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J. Biol. Chem., Vol. 282, Issue 8, 5453-5467, February 23, 2007
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From the Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720
Received for publication, November 14, 2006 , and in revised form, December 19, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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By using 3T3-L1 adipocytes, we originally reported that the -65 E-box present in the FAS promoter, which binds the ubiquitous upstream stimulatory factors (USF-1 and USF-2), mediates insulin activation of the FAS promoter (13, 14). However, by generating mice transgenic for various 5'-deletion and mutant FAS promoter-chloramphenicol acetyltransferase constructs, we found that the -131 FAS promoter construct, containing the -65 E-box, is not sufficient for feeding/insulin-mediated activation of the FAS promoter in vivo. Instead, a region from -278 to -131, containing the -150 sterol regulatory element (SRE), as well as a more upstream region from -444 to -278, containing an additional E-box at -332, are required for high level activation of the FAS promoter by feeding/insulin (15, 16). Furthermore, both the E-box at -65 and the SRE at -150 are required for transcriptional activation of FAS in vivo, as the induction of FAS transcription during fasting/refeeding was abolished in mice transgenic for mutations at these two sites (16).
USFs belong to the class C bHLH/LZ transcription factor family and stimulate transcription by binding to E-boxes (5'-CANNTG-3') as USF-1/USF-2 heterodimers (17, 18). Sterol regulatory element-binding proteins (SREBPs) also belong to the bHLH/LZ family and stimulate transcription by binding to SREs present in the promoters of their target genes (19–23). However, because of an atypical tyrosine residue, which replaces a conserved arginine present in the basic regions of this family of transcription factors, SREBPs can also bind to E-boxes, at least in vitro (24, 25). Nevertheless, we have clearly shown by chromatin immunoprecipitation (ChIP) in transgenic mice that SREBP-1c functions through the -150 SRE and that USFs function through the -65 and -332 E-boxes in vivo (16). Gain-of-function studies using mice doubly transgenic for a truncated active form of SREBP-1a and various FAS-promoter reporter constructs indicate that SREBP-1a, even when overexpressed during fasting, cannot activate FAS transcription through the -65 E-box in a -131 FAS promoter construct (26). Regardless, loss-of-function studies in SREBP-1-/- and USF-1-/- knock-out mice clearly demonstrated that these transcription factors are critical in the regulation of FAS transcription (27–29).
So far, two coregulators for SREBP-1a binding and function have been described. These include Sp1 and NF-Y, both of which seem to be important for mediating a transcriptional response to cellular sterol depletion. For example, SREBP-1a was shown to cooperate with Sp1 on adjacent sites to regulate the FAS (30), low density lipoprotein receptor (31), and acetyl-CoA synthetase-1 genes (32), although a direct physical interaction between SREBP-1a and Sp1 in solution has not been reported. Given the apparent requirements for accessory factors in mediating a maximal response of target genes to SREBP-1a during sterol depletion, the identification of an analogous coregulator for SREBP-1c, the isoform specifically induced during feeding/insulin (15, 33), in mediating the lipogenic response would be an important finding, especially because SREBP-1c is an even weaker transcription factor than SREBP-1a on its own (34). The fact that SREBP-1c only modestly activates the FAS promoter in vitro (26), but dramatically activates FAS transcription in vivo (26, 34, 35), implicates the involvement of other factors that may be absent in vitro.
