Adipose Expression of the Phosphoenolpyruvate Carboxykinase Promoter Requires Peroxisome Proliferator-activated Receptor gamma  and 9-cis-Retinoic Acid Receptor Binding to an Adipocyte-specific Enhancer in Vivo*

Jerry H. DevineDagger , Darrell W. EubankDagger , David E. Clouthier§, Peter Tontonozparallel **, Bruce M. Spiegelmanparallel , Robert E. Hammer§, and Elmus G. BealeDagger Dagger Dagger

From the Dagger  Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, the § Howard Hughes Medical Institute, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050, and the parallel  Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A putative adipocyte-specific enhancer has been mapped to approximately 1 kilobase pair upstream of the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene. In the present study, we used transgenic mice to identify and characterize the 413-base pair (bp) region between -1242 and -828 bp as a bona fide adipocyte-specific enhancer in vivo. This enhancer functioned most efficiently in the context of the PEPCK promoter. The nuclear receptors peroxisome proliferator-activated receptor gamma  (PPARgamma ) and 9-cis-retinoic acid receptor (RXR) are required for enhancer function in vivo because: 1) a 3-bp mutation in the PPARgamma -/RXR-binding element centered at -992 bp, PCK2, completely abolished transgene expression in adipose tissue; and 2) electrophoretic mobility supershift experiments with specific antibodies indicated that PPARgamma and RXR are the only factors in adipocyte nuclear extracts which bind PCK2. In contrast, a second PPARgamma /RXR-binding element centered at -446 bp, PCK1, is not involved in adipocyte specificity because inactivation of this site did not affect transgene expression. Moreover, electrophoretic mobility shift experiments indicated that, unlike PCK2, PCK1 is not selective for PPARgamma /RXR binding. To characterize the enhancer further, the rat and human PEPCK 5'-flanking DNA sequences were compared by computer and found to have significant similarities in the enhancer region. This high level of conservation suggests that additional transcription factors are probably involved in enhancer function. A putative human PCK2 element was identified by this sequence comparison. The human and rat PCK2 elements bound PPARgamma /RXR with the same affinities. This work provides the first in vivo evidence that the binding of PPARgamma to its target sequences is absolutely required for adipocyte-specific gene expression.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cytosolic isozyme of phosphoenolpyruvate carboxykinase (PEPCK,1 EC 4.1.1.32) catalyzes the interconversion of oxaloacetate and phosphoenolpyruvate in adipose tissue and liver, yet PEPCK plays differing roles in the two tissues (1, 2). In liver, PEPCK is a rate-limiting gluconeogenic enzyme. However, white adipose tissue is not gluconeogenic because adipocytes do not express glucose-6-phosphatase and fructose-1,6-bisphosphatase, the enzymes that catalyze the two final steps of gluconeogenesis (2). Radioactive tracer studies in white adipose tissue indicate that PEPCK is involved in glyceroneogenesis, the synthesis of glycerol 3-phosphate from gluconeogenic precursors during fasting (1-3).

Both pathways are induced during fasting, and both are involved in fuel release but in contrasting ways. During a fast, gluconeogenesis provides a supply of glucose, whereas glyceroneogenesis is believed to prevent an all-out release of triglycerides liberated by lipolysis in white adipocytes (3). The role of glyceroneogenesis can be illustrated by comparing the sources of precursors for triglyceride synthesis during feeding and fasting. During feeding, triglycerides are synthesized from dietary fatty acids and glycerol-3-P, a metabolite of dietary glucose. During fasting, a large fraction of the fatty acids released from triglycerides by lipolysis is reesterified with glycerol-3-P in what appears to be a futile cycle of triglyceride degradation and resynthesis (3). Indeed, lipolysis and fatty acid reesterification are both induced during fasting, but a net release of free fatty acids does occur. Because fat cells do not express glycerol kinase they cannot phosphorylate the glycerol released from triglycerides (4). Hence the glycerol-3-P needed for fatty acid reesterification is derived from gluconeogenic precursors via glyceroneogenesis. This reesterification of fatty acids probably prevents ketogenesis during fasting (5). Diabetic ketoacidosis provides an example of the problems that occur with an unregulated release of fatty acids from adipocytes. Since PEPCK and glyceroneogenesis are induced proportionately as a result of the hormonal milieu of fasting, it was proposed that PEPCK is the rate-limiting glyceroneogenic enzyme (2, 5).

Because PEPCK plays different metabolic roles in white adipose tissue and liver we hypothesized that it might be regulated by distinct tissue-specific mechanisms in these two tissues. Extensive work focused upon PEPCK regulation in hepatocytes has revealed that transcription of the gene is under developmental and hormonal control via a complex upstream region within 600 bp of the promoter (1, 6). Much less effort has been directed toward understanding how PEPCK is regulated in adipocytes, but the available data indicate that it is quite different from the mechanisms in hepatocytes. We reported previously that PEPCK DNA upstream of -888 bp is required for expression of the PEPCK promoter in white adipose tissue of transgenic mice (7). In contrast, normal expression of the PEPCK promoter in liver was obtained with DNA elements downstream of -600 bp of the promoter (7). These observations and the fact that the PEPCK gene is a single-copy gene that is transcribed from the same promoter in both tissues indicate that there are separate and distinct hepatocyte- and adipocyte-specific enhancers that control PEPCK gene transcription (6).

