBP, and p300/CBP-associated factor (P/CAF) were described previously (18, 22). Partial cDNA fragments complementary to human CBP, TRAP220, fatty acid-binding protein (aP2) and scavenger receptor (CD36) were amplified by RT-PCR and subcloned into pGEM3Z or pGEMTeasy vector (Promega, Madison, WI). The nucleotide sequences of all PCR-amplified cDNA were verified by sequencing.
Yeast two-hybrid system A yeast two-hybrid screen of a HeLa cell matchmaker cDNA library (CLONTECH, Palo Alto, CA) was performed as we described previously (18) using the DBD and part of the hinge region of hPPAR␥1 [amino acids (aa) 108 to 197] as bait. Approximately 1 ⫻ 106 transformants were screened for interaction with PPAR␥. The plasmids isolated from positive colonies were amplified on a large scale and the nucleotide sequences were determined using an autosequencer.
Glutathione-S-transferase (GST) pull-down assay [35S]methionine-labeled full-length and partial fragments of hPPAR␥ were synthesized in vitro using a TNT-coupled reticulocyte lysate system (Promega). The PCR-amplified partial fragments of KIAA1769 cDNA (aa 460 – 660, 1433–1674, 1566 –1823, and 1824 –2080) and PPAR␥-DBDinteracting protein (PDIP)-1 (aa 337–579) were ligated in-frame into pGEX4T1 (Pharmacia Biotech Inc., Uppsala, Sweden). The GST fusion proteins were purified on glutathione-agarose beads (Sigma, St. Louis, MO), and the interaction assays were performed as described previously (18).
Cell culture, transfection, and luciferase assay CV-1, HeLa, HepG2, and 293 cells were grown as described previously (18). 3T3-L1 preadipocytes were cultured and differentiation to adipocytes was induced as described previously (23). THP-1 cells were grown and differentiation to macrophages was induced as previously described (24). Transient transfection was performed using a calcium phosphate precipitation method as described (18). The total amounts of transfected plasmids were adjusted by adding an empty expression vector in all experiments. Luciferase assays were performed and luciferase activity was normalized by the protein concentration as described previously (18). All transfection experiments were repeated at least twice with identical results. Troglitazone and fenofibrate were provided by Sankyo Co. Ltd. (Tokyo, Japan) and Kaken Co. Ltd. (Tokyo, Japan), respectively.
Tomaru et al. • Identification of Novel Transcriptional Cofactors of PPAR␥
Construction of mutant PDIP1 expression vector A mutant PDIP1␣ vector in which all five LXXLL motifs were substituted to LXXAA (25) was constructed as follows. The cDNA fragments encompassing the first three or the last two LXXLL motifs were excised from pSVSPORT PDIP1␣ by EcoRI/BglII, or BamHI digestion and individually subcloned into pSG5 or pGEM3Z, respectively. Using QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) and oligonucleotides containing point mutations, one of the LXXLL motifs in these fragments was first mutated and the mutant plasmid was amplified on a large scale. Using this mutant plasmid as template, mutation of another LXXLL motif was similarly introduced. By repeating this strategy, all LXXLL motifs were mutated and the wild-type cDNA fragments encompassing LXXLL motifs in pSVSPORT PDIP1␣ were finally substituted by the mutated fragments carrying LXXAA motifs. All mutations were confirmed by sequencing.
Mammalian one-hybrid assay cDNAs corresponding to partial fragments of KIAA1769 (aa 1– 490, 1– 652, 1–1236, 1–1718, 491-1050, 1051–1565, 1566 –2080, and 641-2080) and PDIP1 (aa 1–579 and 1–1238) were amplified by PCR and ligated in-frame into the EcoRI site of pMGAL4DBD (CLONTECH). These constructs were transfected with 3 ⫻ upstream activating sequence-thymidine kinase (UAS-TK) luciferase reporter (18) into 293 cells. The herpes simplex virus VP16 ligated to GAL4DBD (CLONTECH) was used as a positive control. Luciferase activities were measured as described above.
Northern blot analysis [32P]-labeled antisense cRNA probes were synthesized from pGEM3ZKIAA1769, pGEM3ZPPAR␥, pGEMTeasyCD36, pGEM3ZaP2, pGEMTCBP, and pGEMTTRAP220. Human multitissue blots (CLONTECH) were hybridized with the KIAA1769 antisense cRNA probe under conditions described previously (26). Total RNA was isolated from undifferentiated and differentiated 3T3-L1 and THP-1 cells using Isogen (Nippon Gene, Tokyo, Japan). Thirty micrograms of total RNA were electrophoresed and transferred to nylon membranes. Hybridization conditions were as described previously (26).