In this study, by using a combination of tandem affinity purification (TAP) and coimmunoprecipitation, we demonstrate that USF and SREBP-1 directly interact in vivo. We have mapped the interaction domains to the bHLH region of each protein as well as an N-terminal region of SREBP-1c. Furthermore, through cotransfection of USF-1 and SREBP-1c in Drosophila SL2 cells, we show that these two factors synergistically activate the FAS as well as the mGPAT promoters. Importantly, we also show that USF and SREBP bind to the proximal mGPAT promoter in vivo in a manner identical to that of FAS. Functional domain mapping using USF and SREBP deletion constructs indicates that the activation domains of both proteins are required for synergy. Furthermore, the presence of closely spaced E-boxes and SREs in several lipogenic genes suggests involvement of USF and SREBP in mediating the transcriptional response to fasting/refeeding in vivo.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Tandem Affinity Purification—293FT cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. The day before transfection, cells were seeded onto 150-mm dishes to achieve a confluency of 
30–40%. Ten 150-mm dishes were each transfected with 10 µg of SREBP-1c-Myc and 5 µg of pCTAP-A-FLAG-USF-1 or 5 µg of pCTAP-A using Lipofectamine (Invitrogen). Cells were maintained in serum free-media for 16 h following transfection and then replaced with complete media. Approximately 48 h after transfection, cells were harvested by washing twice in PBS followed by scraping. Cells were centrifuged at 1000 x g at 4 °C and resuspended in lysis buffer (Stratagene). The TAP protocol was then performed according to the manufacturer's instructions, except that the streptavidin-binding reaction was incubated overnight. Eluted proteins were boiled in SDS loading buffer and resolved on 7.5% SDS-polyacrylamide gels. The presence of USF-1 and SREBP-1c-Myc was determined by immunoblotting with M2 anti-FLAG (Sigma) and anti-Myc antibodies (4A6, Upstate). For copurification of endogenous SREBP-1 protein with USF-FLAG-TAP, HEK 293F cells were transfected with USF-FLAG-TAP vector or empty TAP vector using 293-Fectin (Invitrogen). Nuclear extracts were prepared according to the method of Andrews and Faller (38) 72 h after transfection and subjected to the TAP protocol. Immunoblotting was performed with anti-FLAG and anti-SREBP-1 antibodies (2A4, Santa Cruz Biotechnology).
Preparation of Mouse Liver Nuclear Extract—Mice were fasted for 48 h and then refed a high carbohydrate, fat-free diet for 18 h. Livers were excised and homogenized in cell lysis buffer (2 M sucrose, 10 mM Tris-Cl, pH 7.4, 25 mM KCl, 5 mM MgCl2, 1 mM EDTA, 10% (v/v) glycerol, 1 mM DTT, protease inhibitor mixture). After centrifugation at 75,000 x g for 60 min, the supernatants were discarded, and nuclei were resuspended in high salt extraction buffer (400 mM NaCl, 10 mM Tris-Cl, pH 7.4, 100 mM KCl, 2 mM MgCl2, 1 mM EDTA, 10% (v/v) glycerol, 1 mM DTT, protease inhibitors) and incubated on ice for 30 min. The extracts were centrifuged for 60 min at 250,000 x g, and supernatants were used as nuclear extracts for subsequent analysis.
Immunoprecipitation—To determine whether SREBP-1 could be coimmunoprecipitated with USF-1, COS-7 cells were cotransfected with 5 µg of pCDNA-FLAG-USF-1 and 10 µgof pCDNA 3.1-SREBP-1a and harvested 48 h after transfection. Cells were washed twice with PBS, scraped, collected by centrifugation at 1000 x g for 10 min, and then lysed by passing through a 21-guage needle 25 times in Triton X-100 lysis buffer (20 mM Tris-Cl, pH. 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM DTT) supplemented with protease inhibitors (Sigma). Lysates were clarified by centrifuging at maximum speed in a tabletop refrigerated centrifuge for 10 min. Antibodies (10 µg/immunoprecipitation) against USF-1 (C-20, Santa Cruz Biotechnology), Sp1 (PEP-2, Santa Cruz Biotechnology), or control IgG were immobilized on protein A-agarose (100 µl) by incubating in 1 ml of Triton X-100 lysis buffer for 2–3 h followed by three washes in the same buffer to remove unbound antibodies. After the last wash, resins were resuspended in 800 µl of Triton X-100 lysis buffer + 200 µlof clarified lysates and incubated overnight with gentle rotation at 4 °C. The following day, immunoprecipitates were washed five times in Triton X-100 lysis buffer followed by two additional washes in the same buffer without Triton X-100. After the last wash, immunoprecipitates were boiled in 50 µlof1x SDS loading buffer, resolved by 7.5% SDS-PAGE, and transferred to nitrocellulose membranes. Immunoblotting was performed with anti-FLAG and anti-SREBP-1 (2A4, Santa Cruz Biotechnology) antibodies as described above. To determine whether USF-1 could be coimmunoprecipitated with SREBP-1c-Myc, cells were transfected with 5 µg of pCDNA 3.1 SREBP-1c-Myc and 10 µgof pCDNA-FLAG-USF-1. Immunoprecipitation was performed as described above except that 10 µg of anti-SREBP-1 antibodies (H160, Santa Cruz Biotechnology) were immobilized on protein A-agarose beads prior to immunoprecipitation.