The family of orphan nuclear receptors, the peroxisome proliferator-activated receptors (PPARs), were discovered during the past decade (8). Briefly, these transcription factors are activated by various hyperlipidemic drugs, xenobiotic compounds, fatty acids and their metabolites, and they regulate genes encoding proteins involved in lipid metabolism (8). In mammalian species there are three PPAR subfamilies: PPARalpha , PPARbeta , and PPARgamma . All three PPARs activate genes through their binding (as heterodimers with retinoid X receptors (RXR)) to single-spaced direct repeat (DR1) hormone response elements (5'-AGGTCANAGGTCA-3') (8). PPARalpha is most abundant in the liver and in various cells of the immune system where leukotriene B4 has been shown to be a natural activating ligand (9). PPARbeta is ubiquitous in its distribution, but its function and natural activating ligands are unknown (8).

Recent investigations have shown that PPARgamma is most abundant in adipose tissue and activated macrophages (10). It is a key factor responsible for differentiation of the adipocyte lineage during development (for review, see Ref. 10), and it is antiinflammatory and causes apoptosis in macrophages (11). Its activating ligands include the thiazolidinedione class of antidiabetic drugs and the natural compound, 15-deoxy Delta 12,14 prostaglandin J2 (12, 13).

The prototype adipocyte-specific enhancer is located 5 kilobase pairs upstream of the aP2 promoter and is activated by PPARgamma /RXR via two DR1 elements (14). We subsequently found two PPARgamma /RXR binding sites, designated PCK1 and PCK2, upstream of the PEPCK promoter (15). PCK1 is centered at -446 bp, and PCK2 is at -992 bp, which is within the region required for PEPCK activation in adipocytes. Transient transfection assays in 3T3-F442A adipogenic cells and NIH-3T3 fibroblasts showed that PCK2 is essential for differentiation-dependent enhancer activity and for transactivation by PPARgamma /RXR (15).

The purpose of this study was to determine whether the region of PEPCK DNA upstream of -888 bp constitutes a bona fide adipocyte-specific enhancer and whether PCK2 is required for its activity in transgenic mice. We also asked whether the proximal DR1 element, PCK1, is important for adipocyte-specific expression of the PEPCK gene. We have identified the 413-bp region between -1242 and -828 bp as an adipocyte-specific enhancer. This enhancer requires an intact PCK2 element, and it functions most efficiently in the context of the PEPCK promoter, suggesting that there are gene- and/or tissue-specific interactions between PPARgamma and other factors involved in regulating transcription. This work provides the first evidence that PPARgamma is required for adipogenic gene expression in vivo.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Transgene Constructs and Production of Transgenic Mice-- The nine transgene constructs utilized in this study are illustrated in Figs. 1A, 2A, and 3. All of the constructs were prepared by standard techniques from the rat PEPCK genomic DNA clone, lambda PC112, described previously (16). The -5000 hGH construct is an EcoRI-BglII fragment extending from -5000 bp to +69 bp inserted into the hGH vector, p0GH (17), digested with EcoRI and BamHI. The -2086 hGH construct, which has been described before (7), is a -2086 bp to +69 bp SstI-BglII fragment inserted into p0GH. The -888 hGH construct, also described previously (7), is a -888 bp to +69 bp Bal-31 deletion end point BglII fragment inserted into p0GH. The remaining six constructs all utilized hGX as the reporter (18). This reporter was made by generating a frameshift mutation in the translated sequence of hGH such that the encoded protein is biologically inactive (18). The -2086 hGX construct is identical to the -2086 hGH construct except for the hGX mutation in the reporter. The -1330 hGX construct is identical except that it terminates at a BsmI site at -1330 bp. The "Enhancer hGX" construct is a -1240 to -830 bp TaqI-BamHI fragment joined to the hGX gene at -90 bp. The promoter in this vector is therefore a minimal promoter (extending to -90 bp) from the hGH gene. Finally, the three genes bearing PPARgamma /RXR binding site mutations, PCK2M, PCK1M, and PCK1+2M, were all generated from the -2086 hGX construct. The PCK2 mutation (PCK2M) was inserted by swapping a -2086 bp to -14 bp SalI-NheI fragment from a PCK2M-CAT (chloramphenicol acetyltransferase) construct (15) for the same fragment in -2086 hGX. The sequence of PCK2M was 5'-GGGATCCTGGTCT-3', whereas the wild type PCK2 sequence is 5'-GGGATAAAGGTCT-3' (the mutated sequences are underlined). PCK1M was introduced via site-directed mutagenesis as described by Kunkel et al. (19). The sequence of PCK1M was 5'-TGACGATCGGCCG-3', whereas the wild type PCK1 sequence is 5'-TGACCTTTGGCCG-3' (note that this DR1 is in the opposite orientation from PCK2). In every case, the transgenes were digested and gel purified from plasmid vectors prior to injection of DNA into fertilized mouse eggs as we described previously (7).

Extraction and Measurement of mRNAs-- Mice were killed by cervical dislocation, and tissues were removed, frozen immediately in liquid nitrogen, and stored at -70 °C. Total RNA was extracted from the frozen tissues by the acid-guanidinium thiocyanate-phenol method (20) using Tri-ReagentTM (Molecular Research Laboratories, Cincinnati, OH) according to the manufacturer's instructions. PEPCK and hGH or hGX mRNAs were then assayed by either one of two methods. The first method was a solution hybridization-RNase protection assay described previously (7, 21) which was designed to assess the average number of mRNA molecules/cell. Briefly, the hybridized, RNase-resistant probe was collected by filtration of a trichloroacetic acid precipitate. Filter-bound radioactivity was then measured by scintillation counting. The molar amount of mRNA was calculated by comparison with a standard curve prepared using known amounts of pure target RNA (PEPCK or hGH). The second method was a standard RNase protection assay utilizing a kit purchased from Ambion (Austin, TX) according to the manufacturer's instructions. This method is more sensitive and allowed for the simultaneous assay of PEPCK, hGH (or hGX), and beta -actin mRNAs in a single tube.

