Regulation of the Action of Steroid/Thyroid Hormone Receptors by Medium-chain Fatty Acids*

Debbie C. ThurmondDagger , Rebecca A. Baillie§, and Alan G. Goodridge

From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Triiodothyronine (T3) causes a 30-fold increase in transcription of the malic enzyme gene in chick embryo hepatocytes; medium-chain fatty acids (MCFAs) inhibit this increase. T3 action is mediated by T3 receptors (TRs) that bind to T3 response elements (T3REs) in this gene's 5'-flanking DNA. In transiently transfected hepatocytes, fragments of 5'-flanking DNA of the malic enzyme gene or artificial T3REs that conferred T3 stimulation also conferred MCFA inhibition to linked reporter genes. Thus, MCFA inhibition may be mediated through cis-acting T3REs and trans-acting TRs, distinguishing MCFA action from that of other fatty acids which act through unique sequence elements. Using binding assays and overexpression of TR, we showed that MCFAs inhibited the transactivating but not the silencing function of TR and did not alter binding of T3 to TR or of TR to T3RE. The C-terminal ligand-binding domain of TR was sufficient to confer stimulation by T3, but not inhibition by MCFA. Inhibition of transactivation by MCFA was specific: ligand-stimulated transcription from T3 or estrogen response elements was inhibited, but that from glucocorticoid or cyclic AMP response elements was not. We propose that MCFAs or metabolites thereof influence the activity of a factor(s) that interacts with the T3 and estrogen receptors to inhibit ligand-stimulated transcription.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Malic enzyme (EC 1.1.1.40) catalyzes the oxidative decarboxylation of malate to pyruvate and CO2, simultaneously generating NADPH from NADP+. In the livers of well fed birds, much of the NADPH generated by this reaction is used for de novo fatty acid synthesis. Malic enzyme activity, like that of other lipogenic enzymes, is regulated by nutritional state (1, 2). When starved chickens are fed or fed chickens are starved, malic enzyme activity, malic enzyme mass, malic enzyme mRNA abundance, and transcription of the malic enzyme gene increase or decrease by similar relative amounts and with time courses consistent with transcription being the primary regulated process (3, 4). These nutritionally induced changes in the expression of the chicken malic enzyme gene can be mimicked quantitatively in chick embryo hepatocytes in culture (5). In the presence of insulin and triiodothyronine (T3),1 transcription of the chicken malic enzyme gene is high, as it is in the livers of fed chicks (6). In the absence of T3 and insulin or in the presence of insulin and T3 plus glucagon or cAMP or medium-chain fatty acids (MCFAs), transcription is low, as in the livers of starved birds (6, 7). Several T3 response elements (T3REs) have been localized in the 5'-flanking DNA of the chicken malic enzyme gene (8-10). Characterization of these T3REs indicates that each contributes differentially to the overall response of the gene to T3 (9).

Hexanoate (C6:0) and octanoate (C8:0) inhibit the T3-induced increase in transcription of the malic enzyme gene in chick embryo hepatocytes within 30 min of their addition; inhibition is reversible upon removal of fatty acid. Inhibition by MCFAs is selective; they have no effect on total transcription in isolated nuclei or on transcription of the genes for glyceraldehyde-3-phosphate dehydrogenase or beta -actin. Inhibition is also specific; 4- and 10-carbon fatty acids and several modified fatty acids have little or no effect (7). These results suggest that the MCFA effect may be relevant biologically. What might that biological relevance be?

Levels of MCFAs in chicken plasma that are high enough to inhibit transcription have not been reported. During starvation, a process that inhibits transcription of the malic enzyme gene, however, fatty acid oxidation is increased. Concomitantly, production of hydroxylated fatty acids similar in chain length to the inhibitory MCFAs also is increased (11-13). In hepatocytes in culture, MCFAs may be converted to hydroxylated fatty acids or metabolites similar thereto, and these may be the intracellular mediators of the inhibition of transcription.

How might MCFAs inhibit transcription of the malic enzyme gene? Hydroxylated long-chain fatty acids activate transcription of the fungal gene for cutinase (14) by stimulating phosphorylation of a trans-acting factor that binds to a specific cis-acting enhancer element (15, 16). Polyunsaturated long-chain fatty acids (PUFAs) stimulate transcription of the acyl-CoA oxidase gene by interacting with the trans-acting factor, peroxisomal proliferator-activated receptor (PPAR) (17), or its adipocyte counterpart, fatty acid-activated receptor (FAAR) (18). These factors bind to peroxisomal proliferator response elements in the acyl-CoA oxidase gene. PUFAs also appear to inhibit expression of the S14 gene via a response element, the PUFA response element; the mechanism for this is not known (19, 20). Long-chain fatty acids inhibit binding of T3 to nuclear TR in cells in culture (21, 22). Both long-chain fatty acids and their acyl-CoA derivatives inhibit binding of T3 to rat liver TR in vitro (23). In intact hepatocytes, however, MCFAs fail to inhibit binding of T3 to TR (7), suggesting that they act by some mechanism other than displacement of T3.

We report here that inhibition of T3-stimulated transcription of the chicken malic enzyme gene caused by MCFAs is mediated by the receptor that binds to T3REs. This mechanism is distinct from that of fatty acids that act through unique sequence elements. Rather than displacing T3 from TR or displacing TR from T3RE, MCFAs inhibit the transactivation function of TR. MCFAs also inhibit estrogen-stimulated transcription through an estrogen response element and the estrogen receptor (ER), but have no effect on ligand-stimulated transcription mediated by glucocorticoid or cAMP response elements.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

