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J. Biol. Chem., Vol. 277, Issue 22, 19554-19565, May 31, 2002

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Sterol Regulatory Element-binding Protein-1 Interacts with the Nuclear Thyroid Hormone Receptor to Enhance Acetyl-CoA Carboxylase- Transcription in Hepatocytes^*

Liya Yin, Yanqiao Zhang, and F. Bradley Hillgartner

From the Department of Biochemistry and Molecular Pharmacology, School of Medicine, West Virginia University, Morgantown, West Virginia 26506

Received for publication, December 10, 2001, and in revised form, January 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In previous work, we characterized a 3,5,3'-triiodothyronine response element (T3RE) in acetyl-CoA carboxylase- (ACC) promoter 2 that mediated 3,5,3'-triiodothyronine (T3) regulation of ACC transcription in chick embryo hepatocytes. Sequence comparison analysis revealed the presence of sterol regulatory element-1 (SRE-1) located 5 bp downstream of the ACC T3RE. Here, we investigated the role of this SRE-1 in modulating T3 regulation of ACC transcription. Transfection analyses demonstrated that the SRE-1 enhanced T3-induced ACC transcription by more than 2-fold in hepatocytes. The effect of the SRE-1 on T3 responsiveness required the presence of the T3RE in its native orientation. In pull-down experiments, the mature form of sterol regulatory element-binding protein-1 (SREBP-1) specifically bound the -isoform of the nuclear T3 receptor (TR), and the presence of T3 enhanced this interaction. A region of TR containing the DNA-binding domain plus flanking sequences (amino acids 21-157) was required for interaction with SREBP-1, and a region of SREBP-1 containing the basic helix-loop-helix-leucine zipper domain (amino acids 300-389) was required for interaction with TR. In gel mobility shift experiments, TR, retinoid X receptor-, and mature SREBP-1 formed a tetrameric complex on a DNA probe containing the ACC T3RE and SRE-1, and the presence of T3 enhanced the formation of this complex. Formation of the tetrameric complex stabilized the binding of SREBP-1 to the SRE-1. These results indicate that SREBP-1 directly interacts with TR-retinoid X receptor in an orientation-specific manner to enhance T3-induced ACC transcription in hepatocytes. T3 regulation of ACC transcription in nonhepatic cell cultures such as chick embryo fibroblasts is markedly reduced compared with that of chick embryo hepatocytes. Here, we also show that alterations in SREBP expression play a role in mediating cell type-dependent differences in T3 regulation of ACC transcription.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In livers of birds and mammals, consumption of carbohydrate in excess of the immediate energy requirements of the animal increases the expression of enzymes involved in the conversion of carbohydrate to triacylglycerols (1). These enzymes include those in glycolysis, such as glucokinase and L-pyruvate kinase; those in fatty acid synthesis, such as acetyl-CoA carboxylase- (ACC)¹ and fatty acid synthase; those in NADPH production, such as malic enzyme; and those in triacylglycerol synthesis, such as glycerol-3-phosphate acyltransferase. For most of the lipogenic enzymes studied, the induction of enzyme levels by dietary carbohydrate is mediated primarily by changes in gene transcription (1).

Several signaling pathways are involved in mediating the stimulatory effects of dietary carbohydrate on lipogenic gene transcription in liver. Increased insulin secretion and glucose metabolism are two important signals mediating this response (1). Another factor signaling changes in carbohydrate status is the active form of thyroid hormone, 3,5,3'-triiodothyronine (T3). Ingestion of a high carbohydrate meal stimulates a rapid increase in the secretion of thyroxine from the thyroid gland and the conversion of thyroxine to T3 in extrathyroidal tissues (2, 3). The resulting increase in T3 concentration in liver activates the transcription of the genes for ACC (4), fatty acid synthase (5), malic enzyme (6), and Spot 14 (S14) (7). Ingestion of carbohydrate also increases the levels of nuclear T3 receptors (TRs) in liver (8). This phenomenon may also contribute to the stimulation of lipogenic gene transcription by dietary carbohydrate. A role of thyroid hormones in mediating the nutritional regulation of lipogenic enzyme expression is supported by the observation that hypothyroidism in high carbohydrate-fed animals causes a marked reduction in expression of ACC, fatty acid synthase, malic enzyme, and S14 in liver (9-11).

ACC catalyzes the pace-setting step of the fatty acid synthesis pathway (1). There are two ACC isoforms that are encoded by distinct genes. ACC (280 kDa) is the major isoform observed in heart and skeletal muscle, where it is thought to function primarily in the regulation of -oxidation of fatty acids (12). ACC (265 kDa) is the principal isoform expressed in tissues that exhibit high rates of fatty acid synthesis such as liver, adipose tissue, and mammary gland. ACC expression is induced by nutritional and hormonal factors that promote high rates of fatty acid synthesis. For example, feeding previously starved chickens a high carbohydrate, low fat diet stimulates an 11-fold increase in transcription of the ACC gene in liver (13). This effect is partially reproduced in primary cultures of chick embryo hepatocytes (CEH) by manipulating the concentration of T3 in the culture medium. The addition of T3 stimulates a 7-fold increase in ACC transcription in CEH (4).

The ACC gene is transcribed from two promoters, resulting in mRNAs with heterogeneity in their 5'-untranslated region (14). These ACC promoters are designated promoter 1 and promoter 2. The increase in total ACC mRNA abundance caused by high carbohydrate feeding in chickens and by T3 in CEH is mediated by alterations in the activities of both promoter 1 and promoter 2, with the latter promoter playing a quantitatively greater role in mediating these responses (15). Additional studies have shown that the stimulatory effect of T3 on promoter 2 activity is mediated by a T3 response element (T3RE) with unique functional and protein binding properties (16). This T3RE enhances ACC promoter activity both in the absence and presence of T3, with a greater stimulation observed in the presence of T3. The results of DNA binding analyses with nuclear extracts from CEH suggest that the T3-independent enhancer activity of the ACC T3RE is mediated by the binding of protein complexes containing LXR·RXR heterodimers and that the increase in enhancer activity caused by T3 treatment is mediated by the binding of a different set of protein complexes. One of these complexes contains TR·RXR heterodimers, and another contains LXR·RXR heterodimers. Based on these observations, we have hypothesized that the ACC T3RE not only mediates T3 regulation of ACC transcription but also ensures a basal level of ACC expression for the synthesis of structural lipids in cell membranes.

In addition to regulation by nutrients and hormones, ACC transcription is controlled by tissue- or cell-specific factors. For example, ingestion of carbohydrate has little or no effect on ACC transcription in heart, kidney, brain, and skeletal muscle (13). Cell type-dependent differences in the regulation of ACC are also observed in cells in culture. T3 regulation of ACC transcription in chick embryo fibroblasts (CEF) is markedly diminished compared with that in CEH (17). The mechanisms responsible for cell type-dependent differences in the regulation of ACC transcription remain to be determined.

Studies analyzing the regulation of other T3-responsive genes have shown that optimal T3 regulation of transcription is dependent not only on the presence of a T3RE but also the presence of accessory elements that bind proteins that are distinct from nuclear hormone receptors (18-23). In isolation, these accessory elements are devoid of T3-responsive activity; they act by enhancing the degree of T3 responsiveness initiated by the T3RE. Some T3 accessory elements bind proteins that are differentially expressed in different cell types and thus play a key role in conferring cell type-dependent differences in T3 responsiveness (18, 22, 23). There is little information on the mechanism by which T3 accessory elements modulate T3 regulation of transcription.

In the present study, we have identified an accessory element that enhances T3 responsiveness of ACC promoter 2 in CEH. This element is located 5 bp downstream of the ACC T3RE and binds sterol regulatory element-binding protein-1 (SREBP-1). We have developed data suggesting that SREBP-1 enhances T3 regulation of ACC transcription by directly interacting with TR·RXR heterodimers bound to the ACC T3RE. In addition, we provide evidence that alterations in SREBP expression play a role in mediating cell type-dependent differences in T3 regulation of ACC transcription.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmids-- DNA fragments used to construct reporter plasmids were named by designating the 5' and 3' ends of each fragment relative to the transcription start site of promoter 2 of the ACC gene. p[ACC108/+274]CAT has been described previously (16). This construct contains ACC sequences between 108 and +274 bp ligated upstream of the CAT gene in KSCAT (24). A block mutation of the SRE-1 between 79 and 72 bp was introduced into p[ACC108/+274]CAT using a polymerase chain reaction-based strategy (25). pBLCAT2 (pTKCAT) was obtained from B. Luckow and G. Schutz (German Cancer Research Center) (26). The cryptic activator protein-1 site located 5' of the multiple cloning site in pBLCAT2 (27) was removed by excising the NdeI/HindIII fragment from this plasmid followed by religation. p[ACC108/82]TKCAT, p[ACC108/66]TKCAT, p[ACC-84/66]TKCAT, and pTKCAT constructs containing mutations in the 108 to 66 bp fragment were made by first annealing complementary synthetic oligonucleotides containing ACC sequences and then inserting them into SphI and SalI site 5' of the thymidine kinase (TK) promoter in pTKCAT.

