Mat1 Inhibits Peroxisome Proliferator-Activated Receptor {gamma}-Mediated Adipocyte Differentiation

Mammalian Cdk7, cyclin H, and Mat1 form the kinase submoduleof transcription factor IIH (TFIIH) and have been consideredubiquitously expressed elements of the transcriptional machinery.Here we found that Mat1 and Cdk7 levels are undetectable inadipose tissues in vivo and downregulated during adipogenesis,where activation of peroxisome proliferator-activated receptor ${gamma}$ (PPAR ${gamma}$ ) acts as a critical differentiation switch. Using bothMat1^–/– mouse embryonic fibroblasts and Cdk7 knockdownapproaches, we show that the Cdk7 complex is an inhibitor ofadipogenesis and is required for inactivation of PPAR ${gamma}$ throughthe phosphorylation of PPAR ${gamma}$ -S112. The results demonstrate thatthe Cdk7 submodule of TFIIH acts as a physiological roadblockto adipogenesis by inhibiting PPAR ${gamma}$ activity. The observationthat components of TFIIH are absent from transcriptionally activeadipose tissue prompts a reevaluation of the ubiquitous natureof basal transcription factors in mammalian tissues.

INTRODUCTION

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INTRODUCTION

The Cdk7 kinase in complex with its cognate cyclin (cyclin H)and a RING domain-containing protein, Mat1, was first characterizedas a cdc2-activating kinase (23, 29, 51), and subsequently identifiedas the kinase submodule of transcription factor IIH (TFIIH)(1, 29, 64, 66). Genetic analyses using temperature-sensitiveor chemical genetic alleles of Cdk7 support the notion thatCdk7 is required for the activating phosphorylation of the T-loopof Cdc2 (45, 46, 75) and Cdk2 (45). However, genetic ablationof Mat1 in mouse cells indicated that while Mat1 is essentialfor embryonic development, it is not required for the viabilityof single cells (44, 63, 65) or for Cdc2 phosphorylation (65).As all studies indicate Mat1 and its orthologs in lower speciesfunction only as part of the Cdk7 kinase complex, and as deletionof murine Mat1 leads to concomitant loss of Cdk7 (44, 65), theresults suggest that Cdk7 kinase activity would not be criticalfor Cdc2 activation or cell viability in murine cells.

As part of TFIIH, the Cdk7 kinase submodule has been implicatedin the phosphorylation of serine 5 of the carboxyl-terminaldomain (CTD) heptapeptide repeat of the large subunit of RNApolymerase II (4, 28). RNA polymerase II CTD Ser 5 phosphorylationhas been suggested to be required for appropriate mRNA processingand associated chromatin modifications (43, 54). While severalstudies especially with the budding yeast (Saccharomyces cerevisiae)ortholog Kin28 (27, 37, 73, 74) support the notion that Cdk7within TFIIH is generally required for normal mRNA transcription,recent analyses in both budding (39) and fission (48) yeastsuggest that Cdk7 kinase activity rather is involved in theregulation of specific transcriptional programs. Consistentwith this notion, the ablation of Mat1 in murine myocardiumresulted in a suppression of genes involved in energy metabolism(65) that is suggested to be mediated through suppression ofthe activity of the coactivator PGC-1.

The Cdk7 kinase complex has also been implicated in the regulationof specific transcription through its ability to phosphorylatea number of transcriptional regulators in vitro, including thenuclear hormone receptors retinoic acid receptors ${alpha}$ and ${gamma}$ , estrogenreceptor ${alpha}$ , peroxisome proliferator-activated receptor ${alpha}$ (PPAR ${alpha}$ ),and PPAR ${gamma}$ (9, 15, 20, 40, 59). The site Cdk7 phosphorylates onPPAR ${gamma}$ 2 in vitro is serine 112 (20), identified originally asan inhibitory mitogen-activated protein kinase site in the N-terminalactivation domain (2, 12, 38). Phosphorylation of PPAR ${gamma}$ -S112inhibits PPAR ${gamma}$ target gene activation by several mechanisms,including decreased ligand binding and impaired ability to recruittranscriptional coactivators (67).

PPAR ${gamma}$ is a member of the nuclear hormone receptor family andis expressed preferentially in adipose tissue (10). Expressionis low in preadipocytes and strongly induced during adipogenesis(14, 55). Adipogenesis is initiated in fibroblasts/preadipocytesthrough the activation of Krox-20, C/EBPß, and PPAR ${gamma}$ transcriptionfactors (17). In this transcriptional regulatory network, PPAR ${gamma}$ has a central role, as its expression is necessary and sufficientfor adipogenesis both in vitro and in vivo (62, 72). PPAR ${gamma}$ activationis enhanced by ligand binding and agonists include high affinitysynthetic ligands such as the antidiabetic thiazolidinediones(TZDs; troglitazone, pioglitazone) and endogenous ligands suchas 15-deoxy- ${Delta}$ ^12,14prostaglandin J₂ (49). In addition to ligandbinding and phosphorylation, coactivators such as PGC-1 andSRC-1 further regulate PPAR ${gamma}$ activity (56).

A putative link between PPAR ${gamma}$ and Cdk7 in vivo was provided bythe observation that mRNAs of PPAR ${gamma}$ target genes were alteredin adipose tissue of mice with mutations in the XPD subunitof TFIIH (20) modeling trichothiodystrophy and featuring fathypoplasia. The altered XPD was proposed to specifically impactCdk7 activity toward PPAR ${gamma}$ -S112 in adipose tissue. This hypothesisis complicated by the variability of the alterations of PPAR ${gamma}$ target mRNAs and PPAR ${gamma}$ promoter occupancy in the analyzed samples(20).

