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Endocrinology, doi:10.1210/en.2005-0450
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Endocrinology Vol. 147, No. 1 377-388
Copyright © 2006 by The Endocrine Society

Isolation and Characterization of a Transcriptional Cofactor and Its Novel Isoform that Bind the Deoxyribonucleic Acid-Binding Domain of Peroxisome Proliferator-Activated Receptor-{gamma}

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

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: tsato{at}showa.gunma-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using the DNA-binding domain (DBD) and hinge region of human peroxisome proliferator-activated receptor (PPAR)-{gamma} 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{alpha} and termed PPAR{alpha}-interacting cofactor 285 (here referred to as PPAR{gamma}-DBD-interacting protein 1 (PDIP1)-{alpha}). 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{alpha} (referred to as PDIP1ß) consisting of 2649 amino acids (295 kDa) in human cultured cell lines by RT-PCR, and 5' rapid amplification of cDNA ends. Ribonuclease protection assay revealed that PDIP1ß mRNA was expressed more abundantly than PDIP1{alpha} mRNA. The C-terminal region of PDIP1 directly binds DBD of PPAR{gamma}, and multiple LXXLL motifs in PDIP1 were not required for the interaction. PDIP1{alpha} and -ß similarly enhanced PPAR{gamma}-mediated transactivation in transfection assays and short interfering RNA targeting PDIP1 mRNA significantly reduced transactivation by PPAR{gamma}. 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{gamma}-mediated transactivation. PDIP1{alpha} and -ß similarly augmented transactivation by PPAR{alpha}, PPAR{delta}, thyroid hormone receptor (TR)-{alpha}1, TRß1, and retinoid X receptor-{alpha}. PDIP1{alpha} also enhanced estrogen receptor{alpha}- and androgen receptor-mediated transactivation, whereas PDIP1ß did not. PDIP1{alpha} showed receptor-specific synergism with activation function-2-interacting coactivators in PPAR{gamma}- and TRß1-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEROXISOME PROLIFERATOR-activated receptor (PPAR){gamma} 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, 2, 3). PPAR{gamma} 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, 2, 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{gamma} include PPAR-binding protein (PBP)/thyroid hormone receptor-associated protein (TRAP) 220/vitamin D receptor-associated protein (DRIP) 205 (6, 7, 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{gamma}-interacting coactivator (PGC)-1{alpha} that binds the DNA-binding domain (DBD) and part of the hinge region of PPAR{gamma} has been isolated (11) and its molecular and physiological functions intensively analyzed (12, 13, 14, 15). PGC-2 was also identified as an amino (N)-terminal AF1-interacting coactivator of PPAR{gamma} (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, 18, 19). Using the yeast two-hybrid 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{alpha}, a key coactivator of PPAR{gamma}, 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{gamma} might exist to exert the pleiotropic function of this NR on the body (1, 2, 3).

Using the DBD/hinge region of human PPAR{gamma} 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{alpha} and is known as PPAR{alpha}-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{gamma} and examined whether functional differences between two PRIC isoforms exist in a variety of nuclear receptor-mediated transactivations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
Expression vectors for human (h)PPAR{gamma}, PPAR{alpha}, TR{alpha}1, TRß1, RXR{alpha}, estrogen receptor (ER)-{alpha} 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{gamma}1 [amino acids (aa) 108 to 197] as bait. Approximately 1 x 106 transformants were screened for interaction with PPAR{gamma}. 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{gamma} 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{gamma}-DBD-interacting protein (PDIP)-1ß (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.

