The Chicken Malic Enzyme Gene: Structural Organization and Identification of Triiodothyronine Response Elements in the 5′-Flanking DNA

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 334, No. 2, October 15, pp. 309–324, 1996 Article No. 0460

The Chicken Malic Enzyme Gene1: Structural Organization and Identification of Triiodothyronine Response Elements in the 5*-Flanking DNA2 Dean W. Hodnett, Dominic A. Fantozzi,* Debbie C. Thurmond, Stephen A. Klautky, Kathleen G. MacPhee, Shawn T. Estrem, Gang Xu, and Alan G. Goodridge3 Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242; and *Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

Received March 4, 1996, and in revised form July 18, 1996

In vivo, feeding stimulates and starvation inhibits transcription of the malic enzyme gene. In chick-embryo hepatocytes in culture, triiodothyronine (T3) stimulates and glucagon inhibits transcription of this gene. As a first step in the characterization of the involved regulatory mechanisms, fragments of genomic DNA spanning the structural and 5*-flanking regions of the chicken malic enzyme gene were cloned. The coding region of the gene is organized into 14 exons and 13 introns and is greater than 106 kb in length. The size of the gene, the number and lengths of the exons, and positions at which introns are inserted into the coding regions are virtually identical in the chicken and rat genes. When transiently transfected into chick-embryo hepatocytes, 5800 bp of 5*-flanking DNA conferred T3 responsiveness to a linked chloramphenicol acetyltransferase (CAT) reporter gene. Using deletion and site-specific mutations of 5*-flanking DNA, we identified a complex T3 response unit that contains one major T3 response element (T3RE) and several minor ones. The major element contains two degenerate copies of the hexamer, RGGWMA, sepa1

GenBank Accession No. U49693. D.W.H. contributed data for Figs. 4 and 6, statistically analyzed the transfection results, and wrote the manuscript; D.A.F. isolated and characterized the genomic clones, contributed the data in Fig. 2, and determined the nucleotide sequence of the 5*-flanking DNA from 0413 to /224 bp; D.C.T. contributed the data for Figs. 5, 7, 9, and 10 and did the DNase I-footprint analysis; S.A.K. constructed the vectors in Fig. 5 and set up the transient transfection system in our laboratory; K.G.M. supervised analysis of the intron–exon boundaries and sequencing of the gene; S.T.E. determined the nucleotide sequence from 04.8 to 02.4 kb; G.X. made and initially tested the first four of the TK-CAT constructs used in the experiments described in Fig. 7. A.G.G. supervised the planning, execution, and interpretation of the experiments and the writing of the paper. 3 To whom correspondence and reprint requests should be addressed. Present address: College of Biological Sciences, Ohio State University, 484 West 12th Ave., Columbus, OH 43210. 2

rated by 4 bp and was a strong repressor in the absence of ligand. Endogenous levels of T3 receptor are sufficient to allow the T3 response elements in the upstream region of the malic enzyme gene to confer responsiveness to T3, suggesting that they are physiologically relevant. q 1996 Academic Press, Inc. Key Words: thyroid hormone; malic enzyme gene; transcription; promoter; avian.

Malic enzyme [L-malate-NADP/ oxidoreductase (decarboxylating), EC 1.1.1.40] catalyzes the NADP/-dependent oxidative decarboxylation of malate to pyruvate and CO2 . Much of the NADPH formed in this reaction is used for fatty acid synthesis. Malic enzyme is a ‘‘lipogenic’’ enzyme; its rate of synthesis and the abundance of its mRNA are increased in liver when starved animals are refed a diet high in carbohydrate (1); regulation is transcriptional (2). In livers of 19- to 21-day-old chick embryos, malic enzyme activity and the rate of fatty acid synthesis are very low. When newly hatched chicks are fed, malic enzyme activity increases about 70-fold. In hepatocytes isolated from 19-day-old chick embryos and incubated in a serumfree, chemically defined medium containing insulin, triiodothyronine (T3) causes an increase in the activity of malic enzyme that quantitatively mimics the increase caused by feeding (3). T3 stimulates mRNA accumulation (1), primarily by increasing the rate of transcription of the malic enzyme gene (4). The first step in the stimulation of transcription by T3 involves binding of T3 to a receptor that is bound to a specific DNA element within the regulated gene (5). T3 appears to stimulate transcription of the malic enzyme gene by such a mechanism because the effect is rapid and does not require on-going protein synthesis (4). We report here the isolation and characterization 309

0003-9861/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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of the structural and 5*-flanking parts of the chicken malic enzyme gene. One major and several minor T3 response elements constitute a T3 response unit nearly 4 kb upstream from the start site for transcription. MATERIALS AND METHODS Materials. Restriction enzymes were obtained from Eastman Kodak (New Haven, CT), Life Technologies (Gaithersburg, MD), New England Biolabs (Beverly, MA), United States Biochemical Corp. (Cleveland, OH), or Boehringer-Mannheim (Indianapolis, IN) and used according to the manufacturer’s instructions. Other enzymes were obtained from the indicated sources: DNase I (Cooper-Worthington, Freehold, NJ); RQ-DNase I (Promega, Madison, WI), RNAse A (Sigma, St. Louis, MO); Moloney murine leukemia virus reverse transcriptase (Life Technologies); T4 polynucleotide kinase, Klenow fragment of Escherichia coli DNA polymerase I and S1 nuclease (Boehringer-Mannheim or Promega); T7 polymerase (Sequenase, United States Biochemical Corp.); DNA polymerase from Thermus aquaticus (Promega); Bst polymerase (Bio-Rad, Richmond, CA). Nucleotides were purchased from Sigma, Pharmacia, or Life Technologies. Radiolabeled nucleotides were obtained from Amersham (Arlington Heights, IL), DuPont-New England Nuclear Corp. (Boston, MA) or ICN (Costa Mesa, CA). D-threo-[dichloroacetyl-1,2-14C]-chloramphenicol was from Dupont-New England Nuclear Corp. Nitrocellulose membranes were from Schleicher and Schuell, Inc (Keene, NH). A nylon-based membrane, GeneScreen, was obtained from DuPont-New England Nuclear Corp. Lipofectin, LipofectACE, and Waymouth medium MD 705/1 were purchased from Life Technologies. E. coli cells of strain DH5a were used for subcloning and large-scale preparations of plasmid DNA; competent cells were obtained from Life Technologies. Agarose (SeaKem LE, NuSieve GTG or SeaPlaque GTG) was purchased from FMC Corp. (Rockland, ME). Unless indicated otherwise, all hormones were from Sigma. All other chemicals were of reagent grade or the best quality commercially available. Plasmid DNA’s pBR322 and pUC19 (P-L Biochemicals), pGEM3 and Bluescript KS/ (Stratagene Cloning Systems, La Jolla, CA), and pIBI31 (Eastman Kodak) were obtained from the indicated sources. A Charon 4A library of chicken genomic DNA (6) was kindly provided by Dr. J. Dodgson (Michigan State University). EMBL3 (American Type Culture Collection, Rockville, MD) and EMBL4 (Dr. H.-J. Kung, Case Western Reserve University) libraries of chicken genomic DNA were obtained from the indicated sources. An EMBL3 library from chicken liver DNA (Clontech) also was screened. Isolation and characterization of chicken malic enzyme cDNA and genomic clones. Hybridization probes were prepared from pGME1 (7), a cDNA complementary to the 3* end of malic enzyme mRNA from the goose uropygial gland, or from pDME1 (8, 9), a malic enzyme cDNA of 1866 bp from duck liver, and used to screen a cDNA library from chick-embryo hepatocytes (10, 11). A chicken malic enzyme cDNA (pCME5) was isolated and its nucleotide sequence determined (M. J. Glynias, B. A. Roe, and A. G. Goodridge, unpublished results). There is 93% sequence identity between the duck and chicken cDNA’s in a 1676-bp overlap that begins at the second in-frame ATG of both cDNA’s. The DNA insert of pCME5 was 1950 bp and did not hybridize to end-labeled poly(U). Full-length mRNA for malic enzyme is 2100 bp (1), so we assumed that the inserts of both pCME5 and pDME1 contained sequences at or near the 5*-end of the mRNA. Chicken genomic libraries were probed with pGME1 DNA and DNA fragments from pCME5 or pDME1 by the plaque hybridization procedure (12). Genomic clones were characterized by restriction mapping and partial determination of their nucleotide sequences. The linear order of the cloned DNA’s along the malic enzyme gene was determined by hybridization to specific fragments of the cDNA, synthetic oligonucleotides corresponding to parts of the cDNA or parts of other genomic clones, and by analyses of nucleotide sequences. Reporter plasmids. Routine subcloning was performed by standard methods (13, 14). We constructed expression plasmids con-