GST-Pulldown Assay—Escherichia coli BL21 cells were transformed with various GST-USF-1 and GST-SREBP-1c fusion constructs. Ampicillin-resistant colonies were grown to an A600 of 0.6 in 250 ml of LB media followed by induction of protein expression by addition of 1 mM isopropyl 1-thio-
-D-galactopyranoside for 4 h. Cells were harvested by centrifugation at 3000 x g for 10 min and resuspended in 9 ml of PBS. Cells were lysed by five 1-min cycles of sonication on ice. Following sonication, glycerol and Triton X-100 were added to concentrations of 10 and 1%, respectively, and incubated with gentle rotation at room temperature for 1 h. Aliquots of the lysates were clarified by centrifugation at maximum speed in a tabletop refrigerated centrifuge. Approximately 500 µg of each lysate containing GST fusion proteins were added to 100 µlof glutathione-agarose resin and brought to a final volume of 1 ml with PBS bead-binding buffer (1x PBS, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM DTT). The lysates were incubated with glutathione-agarose resin for 2–3 h followed by three washes in binding buffer to remove unbound GST fusion proteins. FLAG-USF-1 or SREBP-1c in pCDNA 3.1 was used as DNA templates for in vitro transcription by T7 RNA polymerase, followed by in vitro translation with a rabbit reticulocyte lysate kit (Promega) supplemented with [35S]methionine (2 µl of 10 mCi/ml) in the same reaction. Coupled in vitro transcription and translation reactions were incubated at 30 °C for 90 min. Immobilized GST fusion proteins were resuspended in 1 ml of binding buffer, and 5 µlof in vitro translated and transcribed 35S-labeled USF-1 or SREBP-1c was added. Complexes were incubated overnight at 4 °C with gentle rocking and then washed seven times in 1 ml of binding buffer followed by boiling in 100 µl of SDS-loading buffer. Captured proteins were resolved by 7.5% SDS-PAGE, and the gels were dried and exposed to film to detect the presence of 35S-USF-1 and 35S-SREBP-1 by autoradiography. In parallel experiments to assess the correct size and integrity of GST fusion proteins, the same procedure was followed except that no in vitro transcribed and translated proteins were added to GST complexes, and SDS-polyacrylamide gels were analyzed by Coomassie staining of purified proteins. In experiments where 35S-USF-1 was immunoprecipitated from the reticulocyte lysate reaction, 10% of the in vitro translation reaction was mixed with 1 µg of anti-USF-1 antibodies (C-20, Santa Cruz Biotechnology) in 1 ml of PBS bead-binding buffer and immunoprecipitated overnight. Immune complexes were collected with protein A-agarose, washed five times with PBS bead-binding buffer, and eluted with 100 mM glycine, pH 2.5, for 5 min. The eluates were neutralized with 1 M Tris, pH 7.4, and added to GST and GST-SREBP immobilized on glutathione-agarose beads and processed as described.
For copurification of endogenous SREBP-1 with GST-USF-1-FLAG from mouse liver, nuclear extracts were added to immobilized GST or GST-USF-1-FLAG fusion protein and incubated overnight with gentle rotation. Following extensive washing, bound proteins were eluted with glutathione and then subjected to a second round of purification on anti-FLAG resin (Sigma). Eluted complexes were subjected to SDS-PAGE and Western blotting to detect the presence of GST-USF-1-FLAG and endogenous SREBP-1.
Transfection of SL2 Cells for Functional Studies—Drosophila SL2 cells were maintained as described (39). Approximately 16–20 h before transfection, cells were plated at a density of 0.25 x 106 cells/ml into 48-well plates. Cells were transfected using a standard calcium phosphate precipitation protocol. Each well received the indicated amount of pPAC-USF-1 and/or pPAC-SREBP-1c expression vector (generally 1–25 ng) and 500 ng of luciferase reporter construct along with 10 ng of the control plasmid pAC-5.1/V5-His/lacZ for normalization of transfection efficiency. The total amount of DNA transfected per well was brought to 1 µg with the empty expression vector pPAC-0 (470–500 ng/well). Cells were harvested 48 h after transfection, and luciferase/-galactosidase activities were assayed using the dual-light system (Applied Biosystems). Each transfection was carried out in triplicate.
