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Molecular Endocrinology, doi:10.1210/me.2006-0401
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Molecular Endocrinology 21 (4): 857-864
Copyright © 2007 by The Endocrine Society

Altered Promoter Recycling Rates Contribute to Dominant-Negative Activity of Human Peroxisome Proliferator-Activated Receptor-{gamma} Mutations Associated with Diabetes

Gang Li and Todd Leff

Center for Integrative Metabolic & Endocrine Research, Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201

Address all correspondence and requests for reprints to: Dr. Todd Leff, Department of Pathology, Wayne State University School of Medicine, 111 Lande Building, 550 East Canfield, Detroit, Michigan 48201. E-mail: tleff{at}med.wayne.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The transcription factor peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) plays an important role in regulating lipid and glucose metabolism and improves insulin sensitivity in diabetic patients when activated by thiazolidinedione drugs. Several loss-of-function mutations in PPAR{gamma} have been identified that cause lipodystrophy and diabetes in humans. Because affected individuals are heterozygotes and have one normal PPAR{gamma} allele, it is of interest to know whether these mutations act in a dominant-negative fashion to inhibit the activity of the wild-type (WT) receptor. Here we compare the molecular phenotypes of two previously identified PPAR{gamma} mutations: P467L, reported to be dominant negative; and F388L, reported to be devoid of dominant-negative activity. We developed a competitive chromatin immunoprecipitation assay to measure the relative ability of mutant PPAR{gamma} to compete with WT receptor for binding to a PPAR regulatory element (PPRE)-containing promoter. By determining the ratio of mutant and WT receptors bound to a PPRE over time, we estimated the relative promoter turnover rate of each receptor. This assay demonstrated that PPAR{gamma} bearing the P467L had a reduced promoter turnover rate compared with the F388L receptor, and over time out-competed the WT receptor for promoter binding sites. We propose that the P467L receptor is dominant negative because in a cell containing both WT and mutant receptors, the majority of the PPAR-regulated promoters will be occupied by the transcriptionally defective mutant receptor. In contrast, the F388L mutation lacks dominant-negative activity because its more rapid promoter turnover rate prevented it from out-competing the WT receptor for promoter binding sites.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PEROXISOME PROLIFERATOR-activated receptor-{gamma} (PPAR{gamma}) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. PPAR{gamma} is required for adipocyte differentiation and plays an important role in the regulation of glucose and lipid metabolism and the control of insulin sensitivity (for a reviews see Refs. 1, 2, 3). Reduction of PPAR{gamma} activity by loss-of-function mutations causes lipodystrophy and diabetes in humans. To date, five lipodystrophy-associated mutations in the PPAR{gamma} ligand binding domain have been identified (4, 5, 6, 7, 8). All five mutations V290M, Y355X, F388L, R425C, and P467L are located in the ligand binding domain of the receptor and all affected individuals are heterozygotes and posses a functional wild-type (WT) allele in addition to the mutant allele.

Despite similar clinical manifestations, these mutations show distinct characteristics at the molecular level. Two mutations (P467L and V290M) were found to possess strong dominant-negative activity against the WT receptor (5, 9), suggesting that in cells containing equal amounts of WT and mutant receptors (as would be the case in human subjects), the total amount of PPAR{gamma} activity would be quite low. On the other hand, the F388L and Y355X mutations did not show dominant-negative activity against the WT receptor (6, 7), raising the possibility that in these patients the total amount of PPAR{gamma} activity would not be as severely compromised. In the current paper, we have examined the mechanistic basis for the differences between these groups of mutations by comparing specific parameters of transcriptional activation by the dominant-negative P467L mutant with the F388L mutant.

The interaction of nuclear receptors with the transcription initiation machinery is a dynamic process in which nuclear receptors and their cofactors cycle on and off their target promoters during transcriptional activation (10, 11, 12, 13). This turnover of DNA-bound receptor appears to be necessary for high levels of transcription. Recent studies on the estrogen receptor have suggested that proteasome-mediated degradation of the receptor is essential for normal promoter recycling and ligand-induced transcriptional activation (14, 15, 16). This also appears to be the case for PPAR{gamma}, where transcriptional activity is dependent on proteasome-mediated degradation of the receptor (17, 18).