Materials-- Restriction enzymes were obtained from Life Technologies, Inc., New England Biolabs Inc., U. S. Biochemical Corp., or Boehringer Mannheim. Other enzymes were obtained from the indicated sources: RQ-DNase I (Promega), T4 polynucleotide kinase and Klenow fragment of Escherichia coli DNA polymerase I (Boehringer Mannheim), DNA polymerase from Thermus aquaticus (Promega), and Bst polymerase (Bio-Rad). Nucleotides were purchased from Sigma, Amersham Pharmacia Biotech, or Life Technologies, Inc. Radiolabeled nucleotides, Hyperfilm-HP, ECL Western blotting kits, and donkey anti-rabbit IgG conjugated to horseradish peroxidase (secondary antibody) were obtained from Amersham Pharmacia Biotech. D-threo-[dichloroacetyl-1,2-14C]Chloramphenicol was purchased from NEN Life Science Products. LipofectACE, Waymouth MD 705/1 medium, and E. coli cells of strain DH5alpha were obtained from Life Technologies, Inc. SeaKem LE agarose, NuSieve GTG agarose, and DNA isolation columns (SpinBind) were purchased from FMC Corp. Hormones, fatty acids (as sodium salts), and heparin columns were purchased from Sigma. Polyclonal antibody to chicken TRalpha was purchased from Santa Cruz Biotechnologies (cross-reacts with chicken TRbeta ). Monoclonal antibody to chicken RXR was provided by Pierre Chambon (Université Louis Pasteur/ IGBMC, Illkirch, France). Nitrocellulose membranes were purchased from Millipore Corp.

Plasmids-- Expression plasmids containing 5'-flanking DNA of the chicken malic enzyme gene inserted upstream of the CAT gene in pKSCAT were constructed as described (10). Synthesis of DNA constructs containing fragments of chicken malic enzyme DNA inserted into the multiple cloning site 5' of the promoter of the herpes simplex virus thymidine kinase (TK) gene in pBLCAT2 (24) also has been described (10). Plasmid DR4×5-TKCAT was made by inserting five copies of annealed oligonucleotides encoding a direct repeat element with a 4-bp spacer (AGGTCAnnnnAGGTCA) into the multiple cloning site of pBLCAT2. Structures of plasmid DNAs were confirmed by restriction enzyme mapping and partial sequence analysis.

Construction of pRSV-Luc was described (10). Plasmid CMV-beta GAL (25) was obtained from Richard Maurer (Oregon Health Sciences University). Plasmid [ME-3474/+31]CAT was provided by F. Bradley Hillgartner (West Virginia University). Marc Montminy (Salk Institute) provided pRSV-CREB. Bruno Luckow and Gunter Schutz (German Cancer Research Center, Heidelberg, Germany) provided pBLCAT2. The expression vector for GAL4, pSG424, contained sequences encoding amino acids 1-147 of the GAL4 DNA-binding domain. Plasmid SG424 and the expression plasmid for the GAL4-binding element, pMC110, were obtained from Mark Ptashne (Harvard University). Herbert H. Samuels (New York University) provided the cDNA for chicken TRalpha (cloned into the pET8c expression vector) and pGAL4-cTRalpha (containing the sequences encoding amino acids 120-408 of the ligand-binding domain of chicken TRalpha cloned into pSG424). Ronald Evans (Salk Institute) and Bert W. O'Malley (Baylor College of Medicine) gave us pTREpal-TKCAT and pERE-TKCAT, respectively. Expression vector for human ER (pHEO) was obtained from Geoffrey L. Greene (University of Chicago). Expression plasmids for the GRE linked to TKCAT DNA (pDelta GTCO) and rat GR (pVARO) were gifts from Keith R. Yamamoto (University of California, San Francisco). Ganes Sen (Cleveland Clinic Foundation) provided pCRE-TKCAT.

Cell Culture and Transient Transfection-- Livers of 19-day-old chick embryos were removed, chopped, and treated with collagenase (26). Isolated hepatocytes were separated from red blood cells; resuspended in Waymouth medium MD 705/1 supplemented with penicillin (60 µg/ml), streptomycin (100 µg/ml), insulin (50 nM), and corticosterone (1 µM); and incubated in 35-mm tissue culture dishes in an atmosphere of 5% CO2 in air at 40 °C. Twenty hours after the cells were plated, they were transiently transfected using 40 µg of LipofectACE/well. Each 35-mm dish was transfected with 5 µg of plasmid DNA: p[ME-5800/+31]CAT (10.7 kilobase pairs, 2.5 µg) or a molar equivalent of other test constructs, pCMV-beta GAL or pRSV-LUC (0.5 µg), and pBluescript KS+ (balance). Two 35-mm plates were used for each experimental condition. After 24 h in the transfection medium, the medium was removed by aspiration and replaced with Waymouth medium supplemented with or without 1.6 µM T3 and with or without 1 or 5 mM sodium hexanoate (26). Both of these hexanoate concentrations caused similar degrees of inhibition (data not shown).

CAT, beta -Galactosidase, and Luciferase Assays-- Forty-eight hours after adding T3, the cells were harvested; lysed by three cycles of freezing and thawing; and analyzed for soluble protein content (27) and beta -galactosidase (28) or luciferase (29) and CAT (30) activities. beta -Galactosidase activity was determined by measuring the absorbance at 420 nm following incubation of cell lysates with o-nitrophenyl beta -galactopyranoside. Luciferase activity was determined by measuring the light units emitted following incubation of cell lysates with luciferin. For CAT assays, samples of the cell lysates were heat-treated for 30 min at 60 °C; denatured protein was removed by centrifugation. CAT activity was determined by incubating a portion of cell lysate with acetyl-CoA and [14C]chloramphenicol for 15 h at 37 °C. Incubation mixtures were then extracted with ethyl acetate and subjected to thin-layer chromatography. Conversion to the acetylated product was detected by liquid scintillation spectrophotometry or direct autoradiography using the Packard InstantImager.

Gel Electrophoretic Mobility Shift Assay-- Each oligonucleotide probe contained a 5'-extension and was labeled by a fill-in reaction catalyzed by the Klenow fragment of E. coli DNA polymerase I. Other procedures and the preparation of nuclear extracts were described previously (9).

DNase I Footprint Analysis-- Nuclear extracts were prepared from chick embryo hepatocytes incubated with insulin plus corticosterone plus T3, with or without hexanoate (1 mM). Other procedures were as described previously (10).

