Peroxisome Proliferator-activated Receptor γ Coactivator 1β (PGC-1β), A Novel PGC-1-related Transcription Coactivator Associated with Host Cell Factor

Peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) plays a critical role in regulating multiple aspects of energy metabolism, including adaptive thermogenesis, mitochondrial biogenesis, and fatty acid β-oxidation. Recently, this coactivator of nuclear receptors/transcription factors has been shown to control hepatic gluconeogenesis, an important component of the pathogenesis of both type-1 and type-2 diabetes. We described here the cloning of a novel bona fide homologue of PGC-1, PGC-1β (PGC-1 was renamed as PGC-1α), first identified through searches of new data base entries. Despite the fact that PGC-1α and -1β share similar tissue distributions with highest levels of expression in brown fat and heart, their mRNAs are differentially regulated in the brown adipose tissue upon cold exposure and during brown fat cell differentiation. Like PGC-1α, PGC-1β mRNA levels are increased significantly in the liver during fasting, suggesting a possible role for this factor in the regulation of hepatic gluconeogenesis and/or fatty acid oxidation. Consistent with this, PGC-1β was shown to physically interact and potently coactivate hepatic nuclear factor 4 and peroxisome proliferator-activated receptor α, nuclear receptors that are essential for hepatic adaptation to fasting. Finally, using sequence comparisons between PGC-1α and -1β, we have identified a conserved amino acid motif that serves as a docking site for host cell factor, a cellular protein implicated in cell cycle regulation and viral infection. HCF is shown to bind to both PGC-1α and -1β and augment their transcriptional activity. Peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) plays a critical role in regulating multiple aspects of energy metabolism, including adaptive thermogenesis, mitochondrial biogenesis, and fatty acid β-oxidation. Recently, this coactivator of nuclear receptors/transcription factors has been shown to control hepatic gluconeogenesis, an important component of the pathogenesis of both type-1 and type-2 diabetes. We described here the cloning of a novel bona fide homologue of PGC-1, PGC-1β (PGC-1 was renamed as PGC-1α), first identified through searches of new data base entries. Despite the fact that PGC-1α and -1β share similar tissue distributions with highest levels of expression in brown fat and heart, their mRNAs are differentially regulated in the brown adipose tissue upon cold exposure and during brown fat cell differentiation. Like PGC-1α, PGC-1β mRNA levels are increased significantly in the liver during fasting, suggesting a possible role for this factor in the regulation of hepatic gluconeogenesis and/or fatty acid oxidation. Consistent with this, PGC-1β was shown to physically interact and potently coactivate hepatic nuclear factor 4 and peroxisome proliferator-activated receptor α, nuclear receptors that are essential for hepatic adaptation to fasting. Finally, using sequence comparisons between PGC-1α and -1β, we have identified a conserved amino acid motif that serves as a docking site for host cell factor, a cellular protein implicated in cell cycle regulation and viral infection. HCF is shown to bind to both PGC-1α and -1β and augment their transcriptional activity. nuclear receptor peroxisome proliferator-activated receptor PPARγ coactivator-1 brown adipose tissue herpes simplex virus host cell factor nuclear respiratory factor RNA recognition motif HCF binding motif retinoic acid receptor thyroid receptor glucocorticoid receptor hepatic nuclear receptor 4 glutathione S-transferase DNA binding domain PGC-1-related coactivator The transcriptional function of many nuclear receptors (NRs)1 is regulated by ligand-dependent recruitment of coactivators to the C-terminal ligand binding domain (1Aranda A. Pascual A. Physiol. Rev. 2001; 81: 1269-1304Crossref PubMed Scopus (1210) Google Scholar, 2Rosenfeld M.G. Glass C.K. J. Biol. Chem. 2001; 276: 36865-36868Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). A number of coactivators, including the p160 family, p300/CBP and P/CAF, contain intrinsic histone acetyl transferase activity and regulate transcription by modulating histone acetylation (1Aranda A. Pascual A. Physiol. Rev. 