In the current study, we examined the possibility that lipodystrophy-associated PPAR{gamma} mutations alter the dynamics of the interaction of the receptor with its DNA recognition element. The goals of this study were to determine whether familial partial lipodystrophy (FPLD)-associated PPAR{gamma} mutations affect promoter recycling rates, and to explore the relationship of recycling rate to dominant-negative activity of these mutations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
P467L and F388L PPAR{gamma} Mutants Display Different Degrees of Dominant-Negative Activity
The transcriptional activities of PPAR{gamma} receptors bearing either the P467L or the F388L mutations were directly compared in a transient transfection assay. As shown in Fig. 1AGo, both mutations caused a significant reduction in basal transcriptional activity (in the absence of exogenous ligand). In striking contrast to the defect in basal activity, both mutant receptors were as active as the WT receptor in the presence of saturating amounts of the ligand rosiglitazone. Both mutations reduced the potency of rosiglitazone for transcriptional activation (5, 6).


Figure 1
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  		Fig. 1. P467L and F388L Mutations Show Different Degrees of Dominant-Negative Activity

NIH3T3 cells were transfected with WT PPAR{gamma}, F388L or P467L mutant PPAR{gamma} expression plasmids together with a PPAR-responsive reporter construct (pFATP-luc), and treated with DMSO or rosiglitazone (Rosi) for 16 h. A, Transcription activity of mutant and WT PPAR{gamma} receptors in the absence of exogenous ligand or in the presence of saturating amounts of rosiglitazone. B, NIH3T3 cells were transfected with 1 ng of WT PPAR{gamma} alone (0:1) or 1 ng of WT receptor together with increasing amounts of either WT or mutant receptor as indicated in the absence of added ligand. C, Cells were treated for 16 h with rosiglitazone (Rosi) or vehicle (no ligand) as indicated. Data are means ± SD (n = 3). Statistical significance compared with WT alone within each experiment (Student’s two-tailed t test): *, P < 0.05; **, P < 0.01; ***, P < 0.001. NS, Not significant.

 
To directly compare the dominant-negative activity of the P467L and F388L mutants, a mixing experiment was performed in which increasing amounts of mutant receptors were cotransfected with a fixed amount of WT PPAR{gamma}. The transfection conditions were adjusted so that the complete saturation of transcriptional capacity was achieved as the receptor amount increased (Fig. 1BGo; WT + WT), presumably due to full occupancy of available PPAR{gamma} binding sites. Addition of increasing amounts of the P467L mutant caused a dose-dependent reduction in the activity of the cotransfected WT receptor (Fig. 1BGo; 467 + WT) confirming previous reports that this mutation was dominant negative (5, 9). In contrast, increasing the F388L mutant receptor did not cause a significant change in transcriptional activity of the WT PPAR{gamma} (Fig. 1BGo; 388 + WT). The P467L mutant was not dominant negative at saturating ligand concentrations (Fig. 1CGo). Together, these findings confirm that that in contrast to the P467L mutation, the F388L receptor does not posses dominant-negative activity.

To assess the influence of these mutants in a more natural cellular context, the F388L and P467L mutant receptors were introduced into 3T3-L1 preadipocytes using a high efficiency transfection protocol, and the transcriptional activity of the endogenous aP2 gene was measured during their differentiation into adipocytes. As shown in Fig. 2AGo, after 40 h of differentiation, transfection of WT PPAR{gamma} caused 3-fold increase of endogenous aP2 expression, whereas an equal amount of F388L PPAR{gamma} caused only a slight increase (1.6-fold) in aP2 expression. In contrast, an equal amount of P467L PPAR{gamma} caused a significant suppression of endogenous aP2 expression, demonstrating that this mutation also behaves in a dominant-negative fashion when transcribing an endogenous gene in an adipocyte cell line. As predicted from the results in Fig. 1CGo, the presence of saturating amounts of ligand (20 µM rosiglitazone), abolished the dominant-negative activity of the P467L mutation (Fig. 2BGo). These findings confirm that, in contrast the P467L mutation, the F388L mutation does not function in a dominant-negative fashion, even on an endogenous gene in a cellular context that is more relevant to the physiological function of the receptor.


