Glucocorticoids Regulate Transcription of the Gene forPhosphoenolpyruvate Carboxykinase in the Liver viaan Extended Glucocorticoid Regulatory Unit*

Received for publication, April 15, 2005, and in revised form, July 15, 2005 Published, JBC Papers in Press, August 12, 2005, DOI 10.1074/jbc.M504119200

Hanoch Cassuto‡, Karen Kochan‡, Kaushik Chakravarty§, Hannah Cohen‡, Barak Blum‡, Yael Olswang‡,Parvin Hakimi§, Chuan Xu§, Duna Massillon§, Richard W. Hanson*, and Lea Reshef‡1

From the ‡Department of Developmental Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, 91120 Israeland §the Departments of Biochemistry and Nutrition, Case Western Reserve University, Cleveland, Ohio 44106-4935

The hepatic transcriptional regulation by glucocorticoids of thecytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C)gene is coordinated by interactions of specific transcription factorsat the glucocorticoid regulatory unit (GRU). We propose anextended GRU that consists of four accessory sites, two proximalAF1 and AF2 sites and their distal counterpart dAF1 (�993) and anew site, dAF2 (�1365); together, these four sites form a palin-drome. Sequencing and gel shift binding assays of hepatic nuclearproteins interacting with these sites indicated similarity of dAF1and dAF2 sites to the GRU proximal AF1 and AF2 sites. Chromatinimmunoprecipitation assays demonstrated that glucocorticoidsenhanced the binding of FOXO1 and peroxisome proliferator-acti-vated receptor-� to AF2 and dAF2 sites and not to dAF1 site butenhanced the binding of hepatic nuclear transcription factor-4�

only to the dAF1 site. Insulin inhibited the binding of these factorsto their respective sites but intensified the binding of phosphoryl-ated FOXO1. Transient transfections in HepG2 human hepatomacells showed that glucocorticoid receptor interacts with severalnon-steroid nuclear receptors, yielding a synergistic response of thePEPCK-C gene promoter to glucocorticoids. The synergistic stim-ulation by glucocorticoid receptor together with peroxisome prolif-erator-activated receptor-� or hepatic nuclear transcription fac-tor-4� requires all four accessory sites, i.e. a mutation of each ofthese markedly affects the synergistic response. Mice with a tar-geted mutation of the dAF1 site confirmed this requirement. Thismutation inhibited the full response of hepatic PEPCK-C gene todiabetes by reducingPEPCK-CmRNA level by 3.5-fold and the levelof circulating glucose by 25%.

Transcription of the gene for PEPCK-C2 (EC 4.1.1.32) is acutely con-trolled in a tissue-specific manner by diet and hormones (1, 2). The

interaction between glucagon (acting via cAMP) and glucocorticoidsthat stimulate gene transcription and insulin, which inhibits this proc-ess, determines the level of hepatic PEPCK-C. Glucocorticoids stimu-late transcription of this gene in the liver and kidney cortex (3) andinhibits it in adipose tissue (4); metabolic acidosis induces PEPCK-Cgene transcription in the kidney cortex but has no effect in the liver (3).A number of transcription factors have been implicated in this complex,tissue-specific regulation of PEPCK-C gene transcription (5–8).Glucocorticoids play a particularly important role in coordinating the

control of PEPCK-C gene transcription in a tissue-specific manner.Olswang et al. (9) reported that glucocorticoids repressed PEPCK-Cgene transcription in adipose tissue by interfering with the DNA bind-ing of members of the C/EBP family of transcription factors to specificsites in the gene promoter. Glucocorticoids are also known to inhibit thetranscription of the gene encoding C/EBP� in adipocytes (10). Theinduction of renal PEPCK-C gene transcription by glucocorticoidsrequires an intact and occupied hepatic nuclear factor 1 (HNF-1) bind-ing site, which is the only renal-specific binding site identified to date inthe PEPCK-C gene (7); this site is not required for PEPCK-C gene tran-scription in either the liver or adipose tissue (6, 11, 12).An important advance in understanding the mechanisms by which

glucocorticoids alter the regulation of PEPCK-C gene transcriptionstems from the work of Granner and co-workers (13), who have identi-fied a region of the PEPCK-C gene promoter, which they termed theglucocorticoid regulatory unit (GRU). This region of the gene promoterextends from approximately �455 to �321 and contains three acces-sory protein binding domains (14), one of which (AF2) overlaps a site inthe promoter that is involved in the repression of PEPCK-C gene tran-scription by insulin (15). The AF1 site binds HNF-4� (16), chickenovalbumin upstream transcription factor (COUP-TF) (17), peroxisomeproliferator activated receptor (PPAR�2) (18), retinoic acid receptor(RAR�) (19), and retinoid X receptor (RXR�) (20), whereas AF2 bindsmembers of the Forkhead family of transcription factors, includingFOXO1 (21) andHNF-3� (Foxa2) (22). Recently Stoffel and co-workers(23) reported that the AF2 site in the PEPCK-C gene promoter prefer-ably binds FOXO1 and only minimally HNF-3�. It has been proposedthat C/EBP� interacts at this site with other transcription factors as partof a nucleoprotein complex to regulate PEPCK-C gene transcription(24).Despite the key role played by the GRU in the control of PEPCK-C

gene expression, there is evidence that it is not sufficient to entirelyexplain the effects of glucocorticoids on transcription of this gene. Asegment of the PEPCK-C gene promoter from �540 to �73 bp wasshown to be sufficient to confer full hepatic expression of a transgene inmice (25–27). However, sequences upstream of position�540 play reg-ulatory roles beyond the basal expression of the gene. For example, in a

* This research was supported by United States-Israel Binational Science FoundationGrant 1999346 and by a grant from the Ministry of Health of Israel (to L. R.) and byNational Institutes of Health Grant DK22541 (to R. W. H.). The costs of publication ofthis article were defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Dept. of Biochemistry, Hebrew Uni-versity-Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel. Tel.: 972-2-6758291; Fax: 972-2-6757379; E-mail: reshef@cc.huji.ac.il.

2 The abbreviations used are: PEPCK-C, the cytosolic form of phosphoenolpyruvate car-boxykinase; HSS, hypersensitive site; GR, glucocorticoid receptor; GRE, GR responseelement; GRU, glucocorticoid response unit; RAR, retinoic acid receptor; RXR, retinoidX receptor; PPAR, peroxisome proliferator-activated receptor; PPARE, PPAR responseelement; RT, reverse transcriptase; CAT, chloramphenicol acetyltransferase; FOXO1,Forkhead box class O-1, FKHR, Forkhead receptor; Foxa2, Forkhead box class A-2;HNF-3�, hepatocyte nuclear factor 3�; HNF-4�, hepatocyte nuclear factor 4�.;COUP-TF I and II, chicken ovalbumin upstream transcription factor I and II, respec-tively; ChIP, chromatin immunoprecipitation.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 40, pp. 33873–33884, October 7, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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fashion similar to the endogenous PEPCK-C gene, a transgene driven bya region of the gene promoter from �2000 to �73 is not expressed inthe fetal liver (26, 27), whereas a transgene driven by segment of the genepromoter from 540 to �73 is expressed (28). These findings are sup-ported by results from transient transfection experiments usingHepa1c1c7 mouse hepatoma cells, which mimic the fetal liver. In theseexperiments the activity of a segment of the PEPCK-C gene promoterfrom �600 to �73 has a rate of transcription that is 5-fold higher thanthat of the counterpart 2000-bp gene promoter (28). Finally, glucocor-ticoids failed to stimulate transcription from a 500-bp segment of the ratPEPCK-C gene promoter in HepG2 cells (29), although glucocorticoidswere shown to induce the expression of the endogenous gene in thesecells (30). This suggested that the GRU extends upstream of the regionthat was described originally.This notion is further supported by the discovery of hypersensitive