Chromatin Immunoprecipitation—Chromatin immunoprecipitation experiments on fasted/refed mice were performed as described previously (16). Primer sequences for amplification of the proximal region of the mouse mitochondrial GPAT promoter were 5'-ACAGCCACACTCACAGAGAATGGGGC-3' and 5'-GAAGAGGCAGACTCGGCGTTCCGGAG-3'. This produces a predicted amplicon size of 182 bp.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We also asked whether endogenous SREBP-1c could be "pulled out" of mouse liver by an affinity-tagged version of USF-1. Full-length USF-1 was cloned in-frame with glutathione S-transferase at the N terminus and expressed in E. coli (Fig. 1A, GST-USF-FLAG). GST-USF-FLAG fusion protein was prepared from E. coli lysates and immobilized on glutathione-agarose beads followed by extensive washing. Nuclear extracts were prepared from mice that had been fasted for 24 h or fasted and then refed a fat-free, high carbohydrate diet. This manipulation has been shown to dramatically increase the expression of SREBP-1c in liver (15). Liver nuclear extracts were incubated with GST-bound resin or GST-USF-FLAG-bound resin; the resin was eluted with glutathione after washing, and the eluates were then re-incubated with an anti-FLAG affinity resin. The bound proteins were separated by SDS-PAGE and immunoblotted with anti-FLAG and anti-SREBP-1 antibodies. As shown in Fig. 1D, no signal was observed in either of the purifications with GST alone. However, immunoreactive bands were clearly detected by the SREBP antibody in the GST-USF-FLAG purifications from both fasted and fasted/refed mouse liver. Furthermore, the signal was higher from refed compared with fasted mice, consistent with the known induction of SREBP-1c. These results further confirm the USF-SREBP-1c interaction.
Identification of Domain(s) of USF That Interact with SREBP-1c—To determine whether the interaction between USF and SREBP is direct, we performed a GST pulldown using bacterially expressed USF and in vitro translated SREBP-1c. Full-length USF-1 was expressed as a fusion protein with GST in E. coli (Fig. 2, A and B). Following immobilization of GST-USF-1 fusion protein on glutathione-agarose beads, in vitro transcribed and translated and 35S-labeled SREBP-1c was added for overnight incubation. Following extensive washing, eluted complexes were subjected to SDS-PAGE and autoradiography to detect the presence of 35S-SREBP-1c. As shown in Fig. 2C, 35S-SREBP-1c was readily detectable in the purification with GST-USF-1 but not with GST alone. These results demonstrate that the interaction between USF and SREBP-1c is direct and specific.
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Identification of Domain(s) of SREBP-1c That Interact with USF—The domain structure of the mature form of SREBP-1c is shown in Fig. 3A. The extreme N terminus of SREBP-1c contains the putative activation domain (46, 47), followed by a Pro/Ser-rich region from amino acids 37 to 153 (23). Like USF, the bHLH/LZ region is located at the C terminus. To determine which region(s) in SREBP-1c interact with USF, we generated a series of 5'-deletion constructs linked to GST and expressed them in E. coli (Fig. 3, A and B). Each of these fusion constructs were immobilized on glutathione-agarose beads and incubated with in vitro transcribed and translated 35S-USF-1. As shown in Fig. 3C, USF efficiently copurified with full-length SREBP-1c-(1–436). USF also copurified with a truncated SREBP-1c-(37–436) that contains all regions except the putative activation domain, indicating that the activation domain of SREBP-1c is not required for interaction with USF. This is consistent with our observation that USF also interacts with SREBP-1a (Fig. 1C, left panel); the only difference between the SREBP-1a and -1c isoforms lies within the first exon that includes four unique amino acids for SREBP-1c. However, when the entire region from 1 to 299 of SREBP-1c was deleted, the interaction with USF was lost (Fig. 3C, lane 5), clearly indicating that the N-terminal region between 37 and 299 is required and that the bHLH region alone is not sufficient for interaction with USF.