Figure 2
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  		Fig. 2. The P467L But Not F388L Suppressed Expression of the Endogenous aP2 Gene in 3T3-L1 Adipocytes

Results of quantitative real-time PCR indicated endogenous aP2 gene expression from 3T3-L1 preadipocyte cells transfected with flag-tagged WT, F388L or P467L PPAR{gamma} in the absence (A) or presence of 20 µM rosiglitazone (Rosi) (B). Western blots using anti-flag antibodies demonstrated equal amounts of WT and mutant proteins in each transfection. Data are means ± SD calculated from three independent replicates. Statistical significance was calculated using the Student’s two-tailed t test: *, P < 0.05; **, P ≤ 0.01. Veh, Vehicle; Vec, vector control; NS, not significant.

 
P467L and F388L PPAR{gamma} Mutants Have Different Promoter Recycling Rates
A recently appreciated aspect of transcriptional activation by nuclear receptors is the cyclical nature of their interaction with target gene promoters and the transcriptional initiation machinery. Cyclical turnover of steroid receptors has been well described (10, 11, 12, 19), and it is likely that this is a general aspect of transcriptional activation by all nuclear receptors. To determine whether these PPAR{gamma} mutations affected recycling of PPAR{gamma} on target gene promoters, we developed a competitive chromatin immunoprecipitation (ChIP) assay that allowed us to measure the proportion of WT and mutant receptor bound to a standard PPAR regulatory element (PPRE) in vivo. If a mutation caused a decrease in the promoter recycling rate of PPAR{gamma}, this would be measured as an increase in the proportion of the PPREs occupied by the mutant compared with the WT receptor.

NIH3T3 cells were cotransfected with equal amounts of expression vectors producing hemagglutin (HA)-tagged WT PPAR{gamma} and FLAG-tagged mutant PPAR{gamma}, with a reporter construct containing a single PPRE derived from the ap2 gene enhancer (ap2-PPRE). Transfection conditions were adjusted to insure a large excess of PPAR{gamma} protein relative to the number of PPAR{gamma} binding sites. The functionality of the WT and mutant PPAR{gamma} proteins in this system was confirmed by examining the transcriptional response to rosiglitazone in a standard luciferase assay (data not shown). The ChIP procedure was carried out on these cells at different times after transfection to measure the ratio of HA-tagged (WT) to FLAG-tagged (mutant) PPAR{gamma} bound to the ap2-PPRE at different times after transfection (Fig. 3AGo). The ChIP signal was quantified by real-time PCR as described in Materials and Methods.


Figure 3
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  		Fig. 3. The P467L But Not the F388L Mutant Displays Prolonged Promoter Residence Time in the Absence of Ligand

A, Schematic diagram showing experimental procedure for competitive ChIP to determine promoter recycling rate. B–D, Results of ChIP analyses from the indicated combinations of WT and mutant PPAR{gamma} cotransfections. Ratios of WT and mutant PPAR{gamma} receptor bound to the cotransfected PPRE at three different times after transfection are derived from quantitative real-time PCR. Western blots of transfected PPAR{gamma} proteins demonstrate that amounts of WT and mutant proteins are not differentially altered during the course of the experiment. Data are means of three independent experiments ± SD. *, P < 0.05 compared with 467+WT at 14 h; §, P < 0.005 compared with 467 + WT at 22 h. rosi, Rosiglitazone.