Construction of mutant PDIP1 expression vector
A mutant PDIP1{alpha} 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{alpha} 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{alpha} 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 PDIP1ß (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 x 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{gamma}, 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{alpha} or -ß 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 x 105 cpm/reaction) was hybridized with 10 µg 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 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Whole-cell lysates were then prepared and 50 µg 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{alpha} and -ß 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{gamma} were cotransfected using the same reagent in the absence or presence of 10 µM troglitazone, and luciferase assay was performed after 24 h.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning a protein that interacts with the DBD/hinge region of human PPAR{gamma}
To isolate proteins that interact with PPAR{gamma}, 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{gamma} as bait. Several positive clones were obtained and individually retransformed into yeast in the presence of pGBT9PPAR{gamma}DBD-H. Significant ß-galactosidase activity was observed with clone 1, stronger than that observed between the ligand-binding domain (LBD) of PPAR{gamma} and the receptor-interaction domain of nuclear receptor corepressor (N-CoR) (Fig. 1AGo). No ß-galactosidase activity was observed by the transformation of either pGBT9PPAR{gamma}DBD-H or pACT2 clone 1 alone indicating the specificity of the interaction. Moreover, clone 1 did not interact with the LBD of PPAR{gamma} in the absence or presence of troglitazone (Tro), whereas the interaction between N-CoR and LBD of PPAR{gamma} was significantly reduced by Tro (Fig. 1AGo). 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. 1BGo). 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. 1BGo and 4CGo).



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  		FIG. 1. Cloning a protein that interacts with DBD and part of the hinge region of PPAR{gamma} using the yeast two-hybrid system. A, Interaction between GAL4DBD-fused PPAR{gamma} 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 µM). 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.

 



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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 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{alpha} and -ß. PDIP1ß possessed an additional 576 aa at its N terminus that links to the eighth aa of PDIP1{alpha}. Six LXXLL motifs in PDIP1ß are underlined. The cDNA and amino acid sequences of PDIP1ß will be available in the DDBJ/EMBL/GenBank database under accession no. AB201715. D, An LXXLL motif in the PDIP1ß-specific portion (LXXLLx1) did not interact with PPAR{gamma}. The polypeptide containing an LXXLL motif in the N terminal of PDIP1ß was synthesized as a GST fusion protein, and a pull-down assay was performed with [35S]-labeled full-length PPAR{gamma} in the absence or presence of Tro. E, 5'RACE of PDIP1ß cDNA. The 5' portion of PDIP1ß mRNA was amplified from Marathon-ready HeLa cell cDNA by nested PCR using two sets of adaptor primers and PDIP1ß 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 PDIP1ß 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{alpha} and -ß 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 PDIP1ß mRNA (410 bp) and PDIP1{alpha} mRNA (299 bp) G, The in vitro translation and in vivo expression of two PDIP1 isoforms. [35S]-labeled PDIP1{alpha} and -ß proteins were synthesized using in vitro translation and subjected to SDS-PAGE (left panel). The FLAG-tagged PDIP1{alpha} or -ß 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{alpha} and -ß proteins.

 
GST interaction assays
To confirm whether KIAA1769 interacts with PPAR{gamma} 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. 2AGo). As shown in Fig. 2BGo, [35S]-labeled full-length PPAR{gamma} bound to GST-CN but not to GST-CC or GST itself. We next determined whether GST-CN could interact with domains other than the DBD of PPAR{gamma}. As shown in Fig. 2BGo, 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{gamma} in vivo and in vitro.



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  		FIG. 2. Interaction between KIAA1769 and PPAR{gamma} 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{gamma} 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{gamma} and GST fusion proteins in the absence or presence of Tro was analyzed using a pull-down assay.

 
To examine whether five LXXLL motifs in KIAA1769 are involved in the interaction with PPAR{gamma}, 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{gamma} was analyzed in GST pull-down assays (Fig. 2CGo). As shown in Fig. 2DGo, these polypeptides did not bind PPAR{gamma} in the absence or presence of Tro. These results suggest that five LXXLL motifs were not required for interaction with PPAR{gamma} and that the PPAR{gamma}-interaction domain in PDIP1 was finally mapped to the C-terminal 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. 3AGo, a single 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. 3BGo). Because PPAR{gamma} has been established to play important physiological roles in adipocytes and macrophages (1, 2, 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{gamma}, 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. 3CGo). 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{gamma} exerts pivotal physiological functions.



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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{gamma}, aP2, or CD36. RNA loading is shown at the bottom. Hybridization signals were quantitated using an image analyzer.