RT-PCR RT-PCR was performed to amplify the N-terminal portion of two PDIP1 isoforms. Full-length PDIP1 cDNAs were also amplified by long RT-PCR and subcloned into pSVSPORT1. The PCR products were then subjected to sequencing using an autosequencer.
5⬘-rapid amplification of cDNA end (RACE) 5⬘-RACE was performed using Marathon-Ready HeLa cell cDNA (CLONTECH). Nested PCR amplification was carried out using two sets of adaptor primers (CLONTECH) and gene specific primers for PDIP1␣ or - cDNA. The PCR product was subcloned into pGEMTeasy vector (Promega) and multiple clones were subjected to sequence analysis.
Ribonuclease (RNase) protection assay RNase protection assay was carried out using RNP III RNase protection assay kit (Ambion, Austin, TX) according to the manufacturer’s instruction. An antisense cRNA probe to detect two PDIP1 isoform mRNAs was designed by comparing the PDIP1 cDNA sequences with the genomic sequence obtained from human genome database and prepared by PCR. The [32P]-labeled probe (1 ⫻ 105 cpm/reaction) was hybridized with 10 g poly(A⫹) RNA purified from HeLa, HepG2, THP-1, and 293 cells ON at 42 C and RNase digestion using RNase A and T1 was carried out for 1 h at 37 C. The digested samples were subjected to sequencing gel electrophoresis and hybridization signals were analyzed using an image analyzer (BAS1800 II, Fujifilm, Tokyo, Japan).
Western blot analysis Full-length PDIP1 cDNAs amplified by long RT-PCR were also inserted into p3XFLAG-CMV7 vector (Sigma) and transfected into 293
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cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Whole-cell lysates were then prepared and 50 g of protein were electrophoresed in SDS-PAGE and transferred to Hybond-P membranes (Amersham, Aylesbury, UK). Western blot analysis was carried out as described previously (18) using an anti-FLAG M2 monoclonal antibody (Sigma) and signals were detected using ECL-plus detection reagents (Amersham) according to the manufacturer’s protocol.
Knockdown of PDIP gene by short interfering (si) RNA A siRNA targeting both PDIP1␣ and - mRNA (siPDIP1) was designed and synthesized by Dharmacon (Lafayette, CO). The nucleotide sequence of siPDIP1 was 5⬘-GCUGGCUCAAGAAGUUUCU-3⬘. As a negative control, control duplex VIII (Dharmacon) was transfected in parallel. Increasing amounts of siPDIP1 were introduced into HeLa cells in 60-mm dishes using Lipofectamine 2000 (Invitrogen). After 48 h of siRNA transfection, total RNA was isolated and the expression of PDIP1 mRNA was quantitated by Northern blot analysis as described above. In separate experiments, 24 h after lipofection of siRNA, DR-1 Luc and pKCR2PPAR␥ were cotransfected using the same reagent in the absence or presence of 10 m troglitazone, and luciferase assay was performed after 24 h.
Results Cloning a protein that interacts with the DBD/hinge region of human PPAR␥
To isolate proteins that interact with PPAR␥, we performed a yeast two-hybrid screen of a HeLa cell cDNA library using the DBD and part of the hinge region (DBD-H) of hPPAR␥ as bait. Several positive clones were obtained and individually retransformed into yeast in the presence of pGBT9PPAR␥DBD-H. Significant -galactosidase activity was observed with clone 1, stronger than that observed between the ligand-binding domain (LBD) of PPAR␥ and the receptor-interaction domain of nuclear receptor corepressor (N-CoR) (Fig. 1A). No -galactosidase activity was observed by the transformation of either pGBT9PPAR␥DBD-H or pACT2 clone 1 alone indicating the specificity of the interaction. Moreover, clone 1 did not interact with the LBD of PPAR␥ in the absence or presence of troglitazone (Tro), whereas the interaction between N-CoR and LBD of PPAR␥ was significantly reduced by Tro (Fig. 1A). A computer search suggested that the nucleotide sequence of clone 1 (2681 bp) was apparently identical with that of the C-terminal region of KIAA1769, a human EST (Fig. 1B). The predicted open reading frame of KIAA1769 encoded a 2080-aa protein with a calculated molecular mass of 231 kDa. KIAA1769 possessed amino acid alignments homologous to Rnase B (RNB) (27) and a UvrD/REP helicase motif (28) at aa 688-1000 and 1584 –1991, respectively. KIAA1769 also possessed five LXXLL motifs, a characteristic NR-interaction alignment identified in several AF2-interacting coactivators (29) at positions aa 505, 549, 604, 1443, and 1660 (see Figs. 1B and 4C). GST interaction assays