 
To validate the system, a control experiment was conducted with equal amounts of HA and FLAG tagged versions of WT PPAR{gamma}. As presented in Fig. 3BGo, the ratio of PPRE-bound HA- and FLAG-tagged receptors did not change over the course of the experiment, indicating that the epitope tags did not differentially affect PPAR{gamma} promoter recycling rate. Western blots confirmed that the proportions of WT and mutant protein expressed in the transfected cells did not change over time. When the same experiment was conducted with equal amounts of P467L and WT receptor (Fig. 3CGo), the real-time PCR results demonstrated a significant 3-fold increase in the proportion of PPRE bound to the P467L receptor after 38 h. These results indicate that P467L mutation increased the promoter residence time (i.e. decreased the promoter recycling rate) of the PPAR{gamma} receptor. These findings are in striking contrast to those observed in cells containing an equal mixture of F388L and WT PPAR{gamma} receptors. In this case, there was no change over time in the ratio of promoters occupied by two receptors (Fig. 3CGo), indicating that the F388L mutation did not affect the promoter recycling rate of PPAR{gamma}.

If the dominant-negative activity of the P467L mutation is related to its reduced promoter recycling rate, then conditions that inhibit its dominant-negative activity, such as saturating amounts of ligand (Figs. 1CGo and 2BGo), should reduce its promoter residence time. When the ChIP competition assay was carried out in the presence of a high concentration of rosiglitazone, the ability of the P467L mutant to out-compete WT receptor for promoter binding was abolished (Fig. 3DGo). A possible explanation for this finding is that even though the P467L has reduced affinity for ligand, at concentrations high enough to activate transcription it undergoes an activity-dependent proteolysis similar to that observed for many nuclear receptors including WT PPAR{gamma} (13, 18, 20, 21). Together, these findings are consistent with a model in which the P467L mutant is dominant negative because its reduced promoter recycling rate leads to the majority of the PPAR-regulated promoters being occupied by the transcriptionally defective mutant receptor.

Influence of Protein Stability on PPAR{gamma} Promoter Recycling Rate
Consistent with its apparent lack of dominant-negative activity, the F388L mutation differs from the P467L mutation in that it has a promoter recycling rate similar to the WT receptor. Given that the F388L is also transcriptionally defective in the absence of ligand (Fig. 1AGo) it is unclear why it has a more rapid promoter recycling rate than the P467L mutant. One possibility is that the F388L mutation causes enhanced proteolytic degradation of the receptor that promotes its removal from the template. Proteolytic degradation of transcription factors has been shown to play an important role in promoter recycling of several nuclear receptors (20, 21, 22), including PPAR{gamma} (18).

To determine whether the F388L mutation caused a general increase in proteolytic degradation of the receptor, we compared the degradation rates of mutant and WT PPAR{gamma}. NIH3T3 cells were transfected with constructs expressing FLAG-tagged receptors under the control of a tetracycline-responsive promoter. The rate of PPAR{gamma} protein degradation was measured starting 6.5 h after doxycycline was added to stop PPAR{gamma} expression. As shown in Fig. 4Go, the degradation rates of the WT and P467L receptors were similar, with half-lives of 2.8 and 2.4 h, respectively. In contrast, the F388L receptor was degraded at a significantly more rapid rate (half-life of 1.8 h; P < 0.05 relative to WT). Thus, the F388L but not the P467L mutation caused a significant reduction in the stability of the PPAR{gamma} protein. This increased susceptibility of F388L-PPAR{gamma} to proteolytic degradation could increase its removal from the promoter, effectively substituting for activity-dependent proteolysis. In this context, the low promoter recycling rate of the P467L mutations is due to a combination of a lack of activity-dependent proteolysis and normal protein stability.


Figure 4
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  		Fig. 4. F388L-PPAR{gamma} Protein is Unstable

Whole cell extracts were prepared from NIH3T3 cells transfected with tetracycline-regulated expression vectors producing FLAG-tagged WT or mutant PPAR{gamma} proteins. Six hours after addition of doxycycline to arrest PPAR{gamma} expression, cells were harvested at the indicated times and whole cell lysates were subjected to Western blot analysis. The amount of FLAG-PPAR{gamma} protein expression was normalized to a cotransfected reference protein (ECFP-N1). Expression of each PPAR{gamma} protein time "0" was assigned a value of "100%" (log 2) and the values for the other time points were expressed relative to time "0." Each line represents the mean of three independent experiments. T1/2 values calculated from the slopes are indicated. The degradation rate of the F388L mutant was significantly different from both WT and the P467L receptors (P < 0.05).