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taining 5*-flanking DNA of the malic enzyme gene inserted upstream of the CAT gene in pKSCAT. A fragment of pRSVCAT (15) containing the coding region of the bacterial chloramphenicol acetyltransferase (CAT)4 gene and an intron and polyadenylation signals derived from the small t antigen gene of SV40 was isolated and subcloned into pBluescript KS(/) to form the promoterless CAT expression vector, pKSCAT. A PstI fragment of the malic enzyme gene from 0413 to /224 bp relative to the transcription start site was removed from genomic clone lCME245 (Fig. 1A) and subcloned into pIBI31 to form pIBI31[ME0413//224]. The PstI fragment was removed from pIBI31[ME0413//224] and digested with NlaIII. NlaIII cleaves 3* to the first potential start codon of the malic enzyme gene at /33 bp. Treatment of the resulting fragment with T4 DNA polymerase removed the 3* overhang. HindIII linkers were added, and the fragment was ligated into the HindIII site of pKSCAT to form p[ME0413/ /31]CAT. p[ME05800//31]CAT was constructed by ligation of an EcoRI–SfiI fragment from lCME245 into p[ME0413//31]CAT that had been digested with EcoRI and SfiI; the resulting construct contains contiguous malic enzyme 5*-flanking DNA from 05800 to /31 bp linked to the CAT gene. Expression vectors containing 5*-deletion mutations of p[ME05800//31]CAT were prepared as follows: plasmid DNA’s were digested with NotI, which recognizes a unique site in the multiple cloning site 5* of the malic enzyme flanking DNA, and a restriction enzyme that digested malic enzyme DNA one, two, or three times but did not cut in the rest of the plasmid DNA (Fig. 1B). If the second restriction enzyme cut the flanking DNA two or three times, partial digestion was used to generate fragments with the two or three possible 5* endpoints. The resulting linear DNA’s were treated with T4 DNA polymerase to create blunt ends and then ligated. Fragments of the malic enzyme 5*-flanking DNA were inserted into the multiple cloning site 5* of the HSV thymidine kinase (TK) promoter in pBLCAT2 (16) to form the ME/TKCAT constructs. The 5*-end of the DNA fragment containing the TK promoter was at 0105 bp with respect to the start site for transcription of the TK gene. Fragments of the malic enzyme 5*-flanking DNA were inserted into the BstXI site in the 5* multiple cloning site of p[ME0147/ /31]CAT to form constructs containing putative regulatory DNA from the malic enzyme gene linked to its own minimal promoter. Structures of the resulting plasmid DNA’s were confirmed by restriction enzyme mapping and partial sequence analyses. The luciferase reporter plasmid, pXP1 (17), was obtained from S. K. Nordeen (University of Colorado Health Sciences Center). pXP1 was digested with SmaI and HindIII, both of which cleave the DNA immediately 5* to the luciferase cDNA. The RSV promoter region, a 397-bp fragment, was obtained by digestion of pRSV-CAT with NruI and HindIII and ligated into pXP1 to form pRSV-LUC. F. B. Hillgartner (West Virginia University) provided p[ME03474//31]CAT and p[(ME03474/02715)ME0147//31]CAT. Bruno Luckow and Gunter Schutz (Heidelberg, Germany) provided pBLCAT2 (16; TKCAT). Herbert Samuels (New York University) provided the cDNA for chicken T3 receptor a cloned into the pET8c expression vector. pCMV-bGAL (18) was obtained from Richard Maurer (University of Iowa). Generation of site-specific mutations. Mutations (site-specific or internal deletions) were introduced into double-stranded plasmids by using the Transformer site-directed mutagenesis kit from Clontech Laboratories, Inc. (Palo Alto, CA), a modification of the procedure of Deng and Nickoloff (19). Structures of the inserts were confirmed by nucleotide sequence analyses. Constructs prepared using the polymerase chain reaction. Fragments of malic enzyme gene 5*-flanking DNA were synthesized by

4 Abbreviations used: CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; HSV, Herpes Simplex Virus; MMLV, Moloney Murine Leukemia Virus; RSV, Rous Sarcoma Virus; RXR, retinoid X receptor; T3, L-3,5,3* triiodothyronine; T3RE, T3 response element; TK, thymidine kinase.

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THE CHICKEN MALIC ENZYME GENE the polymerase chain reaction (PCR) by standard methods (20). Recognition sites for SphI and BamHI were added to the 5* and 3* ends of PCR products. PCR-generated fragments were purified using agarose gel electrophoresis, ligated into the SphI/BamHI sites of pBLCAT2 (16), and cloned. Structures of the inserts were confirmed by nucleotide sequence analyses. RNA isolation. Total RNA was isolated from chicken liver or from hepatocytes in culture by guanidinium thiocyanate/phenol/chloroform extraction (21). Poly(A)/ RNA was isolated by oligo-dT cellulose chromatography or batch extraction using the Promega PolyATtract kit. Poly(A)/ RNA was quantitated by UV spectroscopy. Cell culture. Isolated hepatocytes were prepared from the livers of 19-day chick embryos (22), added to 35-mm tissue-culture plates, and incubated in Waymouth medium MD 705/1 supplemented with streptomycin (100 mg/ml), penicillin G (60 mg/ml), insulin (50 nM) (Eli Lilly and Co.; Indianapolis, IN), and corticosterone (1 mM). Transient transfection. DNA’s were transfected as follows: (1) 2.5 mg p[ME05800//31]CAT or an equimolar amount of another reporter plasmid; (2) 0.5 mg pCMV-bGAL, (hormone-neutral b-galactosidase expression vector, [18]) or 0.5 mg pRSV-LUC (hormone-neutral luciferase expression vector); (3) pBluescript or pKSCAT DNA to bring the total amount of transfected DNA to 5.0 mg per plate. Cells were plated on Day ‘‘zero.’’ On Day 1, medium was replaced with one containing a mixture of DNA’s and LipofectACE. On Day 2, the transfection medium was replaced with fresh medium with or without T3 (1.6 mM). On Day 4, hepatocytes were harvested and cell extracts prepared. A detailed description of our transfection procedures has been published (23). Analysis of cell extracts. Cell extracts were analyzed for protein quantity (24), either b-galactosidase (13) or luciferase activities (25) and CAT activity (15). For CAT activity, extracts were heated to 607C for 30 min; denatured protein was removed by centrifugation. Samples of heat-stable protein containing the equivalent of 2 to 50 mg of unheated soluble protein were incubated for 15 h at 377C in 2.6 mM acetyl-CoA, 100 mM Tris–HCl, pH 7.8, 1 mM EDTA, and 12 mM [14C]chloramphenicol. Chloramphenicol and its acetylated products were extracted with ethyl acetate and separated by thin-layer chromatography. Radioactivity in substrate and products was measured by liquid scintillation spectrometry or direct autoradiography using a Packard Instantimager (Packard Instrument Co., Meriden, CT). The results were expressed initially as percentage of substrate converted to acetylated product per microgram unheated soluble protein and then normalized as described in the figure legends. Primer extension of endogenous mRNA. The primer 5*-CCTGCTTGCATGGCGACGGACT-3* (complementary to /20 to /41 bp of the malic enzyme gene) was radiolabeled at its 5* end. Hepatic poly(A)/ RNA (20 mg) isolated from 2-week-old chicks that had been starved for 48 h and then fed for 24 h was incubated with labeled primer (0.2 mg, 5 1 104 cpm) and 200 units of MMLV reverse transcriptase at 377C for 1 h in 20 ml of 125 mM Tris–HCl, pH 8.3, 185 mM KCl, 25 mM DTT, 7.5 mM MgCl2 . Products were analyzed by electrophoresis through an 8% polyacrylamide gel containing 7 M urea. A sequencing ladder generated from M13mp18 and a universal primer was used as a standard for fragment length. S1 nuclease analysis of endogenous mRNA. A 215-bp BstNI– HaeIII fragment spanning potential start sites as determined by primer extension was isolated and labeled with T4 polynucleotide kinase. The labeled probe (0.2 mg, 5 1 104 cpm) was incubated with 20 mg RNA in 20 ml 80% formamide, 0.4 M NaCl, 0.01 M Pipes, pH 6.5, 0.1% SDS, 1 mM EDTA at 427C for 15 h. Samples were digested with 1000 units S1 nuclease at 377C for 1 h in 0.05 M potassium acetate, pH 4.5, 0.3 M NaCl, 1 mM ZnSO4 , 25 mg/ml denatured salmon sperm DNA. HaeIII fragments of fx 174 end-labeled with 32P were used as standards for fragment lengths. S1 nuclease analysis of mRNA from transfected hepatocytes. Hepatocytes were isolated and incubated as described above except that 10-cm tissue-culture plates were used. Transient transfection was