To confirm whether KIAA1769 interacts with PPAR␥ in vitro, a GST pull-down assay was performed. The C-terminal portion of KIAA1769 obtained in the yeast two-hybrid screen was divided into two fragments (CN: aa 1566 –1823 and CC: aa 1824 –2080), and these polypeptides were expressed in Escherichia coli as GST fusion proteins (Fig. 2A). As shown in Fig. 2B, [35S]-labeled full-length PPAR␥ bound to GST-CN
FIG. 1. Cloning a protein that interacts with DBD and part of the hinge region of PPAR␥ using the yeast two-hybrid system. A, Interaction between GAL4DBD-fused PPAR␥ polypeptides (D-H and LBD) and GAL4 activation domain (AD)-fused clone 1 (C1) was analyzed using a yeast two-hybrid assay in the absence or presence of Tro (10 M). The interaction between the GAL4DBD-fused LBD and GAL4AD-fused N-CoR receptor-interaction domain was analyzed in parallel. B, Schematic representation of the structure of KIAA1769 protein. The positions homologous to RNB and UvrD-helicase motifs are shown by shaded and filled boxes, respectively. NR box motifs (LXXLL) are also shown as boxes.
but not to GST-CC or GST itself. We next determined whether GST-CN could interact with domains other than the DBD of PPAR␥. As shown in Fig. 2B, GST-CN bound to the DBD-H but not to A/B domain or LBD in the absence or presence of Tro. These findings clearly indicated that the C-terminal portion of KIAA1769 interacts with the DBD-H of PPAR␥ in vivo and in vitro. To examine whether five LXXLL motifs in KIAA1769 are involved in the interaction with PPAR␥, two KIAA1769 polypeptides containing three or two LXXLL motifs (aa 460 – 660 and 1433–1674) were bacterially expressed, and their interaction with full-length PPAR␥ was analyzed in GST pull-down assays (Fig. 2C). As shown in Fig. 2D, these polypeptides did not bind PPAR␥ in the absence or presence of Tro. These results suggest that five LXXLL motifs were not required for interaction with PPAR␥ and that the PPAR␥interaction domain in PDIP1 was finally mapped to the Cterminal region (aa 1675–1823). Based on the findings obtained by in vitro and in vivo binding assays, we referred to KIAA1769 as PDIP1 in this manuscript. Tissue distribution of PDIP1 mRNA
To elucidate the physiological functions of PDIP1, the expression of the PDIP1 gene in human tissues was analyzed by Northern blot analysis. As shown in Fig. 3A, a single
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FIG. 2. Interaction between KIAA1769 and PPAR␥ in vitro. A, Clone 1 was subdivided into CN and CC and bacterially expressed as GST-fusion proteins. Proper synthesis of the fusion proteins was verified by SDS-PAGE analysis. The positions of the molecular-size markers (kilodaltons) are indicated. B, [35S]-labeled PPAR␥ polypeptides (full, A/B, D-H, and LBD) were synthesized using in vitro translation, and their interaction with equivalent amounts of GST alone, GSTCN, or GSTCC in the absence or presence of Tro was analyzed using a pull-down assay. Input represents 10% of input protein. C, Two polypeptides encompassing LXXLL motifs in KIAA1769 (LXXLLx3 and LXXLLx2) were synthesized as GST-fusion proteins and their proper synthesis was verified by SDS-PAGE analysis. D, The interaction between [35S]-labeled full-length PPAR␥ and GST fusion proteins in the absence or presence of Tro was analyzed using a pull-down assay.