Statistical Analysis-- Statistical significances of differences between matched pairs were determined by the Wilcoxon matched-pairs, signed-rank test (31). S.E. values are provided to indicate the degree of variability in the data.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Identification of the Cis-acting Element Involved in Mediating Inhibition by MCFA-- DNA constructs containing deletions from the 5'-end of the 5'-flanking DNA of the chicken malic enzyme gene were transiently transfected into chick embryo hepatocytes in culture in an effort to localize the inhibitory effect of MCFAs (hexanoate). Cells transfected with the longest construct (p[ME-5800/+31]CAT) responded to T3 with a 14-fold increase in CAT activity, and hexanoate inhibited T3-stimulated CAT activity by 83% (Fig. 1). We have previously reported that 5800 bp of 5'-flanking DNA of the malic enzyme gene contained the sequence element(s) required for stimulation of transcription by T3 (10). The sequence element(s) necessary for inhibition by hexanoate were contained in the same DNA.


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Fig. 1.   Effects of T3 and hexanoate on CAT activity of hepatocytes transfected with constructs containing deletions from the 5'-end of the 5'-flanking DNA of the chicken malic enzyme gene. Chick embryo hepatocytes were transiently transfected using LipofectACE (40 µg/plate), p[ME-5800/+31]CAT (2.5 µg/plate or an equimolar amount of the other constructs), pCMV-beta GAL (0.5 µg/plate), and pBluescript DNA (sufficient to balance DNA/plate to 5.0 µg) and treated with or without T3 and with or without hexanoate (C6 (5 mM), except that 1 mM C6 was used in two experiments). Each point represents the mean ± S.E. of five to eight independent sets of hepatocytes, using at least two independently prepared batches of each plasmid. CAT and beta -galactosidase activities of extracts from T3-treated hepatocytes transfected with p[ME-5200/+31]CAT were 1.6 ± 0.5 percentage of conversion/15 h/µg of protein and (4.4 ± 1.8) × 10-4 A420 units/min/µg of protein, respectively. Left, DNA constructs used in these experiments; middle, effects of T3, expressed as -fold change in CAT activity caused by T3 (+T3/-T3); right, effects of hexanoate. Relative CAT activity of cells treated with T3 and hexanoate is expressed as a percentage of that in cells treated with T3 alone. Statistical significance between means within a column is indicated as follows: a, versus p[ME-2715/+31]CAT (p < 0.05); b, versus p[ME-2715/+31]CAT (p < 0.02); c, versus p[ME-4135/+31]CAT (p < 0.02).

Deletions to -5200, -4135, -3845, and -3474 bp resulted in 16-, 17-, 9-, and 7-fold stimulations by T3, respectively. The degree of inhibition by hexanoate was the same for cells transfected with each of these constructs. Cells transfected with constructs containing 5'-deletions to -2715 bp or to shorter end points did not respond to either T3 or hexanoate. These results confirmed the location of T3 response units (T3RUs) between -4135 and -3845 bp and between -3845 and -2715 bp (9, 10). The upstream T3RU contains four functional direct repeat T3REs at -3883 to -3858, -3833 to -3808, -3809 to -3784, and -3794 to -3769 bp; the 5'-most of these T3REs conferred 90% of the responsiveness of the entire upstream T3RU. The downstream T3RU contains a single T3RE at -3081 to -3056 bp (9, 10). Short internal deletions were made in two constructs (p[ME-5800/+31]CAT and p[ME-4135/+31]CAT) that were responsive to both T3 and hexanoate. Each deletion removed the major functional T3RE in the upstream T3RU; the minor T3REs in the upstream and downstream T3RUs were not deleted. T3 responsiveness decreased by ~50% for both deletion constructs, but the MCFA responses were unaffected. Thus, any construct that conferred a T3 response also conferred inhibition by hexanoate, consistent with the possibility that T3REs themselves might confer inhibition by MCFA.

Constructs that contain the upstream T3RU linked to TKCAT (p[ME-3903/-3617]TKCAT and p[ME-3903/-3703]TKCAT) conferred their responses to T3 via one major and three weak T3REs (9, 10). In cells transfected with these constructs, CAT activity was stimulated by T3 and inhibited by hexanoate (Fig. 2). Deletion of part of the strongest T3RE (p[ME-3868/-3617]TKCAT or p[ME-3868/-3703]TKCAT) caused a large reduction in T3 responsiveness, but did not affect responsiveness to hexanoate. Deletions from the 3'-end of p[ME-3903/-3703]TKCAT to -3733 or -3769 bp did not affect T3 or hexanoate responses. Deletions to -3799, -3823, and -3863 bp gradually reduced responsiveness to T3 from >100-fold to 54-fold, consistent with the gradual loss of weak T3REs; inhibition by hexanoate was unaffected. In this set of experiments, the vector, pTKCAT, conferred a 2-fold stimulation by T3 and 39% inhibition by hexanoate; responses of cells transfected with pTKCAT were detectable in some batches of hepatocytes, but not others (cf. Figs. 3 and 4) and may have been transduced through a cryptic T3RE in pTKCAT (32). In those experiments in which pTKCAT-transfected hepatocytes had small but statistically significant responses to T3 and hexanoate, cells transfected with constructs containing the malic enzyme DNA inserted into pTKCAT were not judged to be responsive to T3 or hexanoate unless the responses were significantly greater than those of cells transfected with the vector alone. In those experiments in which pTKCAT-transfected hepatocytes had no statistically significant response to T3, hexanoate had no inhibitory effect. The results of this set of experiments remain consistent with the hypothesis that inhibition by hexanoate is conferred by T3REs and that inhibition was not localized to a specific T3RE within this region.


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Fig. 2.   Effects of T3 and hexanoate on CAT activity of hepatocytes transfected with constructs containing 5'- and 3'-deletions in the upstream T3 response region of the 5'-flanking DNA of the chicken malic enzyme gene. Chick embryo hepatocytes were transiently transfected with the indicated constructs as described in the legend to Fig. 1 and treated with or without T3 (1.6 µM) and with or without hexanoate (C6, 5 mM). The results are expressed as described in the legend to Fig. 1; each value is the mean ± S.E. of six to eight independent experiments using at least two independently prepared batches of each plasmid. CAT and beta -galactosidase activities of extracts from T3-treated hepatocytes transfected with p[ME-3903/-3733]TKCAT were 4.7 ± 1.7 percentage of conversion/15 h/µg of protein and (3.9 ± 1.8) × 10-4 A420 units/min/µg of protein, respectively. Left, DNA constructs used in these experiments; middle, effects of T3, expressed as -fold change in CAT activity caused by T3; right, effects of hexanoate. Relative CAT activity of cells treated with T3 and hexanoate is expressed as a percentage of that in cells treated with T3 alone. Statistical significance between means within a column is indicated as follows: a, versus pTKCAT (p < 0.02); b, versus pTKCAT (p < 0.05); c, versus p[ME-3903/-3703]TKCAT (p < 0.05).