The cRNA probes were the same for the two different RNase protection assays. The mouse PEPCK probe was transcribed from pmPCR10 using T3 RNA polymerase to synthesize an antisense RNA from a HindIII-digested template. The cRNA product is 411 bp in length, whereas the RNase-protected product is a 326-bp fragment. It was necessary to construct pmPCR10 for these experiments because a mouse PEPCK cDNA had not previously been cloned, and the rat PEPCK cDNAs, although more than 95% identical to mouse PEPCK, were not suitable for RNase protection assays. Polymerase chain reaction primers were designed by comparison of the sequences for rat, human, and chicken PEPCK cDNAs and searching for identical regions. Forward (5'-ATGCGGCCCTTCTTTGGCTA-3') and reverse (5'-TCCACCTCCTTCTCCCAGAA-3') polymerase chain reaction primers were selected and used to amplify mouse genomic DNA. A single product of the predicted size (326 bp) was obtained and inserted into the EcoRV site of pBluescript IISK+ (Stratagene, La Jolla, CA). This sequence corresponds to nucleotides 394-719 in the translated sequence from exon 10 of the rat PEPCK gene.

The cDNA, phGHex2, used for assay of hGH and hGX mRNAs was constructed from a region of exon 2/intron 2 (nucleotides 833-1011, 989-1011 is intron 2). The hGH/hGX cRNA probe was transcribed from phGHex2 using T3 RNA polymerase to synthesize an antisense RNA from a XhoI-digested template. The hGH cRNA transcript is 272 bp in length, whereas the RNase-protected product is a 156-bp fragment.

The beta -actin probe was supplied in the RNase protection assay kit from Ambion. The beta -actin cRNA transcript is 133 bp in length, whereas the RNase-protected product is a 161-bp fragment.

Nuclear Extracts and in Vitro Translated Proteins-- In vitro translation of RXRalpha -SPORT and PPARgamma 2-SPORT (15) plasmids was performed with the TNT SP6-coupled reticulocyte lysate system (Promega) as recommended by the manufacturer. 1 µl of the 50-µl translation product was used in each binding reaction. 3T3-L1 preadipocyte and adipocyte nuclear extracts were prepared as described previously, except that extracts were not dialyzed but were added directly to the binding reaction mixture (15). Final protein concentrations were typically 1-2 µg/µl.

Electrophoretic Mobility Shift Assays-- DNA-protein binding assays were performed in 15-µl incubations as described by Bretz et al. (22). Briefly, nuclear extracts and/or recombinant proteins were incubated in a reaction buffer consisting of 10 mM HEPES (pH 7.9), 1 mM EDTA (pH 8.0), 4% Ficoll, 100 mM dithiothreitol, 1 µg of poly(dI·dC) (Amersham Pharmacia Biotech), unlabeled double-stranded competitor DNA (where specified), and 1 µg of sonicated salmon sperm DNA (Sigma). These reactions were incubated at room temperature for 15 min before the addition of 4 pmol of a 32P-labeled double-stranded DNA probe followed by a second 15-min incubation.

The rat PCK2 probe was prepared as follows. A synthetic double-stranded DNA oligomer (sense strand, 5'-GATCACAACTGGGATAAAGGTCTCG-3'; antisense strand, 5'-GATCCGAGACCTTTATCCCAGTTGT-3') was end labeled by filling the 3'-recessed ends with [alpha -32P] dGTP (DuPont Easytides, 800 Ci/mmol) using the large fragment of Escherichia coli polymerase I (Klenow fragment). The unlabeled rat PCK2 double-stranded oligonucleotide probe thus includes the 13-bp DR1 sequence, six 5'- and two 3'-flanking bp, plus BamHI-compatible extensions at both ends. The human PCK2 and rat PCK1 probes were prepared in the same manner. Their sequences were as follows.

Human PCK2 sense: 5'-GATCACAACTGTTATAAAGGTTTCA-3'

Human PCK2 antisense: 5'-GATCTGAAACCTTTATAACAGTTGT-3'

Rat PCK1 sense: 5'-GATCCATGACCTTTGGCCGTGGGAG-3'

Rat PCK1 antisense: 5'-GATCCTCCCACGGCCAAAGGTCATG-3'

When antiserum was used, binding reactions were incubated with antiserum during the 15 min prior to the addition of the labeled DNA. Sera used included: 1) polyclonal rabbit anti-human RXRalpha (directed against amino acids 214-229) obtained from R. Evans, Salk Institute (23); 2) polyclonal rabbit anti-mouse PPARgamma 2 described previously (24); and 3) normal rabbit serum purchased from Sigma. DNA-protein complexes were resolved on 5% polyacrylamide gels (30:1 acrylamide:bisacrylamide) in 0.5 × TBE (1 × TBE, 50 mM Tris borate (pH 8.3), 1 mM EDTA). The gels were dried and exposed to a Phosphor screen overnight. Quantitation was performed with a PhosphorImaging device (Molecular Dynamics).