A full-length cDNA for chicken SREBP-1 was obtained by screening a chicken liver cDNA library (Stratagene) using a human SREBP-1 cDNA probe (nucleotides 721-1103 relative to the start site of translation) and by 5'-RACE.² The N-terminal amino acid sequence of this chicken SREBP-1 (GenBank^TM accession number AY029224) more closely resembles the 1a isoform than the 1c isoform described for mammalian species (28). The data from RNase protection analyses indicate that other forms of SREBP-1 containing variations in the N terminus are not expressed in chicken cells. An expression plasmid encoding the mature form of chicken SREBP-1 was developed by subcloning a SREBP-1 cDNA fragment encoding amino acids 1-464 into pSV-SPORT1 (Invitrogen) to form pSV-SPORT1-SREBP-1 (1-464). N-terminal and C-terminal truncations of the mature form of SREBP-1 were generated by PCR. PCR products encoding SREBP-1 polypeptides (78-464, 108-464, 238-464, 1-266, 1-340, 1-389, and 300-464) were subcloned into pSV-SPORT1. To generate a plasmid that expresses a fusion protein containing glutathione S-transferase (GST) linked to the mature form of chicken SREBP-1, a SREBP-1 cDNA fragment encoding amino acids 1-464 was subcloned into pGEX-2T (Amersham Biosciences). The structures of all reporter plasmids and expression plasmids were confirmed by restriction enzyme mapping and nucleotide sequence analyses.

pGEM-3Zf()-based plasmids that express full-length chicken TR (1-408) and N-terminal and C-terminal deletion derivatives of chicken TR (1-118, 1-157, 21-408, 51-408, and 120-408) have been previously described (19). The construction of a plasmid that expresses a fusion protein containing GST linked to full-length TR (pGEX-2T-TR (1-408)) has been described (19). The cDNAs for human RXR and human LXR were provided by R. Evans (Salk Institute) and D. Mangelsdorf (University of Texas Southwestern Medical Center), respectively. Expression plasmids for RXR and LXR were developed by subcloning the cDNAs for these receptors into pSV-SPORT1.

Cell Culture and Transient Transfection-- Primary cultures of CEH were prepared as previously described (29) and maintained in serum-free Waymouth's medium MD705/1 containing 50 nM insulin (gift from Eli Lilly Corp.) and 1 µM corticosterone. CEH were incubated on 60-mm Petri dishes (Fisher) at 40 °C in a humidified atmosphere of 5% CO₂ and 95% air. The cells were transfected 6 h after plating, using 20 µg of Lipofectin (Invitrogen), 1.3 µg of p[ACC108/+274]CAT, or an equimolar amount of another reporter plasmid and pBluescript KS(+) to bring the total amount of transfected DNA to 3.0 µg/plate. At 18 h of incubation, the transfection medium was replaced with fresh medium with or without T3 (1.5 µM). At 66 h of incubation, CEH were harvested, and the cell extracts were prepared as described by Baillie et al. (30). CAT activity (31) and protein (32) were assayed by the indicated methods. All of the DNAs used in transfection experiments were purified using the Qiagen endotoxin-free kit.

CEF were obtained from SPAFAS, Inc. (Norwich, CT) and were routinely cultured in DMEM/M199 (Dulbecco's modified Eagle's medium (25 mM glucose) with medium 199 (Invitrogen) in a 1:1 (v/v) ratio, containing 10,000 units/liter penicillin G, 10 mg/liter streptomycin sulfate, and 25 µg/liter amphotericin B supplemented with 5% fetal bovine serum). These cells were transfected with p[ACC108/+274]CAT or p[ACC108/+274]CAT containing a block mutation of the SRE-1 using the calcium phosphate method (33). Briefly, the cells were seeded on T-75 flasks and grown in DMEM/M199 containing 5% fetal bovine serum until 70% confluent. Twenty-four hours before transfection, the medium was changed to DMEM/M199 containing 50 nM insulin and 1 µM corticosterone. This medium was used throughout the experiment. The cells were transfected with 15 µg of reporter plasmid. Exposure to the calcium phosphate/DNA precipitate was for 16 h. Following transfection, the cells were trypsinized and distributed to 60-mm tissue culture plates. T3 was added to the medium at this time. After 48 h of incubation, the cells were harvested, and the extracts were prepared for CAT and protein assays.

Preparation of Membrane and Nuclear Extracts-- All of the procedures were carried out at 4 °C. To prevent proteolysis, a mixture of protease inhibitors (Complete^TM; Roche Molecular Biochemicals) was included in all the buffers. The nuclear extracts were prepared from CEH and CEF by a modification of the method described by Dignam et al. (34). Briefly, CEH from four 100-mm plates or CEF from twelve 100-mm plates were pooled and centrifuged at 1000 × g for 5 min at 4 °C. The resulting cell pellet was homogenized in buffer 1 (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol) using a 20 strokes in a Dounce homogenizer. The homogenate was centrifuged at 1100 × g for 10 min, and the resulting nuclear pellet was washed once in buffer 1. The nuclear pellet was resuspended in Buffer 2 (20 mM Hepes, pH 7.9, 420 mM NaCl, 25% (v/v) glycerol, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol). This suspension was rotated for 30 min and then centrifuged 15,000 × g for 30 min. The resulting supernatant was designated as the nuclear extract fraction. The membrane extract fraction was prepared by centrifuging the supernatant of the original 1100 × g spin for 1 h at 100,000 × g. The resulting membrane pellet was dissolved in Buffer 3 (10 mM Tris, pH 6.8, 100 mM NaCl, 1% (w/v) SDS, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol). The protein content of the nuclear and membrane extracts was determined as described (32).

Gel Mobility Shift Analysis-- Chicken TR, human RXR, and chicken SREBP-1 were translated in vitro using the TNT SP6-coupled reticulocyte lysate system (Promega). Double-stranded oligonucleotides were prepared by combining equal amounts of the complementary single-stranded DNA in a solution containing 10 mM Tris, pH 8.0, 1 mM EDTA followed by heating to 90 °C for 2 min and then cooling to room temperature. The annealed oligonucleotides were labeled by filling in overhanging 5' ends using the Klenow fragment of Escherichia coli DNA polymerase in the presence of [-³²P]dCTP and/or [-³²P]dGTP. The binding reactions were carried out in 20 µl of 20 mM Tris, pH 7.9, 100 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol (v/v), 0.3 mg/ml bovine serum albumin, and 2 µg of poly(dI·dC). A typical reaction contained 20,000 cpm (0.1 ng) of labeled DNA and 5 µl of in vitro translated proteins and was performed at 37 °C for 30 min. DNA and DNA-protein complexes were resolved on 6% nondenaturing polyacrylamide gels at 4 °C in 0.5× TBE (45 mM Tris, pH 8.3, 45 mM boric acid, 1 mM EDTA). Following electrophoresis, the gels were dried and subjected to storage phosphor autoradiography. For competition experiments, unlabeled competitor DNA was mixed with radiolabeled oligomer prior to the addition of nuclear extract. For antibody supershift experiments, nuclear extracts were incubated with antibodies for 30 min prior to addition of the oligonucleotide probe. A mouse monoclonal antibody against SREBP-1 (IgG-2A4) was obtained from the American Type Tissue Collection (Manassas, VA). P. Chambon (Strasbourg, France) kindly provided a monoclonal antibody against RXR. A polyclonal antibody that recognizes chicken TR (FL-408) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The synthetic oligonucleotides that were used as probes in gel mobility shift assays are listed in Fig. 1A and in the legend to Fig. 6.