2001; 81: 1269-1304Crossref PubMed Scopus (1210) Google Scholar). Other coactivators, consisting of heterogeneous proteins with little sequence homology, modulate transcription by acting as protein docking interfaces that recruit histone acetyl transferase-containing complexes or associate with basal transcription factors such as RNA polymerase II holoenzyme (3Freedman L.P. Cell. 1999; 97: 5-8Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). The interaction between NRs and many coactivators requires a conserved LXXLL motif, which is believed to form hydrophobic contacts with the receptors (4Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1712) Google Scholar, 5Westin S. Kurokawa R. Nolte R.T. Wisely G.B. McInerney E.M. Rose D.W. Milburn M.V. Rosenfeld M.G. Glass C.K. Nature. 1998; 395: 199-202Crossref PubMed Scopus (306) Google Scholar). PGC-1 was initially identified as a PPARγ-interacting protein from a BAT library and was subsequently found to associate with an array of NRs and transcription factors (6Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3177) Google Scholar, 7Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B.B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar, 8Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Crossref PubMed Scopus (971) Google Scholar, 9Michael L.F. Wu Z. Cheatham R.B. Puigserver P. Adelmant G. Lehman J.J. Kelly D.P. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3820-3825Crossref PubMed Scopus (549) Google Scholar). Importantly, PGC-1 has been shown to coordinately regulate the program of mitochondrial biogenesis and adaptive thermogenesis in BAT and skeletal muscle, mainly through the coactivation of PPARs and nuclear respiratory factor 1 (NRF1), a nuclear transcription factor that regulates the expression of many mitochondrial genes (6Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3177) Google Scholar, 7Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B.B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar). In transgenic mice, PGC-1 increases mitochondrial biogenesis and β-oxidation of fatty acids in the heart, likely through augmentation of PPARα and NRF1 transcriptional activity (10Lehman J.J. Barger P.M. Kovacs A. Saffitz J.E. Medeiros D.M. Kelly D.P. J. Clin. Invest. 2000; 106: 847-856Crossref PubMed Scopus (1055) Google Scholar). Recently, PGC-1 expression was found to be elevated in fasted liver and several models of type-1 and type-2 diabetes; in addition, PGC-1 can directly control the activation of hepatic gluconeogenesis (11Yoon J.C. Puigserver P. Chen G. Donovan J. Wu Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1560) Google Scholar, 12Herzig S. Long F. Jhala U.S. Hedrick S. Quinn R. Bauer A. Rudolph D. Schutz G. Yoon J.C. Puigserver P. Spiegelman B.M. Montminy M. Nature. 2001; 413: 179-183Crossref PubMed Scopus (1173) Google Scholar). Despite playing an important role in coordinating multiple physiological processes, no close homologue for PGC-1 has been found. We described here the identification of a novel coactivator, which is closely related to PGC-1, that we have termed PGC-1β. Furthermore, we identified HCF, a cellular protein that is involved in herpes simplex virus (HSV) infection and cell cycle regulation (13Wilson A.C. LaMarco K. Peterson M.G. Herr W. Cell. 1993; 74: 115-125Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 14Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (92) Google Scholar), as a novel binding partner that increases the transcriptional activity of both PGC-1α and -1β. Full-length PGC-1β cDNA clone was obtained by ligating the 5′ end of PGC-1β cDNA generated by GENERACER (Invitrogen) to an expressed sequence tag cDNA clone (AA288169) that contains the 3′ end. GST-PGC-1β (N-350) was generated by inserting a PCR fragment coding for the N-terminal 350 amino acids in frame into pGEX-5X1 vector. Fusion constructs between GAL4-DBD and various regions of PGC-1β or HCF were generated by subcloning PCR-amplified cDNA fragments in frame into pCMX-GAL4 plasmid (6Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3177) Google Scholar). N-terminal FLAG-tagged PGC-1β (amino acid 2–1014) was obtained by subcloning the cDNA insert into pCATCH FLAG plasmid (15Georgiev O. Bourquin J.P. Gstaiger M. Knoepfel L. Schaffner W. Hovens C. Gene. 