 
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. 4AGo). 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. 4AGo). As shown in Fig. 4BGo, PCR products of different predicted sizes were amplified in human culture cell lines including THP-1, HeLa, 293, and 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. 4BGo). 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. 4CGo). 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. 4CGo), although full-length PPAR{gamma} did not interact with this LXXLL motif in the absence or presence of Tro in a GST pull-down assay (Fig. 4DGo).

To further confirm the expression of PDIP1ß mRNA in human cells, 5'RACE was performed using HeLa cell cDNA to determine the nucleotide sequence of the 5'-untranslated region of PDIP1ß mRNA. In nested PCR amplification using two sets of adaptor primers and gene-specific primers (GSP) for PDIP1ß, a single band was amplified (Fig. 4EGo). In contrast, we could not obtain PCR products using adaptor primers and PDIP1{alpha}-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. 4EGo).

The relative expression of PDIP1{alpha} and -ß 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. 4FGo, a band corresponding to PDIP1ß mRNA was clearly identified using poly(A+) RNA isolated from HeLa, 293, HepG2, and THP-1 cells, whereas a band representing PDIP1{alpha} mRNA was less faintly observed. No protected fragment was observed using yeast tRNA. These findings together with the results of 5'RACE suggested that PDIP1ß mRNA was more abundantly expressed than PDIP1{alpha} mRNA in these human cell lines.

Full-length cDNAs encoding two PDIP1 isoforms were then subcloned into an expression vector, and in vitro-translated proteins were synthesized and separated by SDS-PAGE. As expected, proteins of expected molecular mass were synthesized by in vitro translation (Fig. 4GGo, 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. 4GGo, 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, we thereafter termed the short and long isoforms as PDIP1{alpha} and -ß, 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 PDIP1ß, was registered (accession no. XM_230961).

PDIP1 potentiated ligand-dependent transactivation by PPAR{gamma} and other members of the NR family in mammalian cells
To evaluate whether PDIP1 affects PPAR{gamma}-mediated transactivation and whether functional differences between PDIP1{alpha} and -ß exist, transient transfection experiments were performed using vectors expressing full-length PDIP1{alpha} and -ß in CV-1 cells. As shown in Fig. 5AGoGo, PDIP1{alpha} cotransfection enhanced the PPAR{gamma}-mediated transactivation of the DR1 reporter in the presence of Tro. PDIP1ß similarly enhanced PPAR{gamma}-mediated transactivation. Mutation of five LXXLL motifs in PDIP1{alpha} did not affect enhancement of transactivation by PPAR{gamma} (Fig. 5BGoGo), further confirming that LXXLL motifs were not functionally required for the enhancement.



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  		FIG. 5. PDIP1 potentiates ligand-dependent transcriptional activation by nuclear receptors. A, PDIP1{alpha} and -ß similarly potentiate PPAR{gamma}-mediated transactivation in transient transfection assays. Increasing amounts of pSVSPORT1PDIP1{alpha} or -ß (0, 0.83, and 1.67 µg/well) were cotransfected with direct repeat with 1-bp spacer (DR-1) Luc (1.67 µg/well) in the presence of pKCR2PPAR{gamma} (83 ng/well). Luciferase activity was measured after 24 h treatment with 10 µM 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{alpha} did not alter its function to enhance PPAR{gamma}-mediated transactivation. All five LXXLL motifs in PDIP1{alpha} were mutated to LXXAA by site-directed mutagenesis, and the wild-type or mutant PDIP1{alpha} in pSVSPORT1 (1.67 µg/well) was cotransfected with DR-1 Luc (1.67 µg/well) and pKCR2PPAR{gamma} (83 ng/well) into CV-1 cells. Luciferase assay was performed as described above. C, Knockdown of endogenous PDIP1 gene attenuated PPAR{gamma}-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{gamma} 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{alpha} and -ß on other nuclear receptor-mediated transactivations. pSVSPORT1 PDIP1{alpha} or -ß (1.67 µg/well) was cotransfected with luciferase reporter vectors (1.67 µg/well) in the presence of receptor expression plasmids (83 ng/well). Increasing amounts of pSVSPORT1PDIP1{alpha} or -ß (0, 0.83, and 1.67 µg/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, 17ß-estradiol. MMTV, Mouse mammary tumor virus; PAL, palindromic TRE; ERE, estrogen response element.

