characterization of novel peroxisome proliferator-activated

Characterization of Novel Peroxisome Proliferator-activatedReceptor � Coactivator-1� (PGC-1�) Isoform in HumanLiver*□S

Received for publication, February 3, 2011, and in revised form, October 14, 2011 Published, JBC Papers in Press, October 18, 2011, DOI 10.1074/jbc.M111.227496

Thomas K. Felder‡, Selma M. Soyal‡, Hannes Oberkofler‡, Penelope Hahne‡, Simon Auer‡, Richard Weiss§,Gabriele Gadermaier§, Karl Miller¶, Franz Krempler�, Harald Esterbauer**, and Wolfgang Patsch‡1

From the ‡Department of Laboratory Medicine, Paracelsus Medical University, 5020 Salzburg, §Department of Molecular Biology,University of Salzburg, 5020 Salzburg, Departments of ¶Surgery and �Internal Medicine, Krankenhaus Hallein, 5400 Hallein, and**Department of Laboratory Medicine, Medical University Vienna, 1090 Vienna, Austria

Peroxisome proliferator-activated receptor � coactivator-1�(PGC-1�) is a transcriptional coactivator that contributes to theregulation of numerous transcriptional programs including thehepatic response to fasting. Mechanisms at transcriptional andpost-transcriptional levels allow PGC-1� to support distinctbiological pathways. Here we describe a novel human liver-spe-cific PGC-1� transcript that results from alternative promoterusage and is induced by FOXO1 as well as glucocorticoids andcAMP-response element-binding protein signaling but is notpresent in other mammals. Hepatic tissue levels of novel andwild-type transcripts were similar but were only moderatelyassociated (p < 0.003). Novel mRNA levels were associated witha polymorphism located in its promoter region, whereas wild-type transcript levels were not. Furthermore, hepatic PCK1mRNA levels exhibited stronger associations with the novelthanwith thewild-type transcript levels. Except for a deletion of127 amino acids at the N terminus, the protein, termed L-PGC-1�, is identical to PGC-1�. L-PGC-1� was localized in thenucleus and showed coactivation properties that overlap withthose of PGC-1�. Collectively, our data support a role ofL-PGC-1� in gluconeogenesis, but functional differences pre-dicted from the altered structure suggest that L-PGC-1� mayhave arisen to adapt PGC-1� to more complex metabolic path-ways in humans.

PGC-1�2 (PPARGC1A) influences transcription in an excep-tional variety of biological pathways including adaptive ther-

mogenesis (1), mitochondrial biogenesis (2), skeletal musclefiber determination and neuromuscular junction formation (3,4), angiogenesis (5, 6), hepatic gluconeogenesis (7–9), fatty acid�-oxidation (10), regulation of clock genes (11), and protectionof neural cells from reactive oxygen species (12, 13). Recentreviews describe the numerous functions of this fascinatingprotein (14–17).Several levels of regulation have been implicated to explain

the diverse roles of PGC-1� and its interactions with distincttranscription factors. For some pathways, expression levels ofPGC-1� and transcription factors coactivated by PGC-1� arecrucial (18). In addition, various signaling pathways targetPGC-1� at the post-translational level. Such modificationsdetailed recently (19) alter the stability of PGC-1� and/or directinteractions with specific factors, thereby enhancing distincttranscriptional programs.Alternative splicing and/or transcription initiation, resulting

in gain or deletion of interacting domains or signaling targets,represents another mode of regulation (15). Several PGC-1�isoforms have been reported in animal models (20, 21). A shortPGC-1� isoform was shown to be coexpressed with wild-typePGC-1� in mouse tissues and in human heart (22). The alter-natively spliced mRNA is translated into a truncated protein,termed NT-PGC-1�, that retains the N-terminal transactiva-tion and nuclear receptor interaction domains and is function-ally active.Knowledge about PGC-1� expression and regulation in

human tissues is limited. However, such information is impor-tant because PGC-1� has been implicated in human disordersas diverse as type 2 diabetes mellitus (23–25) and Huntingtondisease (12, 26–28). Here we report the sequence, subcellularlocalization, and some relevant functional properties of a novelPGC-1� isoform in human liver, termed L-PGC-1�.

EXPERIMENTAL PROCEDURES

Study Subjects—The study included 68 obese but otherwisehealthy female patients. Participants were included if they hadfasting plasma glucose levels �7.0 mmol/liter, C-reactive pro-

* This study was supported by grants from the Fonds zur Förderung der wis-senschaftlichen Forschung (FWF Project P19893-B05), the Wiener Wissen-schafts-, Forschungs-, und Technologiefonds (WWTF Project L507-058),the Land Salzburg, and the Verein für Medizinische Forschung Salzburg,Austria.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1 and S2 and Table S1.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) HQ695733.

1 To whom correspondence should be addressed: Dept. of Laboratory Medi-cine, Paracelsus Medical University, Müllner Hauptstrasse 48, 5020 Salz-burg, Austria. Tel.: 43-662-44823800; Fax: 43-662-4482885; E-mail:[email protected].

2 The abbreviations used are: PGC-1�, peroxisome proliferator-activatedreceptor � coactivator-1�; RLM-RACE, RNA ligase-mediated rapid amplifi-cation of cDNA ends; RPA, ribonuclease protection assay; FOXO1, fork-head box 01A; PCK1, phosphoenolpyruvate carboxykinase 1; RPLP0,ribosomal protein, large, P0; SREBP-1c, sterol regulatory element-bind-ing transcription factor 1c; NR, nuclear receptor; CREB, cAMP-response

element-binding protein; L, liver; NT, novel truncated; PPAR, peroxi-some proliferator-activated receptor; 8-Br-cAMP, 8-bromoadenosine3�,5�-cyclic monophosphate; LXR�, liver X receptor �; MCAD, medium-chain fatty acyl-CoA dehydrogenase; mutHNF4�, mutant HNF4�; G6P,glucose-6-phosphatase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 50, pp. 42923–42936, December 16, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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tein levels �30 mg/liter, no history of diabetes or use of lipid-lowering medication, and no weight changes �3% during theprevious 2months. They underwent a surgical weight-reducingprocedure including a liver biopsy after an overnight fast. Tis-sue samples were collected in RNAlater (Ambion). Study par-ticipants provided informed consent, and study protocols wereapproved by the local ethics committee.Plasmids—Expression plasmids pLXR�, pFOXO1, pFOXA2,