1996; 168: 165-167Crossref PubMed Scopus (41) Google Scholar). All PCR fragments were verified by sequencing. BOSC cells and COS cells were maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. The cells were transfected using FuGENE according to the manufacturer (Roche Molecular Biochemicals). Reporter constructs for HNF4α (3× gAF1 luc) and NRF1 (4× NRF1 luc) were described previously (7Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B.B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3300) Google Scholar, 11Yoon J.C. Puigserver P. Chen G. Donovan J. Wu Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1560) Google Scholar). Luciferase assays were performed 48 h after transfection. HIB1B cells were maintained and induced to differentiate as described (6Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3177) Google Scholar). To induce UCP-1 expression, differentiated cells were treated with 10 μmforskolin for 6 h before RNA isolation. For cold exposure, C57/Bl6 mice were maintained at 4 °C for 6 h before sacrifice. RNA samples were analyzed by Northern hybridization using specific cDNA probes. Binding assays using GST fusion proteins were performed essentially as described (6Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3177) Google Scholar). Ligands were included in some binding reactions as indicated. For coimmunoprecipitation assays, BOSC cells were transiently transfected with various combinations of plasmids and lysed after 36 h in a buffer containing 100 mm Tris (pH = 8.0), 250 mm NaCl, 1% Nonidet P-40, 1 mm EDTA, 1 mm MgCl2, and protease inhibitors. The lysates were incubated with 10 μl of anti-FLAG-Sepharose beads (Sigma) for 1 h at room temperature. The beads were then washed four times with the lysis buffer and resuspended in SDS-PAGE buffer. The samples were then processed for immunoblotting analysis. PGC-1 is not known to have any close homologues. Because a great amount of sequence data has recently been entered into the genomic and expressed sequence tag databases, we searched these databases for transcripts with similarities to the N terminus of PGC-1. In contrast to the C terminus, this domain is uniquely found in rodent and human PGC-1. A partial transcript in the Celera mouse genome database (mCT4723) was found to encode a novel protein sharing a high degree of sequence identity to PGC-1, which we designated PGC-1β (AF453324). To keep nomenclature consistent, we thus renamed PGC-1 as PGC-1α. Sequence analysis revealed that the 3.6-kb cDNA contains an open reading frame of 1014 amino acids that has similarity to PGC-1α across the entire molecule, including three LXXLL motifs at the N terminus and one RNA recognition motif (RRM) at the C terminus (Fig. 1A). Identity between PGC-1α and -1β is especially high in the N-terminal activation domain and the C-terminal RNA binding domain (40 and 48%, respectively; see Fig. 1B). Furthermore, sequence comparison of all PGC-1 family members, including PGC-1-related coactivator (PRC), revealed a novel conserved region containing a tetrapeptide motif (DHDY; amino acid 683–686) that has been previously identified in other proteins as a putative binding site for HCF, a protein involved in the regulation of cell cycle progression and the assembly of a multiprotein transcriptional complex during HSV infection (16Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar,17Andersson U. Scarpulla R.C. Mol. Cell. Biol. 2001; 21: 3738-3749Crossref PubMed Scopus (305) Google Scholar). The function of this motif will be analyzed below. PGC-1β also contains two glutamic/aspartic acid-rich acidic domains, but unlike PGC-1α and PRC, it lacks most of the arginine/serine-rich domain, a region that has been implicated in the regulation of RNA processing (18Monsalve M. Wu Z. Adelmant G. Puigserver P. Fan M. Spiegelman B.M. Mol. Cell. 2000; 6: 307-316Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Human PGC-1β is predicted to share over 70% amino acid sequence identity with mouse PGC-1β and is localized to 5q33. Murine PGC-1β is localized to chromosome 18. BLAST searches also revealed that the PGC-1 family of proteins is also well conserved in species such as chicken, zebrafish, and Xenopus, suggesting that PGC-1 may play important roles common to all of these species, perhaps linked to mitochondrial function