Cells were transfected with constructs containing block or deletion mutations to verify that the T3 and hexanoate effects were inseparable. In two sets of experiments, p[ME-3903/-3703]TKCAT was transfected into hepatocytes; T3 caused 65- and 113-fold increases in CAT activity, and hexanoate reduced the T3-induced activity by 67 and 65%, respectively (Figs. 3 and 4). Mutation of the first half-site (pMUT1[ME-3903/-3703]TKCAT) did not alter significantly either stimulation by T3 or inhibition by hexanoate, compared with that of the nonmutated parent construct (Fig. 3). Cells transfected with constructs containing block mutations in sites 2, 3, 4, 5, or 6 (individually) exhibited significantly reduced responsiveness to T3, but no change in inhibition by hexanoate (Fig. 3). Block mutations in either the upstream (pMUTB[ME-3903/-3703]TKCAT) or downstream (pMUTC[ME-3903/-3703]TKCAT) half-site of T3RE2 or both (pMUTD[ME-3903/-3703]TKCAT) significantly reduced responsiveness to T3, but did not significantly alter responsiveness to hexanoate (Fig. 4). Similarly, deletion of T3RE2 decreased the T3 response by 95%, but inhibition by hexanoate was still 49% (Fig. 4). In all of the experiments reported thus far, the effects of T3 and hexanoate were inseparable, suggesting that any T3RE was sufficient for the hexanoate effect. Cells transfected with a single copy of the strong T3RE, p[ME-3883/-3858]TKCAT, responded to both T3 and hexanoate (Figs. 4 and 5), indicating that T3RE2 by itself is sufficient to confer both stimulation by T3 and inhibition by hexanoate.


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Fig. 3.   Effects of T3 and hexanoate on CAT activity in hepatocytes transfected with DNA constructs containing block mutations in T3RE half-sites. A, natural and mutant sequences of the T3RE half-sites. The numbers refer to the half-sites identified in the T3RU of the chicken malic enzyme gene (10). MUT1 to MUT6 refer to the number of the half-site that was mutated within the 5'-flanking DNA of each construct. B, chick embryo hepatocytes were transiently transfected as described in the legend to Fig. 1 and treated with or without T3 (1.6 mM) and with or without hexanoate (C6, 5 mM). The results are expressed as described in the legend to Fig. 1; each value is the mean ± S.E. of seven to nine independent experiments using at least two independently prepared batches of each plasmid. CAT and beta -galactosidase activities of extracts from T3-treated hepatocytes transfected with p[ME-3903/-3703]TKCAT were 8.8 ± 2.6 percentage of conversion/15 h/µg of protein and (4.4 ± 1.4) × 10-4 A420 units/min/µg of protein, respectively. Left, DNA constructs used in these experiments; middle, effects of T3, expressed as -fold change in CAT activity caused by T3; right, effects of hexanoate. Relative CAT activity of cells treated with T3 and C6 is expressed as a percentage of that in cells treated with T3 alone. Statistical significance between means within a column is indicated as follows: a, versus pTKCAT (p < 0.01); b, versus pTKCAT (p < 0.05); c, versus p[ME-3903/-3703]TKCAT (p < 0.01); d, versus p[ME-3903/-3703]TKCAT (p < 0.05).


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Fig. 4.   Effects of T3 and hexanoate on CAT activity in hepatocytes transfected with constructs containing wild-type and mutant versions of a T3 response region of the chicken malic enzyme gene linked to TKCAT. A, block mutations in half-sites 2U and 2D. Fragment A has the wild-type sequence; fragments B, C, and D contain block mutations in positions II-IV of one (B and C) or both (D) half-sites. B, chick embryo hepatocytes were transiently transfected as described in the legend to Fig. 1 and treated with or without T3 (1.6 µM) and with or without hexanoate (C6, 5 mM). Left, DNA constructs used in these experiments. Middle, effects of T3, expressed as -fold change in CAT activity caused by T3. The results are expressed as described in the legend to Fig. 1 and represent the mean ± S.E. of four to eight independent experiments using at least two independently prepared batches of each plasmid. CAT and beta -galactosidase activities of extracts from T3-treated hepatocytes transfected with p[ME-3903/-3703]TKCAT were 8.1 ± 2.7 percentage of conversion/15 h/µg of protein and (5.7 ± 1.8) × 10-4 A420 units/min/µg of protein, respectively. Right, effects of hexanoate. Relative CAT activity of cells treated with T3 and C6 is expressed as a percentage of that in cells treated with T3 alone. Statistical significance between means within a column is indicated as follows: a, versus pTKCAT (p < 0.01); b, versus pTKCAT (p < 0.02); c, versus p[ME-3883/-3858]TKCAT (p < 0.02); d, versus p[ME-3883/-3858]TKCAT (p < 0.01).


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Fig. 5.   Effects of T3 and hexanoate on CAT activity in hepatocytes transfected with constructs containing different kinds and numbers of T3REs. Chick embryo hepatocytes were transiently transfected as described in the legend to Fig. 1 and treated with or without T3 and with or without hexanoate (C6, 1 mM). Left, DNA constructs used in these experiments. First four columns, relative CAT activities are expressed as described in the legend to Fig. 1; each value is the mean ± S.E. of six to nine independent experiments using at least two independently prepared batches of each plasmid. Relative CAT activities were calculated by setting the CAT activities for T3-treated hepatocytes transfected with pTKCAT to 1.0 and adjusting all other activities proportionately. CAT and beta -galactosidase activities of extracts from T3-treated hepatocytes transfected with pTKCAT were 0.21 ± 0.04 percentage of conversion/15 h/µg of protein and (7.1 ± 1.3) × 10-3 A420 units/min/µg of protein, respectively. Fifth column, effects of T3 are expressed as -fold change in CAT activity caused by T3. Sixth column, the effect of hexanoate is expressed as CAT activity in cells treated with T3 plus hexanoate divided by that in cells treated with T3 alone × 100. The boxed region A is wild-type T3RE2. TREpal contains two copies of a palindromic T3RE, and DR4 contains five copies of a DR4 T3RE, both linked to TKCAT. Statistical significance between means within a column (p < 0.05) is indicated as follows: a, versus pTKCAT; b, versus pDR4-TKCAT.