The observations that the Cdk7 submodule of TFIIH may not beuniversally required for viability and potentially representsa specific transcriptional regulator prompted us to reevaluatethe concept of the Cdk7 submodule as a constitutive ubiquitouskinase complex. Specifically, we were interested in identifyingphysiological circumstances where modulation of Cdk7 submoduleactivity would be used to achieve biological responses throughregulation of the activity of specific transcription factors.The investigation was focused on adipose tissue based on thecritical role of PPAR ${gamma}$ in adipogenesis (62, 72), and the tentativelink to Cdk7 (20), and reveals that the Cdk7 submodule actsas an inhibitor of PPAR ${gamma}$ and as a physiological roadblock toadipogenesis.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Immunostaining of murine tissue and cells in culture. All animal experiments were approved by the Committee for AnimalExperiments of the District of Southern Finland and mice werehoused according to their regulations. Mice were anesthetized,fixed by intracardiac perfusion with 1% paraformaldehyde, andtissue from neck was frozen in OCT medium (TissueTek). Seven-millimetertissue sections were washed in phosphate-buffered saline (PBS),permeabilized using 0.3% PBS-Triton X (Fluka Biochemica), andblocked with PBS containing 5% donkey serum, 0.05% sodium azide,0.2% bovine serum albumin, and 0.3% Triton X. Primary antibodies ${alpha}$ -Mat1 (FL-309; Santa Cruz) and ${alpha}$ -PPAR ${gamma}$ (sc-7273; Santa Cruz) wereincubated in blocking solution overnight and incubated withappropriate secondary antibodies as follows: anti-rabbit Alexa594 and anti-goat Alexa 488 (Molecular Probes). Samples weremounted with Vectashield mounting medium (H-1200; Vector), andanalyzed with a confocal microscope (Zeiss LSM 510; Carl Zeiss).Three-dimensional projections were digitally generated fromconfocal z stacks. Colocalization of signals was assessed fromsingle confocal optical sections.

Immunostaining of cells was done as previously described (63).Immunolabeling was performed with the following antibodies:anti-Mat1 (FL-309; Santa Cruz), anti-PPAR ${gamma}$ (sc-7273; Santa Cruz),anti-phosphorylated PPAR ${gamma}$ -S112 (anti-P $~$ PPAR ${gamma}$ -S112) (PPAR-IF5; Euromedex),anti-C/EBPβ (sc-150; Santa Cruz), and antibromodeoxyuridine(Dako). Secondary antibodies included goat anti-mouse Alexa488 (Molecular Probes) and goat anti-rabbit 549 (Molecular Probe).Stained coverslips were analyzed using Zeiss Axioplan 2 microscopeand Axiovision software. For quantification of PPAR ${gamma}$ -S112- andP $~$ PPAR ${gamma}$ -S112-positive cells in mouse embryonic fibroblasts (MEFs),1,000 to 3,000 Hoechst-positive nuclei were scored.

Western blotting and kinase assays. Western blotting and kinase assays were as described previously(44, 50). Antibodies were as follows: anti-Mat1 (FL-309; SantaCruz), anti-Cdk7 (sc-7344; Santa Cruz), anti-Cdk2 (sc-163; SantaCruz), anti-phospho-Cdk2-T160 (2561; Cell Signaling), anti-phospho-Cdc2-T161(9114; Cell Signaling), antiactin (AC-40; Sigma), anti-PPAR ${gamma}$ (sc-7273; Santa Cruz), anti-P $~$ PPAR ${gamma}$ -S112 (PPAR-IF5; Euromedex),TAF10 (kind gift from L. Tora [52]). Secondary antibodies wereas follows: anti-rabbit horseradish peroxidase (HRP), anti-mouseHRP, and anti-goat HRP (Chemicon International). For kinaseassays, 100-µg portions of cell lysates were immunoprecipitatedwith anti-Cdk7 (50) and assayed for the ability to phosphorylateglutathione transferase (GST)-CTD or GST-Cdk2 (44). For ratfat lysates, adult rats were sacrificed with CO₂ and dislocationof the neck, and fat collected from various locations and snap-frozen.Proteins were isolated from tissue pieces by use of FastPrepFP120 (ThermoSavant) in ELB (150 mM NaCl, 50 mM HEPES, pH 7.4,and 5 mM EDTA with 10 mM β-glycerophosphate, 1 µg/mlleupeptin, 12.5 µg/ml aprotinin, 0.5 mM phenylmethylsulfonylfluoride, and 1 mM dithiothreitol added before use) and centrifugedat 2,000 rpm for 5 min, followed by the collection of supernatantssubsequently used in sodium dodecyl sulfate-polyacrylamide gelelectrophoresis.

Adipocyte differentiation of MEFs and 3T3-L1 cells. Induction of adipogenesis was done as previously described (57).Briefly, 2-day postconfluent MEFs or 3T3-L1 preadipocytes (33)were treated with differentiation medium (Dulbecco's modifiedEagle medium with 10% fetal calf serum supplemented with 10µg/ml insulin, 1 µM dexamethasone, 0.25 mM 3-isobutyl-1-methylxanthine)for 2 days, then for 2 days in medium supplemented with insulin,and finally for 4 days in normal growth medium. For lipid staining,the cells were fixed at day 8 with 10% formalin for 30 min at37°C and stained with Oil Red O. Troglitazone (Cayman ChemicalCompany) treatment was done at 10 µM for 4 to 24 h.