DNA Sequence Analysis-- The rat adipocyte-specific enhancer was sequenced on an automated sequenator from Applied Biosystems in the Institute for Biotechnology Core Laboratory, Texas Tech University. Both DNA strands were sequenced in their entirety. All computer analyses were performed using the Wisconsin Package Version 9.0 software, Genetics Computer Group (GCG), Madison, WI.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of the Systemic Production of hGH on Transgene Expression-- In a previous study using transgenic mice we utilized the hGH gene as the reporter to define tissue-specific elements upstream of the PEPCK promoter (7). A comparison of endogenous PEPCK expression in tissues of normal versus transgenic animals indicated that the high serum concentrations of hGH did not affect transgene expression (7). However, a similar study was reported by McGrane et al. (25) who used bovine growth hormone (bGH) as the reporter. Serum bGH, at high concentrations, caused hyperinsulinemia and significantly repressed both endogenous PEPCK and transgene expression in their mice. Although we did not observe a similar effect with hGH, the use of a biologically active hormone as reporter risks introducing artifacts. We therefore obtained hGX, a reporter encoding a biologically inactive mutant form of hGH, from Dr. Stanley McKnight (18). We used hGX to produce new transgenic mice in which the rat PEPCK promoter was identical to the -2086 to +69 bp construct that we used previously (7). The two "-2086" transgene constructs are shown in Fig. 1A. Transgene expression in white adipose tissue and liver of these two groups of mice was measured by quantitative solution hybridization assay, and the results are shown in Table I and Fig. 1, B and C. There was no statistically significant difference in transgene expression between the hGH and the hGX group in either liver or adipose tissue (p = 0.65 and 0.28 for liver and adipose tissue, respectively). This supports our previous conclusion that hGH does not confound the conclusions and demonstrates that comparisons between hGX and hGH transgenes are valid. It does appear that there could be a slight, albeit statistically insignificant, inhibitory effect of expression in adipose tissue, however. To avoid potential unanticipated artifacts caused by hGH we have prepared all subsequent transgenes with hGX.


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Fig. 1.   An adipocyte-specific element is localized between -1330 and -888 bp. Panel A, the five transgene constructs analyzed are depicted. All constructs are designated by their upstream deletion end points and the reporter gene used. The two different reporter genes were hGH and hGX (designated by the bullet, ), which encodes a biologically inactive protein (18). The filled regions represent PEPCK DNA from the indicated upstream deletion end point to +69 bp in exon 1. This downstream end point is upstream of the initiator methionine codon. The unfilled regions represent the reporter genes (hGH or hGX). The wide lines and boxes represent transcribed DNA (+1 is indicated by the arrow); the thick lines represent upstream DNA. Details of these vectors are given under "Experimental Procedures." Panel B, the number of hGH or hGX transcripts/cell in white adipose tissue from each group of transgenic mice was measured by a solution hybridization assay as described under "Experimental Procedures." The results are presented as the average ± S.E. for each group of mice. The actual values for each mouse are shown in Table I. Panel C, same as panel B except that the results for liver are shown.

                              
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Table I
Transgene expression in individual mice
The amount of mRNA encoded by each of the five transgenes was measured in WAT (white adipose tissues) and livers of individual transgenic mice by a solution hybridization assay. The mRNA levels are presented as molecules/cell. Transgene expression in WAT was not determined (ND) in three mice (66-6-2, 294-6, and 288-5) because there was inadequate tissue available for assay. The average ± S.E. for each transgene is plotted in Fig. 1. The designation number for each individual transgenic mouse is shown in the columns headed "Mouse," and an estimate of the number of transgene copies/haploid genome is shown in parentheses. This latter number was estimated from DNA dot blots. Details of the assays are described under "Experimental Procedures." A least squares analysis of variance was performed on a logarithmic transformation of the data using SAS 1997 on a personal computer. Animal designation number, transgene construct, and tissue type were the factors included in the statistical model. Significance was determined with the Predicted Difference Test in the General Linear Models procedure. Significant differences: a, different from -2086 hGH in WAT (p = 0.05); b, different from -1330 hGX in liver (p = 0.05); c, different from -1330 hGX in WAT (p = 0.02); d, different from all other transgene groups (p = 0.0001); e, marginal difference from -5000 hGH in liver (p = 0.06). There were no other significant differences between groups.

The Adipocyte-specific Element Is Located between -1330 and -888 Base Pairs Upstream of the PEPCK Promoter-- In addition to the two -2086 bp constructs just described, Fig. 1 also shows the structures and expression levels of two new constructs, -5000 bp hGH and -1330 bp hGX, which have not been reported previously. Moreover, the -888 bp hGH construct described previously (7) is shown for comparison purposes.

We concluded previously that an adipose-specific element is localized between -888 bp and -2086 bp because the -2086 but not the -888 transgene (Fig. 1A) was expressed in adipose tissue (Fig. 1B, Table I, and Ref. 7). Considering this observation we prepared the -1330 bp hGX construct (Fig. 1A) and found that it is expressed in both adipose tissue and liver (Fig. 1, B and C). This localized the adipose-specific element between -1330 bp and -888 bp.

The -5000 bp hGH construct was prepared to test whether important regulatory elements are located upstream of -2086 bp, the most distal end point we had tested previously. We used hGH as the reporter in this construct because these animals were prepared prior to our having tested the hGX reporter. Compared with the -2086 hGH mice, transgene expression in the -5000 hGH animals was significantly higher in adipose tissue (p = 0.05), but not liver (p = 0.30), indicating that this upstream region contains one or more stimulatory elements that function in adipocytes. However, transgene expression in the liver of the -5000 hGH animals was significantly higher than in the -1330 hGX mice (p = 0.05). This indicates the presence of a stimulatory "liver element" somewhere between -5000 bp and -1330 bp.

