Identification of a thyroid hormone response element in the ...

Vol. 266, No. 32, Issue of November 15, PP. 21991-21996,1991 Printed in U.S.A .

THEJOURNAL OF BIOLOGICAL CHEMISTRY

0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of a Thyroid Hormone Response Elementin the Phosphoenolpyruvate Carboxykinase (GTP) Gene EVIDENCEFORSYNERGISTICINTERACTIONBETWEENTHYROIDHORMONEANDcAMP cis-REGULATORYELEMENTS* (Received for publication, January 18, 1991)

Marta GiraltS, Edwards A. Park, Austin L. Gurneys, Jinsong Liu, Parvin Hakimi, and Richard W. Hansonn From the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio44106

Transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in the liver is regulated by many hormones including thyroid hormone (T3). Inorder to identify the elements in the promoter which are required for transcriptional induction by T3, we cotransfected a T3 receptor expression vector with a PEPCK-CAT reporter gene into HepG2 cells. Using vectors with deletions in the PEPCK promoter, we identified a single T3 response element (TRE) between positions -332 and -308. This element binds [‘2SI]T3labeled T3 receptor contained in nuclear extracts prepared from rat liver. Furthermore, the P3(I) element (-250 to -234), a previously described cis-sequence involved in mediating the induction of PEPCK gene transcription byCAMP, is also required for the T3 responsiveness of the promoter. In the absence of either the TRE or the P3(I) binding sites, no stimulation of transcription from the PEPCKpromoterby T3 was observed, indicating that both elements are required for the T3 transcriptional regulation. Finally, a synergistic induction of PEPCK gene transcription by T3 and cAMP is described. This interaction requires both T3- and CAMP-responsivecis-acting elements.

Cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK)’ is a key enzyme in gluconeogenesis. PEPCK is present primarily in the liver and kidney, but enzyme activity has also been noted in adipose tissue, the jejunum (Hanson and Garber, 1972) and the mammary gland (Garcia-Ruiz et al., 1983). In theliver, transcription of this gene is stimulated ~~

* This work was supported by Grants DK-21859 and DK-24451 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of a Postdoctoral Fellowship from the “Plan de Formacion de Profesorado y Personal Investigator,” Ministerio de Education y Ciencia, Spain.Present address: Unitat de Bioquimica i Biologia Molecular, Departament de Biquimica i Fisiologia, Universitat de Barcelona, Avda Diagonal, 645, 08028 Barcelona, Spain. § Trainee on the Metabolism Training Program. T To whom correspondence and reprint requests should be addressed. The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; T:,, 3,3’,5-~-triiodothyronine, thyroid hormone; TRE, thyroid hormone response element; CRE, cyclic AMP response element; CREB, CRE-binding protein; C/EBP, CCAAT/enhancer binding protein;8-Br-cAMP, 8-bromo-cyclic AMP; CAT, chloramphenicol acetyltransferase; t p , base pair(s); Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

by several hormones including glucagon (via CAMP), glucocorticoids, and thyroid hormone (Lamers et al., 1982; Loose et al., 1985), whereas it is inhibited by insulin (Granner etal., 1983). Recentstudiesusingtransgenic mice havedemonstrated thata relatively small region of the PEPCK promoter (nucleotides -460 to +73) contains the essential information to confer the appropriate pattern of developmental, tissuespecific, and dietary regulation characteristicof the PEPCK gene (McGrane et al., 1988, 1990). Multiple cis-acting elements in this promoter-regulatoryregion have been found by footprint analysis using proteins prepared from rat liver nuclei and purified transcription factors (Roesler et al., 1989; Park et al., 1990). &Acting elements which account for the CAMP, glucocorticoid, and insulin responses have already been described in this region (Short et al., 1986; Quinn et al., 1988; Bokar et al., 1988; Imai et al., 1990; O’Brien et al., 1990). Furthermore, the recent characterization of the transcriptional induction of the PEPCK gene by cAMP has indicated that two cis-acting elements in the PEPCK promoter, CRE1 and P3(I), are required (Liu et al., 1991). The CRE-1 and theP3(I)elementsbothbindC/EBP(CCAAT/enhancer bindingprotein) while CRE-1 also bindsCREB(CAMPresponsive element binding protein) (Park al., et 1990). Previous studies in this and other laboratories haveshown that T3 stimulates hepatic PEPCK gene expression. It has been demonstrated thatT3increases the rateof transcription of the PEPCK gene in vivo (Loose et al., 1985) and that the time course of this effect parallels the nuclear T3receptor occupancy (Hartong et al., 1987). On the other hand, invitro studiesusinghepatocytesinculture have shownthat T3 modestly increasesPEPCKmRNA levels andcan greatly augment the effect of cAMP on the PEPCK mRNA expression(IynedjianandSalavert, 1984; Hoppneret al., 1986). More recently, it has been shown that T3 treatment of rat FA0 hepatomacells expressing the chicken thyroid hormone receptor (chicken c-erbAa) causes a specific increase in the level of PEPCK mRNA. In that system, T3 also potentiates the cAMP induction of PEPCK mRNA expression (Muiioz et al., 1990). These data suggesta direct effect of thyroid hormone on PEPCKgene transcription aswell as an interaction of T3 with the cAMP transcriptional stimulation of the gene. The aim of thepresentstudy was to identify the ciselements responsible for T3induction of PEPCK gene expression. The effects of T3on gene transcription are mediated by nuclear receptors which are structurally related to the viral oncogene v-erbA and are members of the steroid hormone receptor superfamilyof ligand-responsive transcriptionalfactors (Sap et al., 1986; Weinberger et al., 1986; Evans, 1988).