mRNA species, approximately 10 kb in length, was ubiquitously expressed in multiple human tissues. Among them, a relatively high expression was observed in the heart, liver, skeletal muscle, and pancreas. The origin of a smaller band hybridized with pancreatic mRNA is currently unknown. PDIP1 mRNA was also detected in several tumor cell lines (Fig. 3B). Because PPAR␥ has been established to play important physiological roles in adipocytes and macrophages (1–3), the expression of PDIP1 mRNA in mouse 3T3-L1 cells and a human macrophage cell line, THP-1, was examined. The differentiation of adipocytes and macrophages was monitored morphologically and by measuring the expression of differentiation markers including PPAR␥, aP2, or CD36 mRNAs as reported previously (30). PDIP1 mRNA was detected at similar levels both in undifferentiated and differentiated adipocytes. In THP-1 cells, the expression of PDIP1 mRNA was highest before differentiation and slightly decreased after the induction of differentiation (Fig. 3C). The expression patterns of PDIP1 mRNA in 3T3-L1 cells as well as in THP-1 cells closely resembled those of CBP and TRAP220 throughout their differentiation processes. These
findings demonstrated that PDIP1 mRNA was expressed in a wide variety of tissues in which PPAR␥ exerts pivotal physiological functions. Isolation of a novel isoform of PDIP1 in human macrophages
The human PDIP1 gene is located on chromosome 20q. Although Northern analyses revealed a single PDIP1 mRNA species in multiple human tissues, the human genome database suggested, in addition to the 2080-aa form, the presence of an N-terminal extended form of PDIP1 that is presumably generated by alternative splicing (Fig. 4A). To examine whether the long PDIP1 isoform as well as the short isoform are expressed in several culture cell lines, RT-PCR was performed to amplify the N-terminal portion of PDIP1 using two different sense primers corresponding to the 5⬘untranslated region of each isoform and an antisense primer common to both isoforms (Fig. 4A). As shown in Fig. 4B, PCR products of different predicted sizes were amplified in human culture cell lines including THP-1, HeLa, 293, and
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FIG. 3. Expression of PDIP1 mRNA in human multiple tissues and cultured cell lines. A, Northern blot analysis of multiple human tissue poly(A⫹) RNA was performed using a PDIP1 antisense cRNA probe. The positions of RNA size markers (kilobases) are shown. B, Northern blot analysis of total RNA isolated from several cultured cell lines. Thirty micrograms of total RNA were electrophoresed and transferred to a nylon membrane. Hybridization was performed as described above. RNA loading is shown by ethidium bromide staining of the gel. C, Changes of PDIP1 mRNA levels during the differentiation process of 3T3-L1 preadipocytes and THP-1 cells. The differentiation of 3T3-L1 and THP-1 cells was induced as described above. Total RNA was isolated at the indicated time points and blotted onto nylon membranes. The membranes were hybridized with cRNA probes for PDIP1, CBP, TRAP220, PPAR␥, aP2, or CD36. RNA loading is shown at the bottom. Hybridization signals were quantitated using an image analyzer.
HepG2 cells. In addition, full-length cDNAs encoding the long and short PDIP1 isoforms could be successfully amplified by long RT-PCR using THP-1 macrophage RNA (Fig. 4B). We determined the nucleotide sequences of long and short PDIP1 isoforms and confirmed that the long isoform contains an additional 576 aa at its N terminus that connects to the eighth amino acid of the short isoform of PDIP1 (Fig. 4C). The long PDIP1 isoform therefore consists of 2649 aa with a calculated molecular mass of 295 kDa. An additional LXXLL motif was observed in the N-terminal portion of the long isoform (Fig. 4C), although full-length PPAR␥ did not interact with this LXXLL motif in the absence or presence of Tro in a GST pull-down assay (Fig. 4D). To further confirm the expression of PDIP1 mRNA in human cells, 5⬘RACE was performed using HeLa cell cDNA to determine the nucleotide sequence of the 5⬘-untranslated region of PDIP1 mRNA. In nested PCR amplification using two sets of adaptor primers and gene-specific primers (GSP) for PDIP1, a single band was amplified (Fig. 4E). In contrast, we could not obtain PCR products using adaptor primers and PDIP1␣-specific primers in nested PCR (data not shown). Comparison of the nucleotide sequence of the PCR product with PRIC285 gene sequence in human genome database revealed presence of an additional exon (44 bp) upstream of the exon containing the start codon (Fig. 4E).