The Region between -1240 Base Pairs and -830 Base Pairs Is an Adipocyte-specific Enhancer in Vivo-- Transient transfection assays in cultured cells suggested the presence of a "differentiation-dependent" enhancer between -460 bp and -2086 bp (15). This enhancer required a PPARgamma /RXR-binding element, PCK2, centered at -992 bp (15). In the present study we asked whether this differentiation-dependent enhancer had the characteristics of an adipocyte-specific enhancer. To test this possibility we employed convenient restriction sites to generate the enhancer construct illustrated in Fig. 2A. Analysis of a total of six founder mice bearing the "enhancer" construct showed that it is expressed only in white and brown adipose tissue and not in liver, kidney, or intestine, the only other tissues known to express the PEPCK gene at high levels (Fig. 2, B and C). Panel B shows both PEPCK and transgene expression in nine tissue samples of a single founder animal. The same pattern of expression was seen in the other five founders, but only three tissues are shown for all six animals in panel C. Because the -1240/-830 bp PEPCK region directs transgene expression from a heterologous minimal promoter at a distance of only 90 bp from the promoter, instead of the native 830 bp, we have designated this element as an adipocyte-specific enhancer.


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Fig. 2.   The upstream element is an adipocyte-specific enhancer in vivo. Panel A, the two transgene constructs analyzed are depicted as in Fig. 1. The Enhancer construct consists of PEPCK DNA from -1240 to -830 bp fused at -90 bp of the basal hGH promoter; this fusion is indicated by the thin bent line. The -1330 construct is the same as that shown in Fig. 1. Panel B, PhosphorImage of an RNase protection assay for both hGX and PEPCK mRNAs in total RNA samples isolated from nine tissues of a representative enhancer mouse (founder number 360-3). The RNase-protected products representing the respective mRNAs are designated by labeled arrows. The tissues are: liver (Liv), kidney (Kid), white adipose tissue (WAT), brown adipose tissue (BAT), ileum (Ile), skeletal muscle (SkM), jejunum (Jej), colon (Col), and lung (Lun). Details of the RNase protection assay are described under "Experimental Procedures." Panel C, same as panel B except that hGX mRNA in total RNA samples isolated from liver (L), kidney (K), and white adipose tissue (F) from all six enhancer mice is shown. The individual mouse numbers are indicated. Panel D, same as panel B except that hGX and PEPCK mRNAs in total RNA samples isolated from white adipose tissue from all six enhancer mice and all eight -1330 mice are shown. The far right lane (P) shows the undigested PEPCK and hGX cRNA probes.

During analysis of these "enhancer mice" we noted that the level of expression of the transgene was only approximately 5% of the level obtained with either the -2086/hGX or the -1330/hGX mice. This difference is illustrated in Fig. 2D, which shows the results of an RNase protection assay performed on white adipose tissue RNA from the six enhancer founders and the eight -1330 founders. The average PEPCK mRNA concentration was the same for both groups of animals, but transgene expression was 10-20-fold higher in the -1330 mice. This could be a positional effect of moving the enhancer too close to the promoter for optimal interaction of transcription factors and cofactors. Alternatively, it could be that optimal transcription of the PEPCK gene requires the interactions of enhancer-bound factors with promoter-associated proteins.

Function of the Adipocyte-specific Enhancer in Vivo Requires an Intact PPAR Response Element(PCK2)-- We reported previously the presence of two PPARgamma /RXR binding sites, PCK1 centered at -446 bp, and PCK2 centered at -992 bp, and we hypothesized that expression of PPARgamma , an adipocyte-specific transcription factor, is responsible for turning on PEPCK gene expression via PCK2 during adipogenesis (15).

We therefore asked whether mutations of PCK2 and/or PCK1 will block transgene expression in transgenic mice. Three new groups of transgenic mice, PCK2M, PCK1M, and PCK1+2M, were produced, and transgene expression was compared with the wild type transgene counterpart, -2086/hGX (Fig. 3, A-D). Fig. 3A shows an RNase protection analysis of both PEPCK and hGX mRNAs in white adipose tissue and liver from nine founder mice with the wild type PEPCK construct. The relative level of transgene expression in the liver corresponded to the level of expression in adipose tissue in these mice. By contrast, in seven founder PCK2M mice, the 3-bp mutation in PCK2 eliminated transgene expression in white adipose tissue but not in liver (Fig. 3B). A similar pattern of expression was also seen in brown adipose tissue (not shown). Thus PCK2 is essential for expression of the PEPCK gene in both white and brown adipocytes.


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Fig. 3.   PCK2 but not PCK1 is essential for activity of the adipocyte-specific enhancer. The figure shows PhosphorImages of RNase protection assays for both hGX and PEPCK mRNAs (as in Fig. 2D) in total RNA samples isolated from white adipose tissues and livers of nine -2086 hGX mice (panel A; the same animals used in Fig. 1 and Table I), seven PCK2M mice (panel B), four PCK1M mice (panel C), and six PCK1+2M mice (panel D). The four transgene constructs analyzed are depicted as in Fig. 1A at the top of their respective panels. The two DR1 sites that bind PPARgamma /RXR heterodimers, PCK1 at -446 bp and PCK2 at -992 bp, are indicated by open circles in the upstream DNA regions. Mutations of these elements are depicted by an X over the symbol. The RNase protection assays and PhosphorImage exposures for all four panels were done simultaneously so that all panels are directly comparable.