pSREBP-1c, and pPGC-1� as well as the promoter-luciferasereporter construct pSREBP-1c-LUCwere described earlier (25,29–33). Plasmids pHNF4� and pL-PGC-1� and the enhancedGFP in-frame fusion constructs pL-PGC-1�-GFP and pPGC-1�-GFP were cloned into pcDNA6-V5/His A (Invitrogen) orpEGFP-N1 (Clontech), respectively. pcDNA6 was also used forgenerating human expression plasmids pPPAR� and pPPAR�.The human promoter-luciferase reporter vectors pCD36-Prom-Luc, pMCAD-Prom-Luc, pPCK1-Prom-Luc, pPGC-1�-Prom(2647)-Luc, and pL-PGC-1�-Prom(3518)-Luc were gen-erated in the pGL4 backbone (Promega). Two truncations ofthe promoter region were created from pL-PGC-1�-Prom(3518)-Luc, namely pL-PGC-1�-Prom(1158)-Luc andpL-PGC-1�-Prom(542)-Luc. The predicted binding sites forforkhead box 01A (FOXO1), CREB1 and NR3C1 (glucocorti-coid receptor) of the latter plasmid as well as the AF-2 domain(L374A and L375A) in pHNF4� and pHNF4�-Halo werealtered using the QuikChange site-directed mutagenesis kit(Stratagene). L-PGC-1� was cloned into pCI TPA Art Tet,kindly provided byA.Hartl (34), forDNAvaccination. Plasmidsfor the ribonuclease protection assay (RPA) and Northernprobe generation by in vitro transcriptionwere obtained byT/Acloning of adequate PCR amplicons into the pGEM�-T EasyVector System (Promega). The C-terminal Halo-tagged ver-sions pL-PGC-1�-Halo, pPGC-1�-Halo, pHNF4�-Halo, andpmutHNF4�-Halo used for chromatin isolation and pulldownanalyses were generated in the pHTC HaloTag� CMV-neoVector (Promega). All plasmids were generated by standardmolecular biology cloning techniques and verified by DNAsequencing using the ABI 3500 genetic analyzer (Applied Bio-systems). Primers used for the generation of the respectiveamplicons are given in supplemental Table S1.RNA Analyses—Total RNAwas isolated from Sprague-Daw-

ley rat liver, human liver, andHepG2 cells usingRNeasyMini orMidi kits (Qiagen) and digested with DNase I (Promega).Poly(A�) fractions were prepared using the PolyATract mRNAIsolation System (Promega). The quality of total and poly(A�)-selected RNAwas ascertained by electrophoresis in denaturingformaldehyde-agarose gels. We purchased the FirstChoiceHuman Total RNA Survey Panel and Swiss Webster mouseliver RNA (Applied Biosystems/Ambion) and hepatic totalRNA from dog and rhesus monkey (Biochain).RNA Ligase-mediated Rapid Amplification of cDNA Ends

(RLM-RACE)—The FirstChoice� RLM-RACE kit (AppliedBiosystems/Ambion) was used for RLM-RACE analysesaccording to the manufacturer’s instructions. Briefly, 1 �g oftotal human liver RNA was treated with calf intestine alkalinephosphatase to remove free 5�-phosphates, leaving the 5�-capstructure of full-length mRNA intact. The cap was removed byincubation with tobacco acid pyrophosphatase prior to ligation

of a 45-base RNA adapter oligonucleotide to the non-dephos-phorylated RNA population using T4 RNA ligase (35). cDNAtemplates produced by randomprimed reverse transcription orcommercially available human liver RACE-ready cDNA(Applied Biosystems/Ambion) was used for PCR. Nested prim-ers corresponding to the 5�-RACEadapter sequence or comple-mentary to PGC-1� (see supplemental Table S1) were used toperform the respective outer and inner reactions. PCRproductswere cloned into the pGEM-T Easy vector.RPA—RPAs were performed with the RPA IIITM Ribonucle-

ase Protection Assay kit (Applied Biosystems/Ambion) asdescribed (36). In brief, DNA plasmid templates for in vitrotranscription of 32P-labeled antisense RNA probes for specificPGC-1� transcript sequences (supplemental Table S1) weregenerated as described. 32P-Labeled RNA antisense probeswere synthesized using the Riboprobe� in Vitro TranscriptionSystem (Promega) and [�-32P]CTP (29.6 TBq/mmol; Amer-shamBiosciences). To produce “runoff” transcripts with SP6 orT7 RNA polymerases, plasmids were linearized with endonu-cleases generating 5�-overhangs. In vitro transcribed RNA wasgel-purified, and incorporated radioactivity was determined byliquid scintillation counting (Wallac 1450 Microbeta PLU,EG&GBerthold). Typically, 10�g of total or 500 ng of poly(A�)RNA was hybridized with �5 � 104 cpm 32P-labeled antisenseprobes at 42 °C overnight. After digestion of unprotected RNAwith 0.5 unit of RNase A and 20 units of RNase T1 at 37 °C for30 min, 32P-labeled RNA-RNA hybrids were precipitated, sub-jected to electrophoresis in 5%polyacrylamide gels containing 8M urea, dried, and exposed to the Image Station 2000 Multi-Modal Imaging System (Eastman Kodak Co.) or x-ray films(Kodak).Northern Blot Analyses—Northern blotting was performed

with a NorthernMax� kit (Ambion) using 5 �g of HepG2 or 2.5�g of human liver, skeletal muscle, and kidney poly(A�) RNA(Clontech) per lane separated in 1.1% denaturing agarose gels(37). RNA was transferred to BrightStar�-Plus positivelycharged nylon membrane (Ambion) using a TurboblotterTMsystem (Whatman/Schleicher & Schuell). Membranes werehybridized overnight at 65 °C in Ultrahyb� Northern blot solu-tion (Ambion) with [�-32P]CTP RNA probes complementaryto PGC-1� or L-PGC-1� sequences. Blots were washed at 68 °Cwith low and high stringency buffers and subsequently exposedto Amersham Biosciences HyperfilmTM MP (GE Healthcare).Gene Expression and Genotyping—Equal amounts of RNA

were reverse transcribed (36). Liver and/or hepatic mRNA lev-els of the genes encoding FOXO1 (Hs00231106_m1), glucose-6-phosphatase (G6P;Hs00609178_m1), and phosphoenolpyru-vate carboxykinase 1 (PCK1; Hs00159918_m19) werequantified in duplicate using the TaqMan gene expressionassays (Applied Biosystems) listed in parentheses and the iCy-cler iQ Multi-Color real time PCR detector (Bio-Rad). Acidicribosomal protein RPLP0 (NCBI Reference SequenceNM_001002.3) mRNA was used as an internal standard asdescribed (38). For quantification of PGC-1� transcripts, weused assays spanning exons 1/2 (Hs02026722_m1), 1L/3 (cus-tom-made), and 10/11 (Hs01016719_m1). To minimize theconfounding effects of truncated PGC-1� transcripts asdeduced from Northern analyses, we subtracted exon 1L/3