and/or the regulation of other cellular and developmental processes. PGC-1β mRNA is expressed in a highly tissue-selective manner as shown by Northern hybridization (Fig. 1C). PGC-1β mRNA is most abundantly present in brown adipose tissue and heart, both notable for containing very high concentrations of mitochondria. These results strongly suggest that like PGC-1α, PGC-1β may play an important role in mitochondrial function, respiration, and/or thermogenesis. Two predominant species of PGC-1β mRNA (5 and 9 kb) were detected; this may result from the usage of two alternative polyadenylation signals present in the 3′ end of the PGC-1β gene. Moderate levels of PGC-1β mRNA are also observed in skeletal muscle, liver, and white adipose tissue. PGC-1α was initially identified as a transcription coactivator that controls mitochondrial biogenesis and adaptive thermogenesis in skeletal muscle and brown fat. In contrast to the cold-inducible expression of PGC-1α, the expression of PGC-1β in BAT is not increased in response to cold exposure (Fig.2A). Given its abundant expression in the brown fat, it is possible that PGC-1β is an important factor in the determination and/or differentiation of brown adipocytes. To test whether the expression of PGC-1β is regulated during brown fat differentiation, the mouse brown fat cell line HIB1B was induced to undergo differentiation and examined for the PGC-1β expression (19Klaus S. Choy L. Champigny O. Cassard-Doulcier A.M. Ross S. Spiegelman B.M. Ricquier D. J. Cell Sci. 1994; 107: 313-319Crossref PubMed Google Scholar). Compared with undifferentiated cells, the expression of PGC-1β is elevated on days 3 and 5 of differentiation, parallel with a concomitant decrease in the PGC-1α expression (Fig.2B). Upon treatments with forskolin, an activator of adenylyl cyclase, PGC-1α level is rapidly induced along with the key uncoupling protein of brown fat, UCP-1. In contrast, PGC-1β expression is slightly decreased. These results indicate that PGC-1α and -1β are likely to perform distinct roles in brown fat differentiation and the regulation of other brown fat functions such as adaptive thermogenesis. PGC-1α expression is highly induced in the liver during fasting and has been shown to play a direct role in the activation of hepatic gluconeogenesis in primary hepatocyte culture and rats (11Yoon J.C. Puigserver P. Chen G. Donovan J. Wu Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1560) Google Scholar, 12Herzig S. Long F. Jhala U.S. Hedrick S. Quinn R. Bauer A. Rudolph D. Schutz G. Yoon J.C. Puigserver P. Spiegelman B.M. Montminy M. Nature. 2001; 413: 179-183Crossref PubMed Scopus (1173) Google Scholar). Elevated expression of PGC-1α has also been shown in models of both type-1 and type-2 diabetes (11Yoon J.C. Puigserver P. Chen G. Donovan J. Wu Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1560) Google Scholar). As shown in Fig. 2C, the expression of PGC-1β is significantly increased in the liver during fasting, a pattern that is strikingly similar to the regulation of PGC-1α expression. These results suggest that PGC-1β might be part of the regulatory pathways that activate the hepatic adaptation during fasting, such as the elevation of gluconeogenesis, β-oxidation of fatty acids, and ketogenesis. PPARα and HNF4α are important transcription regulators implicated in hepatic fatty acid oxidation and gluconeogenesis during periods of food deprivation (20Kersten S. Seydoux J. Peters J.M. Gonzalez F.J. Desvergne B. Wahli W. J. Clin. Invest. 1999; 103: 1489-1498Crossref PubMed Scopus (1388) Google Scholar). We therefore asked whether PGC-1β would physically interact with and coactivate these transcription factors. The N terminus of PGC-1β (1), which contains three putative nuclear receptor binding motifs (LXXLL), is able to “pull down” in vitro-translated HNF4α with similar efficiency as PGC-1α (Fig. 3A). Furthermore, the full-length PGC-1β is readily coimmunoprecipitated with HNF4α when coexpressed in BOSC cells, indicating in vivo association of these two proteins (Fig. 3B). In contrast to PPARα and -γ, the recruitment of PGC-1β by RARα and TRβ is highly dependent on their respective ligands (Fig.3A and data not shown). Consistent with a potential role in regulating hepatic gluconeogenesis, PGC-1β potently enhances the transcriptional activity