Western Blot Analysis-- The proteins in nuclear extract and membrane fractions were subjected to electrophoresis in 8% SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (Amersham Biosciences) using an electroblotting apparatus (Owl Scientific). Immunoblot analyses were performed as described in the Western blotting protocol from Santa Cruz Biotechnology. Briefly, the blots were blocked in Blotto (5% nonfat dry milk, 10 mM Tris-HCl, pH 8.0, 150 mM NaCl) at 4 °C overnight and then incubated with monoclonal antibody against SREBP-1 (IgG-2A4) or SREBP-2 (IgG-1D2) diluted to 1 µg/ml in Blotto containing 0.01% (v/v) Tween 20. The results of gel mobility shift experiments employing in vitro translated proteins or chicken liver nuclear extracts indicated that IgG-2A4 and IgG-1D2 reacted with the chicken forms of SREBP-1 and SREBP-2, respectively. After incubation with primary antibody for 1 h at room temperature, the blots were washed in TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) containing 0.01% Tween 20. Next, the blots were incubated with a donkey anti-mouse IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch) diluted 1:10,000 in Blotto, 0.01% Tween 20 for 1 h at room temperature. After washing with TBS, 0.01% Tween 20, antibody-protein complexes on blots were detected using enhanced chemiluminescence (Amersham Biosciences). Chemiluminescence on the blots was visualized using a FluorChem 8000 imager (Alpha Innotech Corporation), and signals for the precursor and mature forms of SREBP-1 and SREBP-2 were quantified using FluorChem V200 software.

RNase Protection Assay-- cDNA fragments for chicken SREBP-1, SREBP-2, and -actin were subcloned into the HindIII and BamHI sites of pBluescript KS⁺ (Stratagene). The SREBP-1 cDNA fragment (300 bp) contained sequences between nucleotides 829 and 1128 relative to the start site of translation. The SREBP-2 cDNA fragment (460 bp) was isolated using reverse transcriptase-PCR. The primers used to generate this fragment were based on a SREBP-2 mRNA sequence reported in the GenBank^TM data base (accession number AJ310769). The 5' boundary of this SREBP-2 fragment was defined by the primer sequence, 5'-agcagggcaaccataagcTGA-3', and the 3' boundary was defined by the primer sequence, 5'-CAGCCAAACCATCCACCTGT-3'. The -actin cDNA fragment (256 bp) contained sequences between nucleotides 4 and 259 relative to the start site of translation. Each subclone was linearized with BamHI, and antisense RNA was transcribed with [-³²P]CTP (specific activity, 3000 Ci/mmol) using bacteriophage T3 RNA polymerase (Promega). Labeled RNAs were purified by polyacrylamide gel electrophoresis. RNA was extracted from CEH and CEF by the guanidinium thiocyanate/phenol/chloroform method (35). RNase protection assays were performed using the RPA II kit (Ambion). Total RNA (20 µg) was hybridized to 4 × 10⁴ cpm of ³²P-labeled RNA at 45 °C for 16 h. The sample was then digested with a mixture of RNase A and RNase T. Protected fragments were separated on 8 M urea, 5% polyacrylamide gels. The gels were dried and subjected to storage phosphor autoradiography. The images were quantified using ImageQuaNT software by Molecular Dynamics.

Protein-Protein Interaction-- GST and GST fusion proteins were expressed in E. coli (BL21, pLysS) and purified using standard techniques (36). Briefly, the bacteria were transformed with pGEX-2T or recombinant pGEX-2T plasmids expressing GST fusion proteins. Overnight bacterial cultures in ampicillin (250 µg/ml) were diluted 1:100 into 250 ml of Luria broth and grown at 37 °C to an A₆₀₀ of 1.0 before induction with 1 mM isopropyl-1-thio--D-galactopyranoside for 60 min. The cells were pelleted and resuspended in 5 ml of buffer A (25 mM Hepes, pH 7.9, 50 mM KCl, 6% (v/v) glycerol, 5 mM EDTA, 5 mM MgCl₂, 1 mM dithiothreitol, 0.05% Triton X-100). The cells were lysed on ice by sonication and centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant was mixed for 1 h at 4 °C on a rotator with 0.5 to 1 ml of 50% glutathione-Sepharose beads (Amersham Biosciences) that were preswollen in buffer A. After absorption, the beads were collected by centrifugation at 4 °C and washed three times with 1 ml of buffer A. The fusion proteins coupled to the glutathione-Sepharose beads were stored at 4 °C as 50% (v/v) slurry in buffer A. The concentrations and sizes of GST and GST fusion proteins were estimated by SDS-PAGE, using a known quantity of molecular weight standards.

L-[³⁵S]Cysteine- or L-[³⁵S]methionine-labeled proteins were prepared by using TNT reticulocyte lysates (Promega). Approximately 2.5 × 10⁴ to 5 × 10⁴ cpm of ³⁵S-labeled protein was incubated with 300 ng of GST fusion protein immobilized on glutathione-Sepharose beads in 300 µl of buffer A for 1 h at 4 °C on a rotator. Where indicated 1 µM T3, 9-cis-retinoic acid, or 22(R)-hydroxycholesterol was included in the binding reaction mixture. In experiments examining the effects of DNA binding on the interaction between TR and SREBP-1 (see Fig. 6), oligonucleotides (30 ng) containing the ACC T3RE and/or ACC SRE-1 were included in the binding reaction. The beads were collected by centrifugation at 4 °C and washed three times with 1 ml of buffer A. The bound proteins were eluted with SDS gel loading buffer and analyzed by SDS-PAGE followed by storage phosphor autoradiography. ³⁵S-Labeled proteins were analyzed by electrophoresis and storage phosphor autoradiography to ensure that similar levels of input radioactivity of the labeled protein were used in the GST binding assays.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A SRE-1 Located Immediately Downstream of a Strongly Active T3RE in the ACC Gene Augments T3 Regulation of Transcription in a Cell Type-specific Manner-- Previous studies with native thyroid hormone-responsive genes have shown that optimal T3 regulation of transcription requires the presence of one or more accessory factor-binding sites in addition to a T3RE (18-23). These accessory elements are usually located within 100 bp of the T3RE. In previous work analyzing the T3 regulation of ACC promoter 2 in CEH, 5'-deletion analyses failed to detect a positive accessory element upstream of the strongly active T3RE between 101 and 86 bp (16). Deletion analyses also failed to detect TR accessory sequences between +31 and +274 bp. We investigated the possibility that sequences immediately downstream of the ACC T3RE contained a TR accessory element. Sequence comparison analysis of the region between 86 and +31 bp revealed the presence of a 10-bp element (80 and 71 bp) that perfectly matched the SRE-1 identified in the rat, hamster, and frog genes for the low density lipoprotein receptor (37). Transient transfection experiments were performed to determine the effects of the SRE-1 on T3 regulation of ACC promoter 2 in CEH. In hepatocytes transfected with a reporter construct containing ACC sequences between 108 and +274 linked to the CAT gene, T3 stimulated a 14.3-fold increase in CAT activity (Fig. 1B). Mutation of sequences between 79 and 72 bp (SRE-1 mut) in the context of the 108 to +274 ACC fragment caused a 53% decrease in T3 responsiveness. This effect was mediated by a reduction in promoter activity in the presence of T3. These data indicate that T3 regulation of ACC transcription in hepatocytes is enhanced by the presence of a SRE-1 located 5 bp downstream of a strongly active T3RE in the proximal region of ACC promoter 2. 

View larger version (34K): [in this window] [in a new window]   Fig. 1.   A SRE-1 between 80 and 71 bp of the ACC gene enhances T3 regulation of ACC transcription in CEH but not in CEF. A, sequence of the chicken ACC gene between 108 and 66 bp. The hexameric half-sites comprising the T3RE are indicated by arrows. The SRE-1 is boxed. The sequence of a block mutation of the SRE-1 (SRE-1 mut) is shown underneath the SRE-1. B and C, p[ACC108/+274]CAT or a construct containing a block mutation of the SRE-1 (SRE-1 mut) in the context of p[ACC108/+274]CAT was transiently transfected into CEH and CEF as described under "Experimental Procedures." After transfection, the cells were treated with or without T3 for 48 h. The cells were then harvested, extracts were prepared, and CAT assays were performed. Left panels, the constructs used in these experiments. The numbers indicate the 5' and 3' boundaries of ACC DNA in nucleotides relative to the transcription initiation site of promoter 2. Right panels, CAT activity of cells transfected with p[ACC108/+274]CAT and treated with T3 was set at 100, and the other activities were adjusted proportionately. The fold stimulation by T3 was calculated by dividing CAT activity for cells treated with T3 (+T3) by that for cells not treated with T3 (T3). The fold responses were calculated for individual experiments and then averaged. The results are the means ± S.E. of five experiments. The results of experiments performed with CEH and CEF are shown in B and C, respectively. The asterisk in B indicates that the fold stimulation by T3 was significantly lower than that of cells transfected with p[ACC108/+274]CAT (p < 0.05).