Liver-specific Human PGC-1� Isoform

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from exon 10/11 containing transcripts for estimation of full-length PGC-1� transcripts.We verified the accuracy of the cus-tomized assay by sequencing the cloned amplicons in threeindividuals. To directly compare measurements of PGC-1�transcript regions, gene segments containing the sequences tar-geted by the respective TaqMan assays were used to constructstandard curves. Data are presented in arbitrary units relative toRPLP0 mRNA. For typing rs12500214, we used a TaqMangenotyping assay (C_11325181_10, Applied Biosystems).Cell Culture and Transfection Experiments—Human hepa-

tomaHepG2 cells were grown as recommended by the supplier(American Type Culture Collection). HepG2 cells cultured in24-well dishes were transfected using Lipofectamine 2000 rea-gent (Invitrogen) as described (25). We used 0.2 �g of reporterplasmids, 0.5 �g of expression plasmids, and 20 ng of pRL-TKplasmid (Promega) as transfection control per well. Cells werecollected 24 h after transfection, and firefly and Renilla lucifer-ase activities were measured with a GloMax Multi DetectionSystem luminometer (Promega) using the Dual-LuciferaseReporter Assay System (Promega). Results are representative oftwo experiments, each performed in quadruplicate, and aregiven as means � S.D. Dexamethasone, 8-bromoadenosine3�,5�-cyclic monophosphate (8-Br-cAMP), WY14643, troglita-zone, 22(R)-hydroxycholesterol, and 9-cis-retinoic acid wereobtained from Sigma-Aldrich and used at the concentrationsindicated.Fluorescence Microscopy—A Zeiss Axioskop microscope

equippedwith anoil immersion�100 objective lens and a videocamera was used for fluorescence and differential interferencecontrast microscopy. HepG2 cells were transfected with plas-mid pL-PGC-1�-GFP or pPGC-1�-GFP. Visualization ofnuclei and mitochondria in living cells was performed with4�,6�-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) DNAstaining andMitoTracker� Red CMXRos (Invitrogen, Molecu-lar Probes), respectively.In Vitro Transcription/Translation—Plasmids pPGC-1� and

pL-PGC-1� and the TNT� Quick Coupled Transcription/Translation System (Promega) were used for in vitro synthesisof wild-type and L-PGC-1�. Briefly, 1 �g of circular plasmidDNA was added to TNT lysate, reaction buffer, RNA polymer-ase, ribonuclease inhibitor, and non-radioactive amino acidsand incubated at 30 °C for 90 min. Samples were denatured insample buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10%glycerol, 50 mM DTT, 0.01% (w/v) bromphenol blue) at 95 °Cfor 5 min, cooled on ice, and subjected to electrophoresis inSDS-polyacrylamide gels.Immunoblotting—Human brain tissue protein extract (Mil-

lipore, catalogue number CL302) and liver nuclear extract(Active Motif, catalogue number 36042) were used for immu-noblotting. Nuclear and cytoplasmic extracts fromHepG2 cellsgrown to confluence in 70-cm2 flasks were prepared using theNE-PERTM kit (Pierce Thermo Fisher Scientific). Protein con-centrations were determined using the BCA Protein Assay(Pierce Thermo Fisher Scientific). Equal amounts of nuclearextracts per lane (15�g) were subjected to SDS-polyacrylamidegel electrophoresis followed by electrotransfer to anAmershamBiosciences HybondTM-P PVDF transfer membrane (GEHealthcare) as described (32). After blocking with 1�TBS con-

taining 5% (w/v) nonfat dry milk for 1 h at 22 °C, membraneswere incubated overnight at 4 °C with monoclonal antibodyPGC-1� (3G6 rabbit mAb, Cell Signaling Technology), poly-clonal IgG PGC-1� (K-15, sc-5816 antiserum, Santa Cruz Bio-technology), or antisera obtained by DNA vaccination. Afterwashing and incubation with the secondary anti-rabbit HRP-linked (Cell Signaling Technology) or anti-mouse IgG HRP-linked antibody (Pierce Thermo Fisher Scientific) for 1 h at22 °C, blots were exposed to SuperSignal West Dura substrate(Pierce Thermo Fisher Scientific), and chemiluminescence sig-nals were recorded using the Kodak Imaging Station 2000MM.Chromatin Immunoprecipitation (ChIP) Assays—We used

theHaloChip System (Promega), which provides a robust alter-native to the standard ChIPmethod by capturing protein-DNAcomplexes from mammalian cells without the need for anti-bodies. The HaloTag protein is fused to the protein of interestvia cloning into aHaloTag vector andmediates a covalent inter-action with a resin-based ligand. C-terminally Halo-tagged ver-sions of L-PGC-1�, PGC-1�, or HNF4� or the empty HaloTagvector was transiently expressed in HEK293 or HepG2 cells for24 h and subsequently treated for 10 min with formaldehyde(1%, v/v) to induce covalent protein-DNA cross-links. Cross-linking was quenched by the addition of glycine to a final con-centration of 125 mM. Cells were pelleted in PBS and frozen at�70 °C for 10min prior to lysis bymechanical disruption using25 strokes of a Dounce homogenizer. Lysates were sonicatedusing a Branson sonicator (13 cycles of 15 s each with 1 min ofcooling on ice between cycles) to shear chromatin to a medianfragment size of �500 bp. Cross-linked complexes containingHaloTag proteins were captured using HaloLink resin accord-ing to the manufacturer’s recommendations. After stringentwashing to remove nonspecific proteins and DNA, capturedDNA fragments were released by heating for 6 h at 65 °C andpurified using a Wizard SV Gel and PCR Clean-Up System(Promega). Purified DNA was subjected to PCR amplificationusing primers spanning the HNF4� binding site in the PCK1promoter (39). Cells transfected with the empty pHTCHaloTag vector were used as a negative control. As an addi-tional control, an aliquot of the L-PGC-1�-Halo lysate wasincubated with blocking ligands, preventing the interaction ofthe Halo-tagged protein with the HaloLink resin.PulldownAnalyses—Weused theHaloTagMammalian Pull-

Down System (Promega) according to the manufacturer’sinstructions. HEK293 cells (2 � 107) were transiently trans-fected with C-terminally Halo-tagged versions of L-PGC-1� orHNF4� as baits to capture interacting proteins. Cells express-ing the HaloTag control vector served as a negative control.Cells were lysed 36 h after transfection, and nuclear extractswere prepared as described (36). HaloTag fusion proteins alongwith their interacting proteins were captured using theHaloLink resin and washed gently. Interacting proteins wereeluted from the resin with SDS elution buffer and subjected toSDS-PAGE followed by electroblotting. Blots were probed withmouse monoclonal HNF4� antibody (sc-101059, Santa CruzBiotechnology) and anti-mouse IgG HRP-linked antibody(Pierce Thermo Fisher Scientific) as secondary antibody.Monoclonal histoneH3 antibody (3H1 rabbitmAb, Cell Signal-




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ing Technology) was used as a loading control of nuclearextracts.DNAVaccination—Female BALB/c mice (6–8 weeks of age;