We next tested constructs containing artificial T3REs, the malic enzyme gene T3RU, and T3RE2 to determine if the response to hexanoate was specific to natural T3REs of the malic enzyme gene or could be mediated by any element that responded to T3 (Fig. 5). Constructs containing T3RE2 (p[ME-3883/-3858]TKCAT) or T3RE2 within the entire T3RU (p[ME-3903/-3703]TKCAT) bestowed 120- and 130-fold responses to T3, respectively. These T3-induced activities were decreased to 35 and 37%, respectively, by hexanoate. Transfection of constructs containing either two copies of the artificial palindromic T3RE or five copies of a consensus direct repeat T3RE linked to TKCAT DNA conferred robust responsiveness to T3 and inhibition by hexanoate (Fig. 5). These results suggest that the inhibitory effect of hexanoate is not limited to sequences from the malic enzyme gene; inhibition by hexanoate is transduced by several kinds of T3REs. Moreover, when sequences containing the T3RUs were inserted into TKCAT in reverse orientation or were linked to the minimal promoter of the malic enzyme gene (-147 to +31 bp) in either orientation and transfected into hepatocytes, T3 stimulation and hexanoate inhibition were conferred (data not shown). The minimal promoter of the malic enzyme gene did not confer responsiveness to T3 or MCFA. Therefore, the T3REs that act as cis-acting elements for the effect of hexanoate can vary greatly in their distance from the start site of transcription, gene of origin, and orientation with respect to the direction of transcription.

Identification of the Trans-acting Factor(s) Involved in Mediating Inhibition by MCFA -- TR is the trans-acting factor common to all the T3REs that conferred MCFA inhibition in transfected cells, suggesting that it also may be the trans-acting factor that mediates the effects of MCFAs. TR acts as a silencer of transcription in the absence of T3 (33). Mutation or deletion of the major functional T3RE (T3RE2) from sequences linked to either a natural or a heterologous promoter causes an increase in basal activity in transfected cells (10). This increase is consistent with loss of the silencing action of TR. Nuclear receptors such as PPAR and chicken ovalbumin upstream promoter transcription factor inhibit T3-stimulated transactivation by T3; they also inhibit TR-mediated silencing of basal activity (20, 34-36). Three constructs, each of which contained the major functional T3RE of the malic enzyme gene, were tested for an effect of MCFA on basal activity (Fig. 6). Plasmid [ME-3903/-3703]TKCAT contains the entire T3RU; pMUT1[ME-3903/-3703]TKCAT contains a block mutation in a nonfunctional direct repeat upstream of the major T3RE of the upstream T3RU; and p[ME-3883/-3858]TKCAT contains a single copy of the major T3RE. When transfected into hepatocytes, all three constructs conferred robust responses to T3 and strong inhibitory responses to hexanoate (Fig. 6). None of these constructs nor the vector itself conferred responsiveness to hexanoate in the absence of T3. This result suggests that the factor that mediates responsiveness to hexanoate does not mediate the silencing action of unliganded TR.


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Fig. 6.   Effects of T3 and hexanoate on CAT activity in hepatocytes transfected with constructs containing wild-type and mutant versions of a T3 response region and wild-type T3RE2. Chick embryo hepatocytes were transiently transfected as described in the legend to Fig. 1 and treated with or without T3 (1.6 µM) and with or without hexanoate (C6, 1 mM). Left, DNA constructs used in these experiments. First four columns, relative CAT activities are expressed as described in the legend to Fig. 1; each value is the mean ± S.E. of seven independent experiments using at least two independently prepared batches of each plasmid. Relative CAT activities were calculated by setting the CAT activities for T3-treated hepatocytes transfected with p[ME-3903/-3703]TKCAT to 1.0 and adjusting all other activities proportionately. CAT and beta -galactosidase activities of extracts from T3-treated hepatocytes transfected with p[ME-3903/-3703]TKCAT were 11.1 ± 3.3 percentage of conversion/15 h/µg of protein and (9.5 ± 3.9) × 10-3 A420 units/min/µg of protein, respectively. Fifth column, effects of T3 are expressed as -fold change in CAT activity caused by T3. Sixth column, the effect of hexanoate on CAT activity is expressed as CAT activity in cells treated with T3 plus hexanoate divided by that in cells treated with T3 alone × 100. The boxed region A is wild-type T3RE2; x indicates a mutation upstream of T3RE2 (MUT1; see Fig. 3) that has little or no effect on T3 responsiveness. No statistically significant differences were observed between means for samples lacking T3, with or without hexanoate.

To transactivate, liganded TR must bind to a T3RE. Previous work suggested that MCFAs do not inhibit transactivation by disrupting binding of T3 to TR (7), suggesting that TR binding to the T3RE or transactivation may be disrupted by MCFAs. DNase I footprint and gel mobility shift analyses were used to test the effect of hexanoate on binding of TR to the T3REs in the upstream T3RU of the malic enzyme gene. Bacterially expressed (37) and partially purified (0.3 µg) TR bound to regions of the T3RU corresponding to T3RE2 and an extension of T3RE-3 (10). Proteins present in nuclear extracts prepared from chick embryo hepatocytes in culture treated with T3 and/or hexanoate for 24 h showed the same protection in these two regions (data not shown). Hexanoate added directly to binding reactions had no effect on binding of TR or other nuclear proteins, indicating that these fatty acids did not have a direct effect on binding of TR to the T3RE. Gel mobility shift analyses indicated that, in nuclear extracts from cells treated with T3, RXR/TR heterodimers bound to the major functional T3RE in a manner similar to that of those in nuclear extracts made from hepatocytes treated with T3 plus hexanoate (data not shown). Thus, hexanoate does not disrupt binding of TR to the T3RE, nor does it disrupt the interaction between TR and its heterodimerization partner, RXR.