Fig. 3C shows the same analysis of the four founder mice bearing the PCK1 mutation. It appears that this mutation decreases, but does not eliminate, transgene expression in either adipose tissue or liver. Unfortunately, we obtained too few mice in this group to determine with certainty whether a PCK1 mutation affects expression in these two tissues. We can, however, conclude that PCK1, unlike PCK2, is not essential for PEPCK expression in adipose tissue.

Fig. 3D shows the analysis of the final group of six founder mice bearing the PCK1 + PCK2 double mutation. There was no transgene expression in adipose tissue, an effect that can be attributed to the PCK2 mutation.

In Adipocyte Nuclear Extracts, Only PPARgamma /RXR Binds PCK2-- We previously used an RXRalpha -specific antibody to show by electrophoretic mobility supershift analysis that RXRalpha is a component of the binding species from adipocyte nuclear extracts. Since that time a PPARgamma -specific antibody has been produced (24). These two antibodies were used in the experiment shown in Fig. 4 to demonstrate that virtually all of the PCK2-binding species contain both PPARgamma and RXR. Moreover, there is no detectable PCK2 binding activity in preadipocyte nuclear extracts which, along with the above data, supports the idea that binding of PPARgamma /RXR heterodimers to PCK2 activates the adipocyte-specific PEPCK enhancer during adipogenesis.


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Fig. 4.   PCK2 is bound exclusively by PPARgamma /RXR heterodimers in adipocyte nuclear extracts. A PhosphorImage of electrophoretic mobility shift reactions using PCK2 as the probe is shown. The sources of added protein were: in vitro translated recombinant PPARgamma and RXRalpha (lanes 1-4), preadipocyte nuclear extracts (lanes 5 and 6), and adipocyte nuclear extracts (lanes 7-10). Other additions to the binding reactions included: no additions, normal rabbit serum (NRS), antiserum to PPARgamma (PPARgamma Ab), or antiserum to RXRalpha (RXRalpha Ab) as indicated. The arrow indicates the migration of PPARgamma · RXRalpha ·PCK2 complexes. Free probe is seen at the bottom of each lane. Details of the assay are described under "Experimental Procedures."

PCK1 and PCK2 Bind Different Factors-- The experiment shown in Fig. 3 shows that only one of the two PPARgamma /RXR binding sites, PCK2, is involved in adipocyte-specific expression of the PEPCK promoter. Because PPARgamma /RXR heterodimers bind PCK1 and PCK2 with approximately equivalent affinities (15) we asked whether the two sites differed in their selectivities for different binding proteins. The mobility shift experiment shown in Fig. 5 indicates that this is indeed the case because: 1) with preadipocyte nuclear extracts there was no detectable binding to PCK2, whereas there were several species bound to PCK1; and 2) with adipocyte nuclear extracts the banding patterns displayed both similarities and differences between PCK1 and PCK2.


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Fig. 5.   PCK2 is selectively bound by factors in adipocyte but not preadipocyte nuclear extracts, whereas PCK1 is bound by factors in both extracts. PhosphorImages of electrophoretic mobility shift reactions using PCK1 and PCK2 as probes are shown. The sources of added protein were in vitro translated, recombinant PPARgamma and RXRalpha (R); 3T3-L1 adipocyte nuclear extract (A); and 3T3-L1 preadipocyte nuclear extract (P). The probes were rat PCK1 (lanes 1-3) and rat PCK2 (lanes 4-6). The arrow indicates the migration of recombinant PPARgamma ·RXRalpha ·PCK2 complexes. Free probe is seen at the bottom of each lane. Details of the assay are described under "Experimental Procedures."

The Adipocyte-specific Enhancer Region Is Conserved in Rats and Humans-- It is likely that DNA-binding proteins in addition to PPARgamma /RXR are involved in making the region between -1242 and -828 bp an adipocyte-specific enhancer. Because important regulatory sequences are often conserved evolutionarily we compared the rat with the human PEPCK promoters and upstream DNAs as part of our strategy to dissect and characterize the enhancer. The dot plot in Fig. 6A shows that there are three significant regions of similarity: the promoter-proximal region of approximately 500 bp; an intermediate region between approximately -600 and -800 bp; and a distal region upstream of -900 bp within the adipocyte-specific enhancer. The exact sequence alignment of the enhancer region is shown in Fig. 6B. This alignment revealed a putative human PCK2 element. This element matches rat PCK2 at 10 of the 13 nucleotides that constitute the DR1. It is clear that the 5'-flanking nucleotides are critical to PPARgamma /RXR binding to DR1 elements (26). The rat and human PCK2s are identical at 11 of 12 5'-flanking nucleotides (whereas rat PCK1 and PCK2 are identical at only 3 of 12, which may explain the differences seen in Fig. 5). To determine whether PPARgamma /RXR binds human PCK2 the mobility shift experiment shown in Fig. 7 was performed. The results of this experiment show that the binding affinities of the rat and human elements are virtually identical.


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Fig. 6.   The rat and human adipocyte-specific enhancers are highly homologous. Panel A, the human and rat PEPCK promoters from -1300 to -1 bp were compared using COMPARE and DOTPLOT in the Wisconsin Package. A dot was placed at every coordinate containing identical nucleotides within windows of 21 bp containing a minimum of 14 matches (stringency). The box encloses the enhancer regions that are shown in panel B. The locations of the PCK2 elements are marked with a solid box on each axis. Panel B, the rat enhancer region defined by the experiment in Fig. 2 was compared with the human PEPCK promoter using GAP and PILEUP in the Wisconsin Package. The optimal alignment is shown here. PCK2 and three additional potential PPARgamma /RXR binding sites on the rat DNA are shaded. Potential C/EBP binding sites, sequences containing the ATTGCGCAAT consensus with up to four mismatches, are overlined for the rat and underlined for the human sequences. The GenBank Accession numbers for the DNAs used in this figure were K03243 (rat) and U31519 (human).