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Two major groups of T3 receptors, designated as a and P forms, have been described, although the functional significance of these different forms is not yet understood (Hodin et al., 1989). However, the two forms display distinct tissuespecific expression, and theP-T3receptor type has been shown to be the predominant form expressed in rat liver (Murray et al., 1988).The specific DNAsequences that mediate transcriptional activation or inhibition in response to T3are known as thyroid hormone response elements (TREs). TREsequences bind T3receptors with high affinity (Glass et al., 1987, 1988) and have been mainly studied in pituitary-specific genes, such as the genes for rat growth hormone (Ye et al., 1988; Glass et al., 1987; Wight et al., 1988; Brent et al., 1989a and 1989b; Norman et al., 1989), prolactin (Forman et al., 1988; Day and Mauer, 1989; Stanley, 1989) and a- and P-thyrotropin (Chatterjee et al., 1989; Carr etal., 1989). Recently, TREs have been found in the 5'-flanking region of two T3-responsive genes expressed in liver malic enzyme (Petty et al., 1990), and the S14 gene (Zilz et al., 1990). In this paper, we report the presence of a unique TRE in the PEPCK promoter which acts together with P3(I), another cis-acting element in thepromoter, to confer T3regulation to the gene. Finally, we show that T3and cAMP act synergistically in stimulating transcription of the PEPCK gene and that this effect depends on functional cis-acting elements. EXPERIMENTAL PROCEDURES

Materials-L-Triiodothyronine was purchased from Sigma. DNAmodifying enzymes and poly(d1). (dC) were obtained from Boehringer Mannheim. ['251]T:l NEX-11OX(2,200 Ci/mmol), [-y-"P]ATP (6,000 Ci/mmol), and [3H]chloramphenicolwere purchased from Du PontNew England Nuclear. Plasmids and Probes-Construction of the 5"deletion mutants of the PEPCK promoter ligated to the chloramphenicol acetyltransferase (CAT) gene has been previously described (Short et al., 1986; Park et al., 1990). Introduction of specific block mutations into the PEPCK promoter was carried out as described in Liu et al. (1990). The CAT expression vector (DelP4)PEPCK-CAT contains an internal deletion between nucleotides -330 and -272 in thePEPCK promot,er. RSV-hT3RP (kindly provided by M. G. Rosenfeld) is an expression vector that contains the human-erbA p form of the T S receptor driven by the Rous sarcoma virus promoter (Glass et al., 1989). Oligonucleotideswere chemically synthesized using an Applied Biosystems 380A DNA Synthesizer. The optimal-TRE oligonucleotide contains the palindromic TRE defined by Glass et al. (1988) flanked by XbaI-compatible ends. The (-332/-308)PEPCK is a 25bp oligonucleotide which corresponds to positions -332 to -308 of the PEPCK promoter. The P3(I)oligonucleotide contains the nucleotides -251 to -234 of the PEPCK promoter, and the CRE-1 oligonucleotide corresponds to nucleotides -94 to -77of the PEPCK promoter. The SV1-CAT vector which contains the enhancerless SV40 promoter driving the CAT gene has been described previously (Bokar et al., 1988). Two copies of the (-332/308)PEPCK-TRE were ligated into the XbaI site which is 5' to the SV1 promoter. Cell Culture and Transfection Assays"HepG2 human hepatoblastoma cellsweregrown in a 1:l mixture of Ham's F-12:Dulbecco's modified Eagle's medium supplemented with 5% calf serum and 5% fetal calf serum. DNA was transfected by calcium phosphate precipitation (Park et al., 1990), and to each plate were added 5 pg of CAT vector, 5 pg of RSV-hT3R6, and 2.5 pg of RSV-Pgal. The latter was included as a control for the efficiency of separate transfections. In order to ensure equivalent transfection efficiency, cells (80-90% confluent) were treated with trypsin, pooled, and added to theprecipitated DNA. The mixture was plated out and leftfor 4-5 h. The cells were then washed twice, and the mediumwas changed toF12:Dulbecco's modified Eagle's medium containing charcoal-treated 10% fetal calf serum (Honvitz and McGuire, 1978 Brentet al., 1988a, 1988b). The cells were incubated for 36-38 h with or without the addition of 100 nM T,. When the cAMP treatment was performed, 0.5 mM 8-Br-CAMP plus 1 mM theophylline were added 16-18 h before harvesting the cells. Analysis of CAT activity in freeze-thaw lysates of the cells was performed as described by Ausubel et al. (1989), using equal amounts of cellular protein for each sample. ['HI