Establishment of Mat1^–/flox primary and immortalized MEFs and deletion of Mat1. MEFs were isolated from embryonic day 12.5 Mat1^–/flox(44) embryos and cultured in Dulbecco's modified Eagle's medium(Gibco) supplemented with 10% fetal calf serum and antibiotics.To generate immortal Mat1^–/flox MEFs, cells from passage3 were infected with a retrovirus encoding residues 302 to 390of p53 (42) and grown as pools following selection with hygromycin(Invitrogen) at 0.2 mg/ml. For the deletion of Mat1, MEFs wereinfected with adenoviruses encoding Cre recombinase (AdCre)(5) or green fluorescent protein (AdGFP) using a multiplicityof infection of 1,500 overnight at 37°C. Subsequent analysesof AdCre (Mat1^–/–)- and AdGFP (Mat1^–/flox)-infectedcells were done 72 h later unless otherwise indicated.

Transfections and reporter gene assays. Transient transfections were performed in MEFs using Superfecttransfection reagent (Qiagen) according to the manufacturer'sinstructions. For PPAR ${gamma}$ activity assays, cells were transfectedwith PPAR ${gamma}$ response element 3 (PPRE₃)-thymidine kinase (TK)-Luc(2) containing three copies of PPRE from acyl coenzyme A oxidasegene linked to the TK promoter (PPRE₃-TK-Luc) and PPAR ${gamma}$ 2 expressionvector(s) (PPAR ${gamma}$ 2/pCMV-SPORT6 from MGC Gene Collection [7] orpSV-SPORT PPAR ${gamma}$ and pSV-SPORT PPAR ${gamma}$ S112A [38], at 30 ng or 30ng and 150 ng, respectively). For luciferase assays, cells wereharvested 48 h after transfection. Relative luciferase activitywas measured with a luciferase assay system (Promega). Valueswere normalized to Renilla luciferase values. Three technicalreplicates were performed from three separate experiments.

For short hairpin RNA (shRNA) knockdown experiments with U2OScells, cells were transfected as follows: with shCdk7 (shRNA-K7/pENTR-H1;3.5 µg) or with shControl (shControl/pENTR-H1; 3.5 µg)plus PPAR ${gamma}$ 2/pCMV (0.5 µg) plus pLL3,7-GFP (0.5 µg)by use of Fugene according to the manufacturer's instructions.Seventy-two hours after transfection, coverslips were fixedand stained as per the immunofluorescence protocol. Quantificationof staining was performed with Image ProPlus 6.1 software, and1,000 GFP-positive cells were counted using Zeiss Axioplan microscopeand software. Sequences for shRNAs were as follows: for shCdk7,CCAATAGAGCTTATACAC; and for shControl, ATCCAAAAGCTGTCTTCGTTCTGCAG.

For small interfering RNA (siRNA) knockdown in MEFs, cells weretransfected using siRNA targeting Cdk7 (siCdk7) or nonspecificsiRNA (siControl) by use of Lipofectamine 2000 (Invitrogen)according to the manufacturer's protocol. Cdk7 siRNA oligonucleotidesand nonspecific siRNA were synthesized by Dharmacon (Lafayette,CO) in a purified and annealed duplex form. The sequences areavailable upon request.

Retroviral transductions. Mat1 cDNA was cut from Mat1/pAHL with EcoRI and SalI and subclonedinto the pBABEpuro vector. Amphotrophic retroviruses were producedin Phoenix 293T cells, and 1 ml of fresh viral medium/well (six-wellplate) of MEFs plus 8 µg/ml Polybrene was used in spintransduction. Selection with puromycin (5 µg/µl)was started 48 h following transduction.

mRNA level assays using quantitative RT-PCR. RNA from MEFs was isolated following 72 h of AdCre (or controlvirus) infection using an RNEasy isolation kit (Qiagen) accordingto the manufacturer's protocol. double-stranded complementaryDNA was amplified using power Sybr green PCR master mix (AppliedBiosystems). Relative mRNA amounts from PPAR ${gamma}$ , aP2, and adiponectinmRNAs were assayed by comparing PCR cycles to GAPDH (glyceraldehyde-3-phosphatedehydrogenase) by use of a 7500 fast real-time PCR (RT-PCR)system software. Samples were normalized to the control genotype.Triplicate samples were run from three separate experiments.Primers used were as follows: for aP2, forward (5'-TCCTGTGCTGCAGCCTTTCTCA)and reverse (5'-CCAGGTTCCCACAAAGGCATCA); for GAPDH, forward(5'-AAGGTCGGAGTCAACGGATT) and reverse (5'-TTGATGACAAGCTTCCCGTT);for PPAR ${gamma}$ 2, forward (5'-TGACCCAGAGCATGGTGCCTTC) and reverse (5'TGTGGCATCCGCCCAAACC);and for adiponectin, forward (5'-GAAGATGACGTTACTACAAC) and reverse(5'-GCTTCTCCAGGCTCTCCTTT).

RESULTS

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RESULTS

Cdk7 and Mat1 levels undetectable in WAT and downregulated during 3T3-L1 adipogenic differentiation. Studies to date have not supported the notion that the mammalianCdk7 submodule would be regulated in cultured cells (8, 41,69, 70) or in tissues (8, 21, 41, 44, 60, 63, 65). To assessthe status of the Cdk7 submodule in adipose tissue, we initiallyused protocols set up during our previous studies (44, 63) toanalyze Mat1 levels by confocal immunofluorescence from frozenmurine white adipose tissue (WAT) sections (Fig. 1A and B).Unexpectedly, no Mat1 staining was detected in adipocyte nuclei,whereas PPAR ${gamma}$ staining was robust (Fig. 1A and B, top). By contrast,Mat1 staining in striated muscle on the same section was readilydetected and coincided with nuclear DAPI (4',6'-diamidino-2-phenylindole)staining on the edges of myofibers (Fig. 1B, bottom), whereasPPAR ${gamma}$ staining was lower than that in WAT.