sites (HSS) in the rat PEPCK-C gene promoter (31). A HSS locus wasidentified that is composed of two adjacent sub-sites, one of whichmapped to positions �999 to �987; this coincides with PPAR bindingdomain in the PEPCK-C gene promoter (18). The second site was spe-cific to PEPCK-C-expressing hepatoma cells and mapped to position�1400 (31). Subsequently it was found in committed and differentiatedadipocytes (32) but not in kidney cells (33). In the current study we havefurther characterized both sub-sites of theHSS B, at�993 and at�1400of the PEPCK-C gene promoter and have found that together these sitestake part in an extended GRU. This extended GRU is liver-specific andplays an important role in the regulation of PEPCK-Cgene transcriptionby glucocorticoids. The glucocorticoid receptor (GR) in the presence ofglucocorticoids interacts with non-steroid nuclear receptors, leading toa synergistic liver-specific stimulation of PEPCK-C gene expression,which requires all four accessory sites. This is supported by the demon-stration that diabetic mice with a mutation in the dAF1 site in theextended GRU of the PEPCK-C gene promoter do not have the samelevel of blood glucose as mice with the intact gene promoter.

EXPERIMENTAL PROCEDURES

Materials

Dulbecco’s modified Eagle’s medium, F-12, and fetal calf serum werepurchased from Biological Industries, Kibutz Beit Haemek, Israel. Bio-synthetic human insulin was obtained from Novo Nordisk (Denmark).Dexamethasone, the synthetic glucocorticoid hormone, was purchasedfrom Teva, Israel Pharmaceutical Industry. Ultraspec, the commercialkit for the preparation of tissue RNA, was purchased from Biotecx Lab-oratories, Inc. (Austin, TX). Nytranmembrane (Schleicher and Schuell)was used for blot hybridization. Radioactive labeling was done using[32P]dCTP 3000Ci/mmol (AmershamBiosciences), and radioactive sig-nals were quantified using phosphorimaging (Fujix BAS 1000, Fuji,Japan). Random hexanucleotide d(N)6 was purchased from RocheApplied Science. Acrylamide and bisacrylamide were from Amaresco(Ohio). Protein G PLUS-agarose beads and antibodies against PPAR�,HNF-4�, HNF-3�, GR, and rabbit IgGwere purchased from Santa CruzBiotechnology (Santa Cruz, CA). Antiserum against FOXO1 (FKHR)and P-FOXO1 (FKHR) were purchased fromCell Signaling Technology(Beverly,MA). Streptozotocin (Zanosar)was fromPharmaciaCorp. andThe Upjohn Co.

Methods

Animals—Transgenic mice were produced harboring the entire un-modified rat PEPCK-C gene flanked 5� by 2000 bp upstream of thetranscription start site and 3� by 1600 bp.Mice with a targetedmutationin the PPAR�2 binding site of the PEPCK-C gene promoter (PPARE)

were generated as described previously (34). All mice were maintainedon a commercial chow diet ((catalog #TK2018SC�F) purchased fromHarlan Teklad (Madison, WI)) in the SPF (specific pathogen-free) unitof the animal facility at the Hebrew University-Hadassah MedicalSchool, Jerusalem. The mutant mice used in the present work werebackcrossed into the C57Bl background (seven generations) to achievea more homogenous genetic background of the population.Transgenic mice were genotyped by PCR, with rat PEPCK-C-specific

primers to exon 9 (the 5� primer; 5�-CTTGTCTACGAAGCTCTCAG)and to exon 10 (the 3� primer; 5�-CGTCCGAACATCCACTC).One�gof DNA was added to the reaction mix (25-�l final volume) containing125 ng of each primer, 10� commercial buffer (with Mg2�), 2.5 mM

dNTP mix, and 1 unit of Taq polymerase. After 33 cycles of amplifica-tion in a programmable thermal controller (MJ research), the PCRproducts were digested with the diagnostic restriction enzyme BglII,specific to a polymorphic site in the endogenous segment generatingdifferential sized products that discriminated between the sizes of theendogenous gene and transgene by electrophoresis on 1.6% agarose gelscontaining ethidium bromide and viewed with UV camera using Quan-tity One software (Bio-Rad).PPARE mice were genotyped using the primers 5�-flanking region

5�-AGCCACTTCTTCTGTACC and 3�-GTAAGCTTTGTTCTGA-CAGG spanning the PPARE region in the PEPCK-C gene. The PCRproduct was digested with XhoI at the diagnostic recognition site toidentify the mice carrying the mutant allele. Mice homozygous for themutant allele are designated PPARE�/�mice. Diabetes was induced inadult male mice (2–7 months) by a single intraperitoneal injection of200 �g/kg of body weight of streptozotocin. Blood glucose levels weretested using a glucose meter (Ascensia Elite, Bayer); after 3–5 days theanimals with a blood glucose concentration of 400 mg/dl and higherwere considered diabetic (35). Some of the diabetic mice used in thisstudy were adrenalectomized 3 days after the injection of streptozoto-cin; they were provided with drinking water that contained 0.9% NaClafter the surgery andwere sacrificed 3 days later. The procedures involv-ing injections of streptozotocin or adrenalectomywere done under lightanesthesia using 0.1 ml/10 g of body weight of 44 mM Avertin (contain-ing 1.25% of tribromoethanol and 2.5% of tertiary amyl alcohol).

Cell Culture, Transfection Conditions, and CAT Assays—HepG2human hepatoma cells were grown in a medium containing a 1:1 mix-ture of Dulbecco’s modified Eagle’s medium and F-12 and 10% fetal calfserum, 100 units of penicillin, and 0.1mgof streptomycin/ml. Cells weretransfected using the calcium phosphate precipitation procedure,essentially according to Chen and Okayama (36) with the slight modi-fications described previously (28) involving 3 �g of supercoiled plas-mid and additional carrier pBlueScript DNA (Stratagene) for a total of10 �g of DNA per 25-mm flask. Where indicated, 1 �g each of GR orHNF-4� expression vectors and 0.5 �g each of expression vectors forRXR� together with PPAR�, RAR�, or COUP-TF were added per flask.The precipitates were left on the cells for 5 h, after which the cells werewashed with phosphate-buffered saline and shocked for 3min with 20%glycerol. The transfection efficiency was monitored by including 0.1 �gof plasmid pEGFP-C1 (Clontech) containing the green fluorescent pro-tein (GFP) gene driven by the cytomegalovirus gene promoter as aninternal standard and quantified by counting GFP-containing cells in ahigh field resolution of a fluorescent microscope. PEPCK-C gene pro-moter activity was determined by measuring the activity of the reportergene product cat, as previously described (28). Dexamethasone (10�7 M)was added to the cells no later than 20 h after transfection, when expres-sion of the reporter gene was barely detectable. Cells were harvested24 h after the addition of dexamethasone. The expression vectors

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included PPAR� and RXR� (37) (from Dr. R. Evans), the rat GR (38)(from Dr. K. Yamamoto), and HNF-4� (from Dr. F. Sladek) (39). Thegeneration of the plasmids containing the cat reporter gene driven bythe rat PEPCK-C gene promoter, which included site-specific muta-tions, has been described previously (40, 41) except for the mutation ofdAF1 and dAF2, which are described here.