Chloramphenicol and butyryl-SCoA were added to each extract. The butyrylated chloramphenicol was extracted by xylenes and quantified by scintillation counting. The CAT activity of different PEPCK-CAT vectors (separate transfection experiments) was corrected for transfection efficiency using the ,&galactosidase activity as a standard. DNA Binding Experiments-Nuclear extracts from rat liver were prepared by the method of Gorsky et al. (1986).For the gel retardation assays, oligonucleotides were labeled using [-y-32P]ATPand T4polynucleotide kinase. The DNA probe (20,000-30,000 cpm) was incubated for 15-20 min at 22 "C with 5 pg of protein from the nuclear extract and 3 pg of poly(d1). (dC) in a final volume of 15 pl containing 20 mM Hepes (pH 7.6), 0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 50mM NaCl, and 20 nM T3. Reactions were loaded onto 5% polyacrylamide gels in 0.5 X TBE (44.5 mM Tris, 44.5 mM borate, 1 mM EDTA). Electrophoresis was performed at 175 V for 90 min at 4 "C. Gelswere analyzed by autoradiography. In the competition experiments, unlabeled oligonucleotides (40 ng/lane) were included in each respective binding reaction. The receptor-DNA complex wasalso demonstrated by labeling the T, receptor with ['251]T,. Binding reactions were performed by incubating aliquots of rat liver nuclear extract (30 pg/lane) with 2 nM [1251]T3 for 60 min at 4 'C. DNA-binding reactions were subsequently performed using the same conditions described above but with 10 ng of unlabeled oligonucleotide per lane and without adding unlabeled T3 in the binding buffer. Electrophoresis was carried out for 90 min at 4 "C using 5% polyacrylamide gels in 6.7 mM Tris, 1 mM EDTA, and 3.3 mM sodium acetate (Lavin et al., 1988). The DNase I footprinting assay was performed as previously described (Roesler et al., 1989). The DNA probe was prepared by endlabeling one strand of DNA using [-y-32P]ATPand T4 polynucleotide kinase and was incubated with 30 pg of rat liver nuclear extract. The oligonucleotide competition was performed by adding 10 or 40 ng of each unlabeled oligonucleotide to the binding reaction. RESULTS