of GR and HNF4α on reporter constructs containing multimerized cognate binding sites (Fig. 3C). Coactivation of GR by PGC-1β is strictly dependent on the presence of glucocorticoid, indicating ligand-dependent recruitment of the coactivator to the receptor; the latter is also observed for TRβ and RARα (data not shown). Although PGC-1β alone has minimal on the reporter plasmids for GR and it significantly HNF4α reporter this is of the presence of nuclear receptors that may bind to the In to PGC-1β is also a of the transcriptional activity of a transcription factor in the control of mitochondrial biogenesis binding and coactivation of several receptors by PGC-1β that PGC-1β regulates the transcriptional activity of multiple likely through direct association with these factors. these factors known to be important in mitochondrial biogenesis, fatty acid and Sequence of all PGC-1 family revealed several conserved of amino acids that may be important for their such region contains a tetrapeptide motif, that is a putative HCF binding motif containing this motif have been shown to associate with HCF, including HSV viral protein and a (16Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar). HCF not bind DNA by it can be by transcription factors such as and functions as a for the assembly of complexes T.M. J. 2000; PubMed Scopus Google Scholar). Although HCF has been found in complex with and the cellular for this factor is therefore possible that PGC-1α and -1β may the recruitment of protein complexes to the binding modulating To test whether PGC-1α and -1β are physically with HCF in FLAG-tagged HCF was with or -1β and analyzed by by As shown in HCF strongly with both PGC-1α and thus a novel interaction partner for PGC-1-related of HCF to PGC-1β increases the transcriptional activity of PGC-1β in a (Fig. the other PGC-1β can be by the N-terminal amino acids of HCF, a domain that has been shown to interact with and potently increases the transcriptional activity of HCF to the (Fig. Coactivation of HCF was also observed for PGC-1α (data not shown). These results that HCF can function in complex with to activate transcription from We described here the identification of a transcription coactivator that is closely related to PGC-1α. PGC-1β has sequence to PGC-1α across the molecule, but it is the of a distinct gene. PGC-1β physically with a number of NRs both in and in vivo and potently their transcriptional activity. of PGC-1β is elevated in the liver during fasting, suggesting like PGC-1α, may play an important role in the activation of hepatic adaptation during fasting. Consistent with this, PGC-1β was found to coactivate and transcription factors that the activation of gluconeogenesis and fatty acid oxidation in the PGC-1β mRNA is abundant in both BAT and heart, that contain and high levels of oxidation. Given that PGC-1β is a coactivator for it is highly likely that PGC-1β regulates mitochondrial function and In contrast to the cold-inducible expression of PGC-1α, the expression of PGC-1β mRNA is not by exposure of to cold or of brown culture with the other PGC-1β mRNA is elevated during brown fat differentiation, suggesting that PGC-1α and -1β perform distinct functions during brown fat and adaptive thermogenesis. The of this new sequence to conserved motifs PGC-1 family that may be important for their We here that all known coactivators in the PGC-1 family, including PGC-1α, -1β, and PRC, contain a docking site for The association between HCF and PGC-1β enhances the transcriptional activity of suggesting that the assembly of complexes may be important in the coactivation of NRs by PGC-1. HCF is a critical component of a complex along with HSV and protein this complex is important for expression during viral infection A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (92) Google Scholar, T.M. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). The activity of HCF is also for cell cycle progression A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (92) Google Scholar). is therefore possible that PGC-1, in complex with HCF and transcription factors such as plays an as role in expression during viral infection and cell perhaps related to energy be in We for reading the We also for reporter Granner for the GR and HNF4α Scarpulla for NRF1 and and Herr for the HCF