If inhibition by hexanoate were mediated by a factor that competed with TR or RXR/TR dimers for binding to the T3RE, inhibition by hexanoate should be decreased in cells that overexpress TR. We tested p[ME-3868/-3703]TKCAT and p[ME-3474/+31]CAT for responsiveness to T3 and hexanoate in the absence and presence of overexpressed TRalpha (Fig. 7). Cells transfected with these constructs have higher basal CAT activities than those transfected with constructs containing an intact T3RE2 or the entire upstream T3RU. Overexpression of chicken TRalpha caused 62- and 100-fold increases in T3 responsiveness of cells transfected with p[ME-3868/-3703]TKCAT and p[ME-3474/+31]CAT, respectively. Hexanoate inhibited the response to T3 to a similar extent whether or not chicken TRalpha was overexpressed. As noted earlier, hexanoate had no effect on CAT activity in the absence of T3. Thus, the factor that mediates the MCFA response may not compete with TR for binding to the T3RE.


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Fig. 7.   Effects of T3 and hexanoate on CAT activity of hepatocytes transfected with constructs containing part of the T3RU linked to TKCAT or part of the 5'-flanking DNA containing the downstream T3RU linked to ME-147/+31CAT, with and without overexpression of chicken TRalpha . Chick embryo hepatocytes were transiently transfected as described in the legend to Fig. 1 with or without overexpression of pRSV-cTRalpha (0.2 µg/plate) and treated with or without T3 and with or without hexanoate (C6, 1 mM). Left, DNA constructs used in these experiments. First four columns, relative CAT activities are expressed as described in the legend to Fig. 1; each value is the mean ± S.E. of six to seven independent experiments using at least two independently prepared batches of each plasmid. Relative CAT activities were calculated by setting CAT activities for T3-treated hepatocytes transfected with p[ME-3868/-3703]TKCAT DNA minus overexpressed TR equal to 100 and adjusting all other activities proportionately. CAT and beta -galactosidase activities of extracts from T3-treated hepatocytes transfected with p[ME-3868/-3703]TKCAT were 4.9 ± 0.8 percentage of conversion/15 h/µg of protein and (2.0 ± 0.3) × 10-4 A420 units/min/µg of protein, respectively. Fifth column, effects of T3 are expressed as -fold change in CAT activity caused by T3. Sixth column, the effect of hexanoate on CAT activity is expressed as CAT activity in cells treated with T3 plus hexanoate divided by that in cells treated with T3 alone × 100. Statistical significance between means within a column (p < 0.05) is indicated as follows: a, versus pTKCAT; b, versus p[ME-147/+31]CAT.

Many TR-associated proteins regulate transactivation by liganded TR (38-40); MCFAs could inhibit transactivation by interacting with such TR-associated proteins. Some of these proteins also interact with ER, but do not interact with GR. To gain insight into the specificity of action by MCFA and to narrow the potential list of factors that might mediate inhibition by MCFA, we tested the effects of hexanoate on cells transfected with constructs that bestow responsiveness to estrogen and glucocorticoids. Single copies of elements that bind these receptors were linked to TKCAT DNA and transfected into hepatocytes with and without overexpression of the cognate receptor. Plasmid ERE-TKCAT conferred 30- and 37-fold responses to beta -estradiol in the absence and presence of overexpressed ER, respectively (Fig. 8). Plasmid GRE-TKCAT conferred 220- and 160-fold responses to corticosterone without and with overexpression of GR, respectively. Therefore, endogenous forms of both ER and GR were functional on these cis-acting elements. Hexanoate inhibited function of the ERE, but not the GRE, with or without overexpression of the cognate receptors. A hormone-activated transcription factor that does not belong to the steroid/thyroid hormone receptor superfamily also was tested. The cis-acting element was a CRE linked to TKCAT DNA. Cells transfected with the CRE construct showed 10- and 23-fold responses to CPT-cAMP (a nonmetabolizable analog of cAMP) with and without overexpressed CRE-binding protein (CREB ), respectively; they did not respond to hexanoate. These results suggest that inhibition by hexanoate has specificity with respect to the involved transcription factor. The actions of TR and ER are inhibited; those of GR and CREB (or other CRE-binding protein) are not inhibited. This distinction may be due to the interaction of TR and ER with a factor that mediates the effect of MCFA; GR and CREB may not interact with that factor.


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Fig. 8.   Effects of estradiol, corticosterone, cyclic AMP, and hexanoate on CAT activity of hepatocytes transfected with constructs containing single copies of the estrogen, glucocorticoid, and cyclic AMP response elements linked to TKCAT, with or without overexpression of the respective receptor or binding protein. Chick embryo hepatocytes were transiently transfected as described in the legend to Fig. 1 with or without overexpression of pHEO (ER), pVARO (GR), and pRSV-CREB (0.5, 1, and 0.5 µg/plate, respectively). Transfected cells were treated with or without beta -estradiol (10 µM), corticosterone (1 µM), and CPT-cAMP (10 µM), respectively, with or without hexanoate (C6, 1 mM). Left, DNA constructs used in these experiments. First four columns, relative CAT activities are expressed as described in the legend to Fig. 1; each value is the mean ± S.E. of six to seven independent experiments using at least two independently prepared batches of each plasmid. Relative CAT activities were calculated by setting CAT activities for individual ligand-treated hepatocytes transfected with the respective element plus or minus overexpressed binding protein equal to 100 and adjusting all other activities proportionately. CAT activities of extracts from ligand-treated hepatocytes were, in order of presentation in the figure, 3.2 ± 0.5, 1.7 ± 0.5, 9.8 ± 0.9, 4.3 ± 1.0, 16.1 ± 2.4, and 23.3 ± 1.7 percentage of conversion/15 h/µg of protein. beta -Galactosidase activities of extracts from ligand-treated hepatocytes were, in order of presentation in the figure, (7.9 ± 1.2), (13.5 ± 2.3), (3.8 ± 1.1), (6.4 ± 1.3), (8.6 ± 1.1), and (2.1 ± 0.6) × 10-3 A420 units/min/µg of protein. Fifth column, effects of ligand are expressed as -fold change in CAT activity caused by ligand. Sixth column, the effect of hexanoate on CAT activity is expressed as CAT activity in cells treated with ligand plus hexanoate divided by that in cells treated with ligand alone × 100. Constructs containing single copies of the estrogen, glucocorticoid, and cAMP response elements were obtained as described under "Experimental Procedures." Statistical significance between means within a row is indicated as follows: a, versus control without hexanoate (p < 0.05).