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FIG. 1. Undetectable Mat1 and Cdk7 in terminally differentiated white adipocytes in vivo. (A) Micrographs from a single frozen section containing WAT and skeletal muscle from the neck of an adult mouse stained with hematoxylin and with Mat1, PPAR ${gamma}$ , and DAPI; micrographs were made using confocal microscopy. Dashed lines indicate areas shown in detail in panel B. (B) To demonstrate the lack of specific nuclear Mat1 staining in WAT, higher magnifications of overlaid double stainings with indicated antibodies are shown, where the presence of Mat1 is visualized as pink and yellow (overlaid) color in muscle nuclei. (C) Western blotting of various fat depots (neck, intraperitoneal [i.p.], and subcutaneous [subc.]) showing adipocyte-specific downregulation of Mat1 and Cdk7. PPAR ${gamma}$ is shown to identify fat tissue and TAF10 is shown for a nuclear loading control.

To confirm the lack of Mat1 with another approach and to extendthe analysis to Cdk7, levels of Mat1 and Cdk7 were analyzedby Western blotting from rat adipose tissue lysates isolatedfrom neck, intraperitoneal, and subcutaneous fat depots. AllWAT sources contained high levels of PPAR ${gamma}$ as well as the TAF10(52) TFIID subunit (Fig. 1C). Consistent with the immunofluorescenceanalysis, no Mat1 or Cdk7 could be detected in any of the fatsamples, although they were readily detectable in control liversamples (Fig. 1C). The lack of detectable amounts of these twoTFIIH subunits was highly unexpected, considering, e.g., theactive mRNA transcription in adipocytes (32, 35). The resultsalso establish adipocytes as the first cell type identifiedto lack detectable levels of these two TFIIH subunits.

To determine whether the lack of expression of Mat1 and Cdk7noted for WAT would be recapitulated in the 3T3-L1 adipocytedifferentiation model (33), Mat1 immunofluorescence stainingwas compared to what was seen for both PPAR ${gamma}$ and C/EBPßduring differentiation (Fig. 2A) As expected (13, 76), C/EBPßwas transiently induced by day 2 and decreased by day 4. PPAR ${gamma}$ was induced from day 0 to day 2 and remained high thereafteras noted before (14). Mat1 staining in turn was high in undifferentiatedpreadipocytes (87.7% Mat1-positive nuclei/Hoechst-positive nuclei)but decreased at day 2 (68.3% Mat1-positive nuclei/Hoechst-positivenuclei) and was almost undetectable at day 4 of differentiation(Fig. 2A). As with the adipose tissue, Western blotting analysisconfirmed the decrease in Mat1 levels as well as in Cdk7 levelsand activity during 3T3-L1 differentiation (Fig. 2B), indicatingthat the 3T3-L1 model was recapitulating events noted for WATin vivo in this regard.

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FIG. 2. Adipocyte differentiation involves the downregulation of Mat1 and Cdk7 levels. (A) Immunostaining of 3T3-L1 cells at days 0, 2, and 4 following induction of adipocyte differentiation with C/EBPβ, PPAR ${gamma}$ , and Mat1 as indicated. Hoechst stain is shown for the day 4 samples. Fractions of Mat1-positive nuclei of Hoechst-positive nuclei are shown as percentages. (B) Western blotting analysis of Mat1, Cdk7, and actin levels on indicated days in control (–) or differentiation (+) medium. (Bottom) Kinase activity in Cdk7 immunoprecipitates (Cdk7 IP kinase) toward GST-Cdk2 from the same samples.

Activation of adipogenesis and adipogenic target genes in Mat1^–/– MEF cultures. The dramatic loss of Mat1 and Cdk7 during adipocyte differentiationsuggested that the presence of the Cdk7 kinase complex mightbe inhibitory for adipogenesis. To investigate this, we utilizedMEFs with a floxed Mat1 allele (63). When Mat1 was depletedfrom immortalized Mat1^–/flox MEFs (Fig. 3A) with the useof AdCre, Mat1 protein was lost within 72 h following infectionin contrast to control (AdGFP)-infected Mat1^–/flox MEFs(Fig. 3A). The depletion of Mat1 resulted in a correspondingdecrease in levels and activity of Cdk7 (Fig. 3B), demonstratingthat Mat1 is required for a functional Cdk7 complex and supportinga role for Mat1 as a stabilizing factor of this complex, assuggested for sciatic nerves in vivo (44).

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FIG. 3. Mat1^–/– MEFs show increased capacity for adipocyte differentiation. (A) Western blotting of Mat1^–/flox MEFs after the indicated number of hours following AdCre or AdGFP infection with indicated antibodies, demonstrating efficient depletion of Mat1 in AdCre-infected MEFs (Mat1^–/–) compared to AdGFP MEFs (Mat1^–/flox). (B) Mat1, Cdk7, and actin levels (Western) and kinase activity in Cdk7 immunoprecipitates (Cdk7 IP kinase) toward GST-CTD and GST-Cdk2 substrates in Mat1^–/flox and Mat1^–/– MEFs. (C) Macroscopic (magnification, x1) and microscopic (magnification, x100) images of Oil Red O-stained of Mat1^–/flox and Mat1^–/– MEF cultures following the indicated treatments. Stained areas represent triglyceride accumulation in MCEs in the induced Mat1^–/– MEF culture. (D) Quantification of adipocyte differentiation as MCE number/10-cm plate following indicated treatments of Mat1^–/flox and Mat1^–/– MEFs as well as Mat1^–/– MEFs transduced with a control (pBABE) or Mat1-expressing (pBABE-Mat1) retrovirus, demonstrating rescue of the adipogenic phenotype. Error bars represent standard deviations from two independent experiments.