Charles River, Sulzfeld, Germany) were immunized via genegun three times at weekly intervals. L-PGC-1�, cloned into pCITPA Art Tet (34), was precipitated onto gold beads (1.6-�mdiameter) with CaCl2 in the presence of spermidine at a loadingrate of 2�g/mgof gold.Mice received a total of 2�g ofDNAperimmunization, divided between two non-overlapping areas, onthe shaved abdomen at a helium pressure of 400 p.s.i. Bloodsamples were taken before and 2 weeks after the third immuni-zation. Mice were boosted by gene gun 11 weeks after the thirdimmunization and exsanguinated 1 week later (34, 40). All ani-mal experiments were approved by the local animal committee.Computational and Statistical Analyses—Linear regression

analyses were performed using log-transformed hepaticmRNAlevels. Effects of genotypes associated with rs12500214 onL-PGC-1� transcript levels were ascertained by analysis of vari-ance adjusted for age and body mass index. Transactivationassays were analyzed by analysis of variance. Allele frequencieswere estimated by gene counting. Agreement with Hardy-Weinberg expectations was tested using a �2 goodness-of-fittest. To identify transcription initiation sites and evolutionarilyconserved regions we used PROMO (41) and rVISTA (42).AccessionNumber—The sequence of the transcript encoding

L-PGC-1� has been deposited in GenBankTM under accessionnumber HQ695733.

RESULTS

Identification and Characterization of Novel PGC-1� Tran-script in Human Liver and HepG2 Cells—Initially, we usedcDNA from various tissues to perform PCR yielding ampliconsevenly spread over the entire human PGC-1� mRNA. Markedreductions of PCR products spanning exons 2–4 relative toother exon-spanning amplicons of comparable length wereobserved in cDNA from liver but not from skeletal muscle orkidney (data not shown). To identify alternative transcripts, weused RLM-RACE, a technique that restricts cDNA amplifica-tion to 5�-capped mRNAs. RACE-ready cDNA prepared fromliver biopsies of three patients served as templates for 5�-RLM-RACE from exon 4. Two distinct amplification products wereobserved in all three subjects (Fig. 1A). Sequencing showed thatthe larger 847-bp amplicon contained an alternative exon 1,termed exon 1L, located within intron 2, that was spliced toexon 3 followed by exon 4. The smaller 637-bp amplicon con-tained the reported wild-type sequence. Similar results wereobtained with RACE-ready human liver cDNA (Ambion) andHepG2 cDNA (data not shown).To fine map the start site(s) of the novel transcript, we per-

formed 5�-RLM-RACE with a nested reverse primer located inexon 1L and observed two amplicons in liver biopsies of foursubjects but no amplicon in brain mRNA (Fig. 1B). Sequencingindicated that the larger fragments differed from the shorterfragments by a 5�-extension of 41 bp (supplemental Fig. S1).Interestingly, the relative intensities of the two bands variedamong individuals. As NCBI dbEST entry BX105309 showedanother exon upstream of exon 1L in human testis, we per-formed PCR with primers located in exon 1L and the putative

upstream exon but did not detect such an exon in liver. More-over, in silico analyses predicted two transcriptional initiationsites in exon 1L with scores of 0.86 and 0.85 that perfectlymatched the sites obtained by RLM-RACE. Hence, the novelliver transcript is generated by variable utilization of two tran-scription start sites in exon 1L. As described previously (37),wild-type transcripts in liver and brainwere also initiated at twoadjacent sites (Fig. 1B).To confirm the expression of exon 1L in human liver and

HepG2 cells, we performed RPAs using a probe spanning theexon 1L/exon 3 junction. Fragments predicted from RACEstudies were protected in liver and HepG2 cells but not in skel-etal muscle or kidney (Fig. 1C). Furthermore, abundance levelsof transcripts with or without exon 1L were comparable.Because significant amounts of 64-nucleotide fragments pro-tected only if exon 1L was spliced to sequences other than theacceptor site of exon 3 were not detected, exon 1L was exclu-sively spliced to exon 3. This conclusion was supported by3�-RACE studies (data not shown). The location of exon 1L inthe genomic PGC-1� context is displayed in Fig. 1D.

Two RPA probes extending from exon 1L or exon 2 to exon 7were used for further characterization of hepatic PGC-1� tran-scripts. Liver and HepG2 RNA contained fully protected frag-ments with both probes (supplemental Fig. S2, A and B), buttranscripts harboring exon 1L were not detected in brain RNA.To determine whether exon 1L-initiated mRNA was subject toalternative splicing similar to transcripts encoding NT-PGC-1�, we performed semiquantitative RT-PCR and sequencedamplicons. Using liver and HepG2 mRNA as templates, weobserved aminor transcript population initiated at exon 1L thatwas alternatively spliced and contained the intron 6 insertion(supplemental Fig. S2C).Next, we determined the full sequence of exon 1L-initiated

transcripts. Exon 1L and exon 13 primers produced ampliconsspecific to HepG2 or liver cDNA compared with control ampli-cons amplifiedwith exon 1 and exon 13 primers and obtained inliver, skeletal muscle, and HepG2 cDNA (Fig. 1E). Sequencingof amplicons verified the exon 1L/exon 3 junction and the reg-ular order of exons 3–13. Northern blots using mRNA and acRNA probe hybridizing to exon 1L revealed two transcripts of�6.4 and �5.3 kb in liver and HepG2 cells but not in kidney orskeletal muscle (Fig. 1F). Using a probe spanning exons 2–7,transcripts of comparable sizes were observed in all mRNAsanalyzed (Fig. 1G). As suggested previously (37), the size differ-ence of 1.1 kb between these mRNA species most likely reflectsusage of alternative poly(A) signals in exon 13. Thus, liver andHepG2 cell transcripts initiated in exon 1L are similar in size towild-type transcripts present in other tissues. Hence, onlyprobes targeting the distinct 5�-regions discriminate the twotypes of transcripts. In liver, HepG2 cells, and kidney and to alesser extent in skeletal muscle, abundant small transcripts of�0.8 kb were observed with the exon 2–7 but not the exon 1Lprobe. Similar data have been reported in rats (43). Because ofthemore effective transfer of shortmRNAby diffusion blotting,their levels are probably greatly overestimated in comparisonwith full-length mRNA.




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Novel Isoform Is Highly Enriched in Human Liver but NotDetectable in Livers of Mice or Higher Mammals—To deter-mine the tissue-specific expression of exon 1L, we quantifiedexon 1L- and exon 1-containing transcripts in 18 human tissues(Fig. 2A). Exon 1- and 2-containing mRNA was expressed inmost tissues but mainly in brain, kidney, liver, skeletal muscle,

and thyroid. In contrast, exon 1L-containing transcripts werestrongly expressed in liver and to a lesser extent in testis. Wetherefore termed the novel transcript L-PGC-1�. As observedwith RPAs, the abundance levels, corrected for amplificationefficiency, of wild-type and exon 1L-containing transcriptswere comparable in human liver.