Identification of a Thyroid Hormone Response Element in the PEPCK Promoter-To determine the cis-acting sequences of the PEPCK promoter responsible for conferring T3transcriptional regulation to the gene, a series of 5'-deletions of the PEPCK promoter (-490/+73), linked to the CAT structural gene, were tested by transient transfection into HepG2 cells. HepG2 cells are a human hepatoblastoma cell line that has been used to study hepatic functions including the effect of thyroid hormone on liver metabolic processes (Javitt, 1990). However, we noted only a marginal T3-dependent increase in transcription from the PEPCK promoter in cells transfected with the (-490)PEPCI(-CAT vector (data not shown). We then co-transfected the PEPCK-CAT vectors with a mammalian expression vector for the human P-T3receptor (RSVhT3RP) which has been used in several cell lines to identify T3response elements (Glass et al. 1989; Chatterjee et al., 1989; Thompson and Evans, 1989). As is shown in Fig. l.4, cotransfection of (-490)PEPCK-CAT with RSV-hT3RP resulted in a 3-fold induction of CAT activity in the presence of 100 nM T3 when compared with cells incubated in the absence of the hormone. Dose-response experiments indicated that maximal T3stimulation of the CAT activity occurred at 100 nM T3, and all subsequent experiments were performed at this concentration. Concentrations of T3 of 0.1 nM, 1 nM, and 10 nM were also tried yielding inductions of 45%, 70%, and 90% of that achieved with 100 nM T3. A similar T3 responsiveness was observed with the (-355)PEPCK-CAT chimeric gene (Fig. LA). However, deletion of 78 bp from -355 to -277 resulted in a complete loss of the T3 induction of transcription from the PEPCKpromoter. Similar results were obtained using progressive downstream deletions. To further assess these data, we constructed a PEPCK-CATvector that contained an internal deletion between positions -330 and -272 in the PEPCK promoter. As is shown in Fig. LA, the (DelP4)PEPCK-CAT vector showed no significant T3 activation. These observations are consistentwith the localization

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FIG. 1. Transcriptional induction by thyroid hormone of PEPCK-CAT constructs. A , PEPCK-CAT expression vectors, con-

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taining several deletions of the PEPCKpromoter, were co-transfected with the T3receptor expression vector RSV-hT3RP into HepG2 cells. DNA transfection and CAT assays were performed as described under “Experimental Procedures.” Results are presented as -fold induction in the presence or absence of 100 nM T, and are theaverage a t least three experiments, each done in duplicate. The basal activity of each CAT vector did not vary more than 20% in the absence of T3. No difference was observed in the activity of vectors transfected with or without the T, receptor expression vector in the absence of T,. There is also a schematic diagram of the protein binding domains previously defined in the PEPCKpromoter (Roesler et al., 1989). B, a SV1-CAT expression vector containing two copies of the (-332/-308)PEPCKTRE was transfected into HepG2 cells in the presence or absence of 100 nM T,. The experiments were performed three times induplicate.

FIG. 2. Synergistic enhancement by thyroid hormone and cAMP of PEPCK gene transcription. Transfection protocol and PEPCK-CAT chimeric genes used in this experiment are described under “Experimental Procedures.” The cells were not treated (control) or treated by the addition of 100 nM T3 (T3), 0.5 mM 8-BrcAMP plus 1 mM theophylline ( C A M P ) ,or a combination of both agents (7’3 + CAMP).Results arepresented as -fold induction relative to thecontrol value and are expressed relative to thecontrol value of the (-49O)PEPCK-CAT, corrected for its transfection efficiency. Values are the means & S.E.M. for three different transfection experiments, each done in duplicate.

of a TRE between positions -330 and -277 in the PEPCK promoter. To test the ability of the (-332/-308) sequence from the PEPCK promoter to function asan independentthyroid response element, we ligated two copies of this sequence into a vector containing the enhancerless SV40 promoter driving the CAT gene. The (-332/-308) sequence was subsequently identified as theT3receptor binding site (Figs. 3 and5). When the parent SV1-CAT and(PEPCK-TRE)SVl-CAT genes were transfected into HepG2 cells, transcription from the PEPCK-TRE genewas stimulated 2.6-foldby100 nM T3, while the SV1-CAT gene was unresponsive (Fig. 1B). These results indicate that the isolated PEPCK-TRE can respond to T3. Synergistic Induction of PEPCK Gene Transcription by Thyroid Hormone and CAMP-In order to analyze whether the addition of T3had any effect on the induction of PEPCK transcription by CAMP,we treated co-transfected cells either with T3, 8-Br-CAMP, or both effectors together. Addition of 8-Br-CAMP caused a 4-fold induction in the CAT activity of the (-490)PEPCK-CAT vector (Fig. 2), which is consistent with the range of induction we have previously observed (Liu et al., 1991). Incubation of the cells in the presence of both T3 and 8-Br-CAMP caused a 19-fold induction, suggesting a synergistic interaction between the effects of TI and CAMP. T o determine if the observed synergism was dependent upon cis-elements within the PEPCK promoter, various chimeric genes containing the PEPCK promoter with specific mutations linked to the structuralgene for CAT were tested (Fig. 2). First, using (DelP4)PEPCK-CAT, a T3unresponsive vec-