 
A prediction of this model is that the promoter recycling rates of the P467L and the WT receptor would be similar if the activity-dependent proteolysis of the WT receptor were inhibited. To test this possibility, we compared the effects of proteasome inhibition on transcriptional activity and promoter residence time of the WT and P467L mutant receptors. As predicted, the proteasome inhibitor MG132 increased the overall amounts of both WT and mutant receptors in NIH3T3 cells, but suppressed their transcriptional activities (Fig. 5AGo). The reduced transcriptional activity, even in the presence of higher amounts of receptor, is consistent with a role for proteolysis in transcriptional activation, and confirms that the degradation of receptor is required for normal levels of PPAR{gamma}-mediated transactivation. Similar inhibition by lactacystin (another proteasome inhibitor) was also observed (data not shown). Also as predicted, the presence of MG132 abolished the difference in promoter residence time between the WT and P467L (Fig. 5BGo). These findings are consistent with a model in which the differences in promoter recycling rates between the P467L and F388L mutations are due to differences in protease susceptibility of the two receptors.


Figure 5
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  		Fig. 5. Inhibition of Proteasome-Mediated PPAR{gamma} Degradation Abolishes the Differences in Promoter Recycling Rates of WT and P467L Receptors

A, Proteasome inhibition by MG132 decreases the transcriptional activity of PPAR{gamma}. NIH3T3 cells were cotransfected with either WT or mutant PPAR{gamma} together with the PPAR-responsive reporter construct pFATP-luc. Cells were treated with MG132 (10 µM) for the last 5 h of the transfection, and cell lysates were analyzed for reporter gene expression and by Western blotting. Data are means ± SD (n = 3) of luciferase activity normalized to a cotransfected ß-galactosidase reference standard. ***, P < 0.0001 compared with the DMSO control. B, Competitive ChIP experiments carried out as in Fig. 3Go, but in the presence of 10 µM MG132.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have examined the molecular phenotype of two different human PPAR{gamma} mutations that cause FPLD and diabetes. Although both mutations have similar clinical characteristics, we show here that they have distinct molecular phenotypes. The P467L mutant displayed dominant-negative activity, strongly suppressing the activity of coexpressed WT PPAR{gamma} receptor in the absence of added ligand (Fig. 1BGo). In the same assay system, the F388L mutation had a simple loss-of-function phenotype without dominant-negative activity. These differences were also observed when the mutant receptors were introduced into cultured adipocytes containing endogenous PPAR{gamma}, and their activities on an endogenous PPAR{gamma} target gene (aP2) were assessed (Fig. 2Go). Together, these results suggest that in patients the F388L mutation causes a much less severe reduction in total PPAR{gamma} activity than the dominant-negative P467L mutation.

The two mutations also showed striking differences in their abilities to compete with WT PPAR{gamma} for a limited number of binding sites in a competitive ChIP assay. In the absence of ligand, P467L-PPAR{gamma} out-competed the WT receptor and over time occupied the majority of receptor binding sites (Fig. 3CGo). Under these conditions, the transcriptionally defective P467L-PPAR{gamma}, effectively suppresses the activity of WT PPAR{gamma} by blocking its access to promoter binding sites. In contrast, F388L-PPAR{gamma} did not out-compete the WT receptor in the competitive ChIP assay and the proportion of PPRE sequences occupied by the mutant and the WT receptor did not change over time (Fig. 3CGo). In this case, with half the PPAR response elements occupied by WT receptor, the overall transcriptional activity would not be lower than 50% of normal and the mutant would not display dominant-negative activity.