FIGURE 1. Identification of L-PGC-1� in human liver. A, 5�-RLM-RACE of hepatic total RNA from three patients (P1–P3) with outer and inner reactions primedfrom exon 4. B, 5�-RLM-RACE of hepatic total RNA from four patients (P1–P4) and human brain total RNA. Primers for outer and inner reactions are indicated byblack and gray arrows, respectively. C, RPA using a probe extending from exon 1L to exon (ex) 3 and total human tissue or HepG2 RNA. Protected fragments areshown below the autoradiograph. D, diagram showing exon 1L (black box), the translation start site for L-PGC-1� (bold arrow), and PGC-1� (plain arrow) in thegenomic context of the PGC-1� locus. E, amplification of L-PGC-1� (1L–13) and PGC-1� (1–13) using HepG2, liver, or skeletal (sk.) muscle cDNA. F and G, Northernblots of mRNA from human tissues and HepG2 cells using probes complementary to exon 1L (F) or extending from exon 2 to exon 7 (G). M, molecular marker;nc, negative control; pc, in vitro transcribed mRNA containing exon 1L and exon 3–7 sequences; nt, nucleotides.




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PGC-1� mRNA is highly conserved among mammals, andexon 1L is located within an evolutionarily conserved region.We therefore ascertained whether exon 1L homologues areexpressed in livers of mammals. Using several primer pairs tar-geting the conserved exon 1L region and exon 3 of the mousegene, we failed to detect transcripts in liver cDNA of overnightfasted mice. In contrast, transcripts spanning exons 1–3 werereadily amplified from the same cDNA. In addition, a probecomplementary tomurine exons 2 and 3protected fragments ofwild-type transcripts as expected (Fig. 2B). However, 92 nucle-otide-containing fragments indicative of splicing of anotherexon (such as exon 1L) to exon 3 were not observed. RT-PCRs

using hepatic RNA from rat, dog, and rhesusmonkey and prim-ers located in fully conserved regions of exon 1L and exon 3produced no amplicons, whereas a product was readilyobtained in human liver. The adequacy of RNAs was verified inassays with universal primers located in exons 11 and 13 asamplicons were obtained in all species (Fig. 2C).In Vitro Transcription/Translation, Immunoblotting, and

Immunocytochemistry—Splicing of exon 1L to exon 3 elimi-nates the start ATG codon of PGC-1� located in exon 1. Thefirst ATG codon, producing an open reading frame, is shiftedinto exon 3, resulting in deletion of the first 127 amino acids. Totest whether such a shorter isoform is translated, we performed

FIGURE 2. Tissue- and species-specific expression of L-PGC-1� transcripts. A, mRNA abundance of exon 1/exon 2- and exon 1L/exon 3-containing tran-scripts relative to RPLP0 mRNA and normalized to amplification efficiency: Columns (error bars) are means (S.D.). B, RPA using total mouse liver RNA and amouse-specific probe extending from exon (ex) 2 to exon 3. Fragments protected in wild-type or possible variant transcripts are shown below the autoradio-graph. C, evolutionary conservation of rat, mouse, dog, and rhesus monkey sequences (relative to human) extending from exon 1 to exon 4 (rVISTA). Primerslocated in exon 1L and exon 3 as indicated by arrows and matching the sequence in each species were used for PCR. Universal primers located in exon 11 andexon 13 were used for validation PCR. Blue, exons; salmon, intronic sequences; green, repeat sequences or transposons. AU, arbitrary units; t., tissue; Ovar, ovary;Sk., skeletal; Sm., small; nt, nucleotides; nc, negative control.




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in vitro coupled transcription/translation reactions withPGC-1� and L-PGC-1�. Both PGC-1� and L-PGC-1� (�91and 77 kDa, respectively) were identified by immunoblottingusing an antibody directed against C-terminal epitopes ofPGC-1� (Fig. 3A). The same antibody recognized two proteinsof sizes attributed to potential post-translationally modifiedPGC-1� and L-PGC-1� in HepG2 nuclear extracts, whereas amonoclonal antibody against an N-terminal epitope detectedPGC-1� and NT-PGC-1� but not L-PGC-1� (Fig. 3, B and C).Antiserum produced by vaccination with DNA encodingL-PGC-1� clearly detected a band (not observed with preim-mune serumof the predicted L-PGC-1� size in nuclear extractsof HepG2 cells and liver, whereas faint bands corresponding tothe size of the wild-type protein were visualized (Fig. 3, D and

E). A comparison of functional domains present in PGC-1�,L-PGC-1�, and NT-PGC-1� is shown in Fig. 3F.To ascertain the subcellular localization of L-PGC-1�, which

contains a nuclear localization signal, we transiently expressedwild-type and L-PGC-1�, both C-terminally tagged with in-frame enhanced GFP, in HepG2 cells. We observed colocaliza-tion of both wild-type and L-PGC-1� with DAPI staining, indi-cating nuclear localization of the new isoform (Fig. 4).The 5�-Sequence of L-PGC-1� Supports Its Transcription—

To substantiate the accuracy of transcription initiation sites inexon 1L, we cloned a � 3.2-kb upstream fragment into areporter vector and observed, in comparison with control, a4-fold increase of transcriptional activity in transient transfec-tions of HepG2 cells (Fig. 5A). In silico analysis predicted a

FIGURE 3. Detection of L-PGC-1� in human liver and/or HepG2 cells and diagram of functional domains of PGC-1�. A, in vitro translated proteins detectedby polyclonal antiserum directed against C-terminal epitopes. B and C, immunoblots of nuclear extracts from HepG2 cells using a polyclonal serum againstC-terminal epitopes (B) and a monoclonal antibody (Ab) against an N-terminal (N-term) epitope (C). D, immunoblots of the HepG2 nuclear extract used in B andC with polyclonal pre- and post-DNA vaccination-based sera. E, immunoblots of nuclear extracts from human liver and nuclear (nuc) and cytoplasmic (cyto)extracts from HepG2 cells using the N-terminal monoclonal antibody (left panel) or DNA vaccination-based antiserum (right panel). F, NT-PGC-1� and L-PGC-1�structures are indicated by bold brackets; interacting surfaces along with interacting partners are depicted by brackets. NES1 and NES2, nuclear export signals;AD, activation domain; L1, L2, and L3, nuclear boxes; SUMO, sumoylation sites; HCB, host cell factor docking site; RS, arginine/serine-rich domains, NL, nuclearlocalization signal; RRM, RNA recognition motif; P, phosphorylation sites; CBP, CREB-binding protein.