Members of the family of proteins that interact with TR and ER, but not GR, bind to the ligand-binding domains of these receptors (40). These proteins were identified in yeast two-hybrid screens using the ligand-binding domain (with no N-terminal or DNA-binding domains) of TR or ER as "bait." The ligand-binding domain of TR, fused to the DNA-binding domain of GAL4, was transfected into hepatocytes with a plasmid that encodes GAL4-binding sites (Fig. 9). In this model, T3-stimulated CAT activity is due to binding of T3 to the hybrid GAL4-TR and not to endogenous TR (Fig. 9). We used p[ME-3903/-3703]TKCAT as a positive control; it conferred both T3 and hexanoate responsiveness. In cells cotransfected with the negative control, pGAL4 (pSG424), and GAL4-CAT, CAT activity was unaffected by T3 or hexanoate. Cotransfection of pGAL4-cTRalpha , the ligand-binding domain of chicken TRalpha fused to pGAL4, with pGAL4-CAT resulted in a robust response to T3, indicating that the truncated TR was capable of binding T3 and transactivating the linked promoter. Cells transfected with the GAL4-TR fusion did not respond to hexanoate. These results suggest that the N-terminal sequence of TR is required for inhibition by hexanoate. They also confirm that binding of T3 to TR is not disrupted by hexanoate.


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Fig. 9.   Effects of T3 and hexanoate on CAT activity in hepatocytes transfected with chimeric GAL4-cTRalpha and a construct containing a GAL4-binding site. Chimeric GAL4-cTRalpha and the construct containing the GAL4-binding site were obtained as described under "Experimental Procedures." Chick embryo hepatocytes were transiently transfected as described in the legend to Fig. 1 and treated with or without T3 (1.6 µM) and with or without hexanoate (C6, 1 mM). pGAL4-DBD (pSG424) and pGAL4-DBD/TR-LBD were cotransfected (0.2 µg each per plate) with the pGAL4-CAT (pMC110) reporter construct. Left, DNA constructs used in these experiments. First four columns, relative CAT activities are expressed as described in the legend to Fig. 1; each value is the mean ± S.E. of six independent experiments using at least two independently prepared batches of each plasmid. Relative CAT activities were calculated by setting CAT activities for T3-treated hepatocytes transfected with p[ME-3903/-3703]TKCAT (T3RUTKCAT) to 100 and adjusting all other activities proportionately. CAT and beta -galactosidase activities of extracts from T3-treated hepatocytes transfected with p[ME-3903/-3703]TKCAT were 22.7 ± 2.3 percentage of conversion/15 h/µg of protein and (6.5 ± 1.3) × 10-3 A420 units/min/µg of protein, respectively. Fifth column, the effect of hexanoate on relative CAT activity is expressed as relative CAT activity in cells treated with T3 plus hexanoate divided by that in cells treated with T3 alone × 100.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The cis-acting elements in the 5'-flanking DNA of the chicken malic enzyme gene that confer inhibition by MCFAs co-localized with the T3REs in this DNA. Furthermore, artificial palindromic and direct repeat T3REs also conferred inhibition by MCFA. MCFAs also inhibited transactivation from an ERE, but had no effect on basal activity in the absence of T3 or estradiol. Thus, the trans-acting factor that binds to T3REs or EREs and participates in the inhibitory effect of MCFA is TR or ER, respectively. The MCFA-regulated factor did not compete with TR for binding to the T3RE, but probably interacted with TR to influence its ability to transactivate linked promoters. Sequences N-terminal to the ligand-binding domain of TR are required for the action of MCFA, presumably because they interact with a MCFA-regulated factor. T3REs are found in many nutritionally regulated genes; T3 stimulates and MCFAs inhibit transcription of both malic enzyme and fatty acid synthase (7). Modulation of the transactivation function of TR bound to a T3RE may represent an effective mechanism for modulation of T3-stimulated transcription of lipogenic genes. MCFAs themselves are unlikely to be physiological regulators of the lipogenic genes in chicks because there is no evidence that plasma concentrations of MCFA in chicks ever reach the required levels. Nevertheless, the specificity of the response with respect to chemical structure, its selectivity with respect to the genes affected, and the rapid onset and reversal of its effects (Ref. 9 and data not shown) are consistent with physiological importance. MCFAs or a compound derived therefrom may be similar in structure to the true inhibitor, the production of which may signal the starved state.

Modulation of the transactivation function of TR without displacement of T3 from TR or of TR from the T3RE has not been described previously as a mechanism by which fatty acids regulate transcription. Long-chain fatty acids, specifically polyunsaturated fatty acids, regulate transcription of some eucaryotic genes by binding to or modifying regulatory proteins, which, in turn, bind to unique DNA elements in the 5'-flanking regions of these genes (17-20). Hydroxy fatty acids also regulate transcription through a unique cis-acting element (14-16). By contrast, our results suggest that MCFAs differ from these other fatty acids in that their inhibitory actions are transduced through the T3RE·TR·T3 complex, rather than by a complex unique to MCFAs.

Proximal promoter elements may play important roles in T3-regulated expression of some genes (41). This is unlikely to confound the interpretation of our results. First, T3REs linked to both TK and minimal malic enzyme promoters are inhibited by hexanoate; the two promoters have quite different structures (10). Second, both estrogen- and T3-stimulated transcription is inhibited by MCFA. Third, MCFAs have no effect on promoter activity in the basal state. Fourth, if hexanoate did regulate activity of a factor bound at a site common to both TK and malic enzyme promoters and that factor mediated both estrogen- and T3-regulated gene transcription, then one would expect fractional inhibition by hexanoate to decrease as T3 responsiveness decreased; it did not.