To study the effects of the depletion of the Cdk7 complex onadipogenesis, the abilities of Mat1^–/flox and Mat1^–/–MEFs to undergo adipogenic differentiation in vitro were compared.In contrast to 3T3-L1 cells, immortalized MEFs generally differentiatepoorly into adipocytes when grown in adipocyte differentiationmedium, but occasional differentiating cells (0 to 2%) formmitotic clonal expansions (MCEs) and subsequently adipocytes(61). Consistent with this, only 7.33 ± 2.5 MCEs per10-cm plate were detected with Oil Red O staining in controlMat1^–/flox cultures. Strikingly, 115 ± 8 MCEs werefound in Mat1^–/– MEFs (Fig. 3C and D), demonstratinga 15-fold increase in adipogenesis. A similar increase in adipogenesiswas observed for Mat1^–/– primary MEFs (see Fig.S1 in the supplemental material). The average sizes (4 to 20cells/MCE) and morphologies of MCEs were indistinguishable,suggesting that the lack of Mat1 does not affect the proliferativeresponse involved in MCE. This did not support the notion thatMat1 would be required for the activation of cell cycle cyclin-dependentkinases, and indeed, enhanced adipogenic properties in Mat1^–/–MEFs were not associated with noticeable changes in MEF proliferationor Cdc2/Cdk2 T-loop phosphorylation acutely following Mat1 deletionat 72 h (see Fig. S2A and S2B in the supplemental material).This suggests that another kinase is phosphorylating Cdk2 andCdc2 in Mat1^–/– MEFs, indicating these cells area useful model to analyze cell cycle-independent functions ofMat1 and Cdk7.

The increased adipogenic response suggested that the loss ofMat1 may be required for cells to progress into adipogenesisand that Mat1 may therefore act as a block to this differentiationprocess. In order to confirm this, Mat1 was reintroduced usingretroviral vectors after which MEFs were again induced for adipocytedifferentiation. Indeed, the expression of Mat1 in MEFs resultedin a decrease in adipogenesis, demonstrating that Mat1 is ableto inhibit adipocyte differentiation (Fig. 3D).

To determine whether the increased adipogenicity of Mat1^–/–MEFs involved PPAR ${gamma}$ activation, Mat1^–/– and Mat1^–/floxMEFs were transfected with a PPAR ${gamma}$ -responsive reporter construct(PPRE₃-TK-Luc [2]) together with a PPAR ${gamma}$ expression plasmid.Interestingly, Mat1^–/– MEFs showed a sixfold increasein relative PPRE₃-TK-Luc reporter activity (Fig. 4A), suggestingthat the loss of Mat1 relieves the inhibition of PPAR ${gamma}$ -mediatedtranscription. The induction of PPAR ${gamma}$ activity was directly attributableto Mat1 loss, as the reintroduction of Mat1 abolished inductionin Mat1^–/– MEFs (Fig. 4A), confirming that Mat1acts as an inhibitor of PPAR ${gamma}$ -mediated transcription.

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FIG. 4. Acquired PPAR ${gamma}$ responsiveness and induction of adipogenic mRNAs in Mat1^–/– and Cdk7 knockdown MEFs. (A) PPAR reporter (PPRE₃-TK-Luc) activity in Mat1^–/flox and Mat1^–/– MEFs transfected as indicated with PPAR ${gamma}$ 2 and Mat1; sample values are compared to those for non-PPAR ${gamma}$ 2-transfected samples. (B) Relative mRNA levels of the adipogenic PPAR ${gamma}$ (1.85-fold), adiponectin (1.70-fold), and aP2 (2.70-fold) genes in Mat1^–/– MEFs compared to values for Mat1^–/flox MEFs. Hatched bars show mRNA levels in Mat1^–/– MEFs where Mat1 was reintroduced by retroviral transduction (pBABE-Mat1). Values for both panels A and B represent averages from three independent experiments, with standard deviations shown by error bars. (C) Western blotting of Cdk7 and Cdk2 (loading control) levels in MEFs transfected with control (siControl)- or Cdk7 (siCdk7)-targeting siRNAs as indicated, demonstrating efficient knockdown of Cdk7 in siCdk7-treated MEFs. (D) Relative mRNA levels of the indicated adipogenic PPAR ${gamma}$ (1.47-fold), adiponectin (2.37-fold), and aP2 (3.23-fold) genes in MEFs transfected with siCdk7 compared to siControl-transfected MEFs from a representative experiment.