T3 regulation of the ACC gene in nonhepatic cells such as CEF is markedly diminished relative to that of CEH (17). Previous studies with other T3-responsive genes have shown that TR accessory proteins play an important role in mediating cell type-dependent differences in T3 regulation of transcription (18-23). Transient transfection experiments were performed with CEF to determine whether the SRE-1 between 80 and 71 bp is involved in mediating differences in T3 responsiveness of the ACC gene between CEH and CEF. In CEF transfected with p[ACC108/+274]CAT, the T3-induced stimulation in CAT activity (2.4-fold) was substantially lower than that observed in CEH (14.3-fold) (Fig. 1, B and C). Mutation of the SRE-1 in the context of p[ACC108/+274]CAT had no effect on T3 responsiveness in CEF (Fig. 1C). These data contrast with those for CEH, demonstrating that mutation of the SRE-1 causes a marked reduction in T3 responsiveness (Fig. 1B). The decreased ability of the SRE-1 to stimulate T3 responsiveness in CEF relative to CEH suggests that this element plays a role in conferring cell type-dependent differences in T3 regulation of ACC transcription. Other sequences within the 108 to +274 fragment also contribute to differences in T3 responsiveness between CEH and CEF, because mutation of the SRE-1 did not eliminate differences in T3 responsiveness between CEH and CEF.

We next asked whether the functional interaction between the ACC SRE-1 and ACC T3RE in CEH required the presence of additional cis-acting sequences. To address this question, we determined whether the ACC SRE-1 could function alone to enhance T3 regulation conferred by the ACC T3RE. CEH were transfected with constructs containing ACC DNA fragments linked to the minimal promoter of the herpes simplex virus TK gene. The TK promoter alone was unresponsive to T3 in CEH (Fig. 2). Appending a DNA fragment containing the ACC T3RE (108 to 82 bp) to TK-CAT caused a 9.6-fold increase in T3 responsiveness (compare fold stimulation by T3 of p[ACC108/82]TKCAT with that of TKCAT). A substantially greater increase in T3 responsiveness was observed (20-fold) when a DNA fragment containing both the T3RE and SRE-1 (108 to 66 bp) was linked to TKCAT. The increase in T3 responsiveness caused by the SRE-1 was due to an increase in promoter activity in the presence of T3. This increase in T3 responsiveness was not due to changes in the spacing between the T3RE and the TK promoter, because T3 responsiveness of a construct containing a block mutation of the SRE-1 (SRE-1 mut) in the context of p[ACC108/66]TKCAT was similar to that of p[ACC108/82]TKCAT. These data indicate that the stimulation of T3-induced ACC transcription by the SRE-1 is mediated by direct interactions between the SRE-1 and T3RE. Appending a DNA fragment containing the SRE-1 alone (84 to 66 bp) to TKCAT had no effect on promoter activity in the absence or presence of T3. This finding indicates that the SRE-1 requires the presence of the T3RE to be transcriptionally active. This conclusion is consistent with results from previous 5'-deletion analyses in the context of p[ACC-4900/+274]CAT demonstrating that deletion of ACC sequences from 84 bp to 59 bp has no effect on ACC transcription in the absence or presence of T3 (16).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   The ACC SRE-1 alone enhances T3 regulation directed by the ACC T3RE. Fragments of the ACC gene containing the T3RE and/or SRE-1 were linked to the minimal TK promoter in TKCAT. CEH were transiently transfected with these constructs and treated with or without T3 as described in the legend of Fig. 1 and under "Experimental Procedures." Left panel, constructs used in these experiments. The numbers indicate the 5' and 3' boundaries of ACC DNA relative to the transcription initiation site of promoter 2. The ACC sequence between 108 and 66 bp is shown in Fig. 1A. Right panel, CAT activity in CEH transfected with p[ACC108/82]TKCAT and treated with T3 was set at 100, and the other activities were adjusted proportionately. The fold stimulation by T3 was calculated as described in the legend to Fig. 1. The results are the means ± S.E. of six experiments. The asterisk indicates that the fold stimulation by T3 for p[ACC108/66]TKCAT was significantly higher than any other construct (p < 0.05).

SREBP-1 Physically Interacts with TR·RXR Heterodimers-- SREs bind a class of basic helix-loop-helix-leucine zipper (bHLH-Zip) factors referred to as sterol regulatory element-binding proteins (SREBPs) (38). SREBPs are synthesized as 125-kDa precursor proteins that are anchored to the endoplasmic reticulum. To become transcriptionally active, precursor SREBP must be translocated to the Golgi, where it encounters two proteases that act in sequence to release the N-terminal segment of SREBP referred to as mature SREBP (39). Mature SREBP is the protein that enters the nucleus and binds the SRE of target genes. In chickens and mammals, SREBPs are expressed from two distinct genes designated as SREBP-1 and SREBP-2 (38, 40). Previous studies employing transgenic animals indicate that SREBP-1 is more effective than SREBP-2 in modulating triacylglycerol synthesis, whereas SREBP-2 is more effective than SREBP-1 in modulating cholesterol synthesis (41, 42).

DR-4-type (half-sites arranged as direct repeats with a 4-bp spacer) T3REs bind TR·RXR heterodimers in a specific spatial arrangement in which RXR contacts the more 5' half-site and TR contacts the more 3' half-site (43). If TR·RXR directly interacts with mature SREBP on the ACC gene, then flipping the orientation of the T3RE may alter the ability of the SRE-1 to enhance T3 regulation of transcription. To investigate this possibility, CEH were transfected with reporter constructs containing the ACC T3RE in the native or flipped orientation. In CEH transfected with p[ACC108/66]TKCAT containing the T3RE in the native orientation, mutation of the SRE-1 inhibited T3 responsiveness by 56% (Fig. 3). In contrast, mutation of the SRE-1 had no effect on T3 responsiveness when the orientation of the T3RE in p[ACC108/66]TKCAT was flipped (compare fold stimulation by T3 of p[ACC108/66]TKCAT containing T3RE flip with that of p[ACC108/66]TKCAT containing T3RE flip and SRE-1 mut). These data suggest that SREBP interacts with TR·RXR in an orientation-dependent manner to enhance T3 regulation of the ACC gene.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   The ability of the ACC SRE-1 to enhance T3 responsiveness requires the presence of the ACC T3RE in its native orientation. Fragments of the ACC gene containing the T3RE in the native or flipped orientation were linked to the minimal TK promoter in TKCAT. CEH were transiently transfected with these constructs and treated with or without T3 as described in the legend of Fig. 1 and under "Experimental Procedures." Left panel, constructs used in these experiments. The numbers indicate the 5' and 3' boundaries of ACC DNA relative to the transcription initiation site of promoter 2. The orientation of the half-sites comprising the T3RE are indicated by the arrows. The ACC sequence between 108 and 66 bp is shown in Fig. 1A. Right panel, CAT activity in CEH transfected with p[ACC108/66]TKCAT and treated with T3 was set at 100, and the other activities were adjusted proportionately. The fold stimulation by T3 was calculated as described in the legend to Fig. 1. The results are the means ± S.E. of five experiments. The asterisk indicates that the fold stimulation by T3 for p[ACC108/66]TKCAT (T3RE in the native orientation) was significantly higher than that of p[ACC108/66 SRE-1mut]TKCAT (T3RE in the native orientation) (p < 0.05).