Although our competitive ChIP assay measures the ability of receptors to compete for DNA binding sites, we interpret this as a reflection of differences in the rate at which a receptor dissociates, or is removed, from the promoter—the recycling rate. Promoter recycling of nuclear receptors appears to be an active process that is required for normal transcriptional activity. For example, it has been reported that the estrogen receptor-{alpha} and its coactivators rapidly cycle onto and off the pS2 promoter in a ligand-dependent manner (11), a phenomena that was subsequently observed for other nuclear receptors (12, 23, 24). This cyclic association of nuclear receptors with responsive promoters appears to be dependent on proteasome activity (13, 25). Although less is known about promoter recycling dynamics of PPAR{gamma}, it has been reported that an activity-dependent proteasome-mediated degradation pathway also plays an important role in determining its transcriptional activity (17, 18).

This model suggests that the apparent reduction in promoter recycling rate of P467L-PPAR{gamma} is due to a defect in activity-dependent proteolysis of the mutant receptor. This possibility is supported by the observation that inhibition of proteasome pathway abolished differences in the promoter recycling rates of the WT and P467L receptors (Fig. 5BGo). The model also presents a possible explanation for why the F388L mutation did not appear to affect promoter recycling rate. If, in addition to reducing basal transcriptional activity, the F388L mutation also caused a general increase in the rate of receptor degradation, the two effects could essentially counteract each other, resulting in a promoter recycling rate similar to WT receptor. In support of this possibility, we observed that F388L-PPAR{gamma} displayed a nearly 2-fold increase in degradation rate compared with the WT and P467L receptors (Fig. 4Go).

Most of the experiments on promoter recycling were carried out in the absence of exogenous ligand. Under these conditions, both mutations are transcriptionally defective (Fig. 1AGo). In the context of the activity-dependent proteolysis model of promoter recycling described above, we must consider the nature of transcriptional activation in the absence of added exogenous ligand. In a transient transfection system, the rather substantial transcriptional activity of PPAR{gamma} in the absence of added ligand is likely due either to a subsaturating amount of an unknown endogenous ligand or to an intrinsic transcriptional activity of unliganded receptor. In either case, transcriptional activity of the receptor in the absence of exogenous ligands would still be affected by an activity-dependent proteolysis as described for ligand-dependent transcriptional activation.

In summary, our results provide a possible explanation for why the P467L mutation of PPAR{gamma} functions in a strong dominant-negative fashion, whereas the F388L mutation does not. We propose that a reduction in the promoter recycling rate of the P467L mutation is a major contributor to its dominant-negative activity and that the lack of dominant-negative activity of the F388L receptor is due to enhanced susceptibility to proteolytic degradation, which causes an increase in its promoter recycling rate.

The fact that the F388L mutation does not posses dominant-negative activity against the WT receptor has significance with regard the role of PPAR{gamma} activity in causing lipodystrophy and diabetes. Patients bearing the F388L mutation would have an overall level of PPAR{gamma} activity that is probably not less than 50% of normal. This is in contrast to cells from patients bearing the dominant-negative P467L mutation, where the overall level of PPAR{gamma} activity is likely to be much lower. Because the clinical manifestation of the heterozygous F388L mutation is quite profound, these results suggest that a comparatively small reduction in PPAR{gamma} activity can have major consequences on adipose tissue biology and metabolic homeostasis. It must be pointed out that we have examined the activity of these mutant receptors on only a small subset of PPAR{gamma} target genes (FATP and aP2), and it is possible that the F388L mutation displays a more defective molecular phenotype in the expression of other PPAR{gamma} target genes. However, the idea that a relatively mild reduction in PPAR{gamma} activity can cause a severe clinical phenotype is supported by recent reports of PPAR{gamma} mutations that generate completely nonfunctional receptors that cannot form dimers with RXR or bind to DNA, yet cause a clinical phenotype similar to that seen in other FPLD patients (7, 26).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
A cDNA encoding full-length human PPAR{gamma}1 was cloned into the eukaryotic expression vector pcDNA4/HisMax-TOPO (Invitrogen, Carlsbad, CA). The F388L and P467L mutations were introduced into this clone using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). The phenylalanine codon (TTT) at position 388 was changed to the leucine codon TTA, and the proline codon (CCG) at position 467 was changed to the leucine codon CTG. The numbering system is based on the amino acid sequence of the human PPAR{gamma}2 for F388L and the PPAR{gamma}1 protein for P467L. The WT and mutant clones were fully sequenced.