RNA Analysis—For the transgenic mice, total RNA from 40-h-fastedmice 10–18 weeks old was extracted using Ultraspec reagent (Biotecxlaboratories, Houston). RT-PCR analysis was performed using randomhexamer primers (Amersham Biosciences) with Moloney murine leu-kemia virus reverse transcriptase (Invitrogen) in the presence of RNasin(Promega) to generate PEPCK-C cDNA. The cDNA was amplifiedusing the same primers indicated above for the genotyping comprisingsequences from exons nine and ten. The PCR protocol involved 20amplification cycles only using radioactive [�-32P]dCTP (3000Ci/mmol). For PPARE mutant mice total RNA was extracted from theliver and kidney using the commercial Ultraspec kit, and Northern blothybridization was done as previously described (34).

New Plasmids Used in the Transfection Studies—The mutated dAF1(PPARE) plasmid (dAF1-mut), derived from the pck-2000-CAT plas-mid (41), was generated by replacing the PPARE site with a syntheticfragment. Briefly, the segment encompassing positions �1445 (HincII)to �598 (HindIII) was amplified in two separate PCR reactions usingprimers containing altered nucleotides in the PPARE site. The PCRproducts were ligated using an artificial XbaI site, and the ligation prod-uct replaced the HincII-HindIII segment of the pck-2000-CAT. Thefirst PCR amplification was performed using the 5�-primer CACGGT-TGACACCCAACAGT (�1448 to �1431), which includes the HincIIsite, and a 3�-primer, ACGTCTAGAATACGTAGCGGCCGCTT-GAACGAGCCGAGAGAAG, containing altered nucleotides (under-lined), introducing an XbaI site and nucleotides from (�1044 to�1063). The second PCR amplification was done using the 5�-primer,GCCGTCTAGAGGTACGCTATTCGCCCGCTGCTCAAGTGT-AGC, containing altered nucleotides (underlined), introducing an XbaIsite and nucleotides from�986 to�970, and a 3�-primer, GGGTGAT-TGTAAGCTTCATTCCG (�606 to�584), which includes theHindIIIsite. This enabled us to replace the segment between �1031 and �987harboring the PPARE site with 36 bp of randomDNA. This plasmidwassequenced for verification. The mutated dAF2 site (dAF2-mut), alsoderived from the pck-2000-CAT plasmid, was generated by replacingthe dAF2 site with a synthetic fragment. Briefly, the section between�1376 to �1345 that contained the dAF2 site was replaced with 33 bpof non-binding DNA, 5�-CGAAGCTTGACCGGCCGGATCCGCGC-CCAGCGA. This replacement created two recognition sites forHindIIIand BamHI, which were used for size verification of the restrictionfragments.

DNase I Footprinting Analysis and Electrophoresis Mobility ShiftAssay—Nuclear proteins were extracted from rat liver as described byGorski et al. (42) withminormodifications (43). The DNase I footprint-ing assay was performed as described previously (43). The autoradiog-raphy density signals of specific bands in the exposed film were quanti-fied using Fluor-STM MultiImager with Multianalyst version 1.1 (Bio-Rad) as previously described (9) and specifically described in the legendto Fig. 2. The electrophoresis mobility shift assays were performed aspreviously described (7). Oligonucleotides of accessory factor 1 (AF1),distal AF1 (dAF1), accessory factor 2 (AF2), and distal AF2 (dAF2) wereprepared by PCR using the corresponding primers; the AF1 site 5�primer was CAGAGCTGAATTCCCTTC, and the 3� primer wasAGCTGTGAGGTGTCAC, generating a 55-bp fragment. The dAF1site 5� primer was GGTTCTTCACAACTGGG, and the 3� primer was

GGGCTACACTTGAGCAGCG generating a 46-bp fragment. TheAF2 site 5� primerwasGGGAGTGACACCTCA, and the 3� primerwasGTGTGCCAGTGGCTGC, generating a 53-bp fragment. The dAF2site 5� primer was GACTTGAAGAGGAAGCCGC, and the 3� primerwas CTGGGGGTCCGTTGGAGC, which produced a 58-bp fragment.Radioactive labeling of the oligonucleotides was performed by including[32P]dCTP in the PCR reaction to a specific activity of 500 cpm/fmolusing 15 fmol/assay.

Chromatin Immunoprecipitation (ChIP) Assay—Zivig-Miller rats(200 g bodyweight) were fasted overnight and sacrificed, and their liverswere removed for the preparation of primary hepatocytes by themethod of Berry and Friend (44) and modified by Leffert (45). Theprimary hepatocytes were allowed to attach to the plastic dish for sev-eral hours, and then the cells were treated overnight with 10�7 M dexa-methasone and/or with 100 �M insulin for the last 2 h and again 5 minbefore fixation. The ChIP assay was performed as previously described(46) and modified byMassillon et al. (47) (see Ref. 48 for specific detailsof the modification used). The DNA isolated by immunoprecipitationwas analyzed by PCR amplification using the same primers indicatedabove for the gel retardation assay for the AF2 and dAF2 accessory sites.The starting chromatin input fraction was diluted 1/1000 and analyzedby PCR simultaneously with the samples. The amplified DNA frag-ments were separated by electrophoresis using 2% agarose gels, stainedwith ethidium bromide.

RESULTS

Evidence for an Extended GRU in HepG2 Cells—The endogenousPEPCK-C gene is not only expressed in HepG2 cells but is inducible byglucocorticoids (30). Therefore, the failure of the hormones to inducethe 500-bp segment of the rat PEPCK-C gene promoter (29) stronglysuggested that sequences outside this segment were required. Weassessed whether the GRU in the PEPCK-C gene promoter extendsupstream of the originally described site by comparing the effect of GRon transcription from the rat PEPCK-C gene promoter in chimericgenes that were driven by segments from �500 to �73 (pck-500-CAT)and �2000 to �73 (pck-2000-CAT) of the gene promoter. The GR inthe presence of dexamethasone stimulated transcription of the pck-2000-CAT gene in HepG2 cells from 3- to 5-fold but had no effect ontranscription of pck-500-CATgene (Fig. 1). Because both pck-500-CATand pck-2000-CAT have recognition sites for the PPAR� or -�, a non-steroid nuclear receptor (18), we tested the response of both gene pro-moters to this receptor. Unlike GR, the heterodimer of PPAR� withRXR� (PPAR/RXR) stimulated transcription fromboth gene promotersabout 8–10-fold (Fig. 1). When GR was transfected together withPPAR/RXR, transcription of the chimeric pck-2000-CAT gene but notthe pck-500-CAT gene was stimulated synergistically (Fig. 1). Thus,although pck-500-CAT failed to respond to GR in the presence of itsligand either when present alone or together with PPAR/RXR, the pck-2000-CAT gene not only responded to the stimulation by GR but alsoresponded synergistically to the combination of GR and PPAR/RXR.