The close proximity of the SRE-1 and T3RE on the ACC gene coupled with functional data indicating a direct and orientation-specific interaction between these regulatory elements (Figs. 1-3) led us to hypothesize that mature SREBP physically interacted with TR·RXR heterodimers to augment T3 regulation of ACC transcription. To investigate this hypothesis, we determined using a pull-down assay whether the mature form of chicken SREBP-1 physically interacted with TR and RXR. In our initial experiments, we tested the ability of in vitro synthesized TR and RXR to bind a bacterially expressed fusion protein containing GST linked to the mature form of SREBP-1. ³⁵S-Labeled TR bound GST-SREBP-1, and the presence of T3 enhanced this interaction (Fig. 4). Inclusion of unlabeled RXR in the binding reaction had no effect on the interaction of ³⁵S-labeled TR with GST-SREBP-1 (data not shown). No interaction was observed between ³⁵S-labeled TR and GST. In contrast to the results for TR, little or no interaction was observed between ³⁵S-labeled RXR and GST-SREBP-1 (Fig. 4). The lack of interaction between RXR and SREBP-1 was confirmed by pull-down experiments employing GST-RXR as the bait and ³⁵S-labeled SREBP-1 synthesized in vitro (data not shown). We also investigated whether SREBP-1 interacted with LXR, because LXR·RXR complexes bind the ACC T3RE in CEH and are postulated to play a role in mediating the T3-induced increase in ACC promoter activity (16). No interaction was observed between ³⁵S-labeled LXR and GST-SREBP-1 (Fig. 4). These data indicate that SREBP-1 selectively interacts with TR.

View larger version (58K): [in this window] [in a new window]   Fig. 4.   The mature form of SREBP-1 specifically binds TR in a T3-regulated manner. Bacterially produced GST or GST linked to the mature form of chicken SREBP-1 (GST-SREBP-1) was immobilized on glutathione-Sepharose beads. These preparations were then incubated with in vitro translated and 35S-labeled TR, RXR, or LXR as described under "Experimental Procedures." The incubations were performed in the absence or the presence of ligand for TR (1 µM T3), RXR (1 µM 9-cis-retinoic acid), or LXR (1 µM 22(R)-hydroxycholesterol) as indicated. After the matrix was extensively washed, the labeled proteins retained on the beads were eluted, resolved by SDS-PAGE, and visualized by storage phosphor autoradiography together with 10% of the total radiolabeled receptor input used in each binding reaction. This experiment was repeated three times with similar results.

We next set out to identify the motifs in TR that interacted with SREBP-1. Pull-down experiments were performed using GST-SREBP-1 as the bait and various truncations of TR labeled with ³⁵S in vitro. Deletion of the first 20 or 50 amino acids from the N terminus of TR had little or no effect on the binding of TR to GST-SREBP-1 (Fig. 5A). When deletion of the N terminus of TR was extended to amino acid 120, binding of TR to GST-SREBP-1 was abolished. To further analyze the interaction between TR and SREBP-1, TR polypeptides containing amino acids 1-157, amino acids 1-118, and amino acids 51-157 were tested for their ability to interact with GST-SREBP-1. A strong interaction was observed between GST-SREBP-1 and a TR polypeptide containing amino acids 1-157. Weaker interactions were observed between GST-SREBP-1 and TR polypeptides containing amino acids 1-118 and amino acids 51-157. Collectively, these data suggest that a TR region containing the DNA-binding domain plus flanking sequences (amino acids 21-157) is required for optimal binding to SREBP-1. Interestingly, interactions between GST-SREBP-1 and TR truncations lacking the ligand-binding domain were not enhanced by the presence of T3. This observation is consistent with the scenario that T3 binding to the ligand-binding domain of TR causes a conformational change that enhances the ability of the N-terminal region of TR to interact with SREBP-1.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   A region of TR containing the DNA-binding domain plus flanking sequences is required for optimal binding to SREBP-1, and a region of SREBP-1 containing the bHLH-Zip domain is required for optimal binding to TR. Wild-type and truncated forms of TR or mature SREBP-1 were translated in vitro in the presence of L-[35S]cysteine- or L-[35S]methionine. 35S-Labeled proteins were then incubated with GST, GST-SREBP-1, or GST-TR bound to glutathione-Sepharose beads as described in the legend of Fig. 4 and under "Experimental Procedures." The incubations were performed in the absence and presence of 1 µM T3. After the matrix was extensively washed, the labeled proteins retained on the beads were eluted, resolved by SDS-PAGE, and visualized by storage phosphor autoradiography together with 10% of the total radiolabeled protein input used in each binding reaction. A, results of experiments using GST-SREBP-1 as the bait protein and full-length and truncated versions of 35S-labeled TR as test proteins. A schematic representation of chicken TR is shown in the upper panel. The DNA-binding domain (DBD) and ligand-binding domain (LBD) are indicated. Embedded within the LBD of TR is a heptad repeat region involved in dimerization. A ligand-induced change in the conformation of the LBD is responsible for providing an interface for interaction with coactivator proteins. B, results of experiments using GST-TR as the bait protein and full-length and truncated versions of mature SREBP-1 as test proteins. A schematic representation of the mature form of chicken SREBP-1 is shown in the upper panel. A region of SREBP-1 enriched in acidic amino acids (amino acids 1-51) mediates interactions with coactivator proteins (55, 56). The bHLH-Zip motif between amino acids 300 and 370 is required for DNA binding (38). The region between amino acids 51 and 213 is enriched in proline and serine residues. These experiments were repeated twice with similar results.

To map the motifs in SREBP-1 that interacted with TR, pull-down analyses were performed using a GST fusion protein containing full-length TR as a bait and various truncations of mature SREBP-1 labeled with ³⁵S in vitro. Consistent with the results of experiments employing GST-SREBP-1 as the bait protein, the full-length, mature form of SREBP-1 (amino acids 1-464) interacted with GST-TR (Fig. 5B). N-terminal deletions of SREBP-1 to amino acid 78, 108, 238, or 300 had little or no effect on the binding SREBP-1 to GST-TR. A C-terminal deletion of SREBP-1 to amino acid 389 also had no effect on the binding of SREBP-1 to GST-TR. In contrast, C-terminal deletions of SREBP-1 to amino acids 349 and 266 abolished the binding of SREBP-1 to GST-TR. These data indicate that a SREBP-1 region containing the bHLH-Zip domain (amino acids 300-389) is required for binding to TR. In contrast to results of experiments analyzing interactions between GST-SREBP-1 and ³⁵S-labeled TR, interactions between GST-TR and ³⁵S-labeled SREBP-1 proteins were not affected by the presence of T3. The latter observation suggests that appending GST to the N terminus of TR blocks ligand-induced conformational changes that facilitate interactions between TR and SREBP-1. A similar observation has been reported for interactions between TR and the homeodomain protein, PBX1 (19).

The TR region between amino acids 21-157 and the SREBP-1 region between amino acids 300 and 389 overlap with motifs mediating DNA binding activity. This prompted us to examine whether the binding of TR and SREBP-1 to their DNA response elements altered the interaction between these proteins. Pull-down assays were carried out with GST-SREBP-1 and in vitro synthesized TR and RXR in the absence or presence of oligonucleotides containing the ACC SRE-1 alone (T3REmut-SRE-1), the ACC T3RE alone (T3RE-SRE-1mut), or both the ACC T3RE and ACC SRE-1 (T3RE-SRE-1). None of the oligonucleotides had an effect on the interaction between GST-SREBP-1 and TR in the absence and presence of T3 (Fig. 6). These results suggest that DNA does not modulate the interaction between SREBP-1 and TR.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   The interaction between TR and SREBP-1 is not altered by the binding of these proteins to DNA. GST or GST-SREBP-1 immobilized on glutathione-Sepharose beads was incubated with in vitro translated and 35S-labeled TR and an equimolar concentration of unlabeled RXR in the absence and presence of T3. Oligonucleotides (30 ng) containing the ACC T3RE and/or SRE-1 were included in some incubations as indicated. After the matrix was extensively washed, the labeled proteins retained on the beads were eluted, resolved by SDS-PAGE, and visualized by storage phosphor autoradiography together with 10% of the total radiolabeled protein input used in each binding reaction. T3RE-SRE-1 (ACC sequences between 108 and 66 bp) contains both the ACC T3RE and SRE-1. T3RE-SRE-1mut contains a mutation of the SRE-1 in the context of the 108 to 66 bp ACC fragment. T3REmut-SRE-1 contains a mutation of the T3RE in the context of the 108 to 66 bp ACC fragment. The sequence of T3RE-SRE-1 and T3RE-SRE-1mut is shown in Fig. 1A. The sequence of T3REmut-SRE-1 is 5'-AGGTGGTTGAATGGAGGTAAAGACTCGCATCACACCACCGCGG-3' (the mutated bases are underlined). Additional experimental details are described in the legend of Fig. 4 and under "Experimental Procedures." This experiment was repeated twice with similar results.

