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Molecular Endocrinology, doi:10.1210/me.2007-0196
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Molecular Endocrinology 21 (11): 2698-2712
Copyright © 2007 by The Endocrine Society

Functional Interaction of Hepatic Nuclear Factor-4 and Peroxisome Proliferator-Activated Receptor-{gamma} Coactivator 1{alpha} in CYP7A1 Regulation Is Inhibited by a Key Lipogenic Activator, Sterol Regulatory Element-Binding Protein-1c

Bhaskar Ponugoti, Sungsoon Fang and Jongsook Kim Kemper

Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Address all correspondence and requests for reprints to: J. Kim Kemper, Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801. E-mail: jongsook{at}uiuc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin inhibits transcription of cholesterol 7{alpha}-hydroxylase (Cyp7a1), a key gene in bile acid synthesis, and the hepatic nuclear factor-4 (HNF-4) site in the promoter was identified as a negative insulin response sequence. Using a fasting/feeding protocol in mice and insulin treatment in HepG2 cells, we explored the inhibition mechanisms. Expression of sterol regulatory element-binding protein-1c (SREBP-1c), an insulin-induced lipogenic factor, inversely correlated with Cyp7a1 expression in mouse liver. Interaction of HNF-4 with its coactivator, peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}), was observed in livers of fasted mice and was reduced after feeding. Conversely, HNF-4 interaction with SREBP-1c was increased after feeding. In vitro studies suggested that SREBP-1c competed with PGC-1{alpha} for direct interaction with the AF2 domain of HNF-4. Reporter assays showed that SREBP-1c, but not of a SREBP-1c mutant lacking the HNF-4 interacting domain, inhibited HNF-4/PGC-1{alpha} transactivation of Cyp7a1. SREBP-1c also inhibited PGC-1{alpha}-coactivation of estrogen receptor, constitutive androstane receptor, pregnane X receptor, and farnesoid X receptor, implying inhibition of HNF-4 by SREBP-1c could extend to other nuclear receptors. In chromatin immunoprecipitation studies, HNF-4 binding to the promoter was not altered, but PGC-1{alpha} was dissociated, SREBP-1c and histone deacetylase-2 (HDAC2) were recruited, and acetylation of histone H3 was decreased upon feeding. Adenovirus-mediated expression of a SREBP-1c dominant-negative mutant, which blocks the interaction of SREBP-1c and HNF-4, partially but significantly reversed the inhibition of Cyp7a1 after feeding. Our data show that SREBP-1c functions as a non-DNA-binding inhibitor and mediates, in part, suppression of Cyp7a1 by blocking functional interaction of HNF-4 and PGC-1{alpha}. This mechanism may be relevant to known repression of many other HNF-4 target genes upon feeding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CYTOCHROME P450 (CYP) 7A1 (CYP7A1) (1) encodes the hepatic microsomal enzyme, cholesterol 7{alpha}-hydroxylase, which is the first and rate-limiting enzyme in the neutral pathway of cholesterol conversion to bile acids (1, 2). The bioactivity of cholesterol 7{alpha}-hydroxylase is regulated mainly at the level of transcription by various effectors, including diurnal rhythm, stress, xenobiotics, cholesterol, and the end-products of the cholesterol catabolic pathway, bile acids (3, 4, 5, 6, 7). CYP7A1 regulation has been also studied as a model for regulation of gene expression by the diet. CYP7A1 transcription is activated in livers of fasted mice and returns to basal levels after feeding (8, 9). Interestingly, the increase in insulin after feeding has been implicated in the suppression of transcription of the human, rat, and hamster CYP7A1 genes (10, 11, 12). Consistent with these studies, the pool size and excretion of bile acids are highly elevated in untreated diabetic humans and animals (13, 14).

An orphan nuclear receptor and hepatic activator, hepatic nuclear factor-4 (HNF-4), is a central regulator of transcriptional networks in the liver and pancreatic ß-cells (15, 16, 17). HNF-4 is one of six genes that are known to cause mature-onset diabetes of the young, implying a critical role of HNF-4 in glucose regulation and diabetes (18). In addition to its role in glucose metabolism, HNF-4 is also involved in regulation of lipid metabolism including regulation of hepatic CYP7A1 expression (5, 6, 19, 20).

Peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}) is a versatile coactivator for numerous nuclear receptors and transcriptional factors, including HNF-4, which regulate diverse biological functions, such as lipid and carbohydrate metabolism and energy homeostasis (21, 22). It has been shown that PGC-1{alpha} substantially increased HNF-4-mediated transactivation of CYP7A1 (5, 6, 8, 9) and that HNF-4/PGC-1{alpha} signaling is inhibited in the suppression of CYP7A1 expression mediated by the xenobiotic orphan nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) (5, 6). Coactivation of HNF-4 activity by PGC-1{alpha} has also been implicated in the nutritional regulation of hepatic gluconeogenic genes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) (21, 23). Using promoter deletion analyses, an HNF-4-binding direct repeat 1 motif, which is highly conserved in the proximal promoters of hamster, rat, and human CYP7A1 genes, was identified as a negative insulin-responsive region (11, 12). However, detailed molecular mechanisms by which increased insulin in the fed animal suppresses transcription of CYP7A1 via the HNF-4-binding site have not been clearly defined.

Sterol-responsive element-binding proteins (SREBPs) are basic-helix-loop-helix-leucine zipper transcription factors that regulate a number of genes involved in cholesterol and fatty acid metabolism in the liver (24, 25, 26). Three forms of SREBP are known, SREBP-1a, SREBP-1c, and SREBP-2. SREBP-1a and -1c are produced from a single gene through alternative transcription start sites, and SREBP-2 is produced from a different gene (24, 25, 27). Both SREBP-1a and -1c preferentially induce expression of key genes involved in fatty acid and triglyceride synthesis, whereas SREBP-2 is a key activator of genes involved in cholesterol biosynthesis (24, 25). Importantly, expression of SREBP-1c is dramatically affected by fasting and feeding, whereas expression of SREBP-2 is only modestly changed (27, 28, 29). SREBP-1c is the predominant form in adult liver and adipocytes, whereas SREBP-1a is abundantly expressed in cultured HepG2 cells (27, 28).

In addition to its role in the activation of the lipogenic program, SREBP-1c has been implicated as a physiological inhibitor of hepatic glucose production (30, 31). Consistent with these earlier studies, SREBP-1c inhibited transcription of PEPCK, a key enzyme in hepatic gluconeogenesis, by interfering with HNF-4 action (32). Because the HNF-4 site in the CYP7A1 promoter has been identified as a negative insulin-responsive sequence and the HNF-4/PGC-1{alpha} pathway plays a critical role in transactivation of CYP7A1 (5, 6, 8, 9), we hypothesized that increased SREBP-1c in the fed state inhibits transcription of CYP7A1 by blocking functional interaction between HNF-4 and PGC-1{alpha}.

We present evidence from in vitro and in vivo studies indicating that a key lipogenic activator, SREBP-1c, functions as a non-DNA-binding transcriptional inhibitor and interferes with the functional interaction of HNF-4/PGC-1{alpha} in the nutritional regulation of CYP7A1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inverse Correlation between Hepatic Expression of Cyp7a1 and SREBP-1c Genes under Different Nutritional Conditions
We first examined the effects of different nutritional conditions on the hepatic mRNA levels of SREBP-1c and Cyp7a1. Mice were fasted for 16 h or fasted for 16 h and then refed for 24 h as described (29), and mRNA levels of the genes were determined by semiquantitative RT-PCR. Fasting decreased, whereas refeeding increased, SREBP-1c mRNA levels (Fig. 1AGo). In contrast, mRNA levels of Cyp7a1 were increased in livers of fasted mice and markedly decreased upon refeeding. The control ß-actin mRNA levels were not changed (Fig. 1AGo).