To further define the region(s) of SREBP-1c that interact with USF, a second series of truncations was generated and fused to GST (Fig. 3, D and E). Full-length GST-SREBP-1c-(1–436) was included as a positive control in this experiment. As shown in Fig. 3F, in vitro translated 35S-USF-1 efficiently interacted with GST-SREBP-1c-(1–436). None of the first three GST-SREBP fusion constructs depicted schematically in Fig. 3D were capable of interacting with USF (Fig. 3F, lanes 4–6), indicating that these N-terminal regions alone are not sufficient for the interaction. Inclusion of the bHLH region of SREBP in the last two constructs shown in Fig. 3D restored interaction with USF (Fig. 3F, lanes 7 and 8), although deletion of the Pro/Ser-rich region had no effect. This indicates that two regions of SREBP-1c are required for binding to USF as follows: an N-terminal uncharacterized region spanning amino acids 154–299, and the bHLH region spanning amino acids 300–370.
To confirm that the interaction between USF and SREBP is direct, we performed an additional experiment in which in vitro translated 35S-USF-1 was first immunoprecipitated from the reticulocyte lysate reaction prior to incubation with full-length GST-SREBP. As shown in Fig. 3G, purified USF-1 still interacted with SREBP-1c, further confirming our results indicating that the two proteins interact directly.
Synergistic Activation of the FAS and GPAT Promoters by USF and SREBP-1c—Our previous studies have suggested that the effects of USF and SREBP-1c on FAS transcription are not independent and mutually exclusive (15, 16, 26). This is supported by the observation that FAS transcriptional activity in vivo is significantly reduced when either factor is missing (27–29) or when their respective binding sites are mutated (16). This is not the case in 3T3-L1 cells, where transfection of either factor alone results in a relatively potent activation of FAS transcription (15, 26). Regardless, because we found that USF and SREBP-1c directly interact in vitro and in vivo, we hypothesized that introduction of both USF and SREBP-1c would result in a synergistic activation of the FAS promoter. The "synergy index" can be defined here as the ratio of the fold activation of the FAS promoter when USF and SREBP-1c are cotransfected together to the sum of their individual fold activations when each factor is transfected separately. Although reports in the literature vary, a synergy index of 1.5–2.5 seems to be indicative of a high degree of functional cooperativity (48).
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2.5-fold), which is consistent with the lack of FAS transcriptional activation in livers of fasted mice when only USF is bound (16). In our previous in vivo experiments using transgenic animals, we demonstrated that mutation of the -65 E-box abolishes induction of FAS transcription in response to fasting/refeeding (16). To determine whether the -65 E-box is required for synergistic activation by USF and SREBP, we used a -444 FAS promoter construct containing a mutation at the E-box that prevents binding of either protein (26). As shown in Fig. 4B, mutation of the E-box at -65 not only abolished synergy between USF and SREBP but also reduced the promoter activity to levels similar to that of the promoterless vector pGL2basic (filled bars). Thus, the -65 E-box is crucial for both basal and stimulated activity of the FAS promoter, and these in vitro results are in agreement with our results in vivo.
To determine whether USF can augment FAS promoter activation by SREBP in a dose-dependent manner, and vice versa, we cotransfected increasing amounts of pPAC-SREBP-1c along with constant pPAC-USF-1 (10 ng/well) and the -444 FAS reporter construct. As shown in Fig. 4C, SREBP-1c alone activated the FAS promoter to a maximum of about 20-fold at 10 ng of expression plasmid/well. However, inclusion of 10 ng/well pPAC-USF-1 boosted the activation by SREBP-1c at all concentrations and reached a maximum of 80-fold at 10 ng of pPAC-SREBP-1c (Fig. 4C). At the highest concentration of SREBP-1c (20 ng/well), we observed a reduction in promoter activity, probably as a result of "squelching." Importantly, no activation of the promoterless pGL2-basic vector by USF or SREBP was observed in these experiments (Fig. 4C, open squares and circles). Overall, these results indicate that SREBP-1c-mediated activation of the FAS promoter is highly synergistic with USF.
In the inverse experiment, we transfected increasing amounts of pPAC-USF-1 in the presence of 10 ng/well pPAC-SREBP-1c. As shown in Fig. 4D, the cotransfection of SREBP-1c significantly boosted activation by USF-1 to a maximum of about 60-fold. The highest synergy was observed at 10 ng of each transcription factor/well, similar to the results shown in Fig. 4C. At this concentration of each factor in both experiments, the fold activation of the FAS promoter obtained when both USF and SREBP were added together was about three times higher than the sum of their individual fold activations, indicating that USF and SREBP are not acting in an additive and independent manner.