SREBP-1 Forms a Tetrameric Complex with TR·RXR Heterodimers on a ACC DNA Fragment Containing Sequences between 108 and 66 bp-- The finding that SREBP-1 physically interacted with TR·RXR when both complexes were bound to DNA raised the possibility that SREBP-1 enhanced T3-induced ACC transcription by forming a tetrameric complex with TR·RXR on the ACC gene. To obtain evidence supporting this proposal, gel mobility shift experiments were performed using in vitro translated TR, RXR, and mature SREBP-1 and an ACC probe containing the T3RE and SRE-1 (T3RE-SRE-1, 108 to 66 bp). Incubation of the T3RE-SRE-1 probe with TR, RXR, and SREBP-1 resulted in the formation of three complexes (Fig. 7A). The results of supershift analyses with antibodies against TR, RXR, and SREBP-1 indicated that the top band contained a SREBP-1·TR·RXR complex, the middle band contained SREBP-1 homodimers, and the bottom band contained TR·RXR heterodimers. To determine whether the top band contained a SREBP-1/TR·RXR trimer or a SREBP-1·SREBP-1/TR·RXR tetramer, gel mobility shift experiments were performed with ACC probes (108 to 66 bp) containing mutations in either the upstream half-site or downstream half-site of the ACC SRE-1. Because binding of SREBP dimers to DNA requires the presence of both SRE-1 half-sites (37), mutation of either ACC SRE-1 half-site would likely prevent the binding of a tetrameric complex. Mutation of either SRE-1 half-site in the 108 to 66 bp ACC probe abolished the binding of the middle and top bands (data not shown). This observation suggests that the top band is a SREBP-1·SREBP-1/TR·RXR tetrameric complex.

View larger version (77K): [in this window] [in a new window]   Fig. 7.   SREBP-1 forms a tetrameric complex with TR·RXR heterodimers on a DNA probe containing ACC sequences between 108 and 66 bp. Gel mobility shift assay were performed as described under "Experimental Procedures" using 32P-labeled DNA probes and in vitro synthesized TR, RXR, and mature SREBP-1. A, a DNA probe containing ACC sequences between 108 and 66 bp (T3RE-SRE-1) was incubated with TR and RXR (1:1 molar ratio) in the presence of mature SREBP-1. In some incubations, in vitro translated proteins were incubated with antibodies against TR, RXR, SREBP-1, or COUP-TF prior to the addition of the probe. B, the T3RE-SRE-1 probe was incubated with increasing amounts of TR and RXR (1:1 molar ratio, 0-2 µl) in the presence of a fixed level of mature SREBP-1 (0 or 2 µl). Unprogrammed lysate was added to bring the total amount of lysate to 4 µl/reaction. T3 (1 µM) was added to binding reactions as indicated. This experiment was repeated three times with similar results. C, experiments were performed using a ACC probe (108 to 66 bp) containing a mutation of the T3RE (T3REmut-SRE-1). The incubation conditions were the same as those described for B. This experiment was repeated twice with similar results. D, ACC DNA fragments (108 to 66 bp) containing the T3RE in the native orientation (T3RE-SRE-1) or the flipped orientation (T3RE Flip-SRE-1) were labeled with 32P to the same specific activity. These ACC probes were incubated with increasing amounts of mature SREBP-1 (0-2 µl) in the presence of a fixed level of TR·RXR (0 or 2 µl). Unprogrammed lysate was added to bring the total amount of lysate to 4 µl/reaction. Similar amounts of radiolabeled T3RE-SRE-1 and T3RE Flip-SRE-1 were used in the binding reactions. This experiment was repeated twice with similar results. The positions of the tetrameric, heterodimeric, and homodimeric protein complexes are indicated by arrows. Nonspecific complexes are indicated by the asterisks. The sequence of the ACC probes are shown in Fig. 1A and in the legend to Fig. 6.

In Fig. 7B, the T3RE-SRE-1 probe was incubated with increasing amounts of TR·RXR in the presence of a fixed concentration of SREBP-1. Total SREBP-1 binding activity (signal of the SREBP-1·SREBP-1/TR·RXR complex plus the SREBP-1 homodimeric complex) was higher in the presence of TR·RXR than in the absence of TR·RXR. The extent of the increase in total SREBP-1 binding activity caused by TR·RXR was greater in the presence of T3 than in the absence of T3. In contrast, total TR·RXR binding activity (signal of the SREBP-1·SREBP-1/TR·RXR complex plus the TR·RXR heterodimeric complex) was not affected by the presence of SREBP-1. These data indicate that SREBP-1 can form a tetrameric complex with TR·RXR on the ACC gene and that the formation of this complex enhances the DNA binding activity of SREBP-1 but has no effect on the DNA binding activity of TR·RXR.

To determine whether tetrameric complex formation on the T3RE-SRE-1 probe and the concomitant increase in SREBP-1 DNA binding activity required the binding of TR·RXR to the T3RE, gel mobility shift experiments were performed using a probe containing a mutation of the T3RE in the context of the 108 to 66 bp ACC fragment (T3REmut-SRE-1). Incubation of this probe with in vitro synthesized SREBP-1 resulted in the formation of a single protein-DNA complex containing SREBP-1 homodimers (Fig. 7B). The addition of increasing amounts of TR·RXR to the binding reaction had no effect on the pattern or intensity of protein binding. These data indicate that tetrameric complex formation on the 108 to 66 bp ACC fragment requires the binding of TR·RXR to the T3RE. The results of experiments employing a probe containing a block mutation of the SRE-1 in the context of the 108 to 66 bp ACC fragment (T3RE-SRE-1mut) indicate that tetrameric complex formation also requires the binding of SREBP-1 to the SRE-1 (data not shown).

The data in Fig. 3 demonstrated that the stimulation of T3-induced transcription by the ACC SRE-1 was dependent on the orientation of the ACC T3RE. The effect of T3RE orientation on SRE-1 activity may be mediated by changes in the ability of SREBP to form a tetrameric complex with TR·RXR on DNA. To investigate this possibility, gel mobility shift experiments were performed using in vitro synthesized proteins and ACC probes containing the T3RE in the native and flipped orientation. ACC fragments (108 to 66 bp) containing the T3RE in the native orientation (T3RE-SRE-1) or flipped orientation (T3RE Flip-SRE-1) were labeled with ³²P to the same specific activity. Equal amounts of each probe were incubated with a fixed concentration of TR·RXR in the presence of T3 and increasing concentrations of SREBP-1. At each concentration of SREBP-1, the abundance of tetrameric complexes (SREBP-1·SREBP-1/TR·RXR) was higher on the T3RE-SRE-1 probe than on the T3RE Flip-SRE-1 probe (Fig. 7C). This observation provides support for a role of tetrameric complex formation in mediating the stimulatory effects of the SRE-1 on T3-induced ACC transcription.

The TR·RXR-induced increase in SREBP-1 binding to the ACC SRE-1 (Fig. 7) may be due to an increase in the affinity of SREBP-1 for the SRE-1 and/or a decrease in the dissociation rate of SREBP-1 from the SRE-1. To determine whether TR·RXR altered the dissociation rate of SREBP-1 from the SRE-1, protein complexes prebound to the labeled T3RE-SRE-1 probe were incubated for various times with a 1,000-fold molar excess of an unlabeled competitor DNA containing the ACC SRE-1 alone (T3REmut-SRE-1). The dissociation rate of SREBP-1 in the SREBP-1·SREBP-1/TR·RXR tetrameric complex was decreased by 61% compared with the dissociation rate of the SREBP-1 homodimeric complex (Fig. 8A). These data indicate that tetrameric complex formation stabilizes the binding of SREBP-1 to the ACC SRE-1. This phenomenon accounts for at least part of the TR·RXR-induced increase in SREBP-1 binding to the T3RE-SRE-1 probe.