tor, we observed a 4-fold induction by 8-Br-CAMP in either the presence or absence of TS.We also analyzed the response of two PEPCK-CAT vectors in which specific block mutations were introduced into the CRE-1 and P3(I) elements, which impair the cAMP responsiveness of the promoter (Liu et al., 1991). The induction of transcription from the PEPCK promoter by 8-Br-CAMP was about 2-fold and 1.5-fold with the P3(I) and CRE-1block mutation, respectively, in agreement with the previous observations. When we analyzed the T3cAMP interaction using the PEPCK promoter with a mutation a t CRE-1, the effect was additive. Surprisingly, the PEPCK promoter with a mutationat P3(I) not only exhibited an impaired transcriptional response to cAMP induction but also was not induced by T3. These results indicate that the synergistic effect of T3 and cAMP on inducing transcription from the PEPCKpromoter is mediated by a synergistic interaction of the T3and cAMP cis-acting elements. In addition, the lack of TSinducibility of the P3(I)block mutation suggests that two cis-sequence elements are required for the T3transcriptional regulation of the PEPCK gene. Identification of Thyroid Hormone Receptor Binding SitesWe next used gel mobility shift assays with nuclear extracts from rat liver to determine whether the T3 receptor bound to one orboth of the sequences required for conferring TB responsiveness to the PEPCKpromoter. A 25-bp oligonucleotide corresponding to positions -332 to -308 of the PEPCK promoter was labeled with 32Pand incubated with rat liver nuclear extracts. A single DNA-protein complex was formed (Fig. 3A). To determine whether this binding was sequencespecific and corresponded to the binding of T3receptor pres-

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FIG.3. Binding of TSreceptor to the PEPCK promoter. A, :"P-labeled (-332/-308)PEPCK oligonucleotide was incubated with nuclear extractsfrom rat liver. Where indicated, unlabeled competitor DNA (40 ng/lane) was included in the binding reaction. B, binding of [1Z511]Tn-labeled T R receptors present in rat liver nuclear extracts to the indicatedunlabeled oligonucleotides. The 32P-labeled (-332/ -308)PEPCK probe was incubated with rat liver nuclear extracts and run in parallel. Gel shift assays were performed as described under "Experimental Procedures." ent in the nuclear extract, a TRE oligonucleotide containing the well defined optimal palindromic T3 receptor binding sequence (Glass et al., 1988) was used as a competitor. A 100fold molar excess of unlabeled optimal TRE competed specifically, whereas no competition was detected using a 100-fold molar excess of either the CRE-1 or the P3(I) DNA probes. To directly identify the protein within the retarded complex, Ts receptors in nuclear extracts from rat liver were labeled with [ 9 ] T 3and thenincubated withunlabeled DNA probes. A single ['251]T3-labeledcomplex was identified using either the optimal TRE of the (-332/-308)PEPCK-TRE sequences (Fig. 3B). The ['251]T3-labeledband co-migrated with the R2P-labeledband monitored in parallel on the same gel. Competition of nonradioactive T3 (100-fold molar excess) demonstrated the specificity of the [12'I]T3 binding to the receptor. [ '2sI]T3-labeled receptors did not enter efficiently intothe gel inthe absence of DNA (no DNA lane). As expected, no ['2sI]T3-labeledband was detected withthe nonspecific sequences CRE-1 or P3(I). Finally, we assessed the location of T3 receptor binding sites in the intact PEPCKpromoter by oligonucleotide competition DNase I footprinting. The optimal TRE and the (-332/-308)PEPCK-TRE oligonucleotides competed for binding at a single region of the PEPCK promoter which corresponded to the defined -3321-308 site (Fig. 4). In contrast, the P3(I)oligonucleotide did not alter thefootprinting at that site, although it competed for binding at other sites, indicating that the PEPCKpromoter contains a unique site capable of binding the T3receptor.