The relative expression of PDIP1␣ and - mRNA in human cells was more precisely quantitated by RNase protection assay using an antisense cRNA probe that distinguishes two isoform mRNAs after RNase digestion. As shown in Fig. 4F, a band corresponding to PDIP1 mRNA was clearly identified using poly(A⫹) RNA isolated from HeLa, 293, HepG2, and THP-1 cells, whereas a band representing PDIP1␣ mRNA was less faintly observed. No protected fragment was observed using yeast tRNA. These findings together with the results of 5⬘RACE suggested that PDIP1 mRNA was more abundantly expressed than PDIP1␣ mRNA in these human cell lines. Full-length cDNAs encoding two PDIP1 isoforms were then subcloned into an expression vector, and in vitrotranslated proteins were synthesized and separated by SDS-PAGE. As expected, proteins of expected molecular mass were synthesized by in vitro translation (Fig. 4G, left panel). Moreover, Western analyses demonstrated that two isoforms of expected molecular mass could be expressed as FLAG-tagged proteins in 293 cells by transient transfection (Fig. 4G, right panel). These findings indicated that two PDIP1 isoforms could be translated in vivo and in vitro and conventional Northern blot analysis could not distinguish two PDIP1 mRNA species because of their size difference and/or uneven expression levels. For convenience,
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FIG. 4. Isolation and expression of a long isoform of PDIP1 generated by alternative splicing. A, Schematic representation of the exon-intron structures of human PDIP1 gene. The exons are indicated by open boxes. Arrows indicate the positions of PCR primers. The positions of start (ATG) and stop codons are indicated. B, RT-PCR analysis of the expression of short and long isoforms of PDIP1 in human cultured cell lines. RT-PCR was performed using primer pairs a/c and b/c to amplify the 5⬘-portions of the short and long PDIP1 isoforms, respectively. The predicted
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sizes of PCR products were 2,163, and 3907 bp (left panel). Full-length cDNA encoding two PDIP1 isoforms was also amplified by long RT-PCR using primer pairs a/d and b/d from THP-1 macrophage RNA (6340 and 7953 bp, respectively) (right panel). C, The amino acid sequences of PDIP1␣ and -. PDIP1 possessed an additional 576 aa at its N terminus that links to the eighth aa of PDIP1␣. Six LXXLL motifs in PDIP1 are underlined. The cDNA and amino acid sequences of PDIP1 will be available in the DDBJ/EMBL/GenBank database under accession no. AB201715. D, An LXXLL motif in the PDIP1-specific portion (LXXLLx1) did not interact with PPAR␥. The polypeptide containing an LXXLL motif in the N terminal of PDIP1 was synthesized as a GST fusion protein, and a pull-down assay was performed with [35S]-labeled full-length PPAR␥ in the absence or presence of Tro. E, 5⬘RACE of PDIP1 cDNA. The 5⬘ portion of PDIP1 mRNA was amplified from Marathon-ready HeLa cell cDNA by nested PCR using two sets of adaptor primers and PDIP1 gene-specific primers (GSP1 and GSP2). The PCR product was electrophoresed and visualized by ethidium bromide staining of the gel. Molecular size markers are indicated. The nucleotide sequence of 5⬘-untranslated region of PDIP1 cDNA will be available in the DDBJ/EMBL/GenBank database under accession no. AB232667. F, RNase protection assay. The position of the cRNA probe (516 bp) corresponding to two PDIP1 cDNAs was schematically shown. This probe distinguishes PDIP1␣ and - mRNA after RNase digestion. Ten micrograms of poly(A⫹) RNA purified from HeLa, 293, HepG2, and THP-1 cells were hybridized with the [32P]-labeled cRNA probe. Yeast tRNA was hybridized in parallel as a negative control. Positions of radiolabeled size markers (Century Marker, Ambion) are indicated. Arrowheads denote specific protected products corresponding to PDIP1 mRNA (410 bp) and PDIP1␣ mRNA (299 bp) G, The in vitro translation and in vivo expression of two PDIP1 isoforms. [35S]-labeled PDIP1␣ and - proteins were synthesized using in vitro translation and subjected to SDS-PAGE (left panel). The FLAG-tagged PDIP1␣ or - expression vector was transfected into 293 cells using lipofection. Whole-cell lysates were prepared, electrophoresed by SDS-PAGE, and blotted onto a Hybond-P membrane. The membrane was immunoblotted using an anti-FLAG monoclonal antibody (right panel). The positions of the molecular size markers (kilodaltons) are indicated. Arrows indicate PDIP1␣ and - proteins.
we thereafter termed the short and long isoforms as PDIP1␣ and -, respectively, in this manuscript. In the rat cDNA database, only PDIP1 cDNA encoding the N-terminal extended form (2926 aa, molecular mass, 330 kDa), homologous to human PDIP1, was registered (accession no. XM_230961).