View larger version (44K): [in this window] [in a new window]   Fig. 8.   The formation of the SREBP-1·SREBP-1/TR·RXR tetrameric complex stabilizes the binding of SREBP-1 to the ACC SRE-1. In vitro synthesized TR, RXR, and mature SREBP-1 were bound to 32P-labeled T3RE-SRE-1 as described under "Experimental Procedures." The binding mixture was then incubated with a 1,000-fold molar excess of unlabeled T3REmut-SRE-1 (A) or T3RE-SRE-1mut (B). At various time points after the addition of competitor DNA, the aliquots of the binding mixture were subjected to nondenaturing gel electrophoresis. Left panel, data from a representative experiment. Right panel, plot of log protein binding activity versus time. The signals for protein-DNA complexes containing SREBP-1·SREBP-1, TR·RXR, or SREBP-1·SREBP-1/TR·RXR were quantitated using a PhosphorImager. The protein binding activities at 0 min were set at 100, and the activities at other time points were adjusted proportionately. The values are the means ± S.E. of three experiments. The positive standard error bar represents the S.E. for the binding of the SREBP-1·SREBP-1/TR·RXR tetrameric complex (), and the negative standard error bar represents the S.E. for SREBP-1·SREBP-1 () or TR·RXR () dimeric complexes. The Kd for SREBP-1 and TR·RXR in dimeric or tetrameric complexes was estimated from the slopes of the straight lines. In A, the t1/2 of SREBP-1 in the SREBP-1·SREBP-1/TR·RXR tetrameric complex was compared with that of the SREBP-1 homodimeric complex. In B, the t1/2 of TR·RXR in the SREBP-1·SREBP-1/TR·RXR tetrameric complex was compared with that of the TR·RXR heterodimeric complex. The sequence of T3RE-SRE-1mut and T3REmut-SRE-1 is shown in Fig. 1A and in the legend to Fig. 6, respectively.

We also investigated whether SREBP-1 binding to the ACC SRE-1 stabilized the binding of TR·RXR to the ACC T3RE. In these experiments, protein complexes prebound to the labeled T3RE-SRE-1 probe were incubated for various times with an unlabeled competitor DNA containing the ACC T3RE alone (T3RE-SREmut). The dissociation rate of TR·RXR in the SREBP-1·SREBP-1/TR·RXR tetrameric complex was not different from the dissociation rate of the TR·RXR heterodimeric complex (Fig. 8B). This finding is consistent with the results in Fig. 7 demonstrating a lack of effect of tetrameric complex formation on the binding of TR·RXR to the ACC T3RE.

SREBP Expression in CEF Is Decreased Relative to That Observed in CEH-- The decreased ability of the ACC SRE-1 to enhance T3 regulation of ACC transcription in CEF relative to CEH may be caused by a reduction in the level of mature SREBP in the former cell type. To investigate this possibility, the concentration of mature SREBP-1 and SREBP-2 was measured in CEH and CEF using Western blot analysis. Mature SREBP-1 and SREBP-2 levels were lower in CEF than in CEH (Fig. 9A). The extent of the reduction in mature SREBP-1 and SREBP-2 in CEF was 68 and 65%, respectively. The concentration of precursor SREBP-1 and SREBP-2 was also lower in CEF than in CEH. SREBP-1 and SREBP-2 mRNA levels were also measured in CEF and CEH using a RNase protection assay. The amounts of SREBP-1 and SREBP-2 mRNA were lower in CEF (64 and 72%, respectively) than in CEH (Fig. 9B). These data support the proposal that alterations in SREBP-1 and SREBP-2 expression mediate the decrease in SRE-1 activity in CEF.

View larger version (22K): [in this window] [in a new window]   Fig. 9.   The expression of SREBP-1 and SREBP-2 is decreased in CEF relative to CEH. CEF and CEH were incubated for 24 h in serum-free medium containing insulin, corticosterone, and T3. The cellular extracts or total RNA was prepared as described under "Experimental Procedures." A, precursor SREBPs and mature SREBPs were measured in nuclear extracts and membrane fractions, respectively, by Western blot analysis. B, the abundance of mRNA for SREBP-1, SREBP-2, and -actin was measured using a RNase protection assay. All of the SREBP-1 mRNAs in CEF and CEH are derived from a single promoter, because 5'-RACE analysis and RNase protection assays using probes from the 5' end of the SREBP-1 cDNA failed to detect 5'-heterogeneity in SREBP-1 transcripts.2 The experimental details for Western blot and RNase protection analyses are described under "Experimental Procedures." These data are representative of three experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous studies have shown that SREBPs play an important role in the regulation of lipogenic genes. In addition to the ACC gene, functional SREs have been identified in the genes for fatty acid synthase (44), ATP-citrate lyase (45), glycerol-3-phosphate acyltransferase (46), and S14 (47). In rat hepatocytes in culture, SREBP mediates the stimulatory effects of insulin and the inhibitory effects of long chain polyunsaturated fatty acids on lipogenic gene transcription (47-52). The results of the present study demonstrate that SREBP-1 also plays a role in mediating the effects of T3 on lipogenic gene transcription. A SRE-1 in promoter 2 of the ACC gene enhances the ability of T3 to activate transcription in CEH.

The data from transfection (Figs. 2 and 3) and protein binding assays (Figs. 4-6) suggest that the stimulatory effect of the SRE-1 on T3-induced ACC transcription is mediated by a direct and T3-inducible interaction between SREBP and TR. We postulate that this interaction facilitates the formation of a SREBP·SREBP/TR·RXR tetrameric complex on the ACC gene. In support of this proposal, in vitro synthesized TR, RXR, and SREBP-1 formed a tetrameric complex on a DNA probe containing the ACC T3RE and SRE-1, and the presence of T3 enhanced the formation of this complex (Fig. 7). Complex formation between TR·RXR and SREBP-1·SREBP-1 stabilized the binding of SREBP-1 to the SRE-1 (Fig. 8). This phenomenon probably accounts for at least part of the increase in ACC transcription caused by tetrameric complex formation. These findings define a new mechanism through which an accessory transcription factor modulates TR activity. Complex formation between TR·RXR and SREBP-1·SREBP-1 may also facilitate the recruitment of coactivator complexes to the ACC gene. Previous studies have shown that both SREBP-1 and ligand-bound TR bind coactivator proteins (53-56). Coactivators regulate transcription by directly interacting with the basal transcriptional machinery, by modulating the interaction between the enhancer-binding protein and the basal transcription machinery, and by modifying chromatin structure.

The ACC T3RE activates transcription not only in the presence of T3 but also in the absence of T3 (16). The increase in transcription conferred by the ACC T3RE in the absence of T3 is mediated by the binding of protein complexes containing LXR·RXR heterodimers. Thus, the ACC T3RE functions as a LXR response element in the absence of T3. The results from transfection analyses indicate that the SRE-1 has no effect on ACC LXR response element activity in the absence of T3 (Figs. 1 and 2). This finding is congruent with the observation that LXR and RXR lack the ability to bind SREBP-1 in a pull-down assay (Fig. 4).

Data from the present study indicate that SREBP must interact with TR·RXR to be effective in regulating ACC transcription in CEH (Fig. 2). Previous studies analyzing the regulation of other SREBP-responsive genes have shown that the ability of SREBP to activate transcription is also dependent on interactions between SREBP and transcription factors that bind DNA elements adjacent to the SRE. For example, SREBP-mediated activation of the genes for farnesyl diphosphate synthase (57), 3-hydroxy-3-methylglutaryl coenzyme A synthase (58), fatty acid synthase (44), ATP-citrate lyase (45), glycerol-3-phosphate acyltransferase (46), and S14 (21) is dependent on a functional interaction between SREBP and nuclear factor-Y (NF-Y) on contiguous DNA-binding sites. In the case of the farnesyl diphosphate synthase gene, the functional interaction between SREBP-1 and NF-Y is associated with the formation of a SREBP-1·NF-Y complex and a concomitant increase in SREBP-1 binding to the farnesyl diphosphate synthase promoter (57). Other studies have shown that SREBP activation of the low density lipoprotein receptor gene is dependent on a functional interaction between SREBP bound to a SRE-1 and Sp1 bound to an adjacent DNA element (59). This interaction between SREBP and Sp1 is associated with an increase in binding of Sp1 to the low density lipoprotein receptor promoter. Further evidence that SREBP directly interacts with NF-Y and Sp1 is provided by the observation that SREBP-1 physically interacts with NF-Y and Sp1 in protein binding assays (58, 60). These findings in combination with the results of the present study suggest that interaction of SREBP with adjacent enhancer-binding proteins is a common mechanism through which SREBP activates gene transcription.