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performed using 12.5 mg p[ME05800//31]CAT, 12.5 mg pKSCAT, and 127 mg LipofectACE per plate. After removal of the transfection medium, the hepatocytes were incubated with insulin plus corticosterone or with insulin plus corticosterone plus T3 for 48 h. RNA transcribed from chimeric genes containing the CAT gene linked to the homologous promoter was extracted from hepatocytes treated with or without T3. RNA transcribed from the endogenous gene or chimeric genes containing the CAT gene linked to the TK promoter was extracted from hepatocytes treated with T3 only. The deoxyoligonucleotide, 5*-GATATATCAACGGTGGTA-3* (/23 to /40 bp relative to start site of translation of the CAT gene), was 5*-end labeled, and used to synthesize single-stranded DNA probe from an M13 template (13). Poly(A)/ RNA (10 to 35 mg) was incubated with 5 1 104 cpm of probe (10 ng) in 30 ml of 80% formamide, 0.4 M NaCl, 0.04 M Pipes, pH 6.4, 1 mM EDTA for 18 h at 427C. Samples were digested for 1 h at 247C with 600 units S1 nuclease per milliliter, 0.28 M NaCl, 50 mM sodium acetate, pH 4.5, 4.5 mM ZnSO4 , 20 mg/ml denatured herring sperm DNA. Digestion products were subjected to electrophoresis on 7 M urea sequencing gels (6–7% Long Ranger, J. T. Baker, Phillipsburg, NJ) and visualized by autoradiography. The M13 template used in the synthesis of the probe was sequenced using the same primer and [a-32P]dATP. The products of the sequencing reaction were added to yeast tRNA and subjected to electrophoresis with the S1 reaction products. Sequence analyses. Except as noted, nucleotide sequences were determined by the dideoxynucleotide method using various DNA polymerases or by an automated procedure using an ABI sequencer in the DNA Core of the University of Iowa. Sequences were compiled and analyzed using Geneworks (Intelligenetics, Inc., Mountain View, CA) or GCG (26). DNase I footprint analysis. Nuclear extracts were prepared from chick-embryo hepatocytes incubated with insulin plus corticosterone plus T3 for 48 h. The extraction buffer contained 0.42 M NaCl and the protease inhibitors leupeptin, benzamidine, aproteinin, and PMSF; the extraction procedure has been described (14). Chicken TRa cDNA in pET8c was expressed in E. coli BL21(DE3)pLYSs and purified from E. coli through the heparin-agarose (Sigma) step (27). Binding reactions contained 10 mM Hepes, pH 7.5, 5% glycerol (vol/ vol), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5 mg poly(dI/dC), 60 mg nuclear extract, and/or 0.03–3.0 mg partially purified chicken T3 receptor a, and 2 1 104 cpm [a-32P]dATP-labeled DNA (13 fmols) in a total volume of 80 ml. Reactions were incubated at 47C for 2 h, followed by 10 min at 207C. DNA–protein complexes were digested with RQ DNase I (Promega) in the presence of 5 mM MgCl2 and 1 mM CaCl2 for 2 min at 207C. The amount of DNase I added to each reaction was adjusted to yield approximately the same degree of digestion with each sample. Digestion was stopped with EDTA. Reaction mixtures were extracted once with phenol:chloroform (1:1 vol/ vol) followed by extraction with chloroform alone. DNA–protein complexes were precipitated by adding 50 ml saturated ammonium acetate and 14 mg tRNA in ethanol, followed by an 80% ethanol wash. Complexes were analyzed on a 6% denaturing polyacrylamide gel (Long Ranger) containing 110 mM Tris base, 110 mM boric acid, 2.4 mM EDTA, 7 M urea, and subjected to autoradiography at 0707C with Hyperfilm-MP (Amersham) for 20 h. Maxam–Gilbert DNA sequencing reactions were performed as described (14). Statistical analyses. Data were analyzed using SAS/STAT software (SAS Institute, Inc., Cary, NC) (28). Significance of differences between pairs of means was determined by the Wilcoxon signed-rank test or the Mann–Whitney test (29). Standard errors of the mean are provided to indicate the degree of variability in the data.

RESULTS

Organization of the coding region. Eleven genomic clones were isolated from the malic enzyme locus in the chicken genome (Fig. 1A). The 5*-most clone,

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FIG. 1. Genomic organization of the chicken malic enzyme gene. (A) Cloned DNA’s isolated from genomic libraries in bacteriophage-l EMBL4 or Charon 4A are depicted as solid lines at top. Exons are shown as solid rectangles with roman numeral designations. The open rectangle corresponds to exon III of the rat gene, but was not isolated from the chicken genome. The chicken cDNA is shown at bottom. The connecting lines show the cDNA sequence associated with each exon. Restriction endonuclease sites within the cDNA are indicated. A, AvaI; E, EcoRI; H, HindIII; R, RsaI; S, SphI; X, XbaI. (B) Restriction map of the 5*-flanking DNA of the malic enzyme gene. The solid and dotted lines represent 5*-flanking and structural DNA, respectively, of the malic enzyme gene. An arrow indicates the major start site for transcription. The indicated restriction sites were used in the construction of 5* deletions. The shaded ovals represent DNAse I-hypersensitive sites.

lCME245, contains the major and minor start sites for transcription (Fig. 2). The 3*-most genomic clone, lCME1, contains a consensus sequence for polyadenylation (AATAAAA) and sequence corresponding to that of the 3* end of the cDNA (results not shown). From the start site for transcription to the polyadenylation signal, the cloned genomic DNA spans more than 106 kb. Most of the clones do not contain overlapping DNA inserts, so the transcription unit is substantially longer than 106 kb. The first intron is more than 33 kb in length (Fig. 1A); several 3*-introns are much shorter. The locations of exon – intron junctions were determined by comparison of sequences derived from the DNA inserts of the genomic DNA clones with those from pCME5 and pDME1. Thirteen of the gene’s fourteen exons were identified and characterized (Fig. 1A, Table I). We did not isolate a genomic clone corresponding to exon III of the rat. The ends of all introns correspond to the GT-AG rule (30), and the sequences comprising the intron/exon junctions generally agree with the consensus sequences for intron/exon junctions (31). Introns 2 – 13 were inserted into the coding

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sequences at the same positions in the chicken and rat genes (32, 33). Transcription initiation in the endogenous gene. Primer extension of poly(A)/ RNA from the liver of a starved-refed chick yielded two major products that corresponded to start sites that were 29 and 30 bp 5* of the A of the first ATG (Figs. 2A and 3). Minor start sites were detected between about 030 and 068 bp upstream from the major start sites. Primer extension of poly(A)/ RNA from T3-treated chick-embryo hepatocytes yielded products that mapped to the same sites (results not shown). In S1 nuclease protection experiments (Fig. 2B), poly(A)/ RNA from the liver of a starved-refed chick or from chick-embryo hepatocytes yielded major species of 70 and 72 nt that mapped to the same major start sites identified in the primerextension analysis. Minor products were similar in size to some of the minor products detected in the primerextension analysis. Sequence analysis of the promoter and 5*-flanking region. The nucleotide sequence of the 5*-flanking