Footprinting Analysis of the Liver-specific HSS Site B—To identifyputative upstream regulatory sites within the PEPCK-C gene promoter(�2000 to �73) that are responsive to glucocorticoids, we focused onthe HSS B, previously described in this region (31). The HSS B locuscomprises a non-liver-specific site centered at position �993 and a liv-er-specific site at position �1400 of the PEPCK-C gene. The non-liver-specific site contains a recognition site for PPAR� or PPAR� (18), whichis required for the expression of a transgene driven by the rat PEPCK-Cgene promoter in adipose tissue of transgenic mice (49). This site wasspecifically mutated in the genome of mice (34), resulting in a total lack

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of expression of the gene for PEPCK-C in the white adipose tissue butnormal expression in the liver and kidney.There is a high degree of sequence identity within the liver-specific

region of HSS B (upper panel of Fig. 1) between the PEPCK-C genepromoter from the rat and the mouse (Fig. 2c). DNase I footprinting ofthis region demonstrated a binding site centered at position �1365 ofthe PEPCK-C gene that was protected by nuclear proteins isolated fromrat liver but not from spleen (Fig. 2a). This sitewas partially protected bynuclear proteins from the liver of 19-day-old fetuses and from 3T3-F442Amouse adipocytes but not proteins from the kidney. To assess theaffinity of nuclear proteins to the protected site, the ratio of the densitiesof a band inside and a band outside the protected region was deter-mined, and an arbitrary ratio of 10 between the densities of the insideand outside bands was set for footprinting performed in the absence ofnuclear proteins. The ratio between the same bands using nuclear pro-teins from the adult liver (which expresses PEPCK-C) was 3.5, whereasthe ratio obtained using spleen nuclear proteins was of 8.2 (close to thevalue obtained without nuclear proteins). Nuclear proteins from thefetal liver gave a ratio of 4.4, from the adipocytes a ratio of 5, and fromthe kidney a ratio of 7.2 (Fig. 2b). These results demonstrate not onlythat the site is specific for PEPCK-expressing tissues (it is absent innuclear proteins isolated from the spleen), but also it had a differentialaffinity for binding nuclear proteins from tissues that express PEPCK-C,with the highest affinity exhibited by the liver and the lowest by thekidney (Fig. 2b). These results agree with those reported for the sub-siteof HSS B in PEPCK-C gene promoter where DNase I sensitivity in theliver (31) and preadipocytes (32), but not in the kidney (33), wasdetected.The sequence of the protected region around�1365 of the PEPCK-C

gene promoter, which was identified by DNase I footprinting, has con-siderable similarity to the AF2 sequence in the GRU (13); on the basis ofthis similarity we termed this site the distal AF2 (dAF2). Likewise, the

non-liver-specific HSS B sub-site, which co-localized with the PPARE(18, 49) and is similar to the AF1 site in the GRU (13), was termed thedistal AF1 (dAF1) site.

Electrophoresis Mobility Shift Assay—To begin characterizing thepatterns of hepatic nuclear proteins that bind to the dAF1 and dAF2sites, we performed mobility shift assays. A similar pattern of bindingbetween the AF1 and dAF1 sites and between the AF2 and dAF2 sites ofthe PEPCK-C gene promoter to nuclear proteins was noted using elec-trophoresis mobility shift assay. There was an efficient competitionobtained only between the analogous sites of each couple. Thus, theband shift of the dAF1 probewas equally competed by the same amountof nonradioactive self-competitor (dAF1 site) or its partner (AF1 site),whereas neither the dAF1 nor AF1 sites competed with binding to thedAF2 probe. Likewise, although the band shift of the dAF2 probe wasefficiently competed by itself and by the AF2 sequence, neither dAF2nor AF2 competedwith the band shift of the dAF1 probe (Fig. 3, a and b,respectively). The similarity in protein binding between the dAF2 andAF2 sites implies that dAF2 site likely constitutes a binding recognitionsite for the HNF-3� (Foxa2) and FOXO1, members of the Forkheadgene family, as found for the AF2 site (50, 51) (23). The similaritybetween dAF1 andAF1 also suggests that non-steroid nuclear receptorsbind to dAF1, as previously established for AF1 (51).To assess the identity of hepatic proteins binding to dAF1 and dAF2,

gel shift analysis was performed using specific antibodies. The dAF1probe was supershifted by the addition of an antibody to HNF-4� to anextent that corresponded to its abundance in hepatic nuclei (52) (Fig.3a). However, the antibody to PPAR� did not affect the binding ofproteins to the dAF1 site. Binding of members of the Forkhead family oftranscription factors was demonstrated using the dAF2 site as a probe(Fig. 3b). The results showed diminished intensity of the bound lowerband (relative to the upper bound band), with antibodies against

FIGURE 1. Transcription from the PEPCK-C genepromoter is stimulated synergistically by aninteraction between GR and non-steroidnuclear receptors. Upper panel, schematic repre-sentation of hypersensitive sites C and B. Site Cencompasses the entire proximal 500 bp of thePEPCK-C rat gene promoter. Site B comprises twosub-hypersensitive sites. The more proximal site,centered at position �993, has been originallyidentified as a PPAR�2 recognition site (18) andlater proven in transgenic mice (49) and through atargeted mutation in mice (34) to constitute anadipose-tissue enhancer of the PEPCK-C gene. Theoccupation of this site is not tissue-specific (9). The5� liver-specific site is centered at position �1365.Lower panel, trans-activation of the PEPCK-C genepromoters by PPAR� and GR in HepG2 humanhepatoma cell line. HepG2 cells were transfectedwith 500 (pck-500-CAT) or 2000 bp (pck-2000-CAT) rat PEPCK-C gene promoters (indicatedabove the histograms) driving the CAT structuralgene. The addition of expression vectors forPPAR� and RXR� (PPAR�) or GR (GR) or both is indi-cated below (�). Dexamethasone (10�7

M) wasadded for 24 h before harvesting the cells trans-fected with GR. The fold stimulation over basalactivity of each of the two gene promoters isexpressed as the mean � S.E. (at least six inde-pendent experiments).

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FOXO1 and phosphorylated FOXO1 (P-FOXO1) and the appearance ofa weak but discrete super shift band with HNF-3� antibody (Fig. 3b).

ChIP Assay—The in vivo occupancy of regulatory sites of theextended GRU by transcription factors was assessed by a ChIP assayusing isolated rat hepatocytes that were treated with hormones. Wenoted a similar binding pattern by transcription factors to both the AF2and dAF2 sites (Fig. 4, a and b) that differed from the binding pattern todAF1 site (Fig. 4c). The addition of dexamethasone stimulated the occu-pation of both the AF2 and dAF2 sites by FOXO1 and PPAR� (Fig. 4, aand b) but failed to stimulate the occupation of dAF1 by either of thesefactors (Fig. 4c). The apparent effect of dexamethasone on the binding ofPPAR�was not expected and equally surprisingwas the lack of responseof HNF-4� to the added hormone (Fig. 4, a and b). We did observe areciprocal effect of glucocorticoids on the binding pattern of these twofactors to dAF1 (Fig. 4c). The hormones stimulated the binding ofHNF-4� but had no effect on that of PPAR�. Unlike the binding offactors described above, the addition of dexamethasone had no effect ofthe binding of HNF3� to either AF2 or dAF1 (Fig. 4, a and c). A mod-erate stimulation of binding of this factor to the dAF2 was noted, buteven then it seemed to be inferior to the stimulation of FOXO1 bindingto this site (Fig. 4b). These results essentially corroborate those byWolfrum et al. (23), who reported on binding of FOXO1 rather thanHNF3� to the AF2 site.

The addition of insulin to the hepatocytes inhibited the effect of dex-amethasone on FOXO1, PPAR�, and HNF-4� (Fig. 4, a–c). In contrast,insulin intensified the binding of P-FOXO1 to all three sites when addedby itself or in the presence of dexamethasone (Fig. 4, a–c). There are two

exceptions; one is the stimulation by dexamethasone of the binding ofP-FOXO1 to dAF1 (Fig. 4c), and the second is the intensified binding ofHNF3� by insulin to dAF2 and dAF1 sites but in this case only in thepresence of dexamethasone (Fig. 4, b and c). Our data suggest thatinsulin globally inhibited the binding of all the factors stimulated bydexamethasone but intensified the binding of P-FOXO1.