To determine whether the functional collaboration between USF and SREBP-1c might constitute a common mechanism for lipogenic gene induction during feeding/insulin, we next asked whether cotransfection of USF and SREBP-1c would result in synergistic activation of the mGPAT promoter as well. Mitochondrial GPAT catalyzes the condensation of fatty acids with the sn-1 position of glycerol 3-phosphate and, like FAS, is strongly induced by feeding/insulin at the transcriptional level (8, 11). For this purpose we used the -1447 mGPAT promoter, which we previously demonstrated to have high activity in 3T3-L1 adipocytes (37) and which has already been shown to be activated by SREBP-1a through an SRE-like element at -64 (51). There are also several E-boxes in this construct, including one at position -321 which is identical in sequence to that of the E-box in the FAS promoter at -65. Despite this observation, USF alone was unable to activate the -1447 mGPAT promoter in SL2 cells (Fig. 5A, circles). However, in the presence of 10 ng/well pPAC-SREBP-1c, USF-1 activated the mGPAT promoter in a dose-dependent manner up to a maximum of 50-fold at a concentration of 10 ng/well (Fig. 5A, squares). Likewise, SREBP-1c alone only weakly activated the mGPAT promoter (Fig. 5B, circles) to a maximum of 9-fold at a concentration of 10 ng/well. However, in the presence of 10 ng/well USF-1, the activation of mGPAT by SREBP was significantly higher at 5 or 10 ng/well (Fig. 5B, squares). No squelching was observed in the experiment shown in Fig. 5B, probably because we used a lower concentration of SREBP-1c plasmid than in the experiments with the FAS promoter. The fold activation of both transcription factors added together was nearly six times higher than the sum of their individual fold activations (Fig. 5C, left panel), indicating a high degree of synergy between USF and SREBP-1c in activation of the -1447 mGPAT promoter.
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The Activation Domains of USF and SREBP Are Required for Synergistic Activation of the FAS Promoter—We hypothesized that synergy between USF and SREBP-1c could potentially arise through two distinct mechanisms. First, because the bHLH regions of both proteins are involved in the physical interaction, synergy between USF and SREBP-1c could arise from recruitment or enhanced binding of SREBP to the promoter through interaction with USF, as suggested by our previous results in vivo. Second, the synergy could also involve recruitment of one or more coactivators by either or both activation domains of USF and SREBP. First, to determine whether the activation domain of USF is required for synergistic activation of the FAS promoter, we made a single deletion construct of USF-1 that has the entire activation domain deleted (amino acids 1–196) but preserves the bHLH/LZ region (amino acids 197–310). We reasoned that if the mechanism of synergy between USF and SREBP-1c results entirely from recruitment of SREBP by USF, removing the activation domain of USF-1 but preserving its bHLH/LZ region would not attenuate the synergy because the 113-amino acid bHLH/LZ domain of USF still both interacts with SREBP-1c (Fig. 2C) and binds E-boxes (52). As shown in Fig. 7A, this truncated USF-1 displayed little transactivation (dotted bar), consistent with others who have reported that the activation domain of USF is indispensable for transcriptional activation (53). We next cotransfected pPAC-USF-1-(197–310) along with full-length SREBP-1c into SL2 cells at the same concentration of each, which gave maximum synergy observed with full-length USF in this experiment. As shown in Fig. 7A, synergy with SREBP-1c was not only abolished with this construct, but cotransfection of the truncated version of USF-1 actually inhibited SREBP-1c-mediated promoter activity by 70% (Fig. 7A, checkered bar). This suggests that the synergy we have observed cannot be attributed solely to cooperative DNA binding and that recruitment of coactivators may be required for maximal activation.