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FOXO1 binding site in the putative promoter. Indeed, cotrans-fection of HepG2 cells with FOXO1 expression plasmidsmark-edly induced reporter gene activity, and two truncated promot-er-reporter constructs retained FOXO1 inducibility (Fig. 5A).Mutagenesis of the predicted FOXO1 core sequence in the0.542-kb promoter construct reduced its activation by FOXO1nearly to the control level (Fig. 5, B and C). Thus, the sequenceimmediately upstream of exon 1L is a functional promoter thatis activated by FOXO1 in vitro. Moreover, FOXO1 andL-PGC-1� mRNA levels in livers of obese subjects are stronglycorrelated (Fig. 5D).Exon 1L along with its 5�-region (harboring rs12500214) is

located in a haplotype block distinct from haplotype blockscomprising the wild-type promoter or the sequence down-stream of exon 2.3 We typed rs12500214 in 68 obese femalesubjects. Genotypes associated with rs12500214 fulfilled Har-dy-Weinberg expectations andwere associated with L-PGC-1�mRNA levels (Fig. 5E). Conversely, no effects of rs12500214 onfull-length PGC-1� mRNA were noted (1.73 � 2.44, 2.11 �

1.96, and 0.60� 0.58 arbitrary units for genotypes GG, GA, andAA, respectively).Glucocorticoids as well as glucagon-PKA signaling are

enhanced in the fasting state, and both signaling pathwaysincrease hepatic PGC-1� expression (9). We therefore investi-gated whether hepatic L-PGC-1� mRNA is also induced bythese signaling cascades. HepG2 cells treated with dexametha-sone showed comparable increases in L-PGC-1� and PGC-1�transcripts and larger increases in the mRNA levels of thePGC-1� targets PCK1 and G6P. At the doses used, even stron-ger effects on L-PGC-1�, PGC-1�, PCK1, and G6PmRNA lev-els were observed with the cAMP analog 8-Br-cAMP (Fig. 6A).Moreover, strong associations of L-PGC-1� and PCK1 tran-script levels were noted in liver biopsies, providing an in vivocorrelate for a role of L-PGC-1� in PCK1 mRNA expression(Fig. 6B). Furthermore, PGC-1� transcript levels displayed onlymodest associations with L-PGC-1� (r 0.3756, p 0.003)and PCK1 transcript levels (r 0.3649, p 0.005). To identifythe respective cis-regulatory sites in the L-PGC-1� promoter,we transfected reporter constructs driven by L-PGC-1� pro-moters of different lengths into HepG2 cells. Again, treatmentof cells with 8-Br-cAMP inducedL-PGC-1�promoter activitiesmore than dexamethasone. Furthermore, the stimulatory

3 T. K. Felder, S. M. Soyal, H. Oberkofler, P. Hahne, S. Auer, R. Weiss, G. Gader-maier, K. Miller, F. Krempler, H. Esterbauer, and W. Patsch, unpublishedobservations.

FIGURE 4. Subcellular localization of PGC-1� and L-PGC-1� in HepG2 cells. Differential interference contrast (DIC) and fluorescence micrographs of HepG2cells transiently transfected with enhanced GFP (eGFP) in-frame fusion constructs pPGC-1�-GFP and pL-PGC-1�-GFP are shown. DAPI or MITO denotes nuclearstaining with 4�,6�-diamidino-2-phenylindole or mitochondrial staining with MitoTracker Red CMXRos, respectively.




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effects of both dexamethasone and 8-Br-cAMP increased withthe extent of promoter truncations (Fig. 6C). In silico analysessuggested putative binding sites for CREB and glucocorticoidreceptors (glucocorticoid response element) at position �368to �348 and �57 to �36, respectively. Indeed, mutagenesis ofthe CREB binding site and the glucocorticoid response elementin the 0.542-kb promoter construct abrogated its activation bythe respective treatment (Fig. 6D).To identify potential transcriptional pathways with distinct

influences on hepatic L-PGC-1� and PGC-1� promoter activi-ties, HepG2 cells were cotransfected with equimolar amountsof L-PGC-1� and PGC-1� promoter-reporter constructs andexpression plasmids encoding various transcription factorsknown to be active in liver (Fig. 6E). PPAR� and nuclear activeSREBP-1c had no stimulatory effect on either reporter con-struct, whereas PPAR� enhanced transcription only from theL-PGC-1� promoter. HNF4� and LXR� activated bothreporter constructs. Comparedwith the PGC-1� promoter, theL-PGC-1� promoter was more strongly trans-activated byFOXO1, whereas the opposite effect was observed for FOXA2.

L-PGC-1� and PGC-1� Have Overlapping CoactivationProperties—To delineate potential functional differencesbetweenPGC-1� andL-PGC-1� inhumansystems,wecomparedtheir coactivationpropertiesbycotransfectionofHepG2cellswithL-PGC-1� and PGC-1� expression vectors along with reporterconstructs driven by promoters of various nuclear receptors(NRs). As PGC-1� was originally identified as a coactivator ofPPAR�, we examined the ability of L-PGC-1� to coactivate theCD36 promoter containing a bona fide PPAR� response elementwith activity in liver (44). L-PGC-1� and PGC-1� showed compa-rable coactivation potencies when equimolar amounts of expres-sion plasmids were used (Fig. 7A). Coactivation of PPAR� andHNF4�, demonstrated previously for PGC-1� (45, 46), was deter-mined with MCAD and PCK1 promoters, respectively. Again,comparable activities for L-PGC-1� and PGC-1� were observed(Fig. 7, B andC). However, only PGC-1� coactivated LXR� at theSREBP-1c gene promoter (Fig. 7D).L-PGC-1� Physically Interacts with HNF4� and Is Recruited

to PCK1 Promoter—As our in vitro reporter gene assays indi-cated that L-PGC1� coactivated the transcriptional activity of

FIGURE 5. Transcriptional activity of exon 1L 5�-upstream region. A, L-PGC-1� promoter-reporter constructs of various lengths were cotransfected with aFOXO1 expression plasmid into HepG2 cells. B, FOXO1 binding site DNA logo (62) and native and mutagenized (MUT) L-PGC-1� promoter (Prom) sequences.C, mutagenesis of the FOXO1 binding site in the 0.542-kb promoter construct abolishes FOXO1-dependent reporter activation in HepG2 cells. D, correlation ofhepatic L-PGC-1� and FOXO1 transcript levels in humans. E, effects of genotypes associated with rs12500214 on L-PGC-1� transcript levels. AU, arbitrary units.Columns are means, error bars are S.D. (A, C) or S.E. (E).