PDIP1 potentiated ligand-dependent transactivation by PPAR␥ and other members of the NR family in mammalian cells
To evaluate whether PDIP1 affects PPAR␥-mediated transactivation and whether functional differences between PDIP1␣ and - exist, transient transfection experiments were
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performed using vectors expressing full-length PDIP1␣ and - in CV-1 cells. As shown in Fig. 5A, PDIP1␣ cotransfection enhanced the PPAR␥-mediated transactivation of the DR1 reporter in the presence of Tro. PDIP1 similarly enhanced PPAR␥-mediated transactivation. Mutation of five LXXLL motifs in PDIP1␣ did not affect enhancement of transacti-
Tomaru et al. • Identification of Novel Transcriptional Cofactors of PPAR␥
vation by PPAR␥ (Fig. 5B), further confirming that LXXLL motifs were not functionally required for the enhancement. To clarify whether PDIP1 is directly involved in PPAR␥mediated transactivation in vivo, the effect of knockdown of PDIP1 was studied by using siRNA targeting endogenous PDIP1 gene (siPDIP1). As shown in Fig. 5C, transfection of
FIG. 5. PDIP1 potentiates ligand-dependent transcriptional activation by nuclear receptors. A, PDIP1␣ and - similarly potentiate PPAR␥mediated transactivation in transient transfection assays. Increasing amounts of pSVSPORT1PDIP1␣ or - (0, 0.83, and 1.67 g/well) were cotransfected with direct repeat with 1-bp spacer (DR-1) Luc (1.67 g/well) in the presence of pKCR2PPAR␥ (83 ng/well). Luciferase activity was measured after 24 h treatment with 10 M Tro. Data represent the mean ⫾ SEM of triplicate determinants. All transfection experiments were repeated at least twice with identical results. B, Mutation of LXXLL motifs in PDIP1␣ did not alter its function to enhance PPAR␥-mediated transactivation. All five LXXLL motifs in PDIP1␣ were mutated to LXXAA by site-directed mutagenesis, and the wild-type or mutant PDIP1␣ in pSVSPORT1 (1.67 g/well) was cotransfected with DR-1 Luc (1.67 g/well) and pKCR2PPAR␥ (83 ng/well) into CV-1 cells. Luciferase assay was performed as described above. C, Knockdown of endogenous PDIP1 gene attenuated PPAR␥-mediated transactivation. A siRNA targeting two PDIP1 isoform mRNAs (siPDIP1) or control siRNA (siCtl) was transfected into HeLa cells by lipofection. The total amounts of transfected siRNA were adjusted by adding siCtl. Forty-eight hours after siRNA transfection, total RNA was isolated, and PDIP1 mRNA level was quantitated by Northern blot analysis. RNA loading is shown by ethidium bromide staining of the gel. In separate experiments, DR-1 Luc and pKCR2PPAR␥ were cotransfected by lipofection after 24 h siRNA transfection. Luciferase assay was performed after 24-h incubation with Tro. D, The cotransfection effects of PDIP1␣ and - on other nuclear receptor-mediated transactivations. pSVSPORT1 PDIP1␣ or - (1.67 g/well) was cotransfected with luciferase reporter vectors (1.67 g/well) in the presence of receptor expression plasmids (83 ng/well). Increasing amounts of pSVSPORT1PDIP1␣ or - (0, 0.83, and 1.67 g/well) were cotransfected with pKCR2AR or ER (83 ng/well). Luciferase assay was performed as described above after 24-h treatment with cognate ligands. LIP, Fenofibrate; cPGI, cyclic prostaglandin I; DHT, dihydrotestosterone; E2, 17-estradiol. MMTV, Mouse mammary tumor virus; PAL, palindromic TRE; ERE, estrogen response element.
Tomaru et al. • Identification of Novel Transcriptional Cofactors of PPAR␥
siPDIP1, but not control siRNA, reduced PDIP1 mRNA levels in HeLa cells and apparently attenuated ligand-dependent activation by PPAR␥. The result suggested that endogenous PDIP1 might be essential for PPAR␥-mediated transactivation in vivo. Because PDIP1 was distributed in a wide variety of human tissues, we next examined whether PDIP1 affects transactivation mediated by other nuclear receptors. As shown in Fig. 5D, PDIP1 augmented the PPAR␣-mediated transactivation of DR1 reporter in the presence of fenofibrate, similar to PDIP1␣. In addition, PDIP1␣ and - enhanced PPAR␦mediated transactivation. Both PDIP1␣ and - could indistinguishably enhance other NR-mediated transactivation including TR␣1, TR1, and RXR␣ in the presence of cognate ligands. Moreover, PDIP1␣ enhanced AR- and ER␣-mediated transactivation in a dose-dependent manner, whereas PDIP1 did not. These findings suggest that PDIP1 could augment a broad range of NR functions and that PDIP1 isoform-specific regulation occurred in AR- and ER␣-mediated transactivation.