For the ChIP and protein stability experiments, the WT, F388L, and P467L cDNA clones were transferred into the tetracycline-responsive eukaryotic expression vector pTRE-shuttle2 (BD Biosciences-CLONTECH, Palo Alto, CA), and a double-FLAG epitope tag (MDYKDHDGDYKDHD) was added to the N terminus of each clone. An additional version of the WT pTRE-shuttle2 clone was constructed containing an N-terminal HA epitope in place of the FLAG epitope tag. The PPAR-regulated promoter used in the ChIP experiments was composed of a 154-bp fragment of the mouse aP2 gene enhancer containing a single PPRE sequence, cloned upstream of a minimal HSV-TK promoter in the luciferase reporter plasmid pGL3. Promoter constructs were verified by DNA sequence analysis.

Cell Culture, Transfections, and Western Analysis
NIH3T3 cells were obtained from the ATCC (Manassas, VA) and maintained in DMEM containing 10% fetal bovine serum supplemented with penicillin/streptomycin at 37 C in 5% CO2. Doxycycline and MG132 were obtained from BD Biosciences-CLONTECH and Sigma (St. Louis, MO).

For transcription assays, NIH3T3 mouse fibroblasts were grown in 96-well plates at 5.5 x 103 cells per well. Cells were transfected with the either WT or F388L or P467L PPAR{gamma} expression plasmids, 1 ng of a ß-galactosidase control plasmid and 35 ng of the PPAR-dependent luciferase reporter pFATP-Luc (three copies of the mouse FATP gene PPRE inserted upstream of the minimal thymidine kinase promoter). Cells were transfected for 4 h with Lipofectamine-plus as described by the manufacturer (Invitrogen) and then treated with dimethyl sulfoxide (DMSO) or the indicated amount of rosiglitazone for 16 h. Luciferase and ß-galactosidase activities were measured in cell extracts using the dual-light assay system (ABI, Foster City, CA) and a 96-well luminometer (Berthold Technologies, Bad Wildbad, Germany). Transfections were performed in triplicate.

For experiments in cultured adipocytes, 3T3-L1 cells were obtained from the ATCC (Manassas, VA) and maintained in DMEM containing 10% fetal bovine serum supplemented with penicillin/streptomycin at 37 C in 5% CO2. 3T3-L1 preadipocytes (9.0 x 105 cells per well of a six-well plate) were transfected with Lipofectamine LTX-plus as described by the manufacturer (Invitrogen) with 1 µg of the tetracycline-regulated PPAR{gamma} expression plasmid, 250 ng of pTET-off (producing the rTA transactivator), and 20 ng of a cyan florescent protein expressing plasmid pECFP-N1 (BD Biosciences-CLONTECH) as a control. Examination of enhanced cyan fluorescent protein (ECFP) florescence after transfection showed that 50–60% of the cells were transfected by this procedure. At 20 h after transfection, media were replaced with the differentiation mixture (DMEM with 10% fetal bovine serum plus 0.5 mM 3-isobutyl-1-methlxanthine, 1 µM dexamethasone, and 10 µg/ml insulin) (27) for 40 h. After harvesting, total RNA was extracted using Purelink RNA purification system (Invitrogen). cDNA was synthesized using the iScript cDNA Synthesis kit according to the manufacturer’s protocol (Bio-Rad, Hercules, CA). Quantitative RT-PCR was carried out to determine endogenous aP2 mRNA levels using an aP2 gene primer pair (5'-AAACGAGATGGTGACAAGCTGGTG-3' and 5'-TGCAAATTTCCATCCAGGCCTC-3') in a Bio-Rad iCycler real-time PCR system. PPIA (peptidylprolyl isomerase A) was used as internal control and the amount of aP2 expression was calculated by the comparative Ct (cycle threshold) method from triplicate reactions. In parallel experiments, cells were treated as described above but harvested for Western analysis to measure expression levels of the transfected PPAR{gamma} proteins.