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HNF4� at the PCK1 promoter, we used ChIP and pulldownassays as additional approaches to directly demonstrate a phys-ical interaction of L-PGC-1� with HNF4�. We transientlytransfected HEK293 cells with expression plasmids encodingthe Halo-tagged versions of PGC-1�, L-PGC-1�, and HNF4�and studied the recruitment of the respected fusion proteins tothe PCK1 promoter. Using DNA fragments captured byHaloTag resins, we performed PCR with a primer pair amplify-ing a 177-nucleotide fragment spanning the predicted HNF4�binding site on the PCK1 promoter (39). Signals of the expectedsizes were obtained for all three constructs, whereas no signalwas noted in cells expressing solely the HaloTag protein (Fig.8A). Next, we performed pulldown experiments with nuclear

extracts from HEK293 cells transiently transfected with emptyvector and Halo-tagged L-PGC-1� or HNF4� expression plas-mids. HNF4� clearly was present in protein complexes isolatedfrom cells expressingHalo-tagged L-PGC-1� but not from cellstransfected with the empty vector or the Halo-tagged HNF4�expression plasmid (Fig. 8B). The latter result was expected asthe Halo-tagged protein covalently binds the HaloTag resin.Previous studies have shown that coactivation of PPAR� and

glucocorticoid receptor by PGC-1� requires an intact AF-2domain (10, 47, 48). To determine whether coactivation ofHNF4� by L-PGC-1� also is dependent on AF-2 function, wemutagenized this domain in HNF4� and its Halo-tagged ver-sion (49) and studied its effect on PCK1 promoter activation. In

FIGURE 6. Induction of L-PGC-1� mRNA by dexamethasone, 8-Br-cAMP, and various transcription factors in HepG2 cells. A, HepG2 cells were incubatedwith 1 �M dexamethasone or 100 �M 8-Br-cAMP for 6 h, and mRNA levels were measured by quantitative real time RT-PCR. B, correlation of hepatic L-PGC-1�and PCK1 transcript levels in humans. C, L-PGC-1� promoter-reporter constructs of various lengths were transfected into HepG2 cells in equimolaramounts and incubated as in A prior to measurement of reporter gene activities. D, mutagenesis (Mut) of glucocorticoid response element (GRE) or theCREB binding site (CREBP) in the 0.542-kb promoter construct abrogates its activation by glucocorticoids or 8-Br-cAMP in HepG2 cells. Columns (errorbars) are means (S.D.) of quadruplicate determinations; asterisks within bars denote p � 0.01 versus control. E, transactivation of L-PGC-1� and PGC-1�promoter-reporter constructs by various transcription factors with reported activity in liver. Asterisks within bars denote p � 0.01 versus control. AU,arbitrary units; Prom-LUC, promoter-luciferase.




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studies of transient transfection of HepG2 cells with HNF4�and mutHNF4� expression plasmids, activation of the PCK1promoter was slightly reduced by the mutated protein in com-parison with the wild-type protein. However, coactivation ofHNF4� by L-PGC-1� was completely abolished by the muta-tion (Fig. 9A). The former result was supported by ChIP assaysshowing an amplification of the HNF4� binding site of thePCK1 promoter in cells expressing the Halo-tagged version ofmutated HNF4� (data not shown). We also performed ChIPassays in HepG2 cells cotransfected with pL-PGC-1�-Halo andpHNF4� or pmutHNF4� (Fig. 9B). Signal intensities of ampli-cons spanning the HNF4� binding site of the PCK1 promoterwere reduced to �50% in cells coexpressing mutant HNF4�compared with cells coexpressing HNF4�. The signals noted inmutHNF4� cells were a consistent finding and may reflect theactivity of endogenous HNF4� present in HepG2 cells. Noamplification products were generated in cells transfected withthe empty vector or after blocking the binding of Halo-taggedL-PGC-1� to the HaloTag resin.

DISCUSSION

Alternative pre-mRNA processing and/or transcript initia-tion substantially enhances the complexity ofmammalian tran-scriptomes as multiple transcripts and proteins with distinctfunctions may be produced from a single gene locus. A system-

atic 5�-end analysis of the human transcriptome using the capanalysis of gene expression approach showed that 58% ofhuman protein-coding transcriptional units had one or morealternative promoters (50). We show here that an alternativepromoter of PGC-1� is used in human liver to produce a noveltranscript that encodes a biologically active protein, termedL-PGC-1�.The structure of the novel transcript was deduced from data

obtained by several complementary methods. Transcriptionalstart sites identified by RLM-RACE were consistent with pre-dictive promoter algorithms, results from Northern blots, andthe demonstration of promoter activity in the immediateupstream region. Wild-type and liver-specific transcripts canbe initiated from two adjacent sites in TATA-less promoters.The novel exon 1L is spliced to the regular acceptor site of exon3 and contains wild-type exons 3–13 in a regular order.Apart from liver, exon 1L is transcribed in testis albeit at a

lower level. However, exon 1L-containing transcripts in testismay differ from the respective liver transcripts as NCBI dbESTentry BX105309 indicates another exon upstream of exon 1L.Testis and brain express the largest amount of variant tran-scripts (51). Although exon 1L is not expressed in human brain,we identified twomajor variant brain transcripts initiated frompromoters distinct from the liver-specific promoter.4 Thus, tis-sue-specific differences in core promoter recognition factors(52) may play a role in PGC-1� transcription. L-PGC-1� tran-scripts can undergo alternative 3�-splicing. The resulting tran-script predicts a protein resembling NT-PGC-1� but devoid oftheN-terminal activation domain.Whether such a protein thatalso lacks the mediator binding site is produced by the humanliver or whether the respective transcripts have other functionsor are targeted to nonsense-mediated mRNA decay (53)remains to be determined.A taxonomic comparison indicated that all main interacting

domains of PGC-1� are highly conserved across recentlydiverged mammalian species (16). Although exon 1L is locatedin an evolutionarily conserved region, L-PGC-1� transcripthomologues were not detected in livers of several mammals.Thus, L-PGC-1� most likely reflects an adaption to more com-plex pathways in humans.Differentially regulated transcription start sites frequently

generate alternativeN termini (54). L-PGC-1�was predicted tolack 127 amino acids at the N terminus. A protein of theexpected size was translated in vitro and detected with specificantibodies in liver and HepG2 cells. The altered structure ofL-PGC-1� likely results in functional changes defined byretained and deleted domains (Fig. 3F). The activation domainfacilitating recruitment of SRC-1 and CREB-binding protein(55) has been mapped to the deleted region. Furthermore, thefirst of three LXXLL motifs, the nuclear export signals, and theGCN5 interaction site mapped to amino acids 1–97 of PGC-1�(56) are deleted in L-PGC-1�. Thus, wild-type and L-PGC-1�may differ in properties related to recruitment of chromatin-modifying factors, intracellular trafficking, coactivation of tran-scription factors, and possibly degradation and post-transla-

4 S. M. Soyal, T. K. Felder, S. Auer, P. Hahne, H. Oberkofler, and W. Patsch,unpublished observations.

FIGURE 7. Functional differences between PGC-1� and L-PGC-1�. HepG2cells were cotransfected with equimolar amounts of PGC-1� or L-PGC-1�expression plasmids (A–D) and CD36 promoter-reporter and PPAR� expres-sion plasmids (A), MCAD promoter-reporter and PPAR� expression plasmids(B), PCK1 promoter-reporter and HNF4� expression plasmids (C), or SREBP-1cgene promoter reporter and LXR� expression plasmids (D). Ligands addedincluded 1 �M troglitazone (A), 10 �M WY14643 (B), and 10 �M 22(R)-hydroxy-cholesterol, and 10 �M 9-cis-retinoic acid (D), respectively. Results aremeans � S.D. Prom-LUC, promoter-luciferase; n.s., not significant. Columns(error bars) are means (S.D.).