 


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  		FIG. 5A. Continued

 
To clarify whether PDIP1 is directly involved in PPAR{gamma}-mediated transactivation in vivo, the effect of knockdown of PDIP1 was studied by using siRNA targeting endogenous PDIP1 gene (siPDIP1). As shown in Fig. 5CGoGo, transfection of siPDIP1, but not control siRNA, reduced PDIP1 mRNA levels in HeLa cells and apparently attenuated ligand-dependent activation by PPAR{gamma}. The result suggested that endogenous PDIP1 might be essential for PPAR{gamma}-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. 5DGoGo, PDIP1ß augmented the PPAR{alpha}-mediated transactivation of DR1 reporter in the presence of fenofibrate, similar to PDIP1{alpha}. In addition, PDIP1{alpha} and -ß enhanced PPAR{delta}-mediated transactivation. Both PDIP1{alpha} and -ß could indistinguishably enhance other NR-mediated transactivation including TR{alpha}1, TRß1, and RXR{alpha} in the presence of cognate ligands. Moreover, PDIP1{alpha} enhanced AR- and ER{alpha}-mediated transactivation in a dose-dependent manner, whereas PDIP1ß 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{alpha}-mediated transactivation.

PDIP1{alpha} and -ß did not possess an autonomous activation domain
To determine whether PDIP1{alpha} and -ß possess intrinsic transactivation domains, PDIP1{alpha} was divided into overlapping eight fragments, and mammalian one-hybrid assays were performed. The function of the N-terminal portion specific for PDIP1ß was also examined in parallel. As shown in Fig. 6Go, full-length PDIP1 {alpha} and -ß stimulated the activity of the UAS-TK reporter by 2.5-fold, whereas none of the divided fragments of PDIP1{alpha} and -ß activated the reporter activity over GAL4DBD alone. These findings suggested that PDIP1{alpha} and -ß did not possess a strong intrinsic activation domain and required the whole structure for its transactivation function.



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  		FIG. 6. PDIP1 requires its whole structure for activation function. Full-length and partial cDNAs of PDIP1{alpha} and -ß were ligated to GAL4DBD and transfected with 3 x 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.

 
Receptor-specific synergism between PDIP1 and the AF-2 interacting cofactors in PPAR{gamma}- and TRß1-mediated transactivation
Because PDIP1 did not show a strong intrinsic activation function, it was speculated that PDIP1 might cooperate 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{gamma} and TRß1. As shown in Fig. 7AGo, PDIP1{alpha} enhanced the PPAR{gamma}-mediated transactivation similar to SRC-1, CBP, or P/CAF. Among these coactivators, PDIP1{alpha} synergistically augmented the ligand-dependent activation by PPAR{gamma} with SRC-1 and at lesser degree with P/CAF. CBP cotransfection revealed no synergistic enhancement of PPAR{gamma}-mediated transactivation with PDIP1. In contrast, PDIP1 synergistically enhanced T3-dependent activation with not only SRC-1 but also CBP and P/CAF in TRß1-mediated transactivation. These findings suggested that PDIP1 might exert receptor-specific synergism with AF-2 interacting coactivators in living cells.



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  		FIG. 7. PDIP1{alpha} showed receptor-specific synergism with AF2-interacting coactivators in PPAR{gamma}- and TRß-mediated transactivation. PDIP1{alpha} (1.67 µg/well) was cotransfected into CV-1 cells with PPAR{gamma} or TRß1 (83 ng/well) in the absence or presence of SRC-1, CBP, or P/CAF expression vectors (1.67 µg/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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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{alpha} (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{gamma} 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{alpha} and has been shown to enhance PPAR{alpha}-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{gamma}. Based on their binding characteristics and molecular mass, we termed the short and long isoforms as PDIP1{alpha} and -ß, 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{gamma}, these motifs in PDIP1 were speculated to be dispensable for interaction with PPAR{gamma}. 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{gamma} in these GST pull-down assays, and mutation of LXXLL motifs in PDIP1{alpha} did not affect its function to enhance PPAR{gamma}-mediated transactivation. Moreover, the third and fourth LXXLL motifs in human PDIP1ß are not conserved in the rat counterpart. We therefore concluded that the C-terminal portion of PDIP1 would function as the PPAR{gamma}-interacting domain.