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PDIP1␣ and - did not possess an autonomous activation domain
To determine whether PDIP1␣ and - possess intrinsic transactivation domains, PDIP1␣ was divided into overlapping eight fragments, and mammalian one-hybrid assays were performed. The function of the N-terminal portion specific for PDIP1 was also examined in parallel. As shown in Fig. 6, full-length PDIP1 ␣ and - stimulated the activity of the UAS-TK reporter by 2.5-fold, whereas none of the divided fragments of PDIP1␣ and - activated the reporter activity over GAL4DBD alone. These findings suggested that PDIP1␣ and - did not possess a strong intrinsic activation domain and required the whole structure for its transactivation function. Receptor-specific synergism between PDIP1 and the AF-2 interacting cofactors in PPAR␥- and TR1-mediated transactivation
Because PDIP1 did not show a strong intrinsic activation function, it was speculated that PDIP1 might cooperate
FIG. 5. Continued
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Tomaru et al. • Identification of Novel Transcriptional Cofactors of PPAR␥
FIG. 6. PDIP1 requires its whole structure for activation function. Full-length and partial cDNAs of PDIP1␣ and - were ligated to GAL4DBD and transfected with 3 ⫻ UAS-TK luciferase reporter into 293 cells. Luciferase assay was carried out after 24 h of transfection. Data represent mean ⫾ SEM of luciferase activity relative to the activity of GAL4DBD that was assigned 1.
with other transcription factors to enhance NR-mediated transactivation. We therefore examined the effects of PDIP1 cotransfection with other AF-2 interacting transcriptional cofactors for PPAR␥ and TR1. As shown in Fig. 7A, PDIP1␣ enhanced the PPAR␥-mediated transactivation similar to SRC-1, CBP, or P/CAF. Among these coactivators, PDIP1␣ synergistically augmented the ligand-dependent activation by PPAR␥ with SRC-1 and at
lesser degree with P/CAF. CBP cotransfection revealed no synergistic enhancement of PPAR␥-mediated transactivation with PDIP1. In contrast, PDIP1 synergistically enhanced T3-dependent activation with not only SRC-1 but also CBP and P/CAF in TR1-mediated transactivation. These findings suggested that PDIP1 might exert receptorspecific synergism with AF-2 interacting coactivators in living cells.
FIG. 7. PDIP1␣ showed receptor-specific synergism with AF2-interacting coactivators in PPAR␥- and TR-mediated transactivation. PDIP1␣ (1.67 g/well) was cotransfected into CV-1 cells with PPAR␥ or TR1 (83 ng/well) in the absence or presence of SRC-1, CBP, or P/CAF expression vectors (1.67 g/well). Luciferase assay was performed after 24 h treatment with cognate ligands. The data represent the mean ⫾ SEM of triplicate determinants. Promoter activity without cotransfection of the coactivator in the presence of the ligand was designated as 1, and the relative activity is indicated below. The experiments were repeated twice with identical results. PAL, Palindromic TRE.
Tomaru et al. • Identification of Novel Transcriptional Cofactors of PPAR␥
Discussion
Recently accumulated evidence indicated that the DBD of NRs functions as an interaction interface for other cellular factors. Some of these DBD-interacting proteins, including PGC-1␣ (11), small heterodimer partner (31), small nuclear ring finger protein (17), GT198 (19), and Tat-binding protein 1 (18) function as coactivators of NRs. Using the DBD-H of PPAR␥ as bait in a yeast two-hybrid screen, we isolated KIAA1769, a human EST, whose function was unknown at the time of isolation. KIAA1769 has subsequently been identified as a component of multiprotein complexes that interact with full-length PPAR␣ and has been shown to enhance PPAR␣-mediated transactivation (21). KIAA1769 was termed PRIC285, probably for the molecular mass of purified rat liver protein (21), although the calculated molecular mass of the rat ortholog of KIAA1769 was 330 kDa. We confirmed earlier findings by others (21) and extended the analysis to isolate and characterize the N-terminal extended isoform generated by alternative splicing by RT-PCR and 5⬘RACE. RNase protection assay revealed that the longer isoform mRNA was predominantly expressed in a subset of human culture cell lines. The predicted molecular mass of the long isoform was 295 kDa. In this yeast two-hybrid system and in vitro interaction experiments, KIAA1769 bound to DBD-H but not to the A/B domain or the LBD of PPAR␥. Based on their binding characteristics and molecular mass, we termed the short and long isoforms as PDIP1␣ and -, respectively, in this manuscript. PDIP1 possessed multiple LXXLL motifs, a characteristic NR interaction alignment identified in many coactivator proteins that bind the AF2 of NR in a ligand-dependent manner (29). Because PDIP1 bound to DBD-H, but not to the LBD of PPAR␥, these motifs in PDIP1 were