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Fig. 7.   PPARgamma /RXRalpha binds rat PCK2 and human PCK2 with equal affinity. A PhosphorImage of electrophoretic mobility shift reactions using either rat or human PCK2 as probe is shown. The source of added protein is indicated at the top of the figure: no lysate, unprogrammed rabbit reticulocyte (RR) lysate, or lysate programmed with mRNAs encoding PPARgamma and RXRalpha . The probes were rat PCK2 (lanes 1-10) and human PCK2 (lanes 11-20). Unlabeled competitors were added as indicated: no competitor; unlabeled rat or human PCK2 at 10-, 50-, and 100-fold molar excess; or a nonbinding mutant rat PCK2 (m) at 100-fold excess. The arrow indicates the migration of PPARgamma ·RXRalpha ·PCK2 complexes. Free probe is seen at the bottom of each lane. Details of the assay are described under "Experimental Procedures."

To assist in dissecting the enhancer, several computer analyses were performed: TESS with the Transfac 3.2 data base at http://agave.humgen.upenn.edu; MAP (Wisconsin Package) with its Transcription Factor Data Base (27); and the Institute for Transcriptional Information object-oriented transcription factor data base at www.ifti.org (28). Not surprisingly this produced a lengthy list of candidate binding sites including elements for C/EBP family members, CTF/NF-1, Sp-1, AP-1, and PPARgamma /RXR. Indeed there were three putative PPARgamma /RXR binding sites in addition to PCK2 (Fig. 6B). One might expect that factors bound at multiple sites could strengthen the enhancer. In support of this idea is the adipocyte-specific enhancer of the aP2 gene which has two PPARgamma /RXR binding sites (29). However, mobility shift assays such as those in Fig. 7 showed that none of these additional sites binds PPARgamma /RXR (data not shown).

Of the remaining candidate factors, the C/EBP family is particularly relevant because they are intimately involved in adipocyte differentiation (30) and regulation of the PEPCK gene in hepatocytes (1). Several of the putative C/EBP sites (Fig. 6B) are highly conserved between rats and humans, suggesting that they could be involved in adipocyte-specific enhancer activity. We are currently investigating which, if any, of these putative sites are functional. It will also be necessary to investigate whether any of the other candidate factors are involved.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Adipocyte-specific PEPCK Enhancer Requires the Binding of PPARgamma /RXR to PCK2-- In this study we have identified as an adipocyte-specific enhancer a 410-bp region located 1 kilobase pair upstream of the PEPCK promoter. Expression of the PEPCK promoter in adipocytes is totally dependent upon the presence of this enhancer. Like that of the aP2 gene (29), the adipocyte-specific enhancer of the PEPCK gene contains a DR1 element, PCK2, that binds a PPARgamma /RXR heterodimer. More importantly, we show that PCK2 must be intact for this enhancer to function. This latter observation, coupled with the observation that PPARgamma and RXR are the only factors in adipocyte nuclear extracts which bind PCK2, provides strong evidence that PPARgamma /RXR is essential for expression of the PEPCK gene in adipocytes.

The adipocyte-specific enhancer of the PEPCK gene is apparently unimportant for tissue-specific expression of the PEPCK gene in liver and kidney because we observed no differences in transgene expression associated with the presence or absence of the enhancer in these tissues. This major regulatory difference between adipose tissue and liver or kidney is in keeping with the functional difference of PEPCK between these tissues.

The Enhancer-Promoter Interaction May Be Gene-specific-- Reporter expression in adipose tissue of the Enhancer hGX mice was only 5-10% that of the -1330 hGX construct. One possible explanation for this is that the spacing between the enhancer and the promoter, which has been modified, is critical for optimal enhancer function. However, the explanation that we prefer is that the PEPCK adipocyte-specific enhancer functions most efficiently in the context of the PEPCK promoter because of gene and/or adipocyte-specific interactions among proteins, perhaps PPARgamma , bound to the enhancer, with promoter-associated transcription factors or cofactors.

Does PCK2 Play a Regulatory Role in Addition to Development?-- Although PCK2 is not involved in the developmental activation of PEPCK in liver, it may be involved in the modulation of PEPCK expression by fatty acids in both liver and adipose tissue. Indeed, various fatty acids induce PEPCK gene expression in adipocytes and cultured hepatoma cells (31). Thus PPARgamma in adipocytes and other PPAR isoforms might serve as fatty acid-regulated transcriptional activators through their binding to PCK2 in hepatocytes. PCK2 could thus serve a dual function: as part of a developmental switch to activate the PEPCK gene during adipogenesis and as a fatty acid-responsive switch to regulate the PEPCK gene in adipocytes (and possibly hepatocytes) subsequent to embryonic and fetal development. It has yet to be determined, however, whether PPARalpha and/or PPARbeta binds to PCK2 in hepatocytes or other cell types.

Is There a Locus Control Region Upstream of -2086 Base Pairs?-- Chalkley and colleagues (32,33) concluded that a hepatocyte-specific element that binds a CREB/ATF factor and Pep A is located at -4800 bp. Our observation that there is a significant difference in liver between the -5000 hGH and the -1330 hGX mice is consistent with this conclusion; however, additional transgenic animals would need to be produced to achieve the statistical power necessary to confirm this issue. Our data do suggest that one or more elements upstream of -2086 bp are involved in PEPCK regulation in adipocytes because there was a significant increase in transgene expression when 5000 bp of upstream DNA was included.