To determine which domain(s) of SREBP-1c are functionally required for synergy with USF, we next generated a series of N-terminal SREBP-1c deletion constructs (Fig. 7B) and cotransfected them along with full-length USF-1 in SL2 cells. The three deletion constructs employed were pPAC-SREBP-1c-(37–436), pPAC-SREBP-1c-(154–436), and pPAC-SREBP-1c-(300–436), which are deleted for the first 36, 153, and 299 amino acids of SREBP-1c, respectively. As shown in Fig. 7B (middle panel), deletion of the N-terminal region of SREBP-1c abolishes its transcriptional activity, despite the fact that these constructs contain the bHLH/LZ region and should therefore retain the ability to bind DNA. This functionally confirms the results of others who have shown that the putative activation domain of SREBP-1c maps to the N-terminal region (46, 47, 54). When cotransfected along with 10 ng/well pPAC-USF-1, which itself resulted in only a very modest 5-fold induction (Fig. 7B, middle panel), synergy of the first two SREBP-1c deletion mutants with USF was abolished under conditions in which full-length SREBP-1c was highly synergistic with USF (filled bar). Notably, these two SREBP-1c mutants, SREBP-1c-(37–436) and SREBP-(154–436), appeared to actually inhibit the modest 5-fold activation by USF. To determine whether these deletion mutants can inhibit activation by USF when USF is transfected at a higher concentration, we cotransfected 20 ng/well pPAC-USF-1 along with 10 ng/well of each SREBP-1c deletion construct (Fig. 7B, right panel). Transfection of 20 ng/well pPAC-USF-1 alone resulted in a more robust 25-fold activation (Fig. 7B, dotted bar), and the first two SREBP-1c deletion constructs displayed a strong inhibition of USF-mediated activation (74 and 81%, respectively). However, the shortest construct, containing only the 136-amino acid bHLH/LZ region of SREBP-1c, had no effect on activation by USF (Fig. 7B, checkered bar). Importantly, the first two constructs can clearly interact physically with USF (as indicated by GST pulldown assay), whereas SREBP-1c-(300–436) was completely unable to interact with USF (Fig. 3C) but still retains the ability to bind DNA (52). These data suggest that the potent inhibition of USF transcriptional activity observed with SREBP-1c-(37–436) and SREBP-1c-(154–436) is not simply because of occupancy of the E-boxes in the FAS promoter by these truncated inactive proteins, but rather that these constructs may interact with USF and function in a dominant negative manner. In addition, these functional deletion studies corroborate the results of our interaction domain mapping and strongly imply that physical interactions between USF and SREBP-1c are required for synergistic activation of the FAS and mGPAT promoters.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The synergistic activation of the mitochondrial mGPAT promoter by USF and SREBP-1c was an intriguing result, because a role for USF in activation of the mGPAT promoter, to our knowledge, has never been investigated. Previous attempts to show activation of the mGPAT promoter by USF might have failed, as USF alone was unable to activate the mGPAT promoter, as shown in our study. This occurred despite the presence of multiple E-boxes, including one at -321 which is identical in sequence to the -65 E-box in the FAS promoter. In this regard, the ability of USF to activate transcription of target genes independently of other transcription factors may be related to the proximity of the E-box to the TATA-box, as has been shown for activation of the adenovirus major late promoter, where USF and TFIID bind cooperatively (60–62). In fact, the -65 E-box in the FAS promoter is approximately the same distance from the TATA-box as in the adenovirus major late promoter. Thus, the lack of activation of the mGPAT promoter by USF alone may be explained by the absence of any E-box motif in close proximity to the TATA-box. Nevertheless, in the presence of SREBP-1c, USF activates the mGPAT promoter in a dose-dependent manner up to 50-fold (Fig. 5A). Because mGPAT is strongly induced by feeding/insulin in a coordinate manner with FAS (8, 11), it is quite possible that interaction between USF and SREBP-1c mediates activation of mGPAT in vivo. In support of this, the pattern of USF and SREBP-1c binding to the mGPAT promoter during fasting/refeeding by ChIP (Fig. 6) was indistinguishable from what we observed previously for the FAS promoter (16).