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FIG.4. DNase I footprinting with oligonucleotide competition. A 564-bp XbaI-BgnI fragment of the PEPCK promoter(nucleotides -490 to +73) was prepared from pBH1.2 by end-labeling the noncoding strand with T 4 polynucleotidekinase and [(J""'P]ATP. The probe was incubatedwith 30 pg of rat liver nuclear extract. The amount of the indicated competitoroligonucleotides added is marked in the figure. Detailed description of the regions protected (showed by boxes in the figure) are provided in Roesler et al., 1989.

ports indicated that T3stimulates PEPCK gene transcription in rat liver (Loose et al., 1985; Hoppner et al., 1986; Hartong et al., 1987). In thisstudy, we have demonstrated a T3 induction of the PEPCK gene transcription thatrequires both T3 and its nuclear receptor and have identified a TRE in the PEPCK promoter at -332 to -308. Moreover, a direct interaction between T3 receptors present in nuclear extracts of rat liver and the (-332/-308)PEPCK oligonucleotide was found by gel shift assay. The demonstration that the DNAprotein complex formed with both the optimal and the(-332/ -308)PEPCK TRE oligonucleotides was specifically labeled with [12'I]T3 provides further evidence that this sequence binds the T3receptor. Several studies involving mutations inthe promoter for the rat growth hormone gene defined a TRE consensus sequence, which contained either direct or inverted repeats of the "halfDISCUSSION site" sequence 5' AGGT(C/A)A 3' (Glass et al., 1988; Brent Thyroid hormone regulates many aspectsof hepatic metab- et al., 1989a, 1989b). The sequence of the T3 receptor-binding olism including a directeffect by T3 on thesynthesis of several site in the PEPCK promoter (antisense strand) contains an regulatory enzymes in lipogenesis and gluconeogenesis (Good- imperfect direct repeat of the TRE consensus sequence (see ridge, 1987). T3also amplifies other hormonal or nutritional Fig. 5 A ) . Furthermore, extensive sequence homology was obsignals to assist in adaptingto starvation,cold, and stress(for served when comparing TRE sequences identified in other review,see Oppenheimer and Samuels, 1983). Previous re- promoters with that in the PEPCK promoter (Fig. 5B).