The efficiency of Mat1^–/– MEFs to differentiateinto adipocytes without the expression of exogenous PPAR ${gamma}$ (Fig.3), and the increased activation of PPAR ${gamma}$ in Mat1^–/–MEFs (Fig. 4A), suggested a sensitization of the PPAR ${gamma}$ pathwayfollowing Mat1 depletion. To investigate this possibility, Mat1^–/–and Mat1^–/flox MEFs were analyzed for the expression ofadipogenic PPAR ${gamma}$ target genes, which are normally expressed atvery low levels in MEFs (31). Interestingly, quantitative RT-PCRanalyses of mRNA levels of three well-characterized PPAR ${gamma}$ targetgenes (the PPAR ${gamma}$ , aP2, and adiponectin genes) demonstrated significantinductions in Mat1^–/– MEFs, whereas no inductionwas noted for Mat1^–/flox MEFs. (Fig. 4B) or for control(GAPDH) mRNA levels in either genotype. Treatment with troglitazone,a potent PPAR ${gamma}$ agonist, caused a subtle further increase in thesegenes in Mat1^–/– MEFs, which was not noticeablein Mat1^–/flox MEFs (data not shown). Again, Mat1 reintroductionwith retroviral vectors was able to restore the expression ofthese adipogenic genes to the baseline level seen for wild-typeMEFs (Fig. 4B). While no Cdk7-independent functions of Mat1have been identified, it was formally possible that the observedconcomitant activations of PPAR ${gamma}$ responses and Cdk7 loss wereindependent phenotypes of Mat1^–/– MEFs. Therefore,to more directly establish the role of Cdk7 in this adipogenicresponse, Cdk7 levels were downregulated using siCdk7 (Fig.4C). Quantitative RT-PCR analyses of the adipogenic genes demonstratedan induction in siCdk7-treated MEFs (Fig. 4D) similar to thatseen for Mat1^–/– MEFs, suggesting that the effectsof Mat1 on adipogenesis are transmitted through the Cdk7 kinase.The results demonstrate that the loss of Mat1 and the subsequentCdk7 activity leads to enhanced PPAR ${gamma}$ activity and thus to increasedadipocyte differentiation.

Mat1 and Cdk7 are required for the inhibitory phosphorylation of PPAR ${gamma}$ at serine 112. The increased adipogenic capacity of Mat1^–/– MEFsis shared by MEFs in which endogenous PPAR ${gamma}$ is replaced withnonphosphorylatable PPAR ${gamma}$ -S112A (57). As PPAR ${gamma}$ -S112 can be phosphorylatedby Cdk7 in vitro (20), it was of interest to analyze whetherPPAR ${gamma}$ -S112 phosphorylation was defective in Mat1^–/–MEFs. For this, PPAR ${gamma}$ 2-transfected Mat1^–/flox and Mat1^–/–MEFs were immunostained for total PPAR ${gamma}$ and PPAR ${gamma}$ phosphorylatedon S112 (P $~$ PPAR ${gamma}$ -S112). Whereas Mat1^–/flox and Mat1^–/–MEFs demonstrated comparable levels of total PPAR ${gamma}$ (Fig. 5A),the levels of P $~$ PPAR ${gamma}$ -S112 were dramatically lower in Mat1^–/–MEFs (Fig. 5A), indicating that Mat1 is required for the inhibitoryphosphorylation on PPAR ${gamma}$ -S112.

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FIG. 5. Mat1 and Cdk7 are required for the inhibitory phosphorylation of PPAR ${gamma}$ at serine 112. (A) Immunofluorescence staining and quantitation (bars) of total PPAR ${gamma}$ and P $~$ PPAR ${gamma}$ -S112 for Mat1^–/– MEFs and Mat1^–/flox MEFs transfected with PPAR ${gamma}$ 2. Bars indicate changes for either total PPAR ${gamma}$ - or P $~$ PPAR ${gamma}$ -S112-positive nuclei in Mat1^–/– MEFs compared to Mat1^–/flox MEFs. (B) GFP, Cdk7, P $~$ PPAR ${gamma}$ -S112, and total PPAR ${gamma}$ immunofluorescence analysis of U2OS cells transfected with knockdown plasmids encoding a scrambled shRNA (shControl) or a Cdk7 shRNA (shCdk7) together with PPAR ${gamma}$ 2 and GFP expression plasmids. Nuclei were detected with Hoechst staining. Note the loss of signal for both endogenous Cdk7 signal (left) and P $~$ PPAR ${gamma}$ -S112 (middle) in shCDK7-transfected cells detected by GFP. (C) Quantitation of total PPAR ${gamma}$ and P $~$ PPAR ${gamma}$ -S112 from the experiment described for panel B and analyzed as for panel A.

To assess the role of Cdk7 directly in the phosphorylation ofPPAR ${gamma}$ -S112, Cdk7 levels were downregulated through introductionof a plasmid expressing shRNA targeting Cdk7 (shCdk7) in U2OSosteosarcoma cells together with a GFP-expressing reporter.As expected, GFP-positive transfected cells demonstrated efficientknockdown of endogenous Cdk7 by immunofluorescence analysis(green cells in Fig. 5B, left [shCdk7]): Cdk7 staining was detectablefor only 3.4% of GFP-positive cells compared to 100% of GFP-negativecells. Similarly, Cdk7 staining was detected in all shControl-transfectedGFP-positive cells (green cells in Fig. 5B, left [shControl]).Subsequently, S112 phosphorylation was compared in shCdk7- versusshControl-transfected cells also cotransfected with PPAR ${gamma}$ (Fig.5B, middle, [P $~$ PPAR ${gamma}$ -S112] and C) and GFP. Whereas PPAR ${gamma}$ -S112 phosphorylationwas readily detectable for 54% of shControl-transfected cells,only 0.22% P $~$ PPAR ${gamma}$ -S112-positive cells could be identified amongshCdk7-transfected cells (Fig. 5B and C). The levels of totalPPAR ${gamma}$ were comparable in shControl- and shCdk7-transfected cells(Fig. 5B, right, and C). This result demonstrates the requirementfor the Cdk7 kinase for the phosphorylation of PPAR ${gamma}$ -S112 incultured cells and provides evidence indicating that the activationof PPAR ${gamma}$ and the increased adipogenic potential of Mat1^–/–MEFs are due to attenuated Cdk7 kinase activity leading to thedecreased phosphorylation of PPAR ${gamma}$ -S112.