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THE CHICKEN MALIC ENZYME GENE

FIG. 2. Transcription initiation in the chicken malic enzyme gene. (A) Primer extension. Chicken liver poly(A)/ RNA or yeast tRNA (20 mg) were analyzed as described under Materials and Methods. Lanes marked G, A, T, C represent the products of sequencing reactions using M13mp18 and universal primer and are used as size markers. Large solid arrow indicates the major primer-extension product; the small arrows indicate minor extension products. Numbers at the right indicate lengths of the individual DNA’s in bp. The major start site for transcription is designated /1. (B) S1 nuclease protection. Poly(A)/ RNA from hepatocytes treated with T3 or from chicken liver or yeast tRNA (20 mg) were analyzed as described under Materials and Methods. Large solid arrow indicates the major product protected from digestion by S1 nuclease. The open arrow indicates full-

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DNA and the 5* end of the coding region of the malic enzyme gene were determined from 04392 bp to /224 bp (Fig. 1B). The proximal promoter region is characterized by a high GC content and the absence of a consensus TATAA box upstream of the major start sites (Fig. 3). This region contains putative DNA binding sites for C/EBP, GATA-1 (noncoding strand), Sp1, and AP-1. Translation initiation sites are located at /31 and /88 bp (both in the same frame). Sequence identity between the nucleotide sequences of the proximal promoter regions of the rat (0886 to /218 bp) and chicken (0886 to /224 bp) genes was 44%. There were no extended regions of high similarity except for that between 0188 to 0101 bp of the chick, which is 73% identical to 0133 to 056 bp (with the insertion of three small gaps) of the rat gene. This is the same degree of identity as that shared by the coding regions of the chicken and rat mRNAs (M. J. Glynias, D. W. Hodnett, K. G. MacPhee, and A. G. Goodridge, unpublished results). Within the region of 73% identity is a perfect 12-bp match (Fig. 3) that includes a consensus site for AP1 and partially overlaps malic promoter element 1 of the rat gene (34). The proximal region of the chicken gene did not have a T3RE similar to that of the rat gene (35). A polypyrimidine tract, interrupted by only three purines, spans the region from 0134 to 086 bp. This region is sensitive to S1 nuclease in its supercoiled form and likely forms non-B DNA structure (36). The rat malic enzyme gene does not contain a polypyrimidine tract in this region. Also of interest in the proximal promoter of the chicken gene is a repeated 9-bp sequence (5*CCCGCAGGA3*). The 5*-ends of these repeats lie at the 3*-end of the polypyrimidine tract (088 bp), and just upstream of the major start sites for transcription (018 bp). A 9-bp repeated sequence (5*TCGCACGGC3*) also is found in the rat gene; the 5*-ends of these repeats begin at 0160 bp and at 021 bp. The rat and chicken repeats share a common core motif, 5*-YCGCASGR-3*. Transcription initiation in chimeric malic enzymeCAT genes. In poly(A)/ RNA from T3-treated hepatocytes transiently transfected with p[ME05800//31]CAT (Fig. 4A), protected species mapped to the same initiation sites as those observed in the endogenous gene, although the relative amount of initiation at /1 was less than that for the endogenous gene (Fig. 4B). We did not detect transcripts from transfected hepatocytes not treated with T3, indicating that the increase in CAT activity caused by T3 was due to an increased abundance of CAT mRNA which, in turn, was likely due to increased transcription. We also mapped sites of transcription initiation from

length, undigested DNA probe. The numbers to the left indicate the lengths of individual DNA’s in bp. The size markers were HaeIII fragments of fx174. The major start site for transcription is at /1.

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Length of Exons and Characteristics of Exon–Intron Junctions in the Chicken Malic Enzyme Gene Exon No.

Length (nt)

5*-splice donor

Intron

3*-splice acceptor

I II III IV V VI VII VIII IX X XI XII XIII XIV

132 134 150 76 162 104 110 98 115 104 144 174 99 ú349

AACAAGgtaggg AGACAGgtaaaa ND ATCAAGgtc AATGAGgtaagt TTCAAGgtatgt TTCAAGgtaaa GGAGAGgtatgt TGAAAGgtaata TAATAgtaatt ACAGAGgtaacc GCTGAGgtactg GTGAGGgtaagt AATAAA

1 2 3 4 5 6 7 8 9 10 11 12 13

ttgcagGGCATG ND tgcagAGGTCT ctccagGCTATT taaaagGCATTG ttccagTTATGG cttcagGAACAG ctacagGCTGCA gctcagGGCGTG tcctagAGTCGC acgtagGGACGT tttcagGTAATA ccgcagAAGAAG

Length (nt) of corresponding exon of rat gene 78 134 150 76 162 105 110 98 114 106 143 174 99 513 (or *1513)

Note. Lengths of the exons in the rat malic enzyme gene were taken from Morioka et al. (33). The length of exon I is based upon the major start site for transcription. Exon nucleotides are in upper-case letters; intron nucleotides are in lower-case letters. Boundaries of exon III were not determined (ND). *The rat expresses two malic enzyme mRNA’s that differ in lengths of their 3*-untranslated regions.

constructs containing minimal promoters linked to upstream regions that confer T3 responsiveness. Cells transfected with p[ME0147//31]CAT had promoter activity but did not show a statistically significant response to T3; a deletion from 0147 bp to 063 bp eliminated promoter activity (results not shown). In hepatocytes transfected with p[(ME04135/02715)ME0147/ /31]CAT, T3 caused a 30-fold increase in CAT activity; transcripts initiated almost exclusively from sites located at about 089 and 074 bp (Fig. 4C). In the absence of T3, no transcripts were detectable. Transcripts derived from p[ME05800//31]CAT and the native gene also used these start sites (Figs. 4A and 4B), but to a lesser extent. These results suggest that sequences between 02715 and 0147 bp may influence the choice of start site. In hepatocytes transfected with TKCAT, a heterologous minimal promoter from the thymidine kinase gene of Herpes Simplex Virus (16), T3 had little or no effect on CAT activity. Transcripts from p[ME04135/02715]TKCAT (Fig. 4D) initiated at one of the expected sites of the TK gene (37). Identification of T3 response elements. T3 caused a 9-fold increase in CAT activity in hepatocytes transfected with p[ME05800//31]CAT (Fig. 5). In cells transfected with constructs with 5* ends at 05200 or 04135 bp, the effect of T3 increased to 12- and 15-fold, respectively. In cells transfected with p[ME03845/ /31]CAT, stimulation by T3 was 8-fold (Fig. 5), suggesting a T3 response element (T3RE) between 04135 and 03845 bp. In hepatocytes transfected with the next smaller deletion, T3-responsiveness fell to 5-fold. Hepatocytes transfected with DNA’s containing 5*-ends from 02715 to 0147 bp, T3 had no effect. These results sug-

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gest that two or more T3RE(s) are located between 04135 and 02715 bp. CAT activity in hepatocytes transfected with p[ME(04135/02715)ME0147//31]CAT was increased 30-fold by T3 (Fig. 6A). Thus, all of the T3 responsiveness bestowed by 05800 bp of 5*-flanking DNA (23fold in this set of experiments) was recovered in the 04135- to 02715-bp fragment. In cells transfected with constructs containing the 03470- to 02715- or 4135to 03471-bp fragments inserted into p[ME0147/ /31]CAT, the T3-induced increase in CAT activity was 4.4-fold or 17-fold, respectively. The next experiments involved 5* deletions of p[ME(04135-/03471)ME0147//31]CAT (Fig. 6A). Deletion to 03845 bp decreased responsiveness to T3 from 17- to 7-fold, consistent with the loss of responsiveness caused by deletion of 04135 to 03845 bp in the context of the entire 5*-flanking DNA (Fig. 5). In cells transfected with a construct that was deleted to 03764 bp, the response to T3 decreased to that for cells transfected with p[ME0147//31]CAT. Several 3* deletions of the 04135- to 03471-bp fragment were then tested (Fig. 6A). Deletion from 03761 to 03471 bp decreased responsiveness from 17- to 5.2fold, despite the fact that activity of the deleted fragment (03764 to 03471 bp) was no greater than that of the minimal promoter. The next 3* deletion removed 03844 to 03761 bp and, compared to the construct containing 04135 to 03761, had no effect on T3-responsiveness. These results are consistent with a T3RE centered at 03761 bp. Subsequent deletion from 03896 to 03844 bp decreased responsiveness to T3 to the same as that for p[ME0147//31]CAT. In sum, the deletion