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which interact with the TRE to induce gene transcription. Thus, the requirement of cis-elements other than the TREs may be a widespread rather than a pituitary-specific charact""->""-> eristic of the molecular mechanisms of T3-regulated transcrip-316 TGGGGGTCAAGGACAGG -332 rPEPCK-TRE antisense strand *. tion. A mechanism in which T3 acting via its receptor AGGTCAAGGTCA consensus sequence enhances the affinity and/or binding of trans-acting factors ( d i r e c tr e p e a t s ) to other cis-regulatory sequences is possible. Support for this suggestion comes from the finding that the levels and/or affinities of liver nuclear proteins bound to the two cissequence elements of the malic enzyme promoter are influB. enced by the thyroid status of the rat (Petty et al., 1989). Several previous studies have indicated a potentiatingeffect rPEPCK-TRE TGGGGGTCAAGGACAGG (-316/-332) antisense strand rME-TRE TTGGGGTTAGGGGAGGA ( - 2 8 1 / - 2 6 5 ) of T3 on the cAMP induction of PEPCK gene expression. rS14-TREl TTGGGGCCTGGCAGC (-2697/-2683) This effect has been described both in vivo (Loose et al., 1985) raMHC-TRE TGGAGGTOACAGGAGGA (-131/-147) antisense strand and in hepatocytes and hepatoma cells (Hoppner et al., 1986; rGH-TRE TCAGGOACGTOACCGCA ( - 1 8 1 / - 1 6 5 ) rPRL-TRE TTGGGGTCAOAAGAGGC ( - 1 5 5 6 / - 1 5 4 2 ) Muiioz et al., 1990). In the present study, we have demonstratedthat T3 and cAMP act synergistically to induce PEPCK gene transcription. To our knowledge, cooperative FIG. 5. Comparison of the nucleotide sequence of various TB interactions between T3 and cAMP have only been described receptor binding sites with that of the PEPCK promoter. A, for transcription from the promoter of the ratgrowth hormone the antisense strand sequence between -316 and -332 of the PEPCK- gene (Copp and Samuels, 1989). We have further demonTRE is compared to theconsensus TRE sequence (Brent et al., 1989a, strated that thiseffect requires functional cis-acting elements 1989b). Homologous nucleotides are indicated by asterisks. B, the for both the T3 and cAMP responses, which excludes the sequences of various native TREs are aligned uersus the PEPCKTRE. The TREsare from the promoters of the malic enzyme ( M E ) possibility that the stimulation of transcription from the T3 (Petty et al., 1990), Spot 14 (Zilz et al., 1990), a-myosin heavy chain promoter was mediated by alterationsin the pathway of ((uMHC)(Izumo and Mahdavi, 1988),growth hormone ( G H ) (Normal cAMP by T3 or vice versa. We suggest that the synergistic et al., 1989), and prolactin (Day and Maurer, 1989) genes from rat. effect of T3 and cAMP is due to interactions between tranNumbers inparentheses indicate the position of the sequences in each scription factors bound to the promoter. The fact that the promoter. P3(I) site is involved in both T3and cAMP responsiveness of the PEPCKpromoter pointsto a key role for the trans-acting The location of the TRE in the PEPCK promoter does not factor(s) bound to this site. Footprinting analysis has shown coincide with any of the previously identified cis-acting ele- that the P3(I)element binds proteins enriched in liver nuclei ments described in the promoter. Aswe have already men- when compared to kidney, spleen, and brain (Roesler et al., tioned, CAMP-responsive sites at CRE-1 andP3(I) arelocated 1989) and that it can bind C/EBP and may also bind other 3' to the TRE,while the glucocorticoid response unit defined members of the C/EBP family (Park et al., 1990). Furtherby Imai et al. (1990) is 5'. However, the PEPCK-TRE se- more, recent experiments with transgenic mice that contain quence overlaps the 5' end of the protein binding domain of the bovine growth hormone gene linked to the PEPCK proP4 (-330 to -270) which wasdefined by footprinting analysis moter with a block mutation in theP3(I) element have using nuclear extracts prepared from rat liver (Roesler et al., suggested an important role for this element in enhancing 1989) (see schematic diagram of the protein binding domains liver-specific expression of thePEPCK promoter.' Thus, in the PEPCKpromoter in Fig. 1).The P4 site contains two P3(I) plays an essential role in conferring both tissue specilow affinity C/EBP binding sites, P4(I) and P4(II) (Park et ficity and hormone inducibility tothePEPCK promoter. al., 1990). Both previous studies indicated the presence of Further investigations willbe required to analyze possible another binding site at the 5' end of P4 which displayed interactions between the T3receptor and members of the C/ different binding properties to the defined P4(I) and P4(II) EBP family. Likewise,the synergistic cooperation between T3 regions. Our results show that this site P4(III) is the T3 and cAMP makes the PEPCK gene an interesting model for receptor binding site in the PEPCK promoter. Furthermore, analyzing how several hormones converge to regulate the the oligonucleotide competition DNase I footprinting (Fig. 4) expression of a single gene. demonstrates that thereis a unique TRE site inthe promoter and also that the P3(I)oligonucleotide, a well defined C/EBP Acknowledgments-We thank M. G . Rosenfeld for the generous binding site, is unable to compete for the binding to the TRE gift of RSV-hT3Rb. We also thank Teiko Kimura for her technical site, whereas it does compete for the binding of proteins to assistence. the P4(I) and P4(II)domains. The PEPCK-TRE can act as an independent thyroid reREFERENCES sponse element when it is ligated to aneutral promoter. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Siedman, However, within the context of thePEPCK promoter, a J. G., Smith, J. A., and Struhl, K. (1989) Current Protocols in second element P3(I) is also required for the T3 induction Molecular Biology, Vol. 1, Wiley Interscience, New York even though this element does not bind the thyroid receptor. Baker, J. A., Roesler, W. J., Vanderbark, G . R., Kaetzel, D. M., Hanson, R. W., and Nilson, J. H. (1988) J . Biol. Biol. 263,19740The requirement of more than a TRE to confer transcrip19747 tional responsiveness to T3has been previously described only G . A., Harney, J. W., Moore, D. D., and Larsen, P.R. (1988) with promoters from pituitary specific genes. Ye et al. (1988) Brent, Mol. Endocrinol. 2,792-798 reported that both the TRE and two cell-specific basal ele- Brent, G . A., Larsen, P. R., Harney, J. W., Koenig, R. J., and Moore, ments are needed for T3 responsiveness of the rat growth D. D. (1989a) J. Biol. Chem. 264,178-182 hormone promoter. Likewise, the involvement of cell-specific Brent, G . A., Harney, J. W., Chen, Y., Warne, R. L., Moore, D. D., and Larsen, P. R. (1989b) Mol. Endocrinol. 3,1996-2004 basal elements has also been suggested for the prolactin promoter (Day and Mauer, 1989). The results of this study indicate that the PEPCKpromoter also requires cis-elements Y. Pate1 and R. W. Hanson, unpublished results. A.

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Thyroid Hormone

Regulates PEPCK Gene Transcription

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