In order to directly assess the role of PPAR ${gamma}$ -S112 phosphorylationin the acquired responsiveness to PPAR ${gamma}$ in Cdk7 knockdown cells,MEFs were transfected with control and Cdk7 siRNAs togetherwith the PPAR ${gamma}$ reporter (PPRE₃-TK-Luc) and either wild-type PPAR ${gamma}$ or a nonphosphorylatable mutant, PPAR ${gamma}$ -S112A. As in Mat1^–/–MEFs, Cdk7 knockdown resulted in a significant increase in PPAR ${gamma}$ activity compared to what was seen for siControl-transfectedMEFs (Fig. 6). However, this difference was not noted when insteadof wild-type PPAR ${gamma}$ the nonphosphorylatable mutant PPAR ${gamma}$ -S112Awas used (Fig. 6), demonstrating that the acquired responsein Cdk7-depleted cells is dependent on PPAR ${gamma}$ -S112.

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FIG. 6. Acquired responsiveness to PPAR ${gamma}$ is dependent on PPAR ${gamma}$ -S112 in Cdk7 knockdown MEFs. PPAR reporter (PPRE₃-TK-Luc) activity in MEFs transfected as indicated with control (siControl) or Cdk7 (siCdk7) siRNAs and either PPAR ${gamma}$ or P $~$ PPAR ${gamma}$ -S112A, demonstrating that in contrast to wild-type PPAR ${gamma}$ , the nonphosporylatable mutant displays similar activities in siCdk7 and siControl MEFs. Values represent averages from three technical replicates from two independent experiments, with standard deviations shown by error bars.

DISCUSSION

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DISCUSSION

The results of this study implicate the Cdk7 submodule of TFIIHin inhibition of adipogenesis and provide evidence indicatingthat the mechanism of this inhibition involves Cdk7-mediatedphosphorylation and inhibition of PPAR ${gamma}$ . The results thereforecontribute to the understanding of adipocyte biology and PPAR ${gamma}$ regulation during adipogenesis and link regulation of the basaltranscription machinery to differentiation in vivo.

The observation that Mat1^–/– MEFs demonstrated increasedactivation of the PPAR ${gamma}$ adipogenic pathway and that this activationwas blocked by reexpression of Mat1 identified Mat1 as a negativeregulator of PPAR ${gamma}$ -mediated transcription. Regarding this, itis interesting to note the recent study on cardiac-specificablation of Mat1, where Mat1 was proposed to function as anactivator of the coactivator PGC-1 (65). While PGC-1 is involvedin PPAR ${gamma}$ -mediated transcription in some systems, it is not aplausible candidate to be involved in PPAR ${gamma}$ regulation in Mat1^–/–MEFs both because PPAR ${gamma}$ -mediated transcription is activated—notrepressed—in Mat1^–/– MEFs and because neitherPGC-1-mediated effects nor expression are detected in MEFs (65).A more direct role for Cdk7 in PPAR ${gamma}$ regulation has been suggestedby the observation that Cdk7 can phosphorylate PPAR ${gamma}$ -S112 invitro (20). This mechanism was more attractive, as phosphorylationof S112 represses PPAR ${gamma}$ activity (2, 12, 38, 67), and MEFs fromPPAR ${gamma}$ -S112A mice demonstrate an increase in adipogenic potentialsimilar to that seen for Mat1^–/– MEFs (57). Consistentwith this, results here from both Mat1-deficient fibroblastsand osteosarcoma cells following Cdk7 knockdown demonstratedthat Mat1 and Cdk7 are required for PPAR ${gamma}$ -S112 phosphorylation.A tentative link between Cdk7 and PPAR ${gamma}$ has been provided previouslyfor XPD mutant mice, which show deregulation of several PPAR ${gamma}$ target genes (20). However, the results are complicated by thefact that in vivo the XPD mutant may deregulate Cdk7 and thereforePPAR ${gamma}$ in a number of tissues, leading not only to primary changesbut also to secondary responses in various tissues. The differencesbetween these results and those present here may be due to,e.g., complexities of in vivo responses or to other proposedfunctions of XPD (16). The results presented here provide evidencethat Cdk7 acts as an inhibitor of adipogenesis and that increasedadipogenic potential following the loss of Mat1 and concomitantlyCdk7 activity are due to decreased PPAR ${gamma}$ -S112 phosphorylationand the subsequent activation of the adipogenic PPAR ${gamma}$ .