Transient Transfection Experiments—We next determined whetherother non-steroid receptors (besides PPAR), such as HNF-4�, RAR�.and COUP-TF 1 and COUP-TF 2, could interact with GR to synergis-tically stimulate transcription from the PEPCK-C gene promoter(�2000 to �73) (Figs. 5 and 6). Synergistic stimulation was noted withHNF-4� and with RAR/RXR, but COUP-TF 1 and 2 inhibited both thebasal level of gene transcription from the PEPCK-C gene promoter andthe activation for transcription by HNF-4� when co-transfectedtogether with HNF-4� (Fig. 5, a and b). In agreement with these results,De Martino et al. (53) recently reported that COUP-TF either cooper-ates or inhibits GR-mediated induction of transcription from thePEPCK-C gene promoter (among others) in HepG2 cells.The data described above suggest that the dAF1 and dAF2 sites in the

extended GRU of the PEPCK-C gene promoter duplicate the AF1 andAF2 sites. Therefore, we next assessed whether all four accessory sitesare required for the glucocorticoid regulation of transcription from thePEPCK-C gene promoter (�2000 to �73). To this end each of the fouraccessory siteswas individuallymutated aswere the twoGRE sites (GRE1 and 2) (13)). A combined mutation of both GRE 1 and GRE 2 sitesabolished not only the response of pck-2000-CAT to GR alone but alsothe synergistic response of GR together with either PPAR/RXR (Fig. 6a)

FIGURE 2. DNase I footprint analysis of the liver-specific sub-HSS B in the rat PEPCK-C gene promoter. Panel a, a 333-bp DNA segment spanning positions �1112 to �1445 ofthe rat PEPCK-C gene was 32P-end-labeled at the 3� site of the fragment. 50,000 cpm of the labeled probe was added per reaction. Incubation was without (0) or with nuclear proteins;10 �g of rat liver incubated with DNase I for 1– 4 min (Liver) and with 20 �g of rat spleen 1 and 4 min. (Spleen). The ratio between the density signals of the distinct two bands withinthe protected dAF2 site and three bands outside, indicated by arrows on the right side of the figure, enabled quantification of the relative protection of the dAF2 site. Panel b, thequantified relative protection of the dAF2 site is shown in the histogram for nuclear proteins from several tissues. The ratio in the absence of proteins was set at 10 (0). The nuclearproteins from the rat tissues are spleen, adult liver (A. liver), 10 �g of fetal liver (F. liver), and 20 �g of kidney. Adipose tissue is from the mouse 3T3-F442A 15 �g of adipocytes (adipose).Panel c, the sequences of the protected dAF2 region of the mouse and rat PEPCK-C gene promoter spanning positions �1405 to �1381 for the mouse and positions �1377 to �1352for the rat are indicated.

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or with HNF-4� (Fig. 6b). This result corroborates earlier findings byGranner and coworkers (13, 14, 54) who noted that a mutation in anysingle element of the GRU only marginally affected the response of thegene promoter to glucocorticoids, and a double mutation of any twoelements of the GRU completely abolished the response to glucocorti-coids. Likewise, we report that a single mutation of each accessory siteonly moderately affected the response of the PEPCK-C gene promoterto GR alone. In contrast, the synergistic response wasmarkedly affectedby a single mutation of any one of the accessory sites. Thus, mutation ofthe AF1 site alone abolished the synergistic response to GR with eitherPPAR/RXR (Fig. 6a) or HNF-4� (Fig. 6b). Similar results were obtained

when the AF2 site in the PEPCK-C gene promoter was mutated (Fig. 6,a and b). Mutation of the dAF1 site had a minimal effect on the stimu-lation of the PEPCK-C gene promoter byGR alone, whereas it markedlyreduced the cooperative stimulation by GR together with either PPAR/RXR or HNF-4� (Fig. 6, a and b). Mutating the dAF2 site in thePEPCK-C gene promoter completely abolished the synergistic responseof the promoter to the GR and PPAR/RXR (Fig. 6a), but it only partiallyreduced the synergistic response of GR and HNF-4� (Fig. 6b). Finally, amutation of either the dAF1 or dAF2 sites in the extendedGRU reducedthe trans-activation of the PEPCK-C gene promoter by HNF-4� alone(Fig. 6b) but did not affect the response to PPAR/RXR alone (Fig. 6a).

FIGURE 3. Electrophoresis mobility shift assays. In panel a the 32P-labeled dAF1 and in panel b the 32P-labeled dAF2 site probes were radioactively labeled by PCR amplificationcontaining [32P]dCTP. The mobility shift assay included 7500 cpm of 32P-labeled probe and 10 �g of nuclear protein extract from rat liver. a, free probe; Fp, no nuclear protein extractadded; �, nuclear protein extract bound to 32P-labeled dAF1. Competitors added: dAF1 �50 M excess (dAF1), AF1 �50 M excess (AF1), AF2 �100 M excess (AF2), and dAF2 �100 M

excess (dAF2). Nuclear protein extract was incubated with HNF-4� antiserum (1 �l) (HNF-4�), HNF-3� antiserum (1 �l) (HNF-3�), GR antiserum (1 �l) (GR), and PPAR� antiserum (1 �l)(PPAR�). b, free probe; Fp, no nuclear protein extract added; �, nuclear protein extract bound to 32P-labeled dAF2. Competitors added: dAF2 �50 M excess (dAF2), AF1 �100 M excess(AF1), dAF1 �100 M excess (dAF1), and AF2 �50 M excess (AF2). Nuclear protein extract was incubated with HNF-3� antiserum (1 �l) (HNF-3�), HNF-4� antiserum (1 �l) (HNF-4�),phosphorylated FOXO1 (FKHR) antiserum (1 �l) (P-FOXO1), and FOXO1 antiserum (1 �l) (FKHR).

FIGURE 4. ChIP assay of AF2, dAF2, and dAF1 accessory sites in the extended GRU of the rat PEPCK-C gene promoter in rat liver in vivo. The association in vivo of hepatic nuclearproteins with AF2, dAF2, and dAF1 sites of the PEPCK-C gene promoter was carried out using ChIP analysis of DNA isolated from hepatocytes of rats fasted overnight. Isolatedhepatocytes were incubated either overnight with dexamethasone (Dex) and/or with insulin for the last 2 h and, additionally, 5 min before fixation as indicated above the figuresfollowed by cross-linking the DNA and associated proteins with formaldehyde. The specific binding of transcription factors to the AF2, dAF2, and dAF1sites of the PEPCK-C genepromoter was identified using antibodies as indicated on the left side of the figures. The amplified DNA was separated by electrophoresis on 2% agarose gels and visualized usingethidium bromide staining. Panel a, the AF2 site comprises positions �437 to �384. Panel b, the dAF2 site comprises positions �1394 to �1335 of the PEPCK-C gene promoter. Panelc, the dAF1 site comprises positions �1088 to �948 of the PEPCK-C gene promoter. Starting input chromatin DNA is shown below as indicated (Input).

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These results establish the requirement of each of the four accessorysites despite their duplication for the synergistic response of PEPCK-Cgene promoter by a combination of GR with either of the two non-steroidal nuclear receptors.