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FIG. 3. Nucleotide sequence flanking the start site for transcription of the chicken malic enzyme gene. The arrow indicates the nucleotide designated as the major start site for transcription. Putative translation start sites are circled. A polypyrimidine tract is boxed. Based on sequence similarity to consensus binding sites, putative binding sites for Sp1 and AP-1 are underlined and indicated. Nine-basepair repeats at 088 and 018 are overlined. The nucleotide sequence corresponding to the oligonucleotide used for primer extension is underlined with a dashed line. Nucleotides from the 5* end of intron 1 are indicated by lower case letters.

experiments described above suggest the presence of one or more T3RE(s) each between 03896 and 03844 bp, 03844 and 03471 bp, and 03470 and 02715 bp. Hepatocytes transfected with the 04135- to 03471bp fragment inserted into p[ME0147//31]CAT in 5* to 3* or 3* to 5* orientations resulted in 17- and 20-fold increases in CAT activity, respectively (Fig. 6A). The 04135- to 02715-bp fragment was then linked to a heterologous minimal promoter of the HSV TK gene and transfected into hepatocytes; T3 caused a 37-fold stimulation of CAT activity (Fig. 6B). The 04135- to 03471-bp region inserted into TKCAT conferred 78and 53-fold stimulation in the 5* to 3* and 3* to 5* orientations, respectively. The malic enzyme T3RE’s

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thus work equally well with promoters that do or do not contain a TATAA box. Furthermore, T3-responsiveness was not altered even though the T3RE’s in some constructs were about 2.5 kb closer to the start site for transcription than in the natural gene or reversed in orientation with respect to the start site. Thus, T3RE’s with enhancer-like function are located between 04135 and 03471 bp. The region from 03903 to 03617 bp was analyzed in the context of the heterologous TK promoter (Fig. 7). Deletion from 03903 to 03868 bp, reduced T3 responsiveness by about 90%. By contrast, deletion from 03617 to 03703 bp had no effect on T3-responsiveness. Cells transfected with a fragment with both 5*- and 3*deletions, 03868 to 03703 bp, responded to T3 to the same extent as cells transfected with a construct containing only the 5*-deletion. Additional 3* deletions to 03769 bp had no effect on the response to T3. Three additional 3*-deletions to 03799, 03823, and 03863 resulted in gradual decreases in T3 responsiveness from 79-fold to 50-, 42-, and 38-fold, respectively. These results suggest the presence of a strong T3RE between 03903 and 03863 bp and weak ones between 03863 and 03733 bp. The nucleotide sequence between 03903 and 03617 bp reveals the presence of several hexamer sequences that closely resemble ‘‘consensus’’ half-sites for T3RE’s (RGGWMA) (Fig. 8). Mutations were introduced at positions II, III, and IV of individual hexamers within p[ME03903/03703]TKCAT (Fig. 9). Mutations of these nucleotides disrupts T3 receptor binding and transcriptional activation by T3 in other T3RE elements (38, 39, 40). Mutations in half-sites 3, 4, 5, and 6 reduced the T3 response by about 50% each without changing basal CAT activity. These half-sites lie between 03863 and 03733 bp where putative weak T3RE’s were identified by 3*-deletions. Mutations in half-site 2D reduced T3 responsiveness by 90%. Mutations in half-site 1 did not cause a statistically significant decrease in T3-responsiveness. Half-site 7 at 03675 was not tested with substitution mutations because a 3*-deletion that removed this region had no effect on T3-responsiveness. T3RE’s are usually composed of two copies of the half-site organized as direct repeats with a 4-nucleotide spacer between half-sites, as inverted repeats with no spacer, or as everted repeats with a 6- or 8-bp spacer (41). Maximal responsiveness requires both half-sites (38, 40). Upstream of half-site 2D, with a 4-nucleotide spacer, is an imperfect direct repeat of half-site 2D (Figs. 8 and 10A). This upstream half-site (2U) was removed in the 5*-deletion that created p[ME03868/ 03617]TKCAT (Fig. 7). A 3-bp block mutation was introduced into half-site 2U in the context of p[ME03903/ 03703]TKCAT (Fig. 10A). Individual mutations of either half-site 2D (mut B) or half-site 2U (mut C) or a double mutation in both half-sites 2D and 2U (mut

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FIG. 4. Transcription initiation from CAT reporter constructs. (A) Templates for the probes used in the S1-nuclease experiments. Templates were first inserted into M13 vectors. The probes were prepared from labeled primers and single-stranded templates as described under Materials and Methods. Solid lines under the templates represent full-length S1 probes. The asterisk indicates the labeled 5*-end. Major transcription start sites are indicated with arrows. The numbers in parentheses indicate distances (in bp) of restriction sites from the start site for transcription in that construct or from the 5* end of the CAT gene. (B) Mapping the transcription start sites for RNA’s transcribed from the endogenous malic enzyme gene and a CAT reporter gene (p[ME05800//31]CAT). Left panel, poly(A)/ RNA (1 mg/lane) from nontransfected chick-embryo hepatocytes. Right panel, poly(A)/ RNA (35 mg/lane) from hepatocytes transfected with p[ME05800//31]CAT and incubated with or without T3. The endogenous and transfected genes were analyzed by nuclease S1 protection using probes specific for the respective genes. Double-headed solid arrows indicate protected species in RNA transcribed from the endogenous gene which are also protected in RNA transcribed from the malic enzyme–CAT fusion gene. The open arrows indicate the positions of full-length, undigested probes. Lanes labeled C, T, A, and G are from dideoxy sequencing reactions using the same primer and template used to prepare the S1 probe for the RNA extracted from transfected cells. The nucleotide sequence around the start site for transcription is indicated at the lower right, with nucleotides at 01 and /1 designated. (C) Mapping of RNA transcribed from a fusion gene in which an upstream fragment of

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D) reduced T3 responsiveness by more than 80%. The reduced T3 responsiveness was due to 5- to 10-fold increases in basal CAT activities; the T3-induced level of CAT activity was unaffected or increased. Deletion of site 2 (03883 to 03858 bp) resulted in an even greater loss of responsiveness to T3 than mutations in both half-sites (Fig. 10B). To test whether the 26-bp fragment from 03883 to 03858 was sufficient for a T3 response, we linked it to the minimal TK promoter. T3 caused a 62-fold increase in CAT activity, similar to that observed with the entire 03903- to 03703-bp region (Fig. 10B). The basal activity with the 26-bp T3RE was only one-sixth of that with the TK promoter alone, consistent with the T3 receptor bound to the T3RE at site 2 being a strong repressor in the absence of ligand. Deletion of the 26-bp fragment (03883 to 03858 bp) from p[ME05800//31]CAT reduced responsiveness to T3 from 9-fold to 4-fold (Fig. 5). In the context of p[ME04135//31]CAT, the deletion reduced responsiveness to T3 from 15-fold to less than 6-fold (Fig. 5). These results confirm an important role for the T3RE at site 2 in T3-responsiveness of the entire 5800 bp of 5*-flanking DNA. Binding of T3 receptor to the T3 response unit. Functional characterization suggested the presence of a strong T3RE at site 2 and weak T3RE’s at sites 3, 4, 5, and 6. We used a DNase I-foot-printing assay to determine if purified T3 receptor and hepatic nuclear proteins bound to these putative T3RE’s (Fig. 11). Purified receptor at 3 mg per assay protected each of the T3RE sites identified by sequence analysis (Fig. 8). There also was protection from DNase I in plasmid DNA, 5* to the insert; protection in the plasmid DNA was flanked by cleavages that were hypersensitive to DNase I. Protection also was observed between 03747 and 03728 bp of the malic enzyme 5*-flanking DNA. This region contains a hexamer (AGGCCA, half-site e, Fig. 8) that lacks upstream or downstream repeats. At 0.3 mg of receptor per assay, binding was evident at sites 2 and 3, but not at site 1, at the overlapping sites 4, 5, and 6, at the site in the plasmid DNA, or at site e. At both 3.0 and 0.3 mg T3 receptor per reaction, hypersensitivity at about 03903 bp was observed. At 0.03 mg per reaction, no binding was detected at any of the sites. When nuclear extract (60 mg) was used, partial pro-