Recently, Jump et al. (21) have reported that SREBP-1c acts in concert with NF-Y to enhance T3 regulation of S14 transcription in rat hepatocytes. An unique feature of this interaction is that it involves DNA elements that are separated by more than 2.3 kb. SREBP-1c (139/131 bp) and NF-Y (104/99 bp) bind DNA elements in the proximal region of the S14 promoter, whereas TR·RXR heterodimers bind a T3-responsive region (2.8/2.5 kb) in the far upstream region of the S14 promoter. The wide separation between the SREBP-1c/NF-Y binding sites and the T3-responsive region suggests that SREBP-1c/NF-Y does not directly interact with TR·RXR on the S14 gene. The interaction between SREBP-1c/NF-Y and TR·RXR on the S14 gene may instead be achieved by intermediary proteins that function as bridges between SREBP-1c/NF-Y and TR·RXR. Thus, the mechanism mediating the interaction between SREBP-1c/NF-Y and TR·RXR on the rat S14 gene appears to be different from the mechanism mediating the interaction between SREBP-1 and TR·RXR on the chicken ACC gene.

In addition to SREBP, other DNA-binding proteins have been shown to modulate TR activity on native genes. For example, heterodimeric complexes containing the homeodomain proteins, PBX and MEIS1, interact with TR·RXR on the malic enzyme promoter to enhance T3 regulation of transcription in CEH (19). In cardiac muscle cells, binding of myocyte-specific enhancer factor 2 to the -cardiac myosin heavy chain gene potentiates the ability of TR bound at an adjacent T3RE to activate transcription in the presence of T3 (23). Protein binding studies indicate that both PBX and myocyte-specific enhancer factor 2 interact with sequences in the A/B region and the DNA-binding domain of TR (19, 23). The results of the present study demonstrate that SREBP-1 also binds the A/B region and DNA-binding domain of TR (Fig. 5). Together, these observations suggest that accessory DNA-binding proteins modulate TR activity by interacting with a specific region of TR. DNA-binding proteins that modulate SREBP activity also interact with a specific region of SREBP. NF-Y (58), Sp1 (60), and TR (Fig. 5) bind the bHLH-Zip domain of SREBP-1. Interestingly, the TR and SREBP-1 regions that contact accessory DNA-binding proteins are distinct from those that interact with coactivator proteins. Coactivators of TR such as CBP/p300, SRC-1/NCoA-1, GRIP1/NCoA-2, and the TRAP/ARC/DRIP complex interact with the ligand-binding domain of TR (53, 54, 61). Coactivators of SREBP-1 such as CBP/p300 and the TRAP/ARC/DRIP complex interact with a N-terminal acidic region in SREBP-1 (55, 56). The observation that coactivators bind TR and SREBP at sites that are distinct from those that bind accessory DNA-binding proteins supports the proposal that complex formation between TR·RXR and SREBP-1·SREBP-1 facilitates the recruitment of coactivators to the ACC gene. Because both TR and SREBP-1 contain coactivator binding sites, we postulate that the TR·RXR/SREBP-1·SREBP-1 tetrameric complex interacts with multiple coactivator proteins, multiple regions within a single coactivator protein, or multiple subunits within a single coactivator complex.

Promoter 2 of the rat ACC gene contains two closely spaced SREs that synergistically interact with an adjacent Sp1-binding site to activate ACC transcription during conditions of sterol depletion (62). In contrast to promoter 2 of the chicken ACC gene, a DNA element resembling a T3RE is not present in the region flanking the SREs in rat ACC promoter. This observation is consistent with results from reverse transcriptase-PCR analyses demonstrating that ACC promoter 2 in rat hepatocytes is not responsive to thyroid hormone status (63). Thus, the role of SREBP in regulating ACC promoter 2 varies depending on the class of animals. This phenomenon may reflect a fundamental difference between avians and mammals in the mechanism by which nutrients and hormones regulate ACC expression in liver.

The results of the present study are the first to suggest that SREBP plays a role in the tissue-specific regulation of T3 action. This supposition is based on the finding that mutation of the SRE-1 inhibits T3 regulation of ACC transcription in CEH but has no effect on T3 regulation in CEF (Fig. 1). The data from Western blot analyses and RNase protection assays (Fig. 9) suggest that alterations in expression of SREBP-1 and SREBP-2 contribute to the difference in SRE-1 activity between CEH and CEF. In intact animals, SREBP-1 and SREBP-2 are expressed in a wide range of tissues, with SREBP-1 being expressed at a substantially higher level in tissues in which lipogenesis is regulated by T3 (i.e. liver) (28, 40, 64, 65). Thus, alterations in SREBP-1 expression likely play a role in mediating tissue-dependent differences in T3 responsiveness of the ACC gene in vivo.

Another lipogenic gene that is activated by T3 in a hepatocyte-specific manner is malic enzyme. In CEH, T3 regulation of malic enzyme transcription is mediated by six T3REs, five of which are clustered in a 109-bp region (3878/3769 bp) referred to as a T3 response unit (24). Flanking the malic enzyme T3 response unit are five accessory elements that play an important role in conferring enhanced T3 responsiveness in CEH relative to CEF (18-20). Interestingly, these accessory elements bind proteins that are distinct from SREBP. Differences between the malic enzyme gene and the ACC gene in the complexity of the T3-responsive region and the nature of the proteins that bind T3 accessory elements suggest that there are gene-specific differences in the molecular mechanism mediating cell type-dependent differences in T3 responsiveness. The reason for the different mechanisms is unclear. One possibility is that the physiological role of malic enzyme does not completely overlap with that of ACC. Malic enzyme furnishes NADPH for fatty acid synthesis, hydroxylation of xenobiotics, and reduction of glutathione, whereas ACC functions exclusively in fatty acid synthesis. Separate regulatory mechanisms may have evolved for ACC and malic enzyme because of subtle differences in the physiological roles of these enzymes.

In a previous work, we have shown that insulin, glucagon, medium chain fatty acids (MCFA), and long chain polyunsaturated fatty acids (PUFA) regulate the ability of T3 to activate ACC transcription in CEH (4, 66). Insulin accelerates the increase in ACC transcription caused by T3, whereas MCFA, PUFA, and glucagon inhibit the effects of T3 on ACC transcription. The observation that the effects of insulin, MCFA, PUFA, and glucagon on ACC transcription are dependent on the presence of T3 coupled with the finding that TR interacts with SREBP on the ACC gene raises the possibility that insulin, MCFA, PUFA, and glucagon regulate ACC transcription by modulating SREBP activity. In rat hepatocyte cultures, insulin increases and PUFA and glucagon decrease the concentration of mature SREBP-1 protein and SREBP-1c mRNA (47, 48, 50-52). Future studies will investigate the role of SREBP in mediating the effects of insulin, MCFA, PUFA, and glucagon on T3 action in avian hepatocytes.

	ACKNOWLEDGEMENT

We thank Dr. Timothy Osborne for providing the cDNA for human SREBP-1a.

	FOOTNOTES

^* This work was supported by Established Investigator Award 9940007N from the American Heart Association and by Grant 2001-35206-11133 from the Cooperative State Research Service/United States Department of Agriculture.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.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Pharmacology, P.O. Box 9142, West Virginia University, Morgantown, WV 26506-9142. Tel.: 304-293-7751; Fax: 304-293-6846; E-mail: fbhillgartner@hsc.wvu.edu.

Published, JBC Papers in Press, March 20, 2002, DOI 10.1074/jbc.M111771200

² Y. Zhang and F. B. Hillgartner, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: ACC, acetyl-CoA carboxylase; CEH, chick embryo hepatocyte(s); CEF, chick embryo fibroblast(s); SRE, sterol regulatory element; SREBP, sterol regulatory element-binding protein; CAT, chloramphenicol acetyltransferase; LXR, liver X receptor; RXR, retinoid X receptor; NF-Y, nuclear factor-Y; TK, thymidine kinase; GST, glutathione S-transferase; T3, 3,5,3'-triiodothyronine; TR, nuclear T3 receptor; T3RE, T3 response element; bHLH-Zip, basic helix-loop-helix-leucine zipper; MCFA, medium chain fatty acids; PUFA, polyunsaturated fatty acids; bp, base pair(s); S14, Spot 14..

	REFERENCES

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