In this study we provide clear evidence for cooperative activation of both the FAS and mGPAT promoters by USF and SREBP. In addition to FAS and GPAT, there is suggestive evidence for a role of both proteins in activation of another lipogenic enzyme gene, acetyl-CoA carboxylase-
(ACC-). Barber et al. (63) used ChIP analysis to show binding of both USF and SREBP-1 to the ovine ACC- promoter. In this study, USF binding did not change significantly between the nonlactating and lactating states, whereas recruitment of SREBP to the promoter during lactation was associated with a strong induction of ACC- transcription. In addition, USF was reported to activate the cardiac ACC- gene through an E-box located in the proximal promoter region (64), and ACC- has been shown by ChIP to be occupied by SREBP-1 at a nearby site, at least in liver (65). Thus, it is possible that the interactions we have observed here might also be involved in synergistic activation of the ACC promoter. Furthermore, the presence of closely spaced E-boxes and SRE-like elements in the promoters of several lipogenic genes (Fig. 8) implicates a possible role for USF and SREBP-1c in mediating the overall transcriptional response to fasting/refeeding in a manner similar to FAS and mGPAT (13–16, 26, 51, 65–74).
The fact that USFs and SREBPs are both highly related bHLH/LZ proteins suggests the obvious possibility that USFs and SREBPs could form a heterodimer on E-boxes, as has already been proposed (63). However, several lines of evidence indicate that this is not the case. First, we did not detect heterocomplex formation between USF and SREBP-1c on the -65 E-box in our earlier gel-shift experiments with either nuclear extracts or recombinant proteins (14). Second, in our ChIP assays, we detected binding of USF, but not SREBP-1, to a truncated -131 FAS promoter containing only the -65 E-box, but lacking the -150 SRE, in transgenic mice (16). Furthermore, in our present domain mapping experiments, we have clearly demonstrated that the isolated bHLH/LZ region of SREBP does not by itself interact with USF (Fig. 3C, lane 5), which would be highly suggestive of heterodimer formation. In addition, others have shown that a truncated SREBP-1 containing only the bHLH/LZ domain efficiently forms homodimers but does not form heterodimers with other Myc family members, including USF and Max (52). It therefore seems likely that if USF and SREBP formed a heterodimer, then the bHLH/LZs of both proteins alone would be sufficient to mediate a stable interaction. Overall, these observations clearly rule out that the mode of interaction between USF and SREBP involves simultaneous binding of each factor to E-boxes.
Together with our previous in vivo data, the present results indicate that the mechanism of synergy may involve both recruitment of SREBP by USF to the FAS promoter followed by interaction of the activation domains of both proteins with yet-to-be identified coactivators. This is supported by our observations that deletion of the N-terminal activation domain of either protein potently inhibits the transactivation ability of its partner. A similar observation has been described for the cooperative interaction between USF and Ets-1 on the human immunodeficiency virus, type 1, enhancer in T cells, where deletion of the activation domain of the latter transcription factor inhibits activation by USF in vitro (75). These authors proposed that recruitment of Ets-1 by USF enables Ets-1 to interact with the basal transcription apparatus. Although the mechanistic details may differ, an analogous situation for the relation between USF and SREBP may explain the results we observe here. Identification of those factors recruited by USF and SREBP-1c would aid in understanding at the molecular level how physical interactions between USF and SREBP lead to synergistic activation of the FAS and mGPAT genes.
In conclusion, we have clearly shown that USF interacts with SREBP-1, and this requires the bHLH domains of both proteins, as well as an additional N-terminal region in SREBP. Cotransfection of USF and SREBP-1c into Drosophila SL2 cells results in a synergistic activation of the FAS as well as the mGPAT promoters, and the activation domains of both proteins are required for synergy. These studies provide a better understanding of the molecular mechanism leading to increased lipogenic gene transcription and lipid storage in feeding/insulin.
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1 To whom correspondence should be addressed: Dept. of Nutritional Sciences and Toxicology, University of California, 219 Morgan Hall, Berkeley, CA 94720. Tel.: 510-642-3978; Fax: 510-642-0535; E-mail: hsul{at}nature.berkeley.edu.
2 The abbreviations used are: FAS, fatty-acid synthase; GPAT, glycerol-3-phosphate acyltransferase; mGPAT, mitochondrial GPAT; USF-1, upstream stimulatory factor-1; SRE, sterol regulatory element; SREBP-1c, sterol regulatory element-binding protein-1c; ChIP, chromatin immunoprecipitation; PBS, phosphate-buffered saline; bHLH/LZ, basic helix-loop-helix/leucine zipper; ACC, acetyl-CoA carboxylase; LDLR, low density lipoprotein receptor; GST, glutathione S-transferase; DTT, dithiothreitol; TAP, tandem affinity purification.
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