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tection was detected at sites 2 and 3. The patterns of protection were similar to that observed with purified receptor but weaker, as would be anticipated due to the low levels of receptor in crude extracts. In addition, binding of nuclear protein to the DNA caused the appearance of five hypersensitive sites. One, at about 03903 bp, was upstream from site 1. Four 3* hypersensitive sites were at 03802, 03788, 03795, and 03779 bp and lie within the overlapping sites 4, 5, and 6. A mixture of purified receptor at 0.3 mg per assay and nuclear extract caused increased protection from DNase I at sites 2 and 3 and enhanced the hypersensitivity at 03903 and 03795 bp. The combination of receptor and nuclear extract had no effect on hypersensitivity at 03802 bp and substantially diminished those at 03788 and 03779 bp. Site 1, the site in plasmid DNA, and a site between 03747 and 03728 bp were protected by 3.0 mg of T3 receptor per assay but were not identified as functional T3RE’s in the transfection assay. This suggests that at high concentrations, T3 receptor may bind to regions that are not physiologically relevant. Sites 2 and 3 were protected by 0.3 mg receptor per assay, both with and without nuclear extract; the sequences protected from DNase I cleavage were the same as those identified by functional assays in hepatocytes in culture. DISCUSSION

Structure and organization of the chicken malic enzyme gene. The chicken and rat malic enzyme genes and their products are similar with respect to intron/ exon structure (33), large size of the primary transcript, nucleotide sequence of the mRNA’s (73% identity), amino acid sequence of the proteins (77% identity), length of each protein-coding exon ({2 bp), and site at which each intron is inserted into the protein-coding sequence. These similarities of organization and sequence have persisted in the coding region of the gene for malic enzyme since the evolution of birds and mammals diverged more than 215 million years ago (42). The significance of sequence conservation in the coding region is likely related to its importance to structural stability and enzymatic function. Why the large size of the gene is conserved is less clear. Synthesis of the greater than 106-kb primary transcript requires nearly 1 h (4). The mRNA is about 2 kb, and discarding

malic enzyme DNA containing T3 response elements is linked to a minimal malic enzyme promoter which, in turn, is linked to the CAT reporter gene. Poly(A)/ RNA (15 mg) was isolated from hepatocytes transfected with p[ME04135/02715]ME0147//31CAT. The solid arrow with a dashed line indicates the position of the major start site of the endogenous gene (/1). Solid arrows with solid lines indicate major start sites from this fusion gene. The open arrow indicates the position of full-length, undigested probe. Lanes labeled C, T, A, and G are from dideoxy sequencing reactions using the same primer and template used to prepare the S1 probe. (D) Mapping of mRNA transcribed from a fusion gene in which an upstream fragment of malic enzyme DNA containing T3 response elements is linked to TK-CAT DNA. Poly(A)/ RNA (10 mg) was isolated from hepatocytes transfected with p[ME04135/02715]TKCAT and treated with T3; total RNA (10 mg) was isolated from nontransfected hepatocytes. The solid arrow represents one of the major start sites (/3) of HSV-TK. Other symbols and letters are defined in the legends to B and C.

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FIG. 5. The effect of T3 on CAT activity in hepatocytes transfected with constructs containing 5*-flanking DNA of the chicken malic enzyme gene linked to a CAT reporter gene. The number at the left of each construct indicates the 5*-end of malic enzyme DNA in nucleotides relative to the major start site for transcription; for all constructs the 3* end of malic enzyme DNA was at /31. Chick-embryo hepatocytes were transiently transfected using LipofectACE (30 to 40 mg/plate) and p[ME05800//31]CAT (2.5 mg/plate or an equimolar amount of the other constructs), pCMVb-GAL (0.5 mg/plate), and pBluescript or pKSCAT (promoterless construct) DNA (sufficient to bring total DNA per plate to 5.0 mg) as described under Materials and Methods. The results were expressed as percentage of [14C]chloramphenicol converted to acetylated chloramphenicol per microgram soluble protein and then corrected for differences in transfection efficiency by dividing by b-galactosidase activity of the same extract (A420 units/mg protein). Relative CAT activities were then calculated by setting the corrected CAT activity for T3-treated hepatocytes transfected with p[ME05200//31]CAT DNA to 100 and adjusting all other activities proportionately. T3 had no effect on bgalactosidase activity. For each construct, ‘‘fold’’ response to T3 was calculated by dividing the corrected CAT activity for hepatocytes treated with T3 (/T3) by that for hepatocytes not treated with T3 (0T3). The ‘‘fold’’ responses were calculated for individual experiments and then averaged; they are not the same as the quotients of the averaged corrected CAT activities. The results are the means { SE of 5 to 8 experiments, each one using an independently isolated batch of hepatocytes. The results represent the testing of at least 2 independently prepared batches of each plasmid. CAT and b-galactosidase activities of extracts from T3-treated hepatocytes transfected with p[ME05200//31]CAT DNA were 1.6 { 0.5 (mean { SE, n Å 8) percent conversion/15 h/mg protein and 4.4 { 1.8 1 1004 (mean { SE, n Å 8) A420 units/min/mg protein, respectively. CAT, chloramphenicol acetyltransferase. Statistical significance between means within a column (P õ 0.05): aversus p[ME04135//31]CAT; b versus p[ME03845//31]CAT; cversus p[ME03474//31]CAT; dversus p[ME05800//31]CAT; eversus pME[04135//31]CAT. Breaks in the solid lines representing 5*-flanking DNA represent deletions from 03883 to 03858 bp.

98% of the primary transcript ‘‘wastes’’ valuable energy. Many other eukaryotic genes are large compared to their corresponding mRNA’s, but very few of these are as large as the malic enzyme gene.

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The physiological signals that regulate the activity of malic enzyme are similar in rat and chicken: high carbohydrate diets, T3, and insulin all stimulate, while starvation and glucagon inhibit malic enzyme activity. The pattern of tissue-specific expression is also similar (43–46). Nevertheless, some of the involved molecular mechanisms are different. For example, feeding a highcarbohydrate diet to starved birds stimulates transcription of the malic enzyme gene (2) and causes an alteration in its chromatin structure (47). In the rat, feeding a high-carbohydrate diet stimulates accumulation of malic enzyme mRNA by a posttranscriptional mechanism (48). Low sequence similarity in the 5*flanking DNA of the chicken and rat genes may explain this difference in molecular mechanism. The absence of a polypyrimidine tract in the promoter of the rat gene also may be an important difference. Transcription of the chicken gene is inhibited markedly when the polypyrimidine tract is deleted (36). Transcription from the promoter of the malic enzyme gene initiates from one major and several minor start sites, consistent with the absence of a TATAA box (49). Unlike many ‘TATAA-less’ promoters, particularly those with well-defined initiator sequences, the nucleotide sequence surrounding the primary start site of the chicken malic enzyme gene does not closely resemble previously described initiator sequences (50). The rat gene for malic enzyme also initiates at one major site and also lacks a TATAA box (51). The region around the start site of the rat malic enzyme gene, however, more closely resembles an initiator element (50) than that of the chicken gene. Characterization of T3RE’s in the chicken malic enzyme gene. The effect of T3 on CAT activity in hepatocytes transfected with p[ME05800//31]CAT varied from 9- to 23-fold in different series of experiments using pCMV-bGAL as a control for transfection efficiency. When pRSV-luciferase was used as the control plasmid, T3-stimulation was about 40-fold (results not shown). Differences between experiments that used different control plasmids appeared to be related to transfection efficiency. Transcription of the natural gene is stimulated 30- to 40-fold by T3 (4). Therefore, the quantitatively important cis-acting elements that confer T3 responsiveness to the malic enzyme gene lie between 02715 and 04135 bp. Sequences in the malic enzyme gene that conferred T3-dependent promoter activity on reporter genes were localized primarily to one far-upstream region. This region colocalizes with a previously identified DNase I hypersensitive site (2) centered at about 03.8 kb. The rat malic enzyme gene contains a similarly placed DNase I hypersensitive site at 04.1 kb (52). To our knowledge, however, far upstream regions of the 5*flanking DNA of the rat malic enzyme gene have not been examined for T3RE’s.