The Role of the dAF1 Site in Vivo in the Response of Hepatic PEPCK-CGene to Diabetes—The findings using HepG2 cells demonstrating therequirement of all four accessory sites for the synergistic response of thePEPCK-C gene promoter to nuclear receptors might be specific to thiscell line. An implication of their physiological significance should comefrom studies in vivo. This was especially important in view of the bulk ofevidence on the role of the GRU that has been derived from the use oftransformed cell lines (13). Using mice, which contain a targeted blockmutation of the dAF1 (PPARE) site in the PEPCK-C gene promoter (34),we determined the effect of a loss of this site on the hepatic expression ofthe gene in response to streptozotocin-induced diabetes.Mice thatwerehomozygous for a mutation in the dAF1 site in the PEPCK-C genepromoter and a mixed population of heterozygous and wild type micewere made diabetic by the injection of streptozotocin. The concentra-tion of blood glucose was determined 3 days after streptozotocin injec-tion (Fig. 7a). Thirteen diabetic mice lacking the dAF1 site in thePEPCK-C gene promoter (�/�) had a lower level of blood glucose ascompared with 18 control littermates comprising a mixture of 8 wildtype and 10 heterozygous mutants (�/ ) (414 � 28.1 mg/dl versus547 � 28.7 mg/dl). When wild-type mice were made diabetic in a par-allel experiment, the level of blood glucose was reduced by about 25%after adrenalectomy (data not shown); this is about the same differencein the blood glucose concentration noted between mice lacking thedAF1 (�/�) and control littermates (�/ ). Apparently, ablation of onlythe dAF1 (of the four accessory sites) in the PEPCK-C gene promoterwas as effective as adrenalectomy in reducing the concentration ofblood glucose in diabetic mice.3 Finally, the mutation of the dAF1 sitecaused a marked decrease (3.5-fold) in the level of PEPCK-C mRNA in

the livers of these mice when compared with the wild type littermates(Fig. 7b); this mutation only marginally altered the renal level ofPEPCK-C mRNA.

The Glucose-mediated Repression of PEPCK-C Gene Is Liver-specific—Toassess thedegreeof liver specificityof thede-inductionofPEPCK-Cgene tran-scription by dietary glucose, we used three independent lines of mice with atransgene consisting of the entire rat PEPCK-Cgene flankedby 2000bpof the5� region and1600bpof the 3� regionof the gene.Herewe showa representa-tiveexperimentassessingtheeffectofglucosefeedingofmiceafterafastof40h.The expression of the transgene in tissues of themice was performed by RT-PCR using a cDNA template of the rat transcript that spans exon sequenceswhich flank both sides of the last intron (intron I (55)). As is shown (Fig. 8),glucose administered by gastric feeding caused the total disappearance ofPEPCK-CmRNAtranscribed from the transgene and amarkeddecrease (butnot disappearance) of the endogenous mRNA for the enzyme in the livers ofmice over a period of 4 h (Fig. 8). This rapid decrease in the level of PEPCK-CmRNAwas liver-specific; therewasnodetectable change inPEPCK-CmRNAin the kidney or adipose tissue of themice caused by glucose feeding. Interest-ingly, thedeceasingrate inthemRNAlevelofthetransgenemarkedlyexceededthatoftheendogenousgene,suggestingthatsequencesoutsideofthetransgenehelp tomoderate the response (Fig. 8).Alternatively, themore rapidde-induc-tion of transcription from the transgene might be related to its rat origin(although the rat and mouse PEPCK-C genes are highly similar (32)). Theseresults establish that the transgene contains sufficient information to elicit thehighly specific hepatic regulation of the PEPCK-Cgene expression.

DISCUSSION

The factors that control the hepatic transcription of the gene forPEPCK-C have been extensively studied over 25 years (1, 2), making itsgene promoter one of the most thoroughly studied of any eukaryotegene promoters. However, much of this research has focused on aregion that is �500 bp 5� of the start site of gene transcription. Thisregion of the gene promoter contains many critical elements that regu-late the response of the gene to diet and hormones (1, 2). However, a3 H. Cassuto, K. Kochan, and L. Reshef, unpublished results.

FIGURE 5. pck-2000-CAT rat PEPCK-C gene promoter is trans-activated by nuclear receptors. Panel a, trans-activation of the PEPCK-C gene promoter by various nuclear receptorsas indicated below without or with GR in HepG2 hepatoma cell line. The cells were transfected with 2000 bp of PEPCK-C gene promoters driving the CAT structural gene. Panel b (inset)represents inhibition by the COUP TF nuclear receptor family as indicated. Expression vectors for PPAR�/RXR� and RAR�/RXR� are designated PPAR� and RAR�, respectively.Dexamethasone (10�7

M) was added for 24 h before harvesting the cells transfected with GR. The -fold stimulation over basal activity of the gene promoter is expressed as the mean �S.E. (at least six independent experiments).

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number of observations over the years have suggested that a segment ofthe gene promoter that extends considerably up-stream from the bet-ter-characterized down-stream region is involved in both the hormonaland tissue-specific control of PEPCK-C gene transcription. For exam-ple, the expression of the gene for PEPCK-C in adipose tissue requires aPPAR�2 binding site that is present at �1000 in the gene promoter (34,49). In addition, there is hepatic-specific HSS that maps at approxi-mately�1400 of the gene promoter, the function of which has not beenstudied in any detail. Finally, the elegant studies by Granner and co-workers (13, 14, 22, 51, 54, 56–60) delineating the GRU in H4IIEC3 rathepatoma cells using a construct that contained the putative GRU butextended only to �500 had the unexplained problem in HepG2 cells,which lacked a response to glucocorticoids using the same gene pro-moter (29). In this report we provide evidence for an extended GRU inthe PEPCK-C gene promoter (from �1400 to �385), which involvesfour individual regulatory elements that share a common sequenceidentity. This extended GRU responds as predicted to nuclear recep-tors, and its function is consistent with the known regulation of

PEPCK-C gene transcription by these hormones. The extended GRU isstrongly supported by the altered response of the PEPCK-C gene to amutated dAF1 site inmice (Fig. 7) and by recent evidence from targetedPPAR� null mice (61).

The Influence of Glucocorticoids and Insulin on the Binding of Tran-scription Factors to the Extended GRU—The presence of an extendedGRU in the PEPCK-C gene promoter requires a re-analysis of the sig-nificance of the binding pattern of transcription factors to this largerregulatory unit in response to hormones. For example, the proximalAF1 and AF2 accessory sites are duplicated in the extended GRU, yield-ing the corresponding distal AF1 and AF2 counterpart sites, thus form-ing two couples, with each couple exhibiting a high degree of sequenceidentity between the proximal and distal partners. It would, thus, bereasonable to assume that these sites would share a common pattern oftranscription factor binding. This is especially important in consideringthe mechanism by which diabetes alters transcription of the gene forPEPCK-C in the liver. The increase in hepatic PEPCK-C gene transcrip-tion noted in diabetes is due not only to the removal of the negative

FIGURE 6. The effect of single mutations of eachaccessory site in the extended GRU of pck-2000-CAT on the trans-activation of this ratPEPCK-C gene promoter by glucocorticoids andnuclear receptors. The upper panel represents ascheme of the extended GRU in the rat PEPCK-Cgene promoter with the two GRE and theupstream four accessory factor sites (spanning theorder from 3� to 5�: AF2, AF1, dAF1, and dAF2). TheHepG2 hepatoma cells were transfected with 2000bp of PEPCK-C gene promoter wild type or thederived gene promoters mutated in each of theaccessory factor sites separately or mutated inboth GRE1 and -2 sites together, designated asAF1mut, AF2mut, dAF1mut, dAF2mut, and GREmutsites in the context of the pck-2000-CAT ratPEPCK-C gene promoter. Panel a, -fold trans-acti-vation by GR alone (GR), PPAR�/RXR� (PPAR�)alone, or with both (PPAR��GR) over basal activ-ity. Dexamethasone (10�7

M) was added for 24 hbefore harvesting the cells transfected with GR.Panel b, the nuclear receptor HNF-4� (HNF4�) wasused instead of PPAR�/RXR�. Otherwise, all is thesame as in panel a. The -fold stimulation by nuclearreceptors over basal transcription activity of eachconstruct of the pck-2000-CAT, taken as one, rep-resents the mean � S.E. for at least six independ-ent experiments (in both a and b).