Although siRNA targeting PDIP1 mRNA significantly reduced PPAR{gamma}-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{gamma}-mediated transactivation with SRC-1 that interacts with the AF2 of PPAR{gamma} in a ligand-dependent manner. These characteristics of PDIP1 resembled those of PGC-1{alpha}, a key coactivator of PPAR{gamma} in adipogenesis, thermogenesis, and glyconeogenesis in the liver and muscles (12, 13, 14, 15). PGC-1{alpha} has been shown to bind the DBD-H of PPAR{gamma} and possess no potent intrinsic activation domain (11). PGC-1{alpha} could enhance the docking of coactivators such as SRC-1 by altering the conformation of PPAR{gamma} (33). These results, taken together, raised the possibility that PDIP1 may function in a manner similar to PGC-1{alpha} that facilitates the recruitment of other coactivator proteins to PPAR{gamma}, thereby activating target gene transcription. Interestingly, PDIP1{alpha} showed receptor-specific synergism with AF2-interacting coactivators in PPAR{gamma}- and TRß-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{gamma} in mammalian cells. Although PDIP1 bound to the DBD-H of PPAR{gamma}, it possibly binds the AF2 of other NRs through multiple LXXLL motifs in a ligand-dependent manner. PGC-1{alpha} has recently been shown to interact with the AF2 of PPAR{alpha}, ER{alpha}, and RXR through the LXXLL motif that was not required for the PPAR{gamma}-interaction (34, 35, 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{alpha}- and AR-mediated transactivation.

PGC-1{alpha} has been shown to be expressed in a highly tissue-specific 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{gamma} exerts diverse biological functions (1, 2, 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.


   Footnotes
 
This work was supported by grants-in-aid for scientific research from the Ministry of Health and Labor (to T.S. and M.M.).

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, glutathione-S-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{gamma}-DBD-interacting protein 1; PGC, PPAR{gamma}-interacting coactivator; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; PRIC, PPAR{alpha}-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.

Received April 18, 2005.