Chalkley and colleagues also suggested that the element at -4800 bp may be a liver-specific locus control region that causes transgene expression to be proportional to the number of integrated copies (32, 33). Linear regression analysis of the data in Table I revealed a significant correlation between copy number and transgene expression in both liver (r2 = 0.79) and white adipose tissue (r2 = 0.81) of the -5000 hGH mice. There was a weaker, albeit significant, correlation in the liver (r2 = 0.62) and white adipose tissue (r2 = 0.64) of the -2086 hGX mice. This finding is consistent with but does not prove the presence of liver and adipose tissue locus control regions upstream of the PEPCK gene.

What Is the Function of PCK1?-- The data presented here suggest that PCK1, the proximal PPARgamma /RXR binding site, is not required for PEPCK expression in vivo, even though it is also bound by PPARgamma /RXR heterodimers in vitro (Figs. 3 and 5). A possible explanation was suggested by electrophoretic mobility shift experiments that revealed that PCK2 is highly selective in its binding to PPARgamma /RXR in adipocyte nuclear extracts, and it is unoccupied in preadipocyte nuclear extracts (Figs. 4 and 5). PCK1, on the other hand, is bound by several factors in both preadipocyte and adipocyte nuclear extracts (Fig. 5). This difference in factor selectivity is probably related to contextual differences in the flanking nucleotide sequences and/or the nonconsensus half-site differences between the two DR1s. The 5'- flanking sequence of PCK2 is more similar to an optimal binding sequence for PPARgamma /RXR (26) compared with PCK1. Also, PCK1 is in the opposite orientation from PCK2, and this inverted polarity could potentially contribute to its function in vivo. Nonetheless, the available data indicate that PCK1 and PCK2 have differing functions. We previously pointed to the fact that PCK1 is the same as the AF-1 site identified by Granner and colleagues (34) as part of a complex glucocorticoid-responsive unit in hepatoma cells. They demonstrated that with hepatoma cell extracts PCK1/AF-1 is occupied by several factors including RAR, RXR, HNF4, and COUP-TF (35, 36). An interesting enigma is the fact that glucocorticoid hormones induce PEPCK transcription in hepatocytes but repress it in adipocytes (1, 2). Perhaps it is the tissue-specific difference in the factors bound to PCK1/AF-1 which renders the complex glucocorticoid response unit an activator in hepatocytes and an inhibitor in adipocytes.

What Do the Similarities between the Rat and Human PEPCK Promoters and Flanking DNAs Suggest?-- The simplest suggestion is that the conserved regions point to transcription factor binding sites that are critical to some aspect of PEPCK expression. It is impossible to ascribe adipocyte-specific functions to any region other than PCK2 because the PEPCK gene is regulated by numerous hormones and other factors and is expressed in adipocytes, hepatocytes, intestinal epithelium, and proximal tubule epithelium of the kidney. Based upon the available evidence (1, 6) we suggest that the proximal 500-bp region is responsible for liver- and kidney-specific expression and for the complex regulation by cAMP and other factors. We also suspect that this region interacts with the adipocyte-specific enhancer to cause the effect shown in Fig. 2D. There is no information available to suggest a function of the intermediate region between -600 and -800 bp. This will require further investigation. The distal homology we have defined here is an adipocyte-specific enhancer. This region must also be the target of future studies.

Summary-- We have found a second example of an adipocyte-specific enhancer; the first example is the aP2 gene (14). More importantly, we have provided the first in vivo evidence that the function of an adipocyte-specific enhancer is absolutely dependent upon the binding of PPARgamma /RXR to its cognate binding site. Studies are now under way to define the minimal enhancer and to understand the molecular mechanisms that modulate and mediate the adipocyte-specific actions of PPARgamma and RXR.

    ACKNOWLEDGEMENTS

We thank Ida Schaefer, Joan Clapper, and Matthew Laverdiere for technical assistance; Drs. Walter Wahli and Simon C. Williams for helpful comments on the manuscript; Dr. John J. McGlone for guidance with statistical analyses; Dr. G. Stanley McKnight for the hGX vector; and Dr. Ronald M. Evans for the antibody directed against RXRalpha and the RXRalpha -SPORT expression vector.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R01GM39895 and Texas Advanced Research Program Grant 010674 (to E. G. B.) and by the Howard Hughes Medical Institute and Perot Family Foundation funds (to R. E. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) K03243 for rat PEPCK promoter with PCK1 and PCK2, K03248 for rat PEPCK exon 10, and M13438 for hGH exon 2/intron 2.

Present address: Howard Hughes Medical Institute, Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75235-9050.

** Present address: The Salk Institute for Biological Studies, La  Jolla, CA 92037.

Dagger Dagger To whom correspondence to should be addressed. Tel.: 806-743-2705; Fax: 806-743-2990; E-mail: bioegb{at}ttuhsc.edu.

    ABBREVIATIONS

The abbreviations used are: PEPCK, cytosolic phosphoenolpyruvate carboxykinase; bp, base pair(s); PPAR(s), peroxisome proliferator-activated receptor(s); RXR, 9-cis-retenoic acid receptor; DR1, single-spaced direct repeat; hGH, human growth hormone; hGX, frameshift mutant of hGH encoding a biologically inactive protein; bGH, bovine growth hormone.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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