While the significance of PPAR ${gamma}$ -S112 phosphorylation as a repressorof adipogenesis has been clearly demonstrated both in vitro(38, 67) and in vivo (57), it is not clear at what stage ofadipogenesis it is important. The critical PPAR ${gamma}$ inhibition throughS112 phosphorylation is expected to be spatially and temporallyrestricted, as analysis of total cellular PPAR ${gamma}$ by Western blottinghas not demonstrated alterations in S112 phosphorylation duringadipogenesis (58) or in adipose tissue (20). Inhibition of PPAR ${gamma}$ activity by extracellular signal-regulated kinase 1/2-mediatedPPAR ${gamma}$ -S112 phosphorylation (2, 12, 38, 67) would be expectedto be limited to the first hour of adipogenic differentiationbased on lack of detectable extracellular signal-regulated kinase1/2 activity thereafter (55). Inhibition of PPAR ${gamma}$ activity byCdk7-mediated PPAR ${gamma}$ -S112 phosphorylation could occur either inpreadipocytes (where PPAR ${gamma}$ is expressed at low levels [55]) ormore likely at early stages of adipogenesis during PPAR ${gamma}$ inductionprior to Cdk7 loss (Fig. 2). As a subunit of the basal TFIIH,the Cdk7 submodule is optimally poised to phosphorylate PPAR ${gamma}$ on critical target gene promoters. For further characterizationof the critical time and target genes of S112 phosphorylation-mediatedinhibition of PPAR ${gamma}$ activity, it would be useful to analyze PPAR ${gamma}$ -S112 $~$ Pdistribution on PPREs of target genes during adipogenesis. Thenotion that PPAR ${gamma}$ -S112 phosphorylation and subsequently PPAR ${gamma}$ activity shows target gene specificity is supported by the phenotypeof the PPAR ${gamma}$ -S112A mouse (57), demonstrating similarities (increasedinsulin sensitivity) and differences (no weight gain) to whatis seen for activation of PPAR ${gamma}$ with TZD agonists. This specificityis also interesting from a therapeutic point of view, as ithas been suggested that modulation of S112 phosphorylation mightprovide an improved alternative treatment modality to the currentlywidely used activation of PPAR ${gamma}$ with TZDs (19, 57).

Investigation of adipocyte differentiation has been significantlystrengthened by the widely used preadipocyte lines NIH 3T3-L1and NIH 3T3-F442A (33, 34). More recently, important new toolshave been provided through gene knockout MEFs, most of whichresult in reduced adipogenicity, often detected only after theoverexpression of PPAR ${gamma}$ (reviewed in reference 26). The identifiednegative regulators of adipogenesis whose deletion leads toenhanced adipogenicity similar to what is seen for Mat1 areGATA3 (71), p107/p130 (18), and E2F4 (24). Based on currentunderstanding, the mechanisms involved appear to be less directlyrelated to PPAR ${gamma}$ transcriptional activation (reviewed in reference26), and therefore Mat1^–/– MEFs may provide a usefultool for the characterization of critical regulatory eventsduring the transcriptional program of adipogenesis and celllineage determination.

The implications of this study lie not only in characterizingmechanisms of adipocyte differentiation but additionally inproviding insight into complexities of physiological roles ofbasal transcription factors. Based on several genetic models(47, 73-75) and expression studies (8, 11, 21, 41, 60, 69, 70),Cdk7 kinase activity has been considered as ubiquitous and essential.In this study, we found that both Mat1 and Cdk7 levels weredownregulated in adipose tissue, reminiscent of what was seenfor some TFIID subunits in other physiological settings (22,25, 30). It will be of interest to characterize the mechanismsinvolved in the regulation of Mat1 and Cdk7 levels in more detail;based on observations of a C-terminally truncated Mat1 in ATRA-treatedHL60 cells (36), it is plausible that the observed decreasein Mat1 is due to ubiquitin-mediated proteolysis leading todestabilization of Cdk7, consistent with results here and previouslynoted for sciatic nerves in vivo (44).

The loss of the Mat1 and Cdk7 subunits of TFIIH in adipose tissuesuggests that TFIIH can function in tissue- and differentiation-specificforms. Recent studies of the Drosophila embryo demonstrate thatthe Cdk7 kinase complex and core TFIIH are localized to differentcellular compartments (16) and transcriptional loci during development(3). Cdk7 kinase activity has also been shown to be dispensablefor the nucleotide excision repair function of TFIIH (6, 53,68) as well as for the recently identified TFIIH coactivatorfunction (21). The lack of detectable Mat1 or Cdk7 in adipocytessuggests that the apparently active mRNA transcription in adipocytes(32, 35) is independent of Mat1 and Cdk7 TFIIH subunits. Fromthese recent findings on TFIIH and TFIID, it is beginning toemerge that differentiated cells may in fact contain very diversecompilations of the core transcription machinery.

ACKNOWLEDGMENTS

We are grateful to V. K. K. Chatterjee for providing the PPRE₃-TK-Lucconstruct, to Bruce Spiegelman for the pSV-SPORT PPAR ${gamma}$ and pSV-SPORTPPAR ${gamma}$ S112A plasmids, to Laszlo Tora for providing the TAF10 antibody,and to Yuan Zhu and Luis Parada for AdCre and AdGFP constructs.We thank Jenny Bärlund, Outi Kokkonen, Saana Laine, andSari Räsänen for technical assistance and SusannaRäsänen for excellent animal husbandry. BiomedicumHelsinki Molecular Imaging Unit, Biomedicum Virus Core, BiocentrumHelsinki Systems Biology Initiative (MGC Gene Collection), andBiomedicum Genomics are acknowledged for services. Kari Vaahtomeri,Tea Vallenius, and Thomas Westerling are acknowledged for commentingon the manuscript.

This work was supported by Nylands Nation Foundation, Diabetestutkimussäätiö,Finnish Cultural Foundation, Research and Science Foundationof Farmos, Academy of Finland, EU FP6 Program (ENFIN), FinnishCancer Organizations, and Sigrid Juselius Foundation. K.H. isa graduate student at Helsinki Biomedical Graduate School.

FOOTNOTES

* Corresponding author. Mailing address: Genome-Scale Biology Program and Institute of Biomedicine, Biomedicum Helsinki, 00014 University of Helsinki, Helsinki, Finland. Phone: 358-9-1912 5555. Fax: 358-9-1912 5554. E-mail: Tomi.Makela{at}helsinki.fi

Published ahead of print on 3 November 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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