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regulation exerted by insulin but also to the chronically increased levelof circulating glucocorticoids and, in turn, the strong stimulation ofgene transcription.Previous studies have shown that the AF1 site binds the hepatic-

enriched orphan receptors HNF-4 (16), COUP-TFII (17), PPAR�2 (18),the RAR� (19), and RXR� (20). The AF2 site binds members of theForkhead family includingHNF-3� (Foxa2) (50, 51, 54), FOXO1, and itsphosphorylated form (21). Recently, Wolfrum et al. (23), using a ChIPassay, reported that the AF2 site primarily binds FOXO1. Our findingsessentially corroborate theirs (Fig. 4).Beyond their similarity in sequence and binding specificity in gel shift

assays, the striking similarity of AF2 and dAF2 sites of the PEPCK-Cgene promoter is clearly established by the ChIP assay, where the pat-tern of binding proteins to these two sites markedly differs from thepattern associated with the dAF1 site. Thus, although dexamethasonestimulates the binding of FOXO1 to the AF2 and dAF2 sites but not todAF1 site, the hormones stimulate the binding of HNF4� only to dAF1but not to the AF2 or the dAF2 sites. These ChIP results, which clearlydiscriminated between the dAF1 site and AF2 and dAF2 sites, weremade possible because of the relatively remote distance of dAF1 sitefrom the other two sites. In contrast, the AF1 site is, in fact, successivelyadjacent to AF2 site.

It is important to note that a ChIP assay does not necessarily provideevidence of DNA binding but, rather, documents complexes of proteinsultimately associated with the transcription factor(s) that binds to anamplified DNA fragment. Therefore, this assay determines not onlyfactors that directly bind to a specific site on the PEPCK-C gene pro-moter but also those associated with the site via protein-protein inter-actions that occur off the promoter. Such is the case with the ChIPanalysis, which indicates intensified binding of PPAR� to complexesformed at bothAF2 and dAF2 sites upon the addition of dexamethasone(Fig. 4, a and b). The similarity of AF2 and dAF2 sites is even morestriking when compared with the different glucocorticoid-induced pat-tern of binding factors to the dAF1 site. Thus, the hormones induced thebinding to dAF1 of HNF4� but not PPAR� or FOXO1 (Fig. 4c).

The ChIP analysis shown in Fig. 4 suggests that insulin treatment ofisolated hepatocytes causes an association of P-FOXO1 with the AF2,dAF2, and dAF1 sites of the PEPCK-C gene promoter (Fig. 4). It is, thus,likely that the phosphorylation of FOXO1 stimulated by the addition ofinsulin disrupts a complex formed between FOXO1 and other tran-scription factors such as PPAR� or HNF-4�, which occurs at the AF2,dAF2, and dAF1 sites, respectively, of the extended GRU. It is widelyaccepted, however, that insulin-induced phosphorylation of P-FOXO1(via its activation of protein kinase B) results in a rapid exodus of thetranscription factor from the nucleus and its subsequent degradation inthe cytoplasm (62). By this model the phosphorylation of FOXO1 in thepresence of insulin would cause its removal from the complex of tran-scription factors, resulting in an inhibition of PEPCK-C gene transcrip-tion. However, Tsai et al. (63) have shown that �25% of the P-FOXO1remains in the nucleus one h after insulin treatment of hepatocytes.Thus, although insulin does cause a major redistribution of transcrip-tion factors within the cell, a significant fraction of the P-FOXO1remains in the nucleus after insulin addition. The authors also notedthat a mutation replacing leucine at position 375 in FOXO1, which iscritical for its exodus from the nucleus, with alanine does not alter theinsulin inhibition of transcription from the insulin-like growth factor-binding protein 1 gene promoter. Taken together, these findings indi-cate that although insulin-induced phosphorylation of FOXO1 doescause the nuclear export of a large fraction of this transcription factor,nuclear export is not required for inhibition of gene transcription.P-FOXO1 binding to the PEPCK-C gene promoter is physiologicallysignificant, since it has been shown that P-FOXO1 inhibits transcriptionof this gene (64). It is attractive to propose that by inducing the phos-phorylation of FOXO1, insulin triggers a disruption of an active tran-scription complex by ablating the association of specific transcriptionfactors to the extended GRU of the PEPCK-C gene promoter.Finally, insulin can also inhibit PEPCK-C gene transcription via other

factors that bind at different sites on the promoter, i.e. the LAP/LIPswitch (60) and the SREBP1c/SP1 switch (48), which have beendescribed in detail elsewhere (48, 60). This provides a redundancy oftranscriptional response of the PEPCK-C gene promoter to insulin,thereby insuring that the gene for PEPCK-C is not transcribed whenglucose is available and the level of insulin is elevated in the blood.

In Vivo Analysis of Critical Elements in the Extended GRU—Thedetailed analysis of the function of the PEPCK-C gene promoter hastraditionally involved the use of transformed cell lines (such as H4IIEC3andHepG2hepatoma cells), which do not necessarily predict the in vivosituation. The role of proposed regulatory elements in the extendedGRU is best tested in animal models, where the response of thePEPCK-C gene promoter to diet and hormones can be studied underphysiologically appropriate conditions. A major assumption of ourmodel is that each of the four accessory sites in the extended GRU is

FIGURE 7. A targeted mutation of the dAF1 site of the PEPCK-C gene (PPARE mice)affects the expression of the gene for PEPCK-C in the liver and the level of bloodglucose in diabetic mice. Panel a, 13 homozygous (dAF1) PPARE mice (�/�) and amixed population of 10 heterozygous and 8 wild type mice (PPARE) (�/ ) were madediabetic by streptozotocin (STZ) injection. The concentration of blood glucose was deter-mined 3 days later, and its mean level � S.E. is shown. The difference between the twogenotypes ((dAF1) (PPARE) mutants (�/�) compared with (dAF1) PPARE (�/ )) was24.3% (significant at p � 0.025). Panel b, Northern hybridization assays using 10 �g oftotal RNA from liver and kidney of streptozotocin diabetic mice were quantified by deter-mining the abundance of PEPCK-C mRNA relative to that of �-actin. The histogramsshow the means � S.E. of the results from four mice each of wild type (�/�) (filled boxes)and mutant (�/�) (empty boxes). The 3.5-fold difference in the hepatic abundance ofPEPCK-C mRNA between the wild type and mutant diabetic mice was significant (p �0.037). The renal abundance of PEPCK-C mRNA was not significantly affected by themutation.