The superfamily of steroid/thyroid hormone receptors is subdivided into two major classes. Class I includes GR and ER; these receptors are cytosolic until bound by ligand. Class II includes TR, RXR, and PPAR, receptors that are nuclear in the absence or presence of ligand (42). The interaction of estrogen with ER triggers the translocation of the complex to the nucleus, where estrogen-bound ER binds to an ERE present in the DNA (43). Because both TR and ER are affected by MCFA, it seems unlikely that MCFA acts cytosolically to disrupt the interaction of estrogen and ER, preventing ER from reaching the nucleus. The mechanisms by which MCFAs inhibit estrogen- and T3-induced transcription are probably the same.

Inhibition of the action of ER by MCFA is consistent with the hypothesis that RXR and PPAR, a known heterodimerization partner for TR at T3REs of the malic enzyme gene and a potential partner for TR, respectively, are not targets for MCFA because ER dimerizes with itself and not RXR or PPAR (42, 43). Overexpression of TR should have increased the number of TR/TR dimers bound to T3REs. Overexpression of TR, however, failed to alter inhibition by MCFA, additional evidence that potential heterodimerization partners such as RXR and PPAR did not mediate the inhibitory action of MCFA. Furthermore, fatty acids with carbon chain lengths shorter than 10 are poor activators of PPAR (44). The ligand-binding domains of both ER and TR interact with TR-interacting proteins (TRIPs); TRIPs do not interact with GR (41). The specificity of the inhibitory action of MCFAs for ER or TR, but not GR, suggested that one or more of these TRIPs might be the target for MCFA; modification or displacement of a TRIP could decrease transactivation by ER or TR. TRIPs, however, bind to the ligand-binding domain of TR or ER, and this part of TR was not sufficient to confer inhibition by MCFA even though it was sufficient for T3 responsiveness. TRIPs thus seem unlikely to be the MCFA-regulated factor that interacts with TR or ER.

T3 responsiveness of hepatocytes transfected with constructs containing all or parts of the T3RU, mutant versions of T3REs, or artificial T3REs varied from <5-fold to >1500-fold. These differences in responsiveness are due to novel proteins that bind to the different T3REs and differentially influence responsiveness (9), differences in binding of RXR/TR to T3REs of different sequences (42), and artificial overexpression of TR. Despite the resulting broad range of promoter activities, MCFA always resulted in inhibitions of 50-80%. Within individual sets of experiments, the range for inhibition was even narrower, despite a wide range in T3 responsiveness (e.g. Fig. 2). Thus, MCFAs may modify function of a factor that has the same relative regulatory effect on RXR/TR heterodimers bound at any T3RE, whether the intrinsic ability of that T3RE to transactivate a linked promoter is large or small.

MCFA action appears to require that part of TR that is N-terminal to the ligand-binding domain because the N-terminal (A/B) and DNA-binding domains were absent from the GAL4-TR chimera that responded to T3, but not MCFA. MCFA did not interfere with the T3RE-TR or RXR-TR interactions, suggesting that the DNA-binding domain and the N- and C-terminal regions required for heterodimerization were not targets for interaction with the factor that mediates the effect of MCFA. This leaves the N-terminal A/B domain of TR as a putative interaction site for the factor that mediates action of MCFA. A number of TR accessory factors have been identified and are required for full transactivation of the T3RE-bound receptors with which they interact. The TR accessory factors that have been characterized interact with activation function domains in the ligand-binding region of TR and other members of the superfamily of steroid/thyroid hormone receptors (45-50). Because the MCFA effect does not appear to be mediated by this region of TR, the known TR accessory factors are not promising candidates for factors regulated by MCFA. Analogous factors may interact with the N terminus, and MCFAs may target one or more of these factors, thus eliciting its inhibitory response.

    ACKNOWLEDGEMENTS

We are indebted to Drs. Richard Maurer (pCMV-beta GAL), Ronald Evans (pTREpal-TKCAT), Bert W. O'Malley (pERE-TKCAT), Geoffrey L. Greene (pHEO), F. Bradley Hillgartner (pRSV-CREB), p[ME-3474/+31]CAT), Marc Montminy, Mark Ptashne (pSG424 and pMC110), Herbert H. Samuels (pET8cTRalpha and pGAL4-cTRalpha ), Keith R. Yamamoto (pDelta GTCO and pVARO), Ganes Sen (pCRE-TKCAT), and Pierre Chambon (monoclonal antibody raised against chicken RXR) for the indicated plasmids and antibody. We thank Drs. Catherine Mounier and Thomas Carlisle for helpful discussions.

    FOOTNOTES

* This work was supported in part by Grant DK 21594 from the National Institutes of Health and by the Core Facilities of the Diabetes and Endocrinology Research Center (supported by National Institutes of Health Grant DK 25295) of the University of Iowa.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a fellowship from the American Heart Association, Iowa Affiliate. Present address: Dept. of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242.

§ Present address: Dept. of Human Ecology, University of Texas, Austin, TX 78712-1097.

To whom correspondence should be addressed: College of Biological Sciences, Ohio State University, 484 W. 12th Ave., Columbus, OH 43210-1292. Tel.: 614-292-1627; Fax: 614-292-1538; E-mail: goodridge.4{at}osu.edu.

1 The abbreviations used are: T3, triiodothyronine; T3RE, T3 response element; T3RU, T3 response unit; TR, T3 receptor; TRIP, TR-interacting protein; MCFA, medium-chain fatty acid; PUFA, polyunsaturated long-chain fatty acid; PPAR, peroxisomal proliferator-activated receptor; ER, estrogen receptor; ERE, estrogen response element; GR, glucocorticoid receptor; GRE, glucocorticoid response element; RXR, retinoid X receptor; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; CMV, cytomegalovirus; beta GAL, beta -galactosidase; RSV, Rous sarcoma virus; LUC, luciferase; ME, malic enzyme; CRE, cAMP response element; CREB, CRE-binding protein; DR4, direct repeat T3RE with a 4-bp spacer; bp, base pair(s); CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3':5'-monophosphate; DBD, DNA-binding domain; LBD, ligand-binding domain.

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Top
Abstract
Introduction
Procedures
Results
Discussion
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