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FIG. 6. The effect of T3 on CAT activity of hepatocytes transfected with upstream regions of the chicken malic enzyme gene linked to homologous and heterologous minimal promoters. Fragments of malic enzyme DNA were isolated from p[ME05800//31]CAT by restriction enzyme digestion and linked to minimal promoters. Numbers indicate the 5* and 3* ends of each fragment relative to the major start site for transcription. Hepatocytes were transiently transfected with these constructs as described in the legend to Fig. 5 except that 0.2 mg pCMVbGAL DNA were used per plate. The results are expressed as described in the legend to Fig. 5 and represent 6–11 independent experiments using at least 2 independently prepared batches of each plasmid. Relative CAT activities were calculated by setting the corrected CAT activities for T3-treated hepatocytes transfected with p[ME05800//31]CAT DNA to 100 and adjusting all other activities proportionately. (A) Responses of hepatocytes transfected with 5*-flanking DNA of the malic enzyme gene linked to a minimal promoter from the malic enzyme gene. CAT and b-galactosidase activities in extracts from T3-treated hepatocytes transfected with p[ME05800/ /31]CAT DNA were 5.2 { 0.8 (mean { SE, n Å 12) percent conversion/15 h/mg protein and 1.3 { 0.3 1 1004 (mean { SE, n Å 12) A420 units/min/mg protein, respectively. (B) Responses of hepatocytes transfected with 5*-flanking DNA of the malic enzyme gene linked to a minimal TK promoter. CAT and b-galactosidase activities of extracts from T3-treated hepatocytes transfected with p[ME05800//31]CAT DNA were 4.3 { 1.3 (mean { SE, n Å 6) percent conversion/15 h/mg protein and 1.4 { 0.4 1 1004 (mean { SE, n Å 6) A420 units/min/mg protein, respectively. Statistical significance between means within a column (P õ 0.05): aversus p[ME04135/02715]ME0147//31CAT; b versus p[ME04135/03471]ME0147//31CAT; cversus p[ME03764/03471]ME0147//31CAT; dversus p[ME04135/03896]ME0147/ /31CAT; eversus p[ME0147//31]CAT; fversus p[ME04135/02715]TKCAT.

The major cis-acting regulatory element involved in T3 responsiveness of the malic enzyme gene contains an imperfect direct repeat of two hexamers spaced by 4 bp, similar to that of T3RE’s of the direct-repeat type (38, 40). The strong T3RE in site 2 of the upstream T3 response unit of the chicken malic enzyme gene is similar in structure to the T3RE’s of the rat malic en-

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zyme (35, 38) and rat S14 (53) genes in that they all have G’s at positions II and III and an A at position VI (Fig. 12). For each of these T3RE’s, position I contains a conserved A or G, and positions IV and V are not conserved. In liver, T3RE’s in the direct-repeat orientation selectively recognize homodimers of the T3 receptor and

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FIG. 7. The effect of T3 on CAT activity of hepatocytes transfected with constructs containing 5*- and 3 *-deletions in the upstream T3response region of the 5*-flanking DNA of the malic enzyme gene. Malic enzyme DNA fragments containing the indicated 5* and 3* ends were synthesized by PCR and linked to the TK promoter. Hepatocytes were transiently transfected with these constructs as described in the legend to Fig. 5. The results are expressed as described in the legend to Fig. 5 and represent the mean { SE of 6–9 independent experiments using at least 2 independently prepared batches of each plasmid. Relative CAT activities were calculated by setting the corrected CAT activities for T3-treated hepatocytes transfected with p[ME03903/03703]TKCAT DNA to 100 and adjusting all other activities proportionately. CAT and b-galactosidase activities of extracts from T3-treated hepatocytes transfected with p[ME03903/03703]TKCAT DNA were 5.1 { 1.9 (mean { SE, n Å 9) percent conversion/15 h/mg protein and 2.0 { 0.5 1 1004 (mean { SE, n Å 9) A420 units/min/mg protein, respectively. TK, thymidine kinase. Statistical significance of comparisons made within a column (P õ 0.05): aversus p[ME03903/ 03617]TKCAT; bversus p[ME03903/03703]TKCAT.

heterodimers of T3 receptor with RXR (54) or RXRrelated factors (55). RXR-TR heterodimers bind to response elements with defined polarity—5*-RXR-TR-3*

FIG. 8. Nucleotide sequence of one of the T3-response regions of the malic enzyme gene. Nucleotides are numbered from the major start site for transcription. Underlined and overlined sequences are motifs that are similar to the sequence of a consensus T3RE halfsite, RGGWMA, and are referenced in the text by their site or halfsite numbers or letters. Half-site 2U is an imperfect repeat 4 bp upstream of half-site 2D.

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(56). The 5* hexamers of the FUR-12 T3RE of the rat S14 gene (53; Fig. 12) and of site 2 in the chicken malic enzyme gene share a nonconsensus G at position IV. Furthermore, homodimers of the T3 receptor do not bind to the T3RE in FUR 12 of the S14 gene in vitro, whereas heterodimers of RXR and T3 receptor do bind. Thus, T3 receptor may not bind to half-site 2U, the 5* half-site, and heterodimers of T3 receptor and another trans-acting factor, such as RXR, may be involved in transactivation by T3 from this site. Why do mutations in site 2 have such large effects on T3 responsiveness of linked promoters, when mutations to sites 1, 3, 4, 5, 6, and 7 have such small effects? The sequences of the half-sites of the T3RE at site 2 are organized such that a functional 3* half-site for T3 receptor is downstream of a half-site that may bind a non-T3 receptor partner (Fig. 13). For putative sites 1, 3, 4, and 7, the 5* half-site has a nucleotide sequence that would bind T3 receptor more effectively than that of the 3* half-site (the one that should bind T3 receptor). Furthermore, the 3* half-site contains nucleotides at positions II, III, or VI that would appear to preclude receptor binding (38). For site VI, AGGTAA may not be an effective half-site (40); in addition, the 5* halfsite has a G at position VI that would make it less effective (38). For site 5, an obvious reason for its lack of robust activity is not apparent. The downstream half-site appears equivalent in sequence to that of site

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zyme T3 response unit that we have characterized (03883 to 03769 bp) is composed of one very active site and four weakly active sites which act together to bestow a high level of T3 responsiveness. In chick-embryo hepatocytes, activation of transcription of the transfected genes by T3 required only endogenous cellular factors; responsiveness to T3 did not depend on cotransfection of an expression vector for the T3 receptor. In this respect, our system differs from those used by many investigators studying the action of T3. In rat hepatocytes in culture (58), NIH 3T3 cells

FIG. 9. The effect of T3 on CAT activity in hepatocytes transfected with DNA constructs containing block mutations in putative T3RE half-sites. (A) Natural and mutant sequences of the putative halfsites. The numbers refer to the half-sites indicated in the legend to Fig. 8. Mutations were introduced into p[ME03903/03703]TKCAT DNA by oligonucleotide-directed mutagenesis. (B) Effect of T3 on CAT activity in hepatocytes transfected with mutant reporter plasmids. MUT1 to MUT6 refer to the number of the half-site that was mutated within the 5*-flanking DNA of each construct. Hepatocytes were transfected with these constructs as described in the legend to Fig. 5. The results are expressed as described in the legend to Fig. 5 and represent the mean { SE of 11–15 independent experiments using at least 2 independently prepared batches of each plasmid. Relative CAT activities were calculated by setting the corrected CAT activities for T3-treated hepatocytes transfected with p[ME03903/ 03703]TKCAT DNA to 100 and adjusting all other activities proportionately. CAT and b-galactosidase activities in extracts from T3treated hepatocytes transfected with p[ME03903/03703]TKCAT were 6.5 { 2.1 (mean { SE, n Å 15) percent conversion/15 h/mg protein and 2.8 { 1.0 1 1004 (mean { SE, n Å 15) A420 units/min/mg protein, respectively. Statistical significance within a column (versus p[ME03903/03703]TKCAT): aP õ 0.01; bP õ 0.05.