For Western analysis of PPAR{gamma} protein amounts, NIH3T3 cells (1.0 x 106 cells/well of a six-well plate) were transfected with Lipofectamine-plus as described by the manufacturer (Invitrogen) with 30 ng of the tetracycline-regulated PPAR{gamma} expression plasmids, 7.5 ng of pTET-off (producing the rTA transactivator), and 1 ng of a cyan florescent protein expressing plasmid pECFP-N1 (BD Biosciences-CLONTECH) as a control. Fourteen hours after transfection, doxycycline was added to a final concentration of 1 µg/ml to turn off PPAR{gamma} expression. Cells were harvested at the indicated intervals, beginning 6.5 h after the addition of doxycycline. In parallel experiments, we determined that PPAR{gamma} mRNA was rapidly degraded, with only 10% of maximal levels remaining after 5 h of doxycycline treatment (data not shown). In some experiments, doxycycline was not added and cells were treated with 10 µM MG132 5 h before harvest (Fig. 5Go). For harvesting, cells were washed once in PBS and then lysed in a buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 30 mM NaF, and 1 mM Na3VO4. Protein concentrations were determined by Bradford assay (Bio-Rad), and equal amounts of protein were analyzed by SDS-PAGE. Western blots were performed by using monoclonal anti-FLAG-M2-peroxidase (horseradish peroxidase)-conjugated antibody (Sigma).

ChIP and Quantitative Real-Time PCR
NIH3T3 cells (2 x 107) were transfected by electroporation at 220 V and 950 µF with 20 µg total DNA, containing 4 µg of pGL3-aP2 and 3 µg of an equal mixture of HA-tagged WT and FLAG-tagged mutant PPAR{gamma} expressing plasmids. As a control, 0.5 µg of pECFP-N1 was included in the transfection. After electroporation cells were plated and cultured for the indicated times and then harvested for ChIP assays. Each ChIP was carried out with 1 x 107 cells. ChIP was carried out according to manufacturer’s protocol (Upstate Biotechnology, Lake Placid, NY). Immunoprecipitated DNA fragments were purified using the QIAquick PCR Purification kit (QIAGEN, Valencia, CA) and subjected to PCR analysis and measured by quantitative real-time PCR using primers that recognized the promoter of the transfected aP2 reporter but not the endogenous aP2 gene enhancer sequences.

Relative promoter occupancy by HA-tagged WT PPAR{gamma} and FLAG-tagged mutant PPAR{gamma} at different times after transfection was determined by comparing the relative amounts of aP2 promoter fragment immunoprecipitated by the HA or FLAG antibodies. Using the Ct method, the amount of aP2 promoter fragment in each immunoprecipitate was normalized to the Ct value from the input sample from the same time point using the 2–{Delta}{Delta}Ct formula. This value was then corrected for the amount of nonspecific signal by subtracting the normalized nonspecific-IgG signal. For each time point, the normalized value of the FLAG immunoprecipitate (mutant PPAR{gamma}) was divided by the normalized value of the HA immunoprecipitate (WT PPAR{gamma}) to give the ratio shown in the graphs in the ChIP experiments.


   FOOTNOTES
 
This work was supported by the American Diabetes Association and the Michigan Tri-Technology Corridor Fund (to T.L.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 16, 2007

Abbreviations: ChIP, Chromatin immunoprecipitation; Ct, cycle threshold; DMSO, dimethylsulfoxide; ECFP, enhanced cyan fluorescent protein; FPLD, familial partial lipodystrophy; HA, hemagglutin; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR regulatory element; WT, wild type.

Received for publication September 25, 2006. Accepted for publication January 8, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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NURSA Molecule Pages Link:

Nuclear Receptors:   PPARγ
Ligands:   Rosiglitazone



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