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tional modifications. PGC-1� shares an N-terminal activationdomain, an arginine/serine-rich domain, and an RNA bindingdomain with the other protein family members PGC-1� andPGC-related coactivator (17). The homology of L-PGC-1�withboth of these other proteins is therefore restricted to the Cterminus.L-PGC-1� is located mainly in the nucleus and therefore

likely to be transcriptionally active. To this end, we used humansystems to delineate functional differences between PGC-1�and L-PGC-1�. The finding that L-PGC-1� coactivatedPPAR�-mediated transcription was not unexpected becausethe docking surface for PPAR� is retained in the hepatic iso-form. Furthermore, both proteins coactivated PPAR�- andHNF4�-mediated transcriptional activation, but only PGC-1�effectively coactivated LXR� at the SREBP-1c gene promoter.As the interaction of PGC-1� with HNF4� is central to hepaticgluconeogenesis, we extended the studies of coactivation ofHNF4� by L-PGC-1� and used ChIP and pulldown assays todemonstrate a direct physical interaction between the two pro-teins at the PCK1 promoter. In addition, mutational studiessuggested that coactivation of HNF4� by L-PGC-1� requiresan intact AF-2 site. The crystal structure of HNF4� in a com-plex with a PGC-1� fragment containing all three LXXLLmotifs, also termed NR boxes, has been resolved recently (57).Only one of the three LXXLLmotifs was bound at the canonicalbinding pocket. However, the bound LXXLL motif was not a

selected box but represented an averaged structure of morethan one NR box. Functional studies showed a main role of NRboxes 2 and 3 in binding and coactivation of HNF4�. As NRboxes 2 and 3 are retained in L-PGC-1�, its coactivation ofHNF4� is plausible. An intact NR box 2 also was necessary forcoactivation of LXR�, but LXR� and other NR box 2-depen-dent NRs such as PPAR� and glucocorticoid receptor coacti-vated by PGC-1� differed in that coactivation of the formerwasnot affected by removal of a repressor binding to NR box 3 (30,31, 58, 59). Small differences in NR binding pockets can createlocal environments that allow NR-specific recruitment ofcoactivators (57). Therefore, it is possible that NR box 1 and/oradditional factors recruited via the N-terminal region (deletedin L-PGC-1�) are required for effective coactivation of LXR�.PGC-1� plays a central role in themetabolic adaptions of the

liver to fasting. In PGC-1�-deficient mice, the program of hor-mone-stimulated gluconeogenesis is defective, whereas consti-tutively activated gluconeogenesis is maintained (60). Ourstudies in HepG2 cells suggest that the hormonal changes thatincrease hepatic PGC-1� expression also enhance L-PGC-1�expression. Like PGC-1� mRNA, L-PGC-1� mRNA increasedin response to glucocorticoids and CREB signaling. As sug-gested by the promoter studies, FOXO1 may play an evengreater role in L-PGC-1� than in PGC-1� transcription. Fur-thermore, the coactivation of PCK1 transcription and thestrong correlation with PCK1 transcripts also argue for a role of

FIGURE 8. L-PGC-1� is recruited to PCK1 promoter and physically interacts with HNF4�. A, chromatin from HEK293 cells transfected with Halo-taggedexpression plasmids as indicated was captured using HaloTag resin and analyzed by PCR amplifying a fragment spanning the predicted HNF4� binding site inthe human PCK1 promoter. B, nuclear extracts from HEK293 cells transfected with Halo-tagged expression plasmids as indicated were subjected to pulldownwith HaloTag resin followed by immunoblotting with a monoclonal HNF4� antibody. Histone-3 (H3) antibody was used as a loading control for nuclearextracts. WB, Western blot; neg., negative; IP, immunoprecipitation.

FIGURE 9. Physical and functional interactions between L-PGC-1� and HNF4� depend on intact AF-2 domain in HNF4�. A, HepG2 cells were cotrans-fected with PCK1 promoter-luciferase reporter constructs and HNF4� or mutHNF4� expression constructs with or without L-PGC-1� expression constructs.Columns (error bars) are means (S.D.). -Fold induction is relative to promoter activity in cells transfected solely with the promoter-luciferase constructs.B, HEK293 chromatin from cells transfected with Halo-tagged L-PGC-1� and HNF4� or mutHNF4� expression constructs was isolated by HaloTag resin.Captured DNA was analyzed by amplifying the predicted HNF4� binding site in the human PCK1 promoter by PCR. Prom-LUC, promoter-luciferase.




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L-PGC-1� in gluconeogenesis. Studies inmice have shown thatacetylation of PGC-1� by GCN5 represses its ability to inducegluconeogenic gene expression (56), whereas deacetylation bySIRT1 enhances its activity on gluconeogenic genes (61). As thebinding site for GCN5 is most likely deleted in L-PGC-1�, it isintriguing to speculate that the regulation of its functionalactivity is not or is less dependent on deacetylation by SIRT1.Like PGC-1�, L-PGC-1� coactivated PPAR�-mediated tran-scription. As PPAR� trans-activated the L-PGC-1� promoter, afeed forward loop may be created that enhances fatty acid oxi-dation, thereby supporting hepatic ATP production in the fast-ing state.In conclusion, we have identified an alternative PGC-1�

transcript that appears to be specific for human liver andencodes a functional protein that lacks 127 amino acids at theNterminus. In vivo correlations between L-PGC-1� transcriptlevels and hepatic mRNA levels encoding distinct transcriptionfactors together with our trans-activation studies in HepG2cells suggest overlapping transcriptional networks regulatingthe expression of L-PGC-1�, PGC-1�, and their downstreamtargets. Collectively, our data suggest a role of L-PGC-1� inhepatic gluconeogenesis, but further studies are needed torationalize differences in regulation and function betweenPGC-1� and L-PGC-1�.

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and Wolfgang PatschRichard Weiss, Gabriele Gadermaier, Karl Miller, Franz Krempler, Harald Esterbauer

Thomas K. Felder, Selma M. Soyal, Hannes Oberkofler, Penelope Hahne, Simon Auer,) Isoform in Human Liverα (PGC-1αCoactivator-1

γCharacterization of Novel Peroxisome Proliferator-activated Receptor

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characterization of novel peroxisome proliferator-activated

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