speculated to be dispensable for interaction with PPAR␥. Comparison of the amino acid sequences of six LXXLL motifs in PDIP1 with those identified in other coactivators revealed that the amino acids in and around the LXXLL motifs in PDIP1 did not fit well with the proposed alignment rules (32). Indeed, three polypeptides containing these LXXLL motifs did not bind PPAR␥ in these GST pull-down assays, and mutation of LXXLL motifs in PDIP1␣ did not affect its function to enhance PPAR␥-mediated transactivation. Moreover, the third and fourth LXXLL motifs in human PDIP1 are not conserved in the rat counterpart. We therefore concluded that the C-terminal portion of PDIP1 would function as the PPAR␥-interacting domain. Although siRNA targeting PDIP1 mRNA significantly reduced PPAR␥-mediated transactivation in cell culture system, PDIP1 did not possess a strong autonomous activation domain, and the whole structure was required for its transactivation function in the present mammalian one-hybrid assay. Although PDIP1 possessed amino acid alignments homologous to the RNB motif and the UvrD/REP helicase motif, the functional significance of these motifs is currently unknown. PDIP1 could synergistically augment the PPAR␥mediated transactivation with SRC-1 that interacts with the AF2 of PPAR␥ in a ligand-dependent manner. These characteristics of PDIP1 resembled those of PGC-1␣, a key coactivator of PPAR␥ in adipogenesis, thermogenesis, and gly-
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coneogenesis in the liver and muscles (12–15). PGC-1␣ has been shown to bind the DBD-H of PPAR␥ and possess no potent intrinsic activation domain (11). PGC-1␣ could enhance the docking of coactivators such as SRC-1 by altering the conformation of PPAR␥ (33). These results, taken together, raised the possibility that PDIP1 may function in a manner similar to PGC-1␣ that facilitates the recruitment of other coactivator proteins to PPAR␥, thereby activating target gene transcription. Interestingly, PDIP1␣ showed receptor-specific synergism with AF2-interacting coactivators in PPAR␥- and TR-mediated transcription. PDIP1 might preferentially cooperate with different coactivator complexes with histone acetyltransferase activities in a receptor-specific fashion. PDIP1 also functions to potentiate the ligand-dependent transactivation of NRs other than PPAR␥ in mammalian cells. Although PDIP1 bound to the DBD-H of PPAR␥, it possibly binds the AF2 of other NRs through multiple LXXLL motifs in a ligand-dependent manner. PGC-1␣ has recently been shown to interact with the AF2 of PPAR␣, ER␣, and RXR through the LXXLL motif that was not required for the PPAR␥-interaction (34 –36). Further studies are required to clarify whether these LXXLL motifs in PDIP1 are necessary for interaction with other NRs. Such differential interaction may explain the PDIP1 isoform-specific enhancement of ER␣- and AR-mediated transactivation. PGC-1␣ has been shown to be expressed in a highly tissuespecific manner and to be strongly induced by physiological stimuli such as cold exposure and fasting (11). In contrast, PDIP1 was ubiquitously expressed in human tissues. A relatively high PDIP1 gene expression was observed in tissues such as the heart, liver, skeletal muscles, and pancreas as well as 3T3-L1 adipocytes and human THP-1 macrophages in which PPAR␥ exerts diverse biological functions (1–3). Interestingly, the expression pattern of PDIP1 during the differentiation process of 3T3-L1 preadipocytes resembled the pattern of CBP and TRAP220. Recent studies showed that TRAP220 and CBP are indispensable for the differentiation of preadipocytes to mature adipocytes in vivo (37, 38). Although the specific roles of TRAP220 or CBP in macrophage differentiation have not yet been established, PDIP1 might also be involved in the differentiation processes of adipocytes and macrophages. Further studies using a PDIP1 knockout mouse are underway to elucidate the physiological functions of PDIP1 in multiple tissues. Acknowledgments
We thank Dr. Ronald M. Evans (Howard Hughes Medical Institute, The Salk Institute for Biological Studies, San Diego, CA) and Dr. Yoshihiro Nakatani (Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA) for providing materials. We also thank Dr. Takashi Minegishi and Mitsuyoshi Utsugi (Gunma University Graduate School of Medicine) for providing 293 and THP-1 cells, respectively. Received April 18, 2005. Accepted October 10, 2005. Address all correspondence and requests for reprints to: Teturou Satoh, M.D., Ph.D., Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi 371-8511, Japan. E-mail:
[email protected].
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This work was supported by grants-in-aid for scientific research from the Ministry of Health and Labor (to T.S. and M.M.).
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