2, and the only deviation from consensus in the upstream site is at positions IV and V where heterogeneity appears well tolerated. The combination of two nonconsensus nucleotides at positions IV and V of the upstream half-site may render this site relatively ineffective. Alternatively, flanking nucleotides may play an important role. For other putative half-sites that did not function as T3RE’s (a–e), an ‘‘imperfect’’ direct repeat was not found or was less similar to the consensus T3RE half-site than those in sequences that conferred twofold effects. This region of the 5*-flanking DNA lacks perfect or imperfect repeats of the everted or inverted type (41). The hormone-responsive regulatory regions of some genes are composed of several individual elements, some that bind hormone receptors and others that bind nonreceptor trans-acting factors (57). The malic-en-

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FIG. 10. The effects of T3 on CAT activity in hepatocytes transfected with constructs containing wild-type and mutant versions of a T3 response region of the malic enzyme gene linked to TKCAT DNA. (A) Block mutations in site 2. Fragment A has the wild-type sequence; fragments B through D contain the indicated block mutations. Mutations were introduced into p[ME03903/03703]TKCAT by oligonucleotide-directed mutagenesis. (B) Effect of T3 on CAT activity of hepatocytes transfected with various constructs. Transient transfection was carried out as described in the legend to Fig. 5, except that pRSV-LUC was used to control for transfection efficiency instead of pCMV-bGAL in three experiments. The results were similar regardless of which control plasmid was used, so the data were pooled after normalization to relative CAT activity in hepatocytes transfected with a construct containing fragment A. The results are expressed as described in the legend to Fig. 5 and represent the means { SE of 6–10 independent experiments using at least 2 independently prepared batches of each plasmid. CAT, b-galactosidase, and luciferase activities in extracts of T3-treated hepatocytes transfected with p[ME03903/03703]TKCAT were 11.4 { 2.6 (mean { SE, n Å 10) percent conversion/15 h/mg protein, 6.0 { 2.0 1 1004 (mean { SE, n Å 7) A420 units/min/mg protein, and 1.8 { 0.6 1 104 (mean { SE, n Å 3) light units/mg protein, respectively. Statistical significance of comparisons made within a column: aversus p[ME03903/ 03703]TKCAT (P õ 0.01); bversus p[ME03883/03858]TKCAT (P õ 0.05); cversus pMUTD [ME03903/03703]TKCAT (P õ 0.01); dversus pTKCAT (P õ 0.01); eversus p[ME03903/03703]TKCAT (P õ 0.05); f versus pMUTD [ME03903/03703]TKCAT (P õ 0.05).

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FIG. 12. Nucleotide sequences of direct-repeat-type T3RE’s from genes for rat malic enzyme (35), chicken malic enzyme (this paper), and rat S14 (54). Arrows indicate imperfect direct repeats of six nucleotides. Nucleotide numbers are assigned relative to the major start sites for transcription. Nucleotides marked by asterisks are identical to those of the chicken malic enzyme gene. Boxes connect nucleotides conserved between all four T3REs.

FIG. 11. Binding of partially purified T3 receptor and nuclear proteins to DNA from a region of the malic enzyme gene containing T3 response elements. A 201-bp DNA fragment (03903 to 03703 bp; 13 fmols) was end-labeled with 32P and incubated with DNase I and the indicated amounts of chicken T3 receptor a (lanes 7–9) or 60 mg nuclear extract (lane 11) or 0.3 mg T3 receptor a plus 60 mg nuclear extract (lane 10) as described under Materials and Methods. Chicken T3 receptor a (28) was expressed in E. coli and partially purified as described under Materials and Methods. Nuclear extract was prepared from chick-embryo hepatocytes incubated in culture with T3 for 24 h. Lanes 1 to 4 contain the products of Maxam–Gilbert sequencing reactions using the same fragment of DNA. Lane 5 was incubated without DNase I, T3 receptor, or nuclear extract. Lanes 6 and 12 were incubated with DNase I and without T3 receptor or nuclear extract. Nuclear extracts from cells incubated in the absence of T3 gave the same patterns with or without added T3 receptor. Cleavage sites that show increased sensitivity to DNase I are designated with lower case letters: a, 03903 bp; b, 03803 bp; c, 03795 bp; d, 03788 bp; e, 03779 bp.

(59) and COS-7 cells (51), for example, overexpression of the T3 receptor is required to elicit a response to T3 from a transfected gene. There is substantial variability in the nucleotide sequences that will bind T3 receptors and mediate a transcriptional response (41). If the intracellular concentration of the T3 receptor is sufficiently higher than that of a normal cell, receptor may bind to sequence elements that are not physiologically relevant T3RE’s. In vitro binding of purified T3 recep-

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tor to nonfunctional malic enzyme sequences and plasmid sequences may be an example of this phenomenon. Furthermore, the use of cell-types not normally responsive to T3 or that do not normally express the gene of interest may confound results, because those cells may contain (or lack) tissue-specific factors that regulate function of the T3 receptor or expression of tissue-specific genes. Our system of chick-embryo hepatocytes avoids these potential artifacts and provides a more physiologically relevant system with which to identify DNA regulatory elements that confer T3 responsiveness. T3 is an important regulator of other ‘‘lipogenic’’ genes (60). T3-induced expression of the rat S14 gene is mediated by multiple T3RE’s located within a T3dependent DNase I hypersensitive site that is far upstream of the start site for transcription (53, 58). The S14 T3-response unit is similar to that of the chicken malic enzyme gene in that multiple T3RE’s are required to elicit maximal T3 induction. The malic enzyme T3RE’s and the S14 T3RE’s are functionally different, however, because the major T3RE in the malic enzyme gene is capable of substantial induction by itself, but the S14 T3RE’s are inactive individually; multiple T3RE’s are required for responsiveness. Differences in the intracellular signaling pathways of chicken and rat hepatocytes or in nucleotide sequences

FIG. 13. A comparison of the sequences of putative T3RE’s in the T3 response region of the chicken malic enzyme gene. The underlined hexamer was identified because it had the same sequence as a consensus T3RE half-site, RGGWMA. Imperfect repeats 4 bp upstream or downstream of these half-sites also are indicated. The nucleotides are numbered with respect to the major start site for transcription.

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of malic enzyme and S14 T3RE’s (Fig. 12) may be responsible for differences in the mechanisms by which each T3 response unit regulates expression of its respective gene.

21.

ACKNOWLEDGMENTS

23.

We thank the following individuals, each of whom made substantial technical contributions to the identification of intron–exon boundaries and/or determined the nucleotide sequence of portions of the malic enzyme gene or cDNA: Emily S. Bishop, Thomas C. Carlisle, Xin Chen, Manuel J. Glynias, Cinder L. Krema, Charles L. Leeck, Min-Ling Liu, Kevin Martin, Peggy Myung, Brian C. Potts, Lisa M. Salati, Dennis A. Savaiano, Susan R. Stapleton, and Timothy R. Winters. This work was supported by Grant DK 21594 from the National Institutes of Health and by the Core Facilities of the Diabetes and Endocrinology Research Center (DK 25295) of the University of Iowa. D.W.H. was supported as a postdoctoral trainee by National Institutes of Health Grant DK 07018.

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