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FIGURE 8. The effect of glucose feeding on the levels of PEPCK-C mRNA in the livers of fasting transgenic mice containing a rat PEPCK-C transgene. Panel a, a schematicillustration of the rat transgene in transgenic mice. The linear black boxes indicate exons, and open boxes indicate introns and flanking regions. �1, transcription start site; A�,polyadenylation signal. The region of the RT-PCR products of the transgene and endogenous gene and its polymorphic BglII site is indicated below. Panel b, RT-PCR analysis of RNAfrom three fasted transgenic mice (numbered 1–3) and three fasted and re-fed with glucose (4 – 6). Total RNA was extracted from the liver (L), kidney (K), and white adipose tissue (A)and processed by RT-PCR. The cDNA samples were amplified using PEPCK-C-specific primers from exons 9 and 10, as specified under “Experimental Procedures.” The amplifiedRT-PCR products were digested with BglII, generating the transgene segments sizes 202 and 99 bp, whereas the amplified segment of the endogenous gene, which has two BglII sites,yielded three segments, sizes 148, 98, and 54 bp. Only the larger bands, 202 bp of the rat transgene and 148 bp of the endogenous gene, are shown. On the left are the markers: R, theproduct of the rat gene; M, the product of the endogenous mouse gene.

FIGURE 9. Model of the proposed conformational changes in the extended GRU of the PEPCK-C gene promoter in response to dexamethasone or insulin. The regulatory sitescomprising the extended GRU (including the AF3 site that resides downstream of the GRE2) are shown as mapped to the PEPCK-C gene promoter in the upper portion of the figure.In the present manuscript we analyzed the GRE1 and -2 and the accessory sites residing upstream of the two GREs. The addition of dexamethasone (Dex) to primary rat hepatocytesor human hepatoma HepG2 cell line supplemented with GR causes the formation of a complex associated with a conformational bend of the DNA that juxtaposes the AF2 and dAF2 sites atthe end of the bended DNA and the AF1 and dAF1 sites within the bended DNA. This alignment attracts co-activators that recruit the transcriptional machinery. The addition of insulin untiesthe complex by phosphorylation of the bound FOXO1 and by releasing the entire bound transcription factors, thus arresting the gene transcription. Pol II, polymerase II.

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essential for the regulation of hepatic PEPCK-C gene transcription bynuclear receptors in vivo. The best example of this regulation is theenhanced glucose production characteristic of the diabetic animalsince, according to our model, a mutation in any one of the four criticalsites in the GRU should not only affect the abundance of hepaticPEPCK-C mRNA but potentially also alter the circulating glucose levelof diabetic mice. Mice with a targeted mutation in the dAF1 provide arigorous test for this assumption, since we previously reported that thismutation did not alter the level of PEPCK-CmRNA in their livers duringfasting (34). Moreover, streptozotocin-induced diabetes raises the con-centration of glucocorticoids in the blood, thereby allowing us to testthe response of fed, rather than fasted mice to a targeted ablation of thedAF1 site in the PEPCK-C gene promoter. Mutating the dAF1 sitemarkedly reduced the level of hepatic PEPCK-C mRNA (by 3.5-fold)and caused a significant decrease in the level of circulating glucose(about 25%). The large difference between the effect of the mutation onthe hepatic level of PEPCK-CmRNAand itsmoremoderate consequenteffect on blood glucose noted in these mice could also be due to theinduction of renal PEPCK-C gene expression via the increased meta-bolic acidosis that occurs during diabetes. The regulatory region in thePEPCK-C gene promoter that responds to acidosis (HNF-1) is about600 bp downstream of the dAF1 and is independent of dAF1 because atransgene harboring only 362 bp of the 5� flanking region of the ratPEPCK-C gene is fully responsive to acidosis in transgenic mice (7).Moreover, renal proximal tubules are not insulin-responsive. The resultis that during diabetes the contribution of renal gluconeogenesis to thecirculating blood glucose level is greatly enhanced (65). Because thedAF1 mutation has little effect on renal PEPCK-C gene expression, it islikely that the kidney contributes significantly to the elevated concen-tration of blood glucose noted in these mice. On the other hand, themarked effect of the dAF1 mutation on the hepatic expression ofPEPCK-C gene compared with its marginal effect on expression of thegene in the kidney strongly supports the requirement of all four acces-sory sites for the synergistic response of the hepatic PEPCK-C genepromoter to nuclear receptors.Recall that the synergistic stimulation of the PEPCK-C gene pro-

moter by the cooperation of GR with non-steroid nuclear receptors ismarkedly affected by a single mutation of each of the accessory sites. Arecent article by Bernal-Mizrachi et al. (61) lends strong support to theexistence of such cooperation between GR and nuclear receptors indiabetes. Thus, these authors could generate diabetes by treating micewith glucocorticoids only in PPAR��/� mice but totally failed to do soin PPAR��/� mice. Likewise, the level of hepatic PEPCK-C mRNAincreased considerably by this glucocorticoid treatment, but only in amanner dependent on the presence of PPAR�. These findings, there-fore, explain the effect of the dAF1 site-targeted mutation in thehomozygous mutant diabetic mice that we have obtained.It is intriguing that a recent report from the same group (66) has

shown that unlike diabetes, PEPCK-C gene expression was not affectedby the presence or absence of PPAR� in fasted mice. This is similar toour previous findings that the hepatic level of PEPCK-CmRNAwas notaffected in fasted mice with a targeted dAF1 mutation (34).Similar results were obtained using transgenic mice containing the

rat PEPCK-C transgene that had been mutated in the AF2 site. Themutation of the AF2 site, one of the four accessory factors binding sitesin the extended GRU of the PEPCK-C gene promoter, results in theinhibition of the diabetes-induced increase of PEPCK-C transgene tran-scription in the livers of transgenicmice and renders the PEPCK-C genepromoter refractory to insulin (41).

A Proposed Model for the Integrated Function of an Extended GRU—Wehave noted that the synergistic response of the PEPCK-C gene promoter toglucocorticoids requires the presence of each of the four accessory sites thatinclude twoduplicated sites inwhich thealignmentof thedistal accessory sitesisopposite to thatof theproximal sites; close inspectionof thealignmentof theaccessory sites indicates a “macro” palindrome. One possible mechanism toexplain the synergistic responseof thePEPCK-Cgenepromoter to glucocorti-coids involves a conformational bendof theDNA in the region containing thefouraccessorysites.Thiswould juxtapose theoccupiedAF2sitewith thedAF2site at the two ends of the bent region and the AF1 and dAF1 sites within thebent loop (Fig. 9). The close juxtaposition of these sites would facilitate therecruitmentof therequiredfactors intoacomplexthatresults inthesynergisticresponse of the gene promoter to nuclear receptors. The feasibility of the pro-posedmodel is currentlybeing testedby introducing transgenes intomice thatcontainmutations ineachofthefourbindingregionsoftheproposedextendedGRU in the PEPCK-Cgene promoter.

Acknowledgement—We are grateful to Dr. Oded Meyuhas for many fruitfuldiscussions.

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Glucocorticoid Regulation of PEPCK-C Gene Transcription

33884 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 40 • OCTOBER 7, 2005

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ReshefLeaYael Olswang, Parvin Hakimi, Chuan Xu, Duna Massillon, Richard W. Hanson and

Hanoch Cassuto, Karen Kochan, Kaushik Chakravarty, Hannah Cohen, Barak Blum,Carboxykinase in the Liver via an Extended Glucocorticoid Regulatory UnitGlucocorticoids Regulate Transcription of the Gene for Phosphoenolpyruvate

doi: 10.1074/jbc.M504119200 originally published online August 12, 20052005, 280:33873-33884.J. Biol. Chem. 

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