Accepted for publication October 10, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rosen ED, Spiegelman BM 2001 PPAR{gamma}: a nuclear regulator of metabolism, differentiation, and cell growth. J Biol Chem 276:37731–37734[Free Full Text]
  2. Rangwala SM, Lazar MA 2004 Peroxisome proliferator-activated receptor {gamma} in diabetes and metabolism. Trends. Pharmacol Sci 25:331–336
  3. Desvergne B, Michalik L, Wahli W 2004 Be fit or be sick: peroxisome proliferator-activated receptors are down the road. Mol Endocrinol 18:1321–1332[Abstract/Free Full Text]
  4. Glass CK, Rosenfeld MG 2000 The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14:121–141[Free Full Text]
  5. Smith CL, O’Malley BW 2004 Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev 25:45–71[Abstract/Free Full Text]
  6. Rachez C, Suldan Z, Ward J, Chang CP, Burakov D, Erdjument-Bromage H, Tempst P, Freedman LP 1998 A novel protein complex that interacts with the vitamin D3 receptor in a ligand-dependent manner and enhances VDR transactivation in a cell-free system. Genes Dev 12:1787–1800[Abstract/Free Full Text]
  7. Zhu Y, Qi C, Jain S, Rao MS, Reddy JK 1997 Isolation and characterization of PBP, a protein that interacts with peroxisome proliferator-activated receptor. J Biol Chem 272:25500–25506[Abstract/Free Full Text]
  8. Yuan CX, Ito M, Fondell JD, Fu ZY, Roeder RG 1998 The TRAP220 component of a thyroid hormone receptor-associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion. Proc Natl Acad Sci USA 95:7939–7944[Abstract/Free Full Text]
  9. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-{gamma}. Nature 395:137–143[CrossRef][Medline]
  10. Mizukami J, Taniguchi T 1997 The antidiabetic agent thiazolidinedione stimulates the interaction between PPAR {gamma} and CBP. Biochem Biophys Res Commun 240:61–64[CrossRef][Medline]
  11. Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM 1998 A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92:829–839[CrossRef][Medline]
  12. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, Troy A, Cinti S, Lowell B, Scarpulla RC, Spiegelman BM 1999 Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98:115–124[CrossRef][Medline]
  13. Yoon JC, Puigserver P, Chen G, Donovan J, Wu Z, Rhee J, Adelmant G, Stafford J, Kahn CR, Granner DK, Newgard CB, Spiegelman BM 2001 Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413:131–138[CrossRef][Medline]
  14. Puigserver P, Rhee J, Lin J, Wu Z, Yoon JC, Zhang CY, Krauss S, Mootha VK, Lowell BB, Spiegelman BM 2001 Cytokine stimulation of energy expenditure through p38 MAP kinase activation of PPAR{gamma} coactivator-1. Mol Cell 5:971–982
  15. Lin J, Wu H, Tarr PT, Zhang CY, Wu Z, Boss O, Michael LF, Puigserver P, Isotani, E, Olson EN, Lowell BB, Bassel-Duby R, Spiegelman BM 2002 Transcriptional co-activator PGC-1{alpha} drives the formation of slow-twitch muscle fibers. Nature 418:797–801[CrossRef][Medline]
  16. Castillo G, Brun RP, Rosenfield JK, Hauser S, Park CW, Troy AE, Wright ME, Spiegelman BM 1999 An adipogenic cofactor bound by the differentiation domain of PPAR{gamma}. EMBO J 18:3676–3687[CrossRef][Medline]
  17. Moilanen AM, Poukka H, Karvonen U, Hakli M, Janne OA, Palvimo JJ 1998 Identification of a novel RING finger protein as a coregulator in steroid receptor-mediated gene transcription. Mol Cell Biol 18:5128–5139[Abstract/Free Full Text]
  18. Ishizuka T, Satoh T, Monden T, Shibusawa N, Hashida T, Yamada M, Mori M 2001 Human immunodeficiency virus type 1 Tat binding protein-1 is a transcriptional coactivator specific for TR. Mol Endocrinol 15:1329–1343[Abstract/Free Full Text]
  19. Ko L, Cardona GR, Henrion-Caude A, Chin WW 2002 Identification and characterization of a tissue-specific coactivator, GT198, that interacts with the DNA-binding domains of nuclear receptors. Mol Cell Biol 22:357–369[Abstract/Free Full Text]
  20. Puigserver P, Spiegelman BM 2003 Peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}): transcriptional coactivator and metabolic regulator. Endocr Rev 24:78–90[Abstract/Free Full Text]
  21. Surapureddi S, Yu S, Bu H, Hashimoto T, Yeldandi AV, Kashireddy P, Cherkaoui-Malki M, Qi C, Zhu YJ, Rao MS, Reddy JK 2002 Identification of a transcriptionally active peroxisome proliferator-activated receptor {alpha}-interacting cofactor complex in rat liver and characterization of PRIC285 as a coactivator. Proc Natl Acad Sci USA 99:11836–11841[Abstract/Free Full Text]
  22. Yang XJ, Ogryzko VV, Nishikawa J, Howard BH, Nakatani Y 1996 A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382:319–324[CrossRef][Medline]
  23. Satoh T, Toyoda M, Hoshino H, Monden T, Yamada M, Shimizu H, Miyamoto K, Mori M 2002 Activation of peroxisome proliferator-activated receptor-{gamma} stimulates the growth arrest and DNA-damage inducible 153 gene in non-small cell lung carcinoma cells. Oncogene 21:2171–2180[CrossRef][Medline]
  24. Utsugi M, Dobashi K, Ishizuka T, Endou K, Hamuro J, Murata Y, Nakazawa T, Mori M 2003 c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production. J Immunol 171:628–635[Abstract/Free Full Text]
  25. Schaufele F, Chang CY, Liu W, Baxter JD, Nordeen SK, Wan Y, Day RN, McDonell DP 2000 Temporally distinct and ligand-specific recruitment of nuclear receptor-interacting peptides and cofactors to subnuclear domains containing the estrogen receptor. Mol Endocrinol 14:2024–2039[Abstract/Free Full Text]
  26. Satoh T, Feng P, Kim UJ, Wilber JF 1993 Identification of thyrotropin-releasing hormone receptor messenger RNA in the rat central nervous system and eye. Mol Brain Res 19:175–178[Medline]
  27. Mian S 1997 Comparative sequence analysis of ribonucleases HII, III, II PH and D. Nucleic Acids Res 25:3187–3195[Abstract/Free Full Text]
  28. Gilchrist CA, Denhardt DT 1987 Escherichia coli rep gene: sequence of the gene, the encoded helicase, and its homology with uvrD. Nucleic Acids Res 15:465–475[Abstract/Free Full Text]
  29. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  30. Moore KJ, Rosen ED, Fitzgerald ML, Randow F, Andersson LP, Altshuler D, Milstone DS, Mortensen RM, Spiegelman BM, Freeman MW 2001 The role of PPAR-{gamma} in macrophage differentiation and cholesterol uptake. Nat Med 7:41–47[CrossRef][Medline]
  31. Nishizawa H, Yamagata K, Shimomura I, Takahashi M, Kuriyama H, Kishida K, Hotta K, Nagaretani H, Maeda N, Matsuda M, Kihara S, Nakamura T, Nishigori H, Tomura H, Moore DD, Takeda J, Funahashi T, Matsuzawa Y 2002 Small heterodimer partner, an orphan nuclear receptor, augments peroxisome proliferator-activated receptor {gamma} transactivation. J Biol Chem 277:1586–1592[Abstract/Free Full Text]
  32. Savkur RS, Burris TP 2004 The coactivator LXXLL nuclear receptor recognition motif. J Pept Res 63:207–212[CrossRef][Medline]
  33. Puigserver P, Adelmant G, Wu Z, Fan M, Xu J, O’Malley B, Spiegelman BM 1999 Activation of PPAR{gamma} coactivator-1 through transcription factor docking. Science 286:1368–1371[Abstract/Free Full Text]
  34. Vega RB, Huss JM, Kelly DP 2000 The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor {alpha} in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Mol Cell Biol 20:1868–1876[Abstract/Free Full Text]
  35. Tcherepanova I, Puigserver P, Norris D, Spiegelman BM, McDonnell DP 2000 Modulation of estrogen receptor-{alpha} transcriptional activity by the coactivator PGC-1. J Biol Chem 275:16302–16308[Abstract/Free Full Text]
  36. Delerive P, Wu Y, Burris TP, Chin WW, Suen CS 2002 PGC-1 functions as a transcriptional coactivator for the retinoid X receptors. J Biol Chem 277:3913–3917[Abstract/Free Full Text]
  37. Ge K, Guermah M, Yuan CX, Ito M, Wallberg AE, Spiegelman BM, Roeder RG 2002 Transcription coactivator TRAP220 is required for PPAR{gamma}2-stimulated adipogenesis. Nature 417:563–567[CrossRef][Medline]
  38. Takahashi N, Kawada T, Yamamoto T, Goto T, Taimatsu A, Aoki N, Kawasaki H, Taira K, Yokoyama KK, Kamei Y, Fushiki T 2002 Overexpression and ribozyme-mediated targeting of transcriptional coactivators CREB-binding protein and p300 revealed their indispensable roles in adipocyte differentiation through the regulation of peroxisome proliferator-activated receptor {gamma}. J Biol Chem 277:16906–16912[Abstract/Free Full Text]



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