Figure 1
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  		Fig. 1. Inverse Correlation between Hepatic Expression of Cyp7a1 and SREBP-1c Genes under Different Nutritional Conditions in Mice in Vivo

A and B, Livers from three mice per each group of mice fed normal chow (Normal), fasted for 16 h (Fasting), or fasted for 16 h and then refed for 24 h (Refeeding) were collected for RT-PCR and Western blotting; A, total RNA was isolated and subjected to semiquantitative RT-PCR using primers specific for the indicated genes; B, nuclear extracts were prepared and levels of the SREBP-1c and HNF-4 were detected by Western blotting. For a loading control, ß-actin levels were also detected. The position of nuclear SREBP-1c is indicated by an arrow, and the asterisk indicates a nonspecific band. C, Mice were fasted (Fs) for 0, 1, 3, 6, or 24 h or fasted for 24 h and then fed (Fd) for 1, 3, or 6 h, and total RNA was isolated and analyzed by quantitative real-time RT-PCR using primer sets for indicated genes. PEPCK mRNA levels were also measured to serve as a control for the fasting/feeding protocol.

 
SREBP-1c is synthesized as a precursor form bound to the endoplasmic reticulum, and after proteolytic cleavage, mature SREBP-1c enters the nucleus and activates transcription of target genes (24). Therefore, we also examined the effects on the levels of the nuclear mature form of SREBP-1c by Western blotting. Levels of the nuclear SREBP-1c were not detectable in livers of fasted mice but substantially increased in livers after refeeding of fasted mice, whereas those of ß-actin and HNF-4 were not markedly changed under these experimental conditions (Fig. 1BGo).

We further examined the time course of the changes in mRNA levels of SREBP-1c and Cyp7a1 in mouse liver during fasting and refeeding of fasted mice by quantitative real-time RT-PCR. The mRNA levels of the PEPCK gene, a key gene in hepatic gluconeogenesis (23, 33, 34), were also monitored as a control. Mice were fasted for 0, 1, 3, 6, or 24 h or were fasted for 24 h and refed for 0, 1, 3, or 6 h. PEPCK mRNA levels in liver progressively increased upon fasting and decreased rapidly to nearly undetectable levels by 3 h after refeeding (Fig. 1CGo) as expected (21, 23, 34). In contrast, the mRNA levels of SREBP-1c progressively decreased during fasting and increased to levels near those in the mice before fasting by 6 h of refeeding (Fig. 1CGo). Cyp7a1 mRNA levels were progressively increased during fasting and rapidly decreased after refeeding. These results demonstrate an inverse correlation between the expression of the SREBP-1c and Cyp7a1 genes during feeding and fasting.

SREBP-1c Inhibits the Interaction between HNF-4 and PGC-1{alpha}
We and others showed that the HNF-4/PGC-1{alpha} transactivation pathway is critical for CYP7A1 expression (5, 6, 8, 9). Therefore, to determine whether SREBP-1c inhibits the HNF-4/PGC-1{alpha} transactivation of CYP7A1, we asked first whether SREBP-1c inhibits the interaction between HNF-4 and PGC-1{alpha} in cultured cells. Cos-1 cells were transfected with expression plasmids for HNF-4 and PGC-1{alpha} in the absence or presence of SREBP-1c, and expression of the proteins was detected by Western blotting (Fig. 2AGo). Whole-cell extracts were immunoprecipitated with HNF-4 antibody or control IgG. PGC-1{alpha} was detected in the anti-HNF-4 precipitates but not in IgG precipitates, and expression of SREBP-1c decreased levels of PGC-1{alpha} in the anti-HNF-4 immunoprecipitates (Fig. 2BGo). These results suggest that HNF-4 interacts with PGC-1{alpha} and that SREBP-1c inhibits this interaction in cells.


Figure 2
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  		Fig. 2. SREBP-1c Inhibits the Interaction between HNF-4 and PGC-1{alpha} in Cells and in Mouse Liver

A and B, Overexpression of SREBP-1c decreased the interaction between HNF-4 and PGC-1{alpha} in cells. Cos-1 cells were cotransfected with expression plasmids for HNF-4, PGC-1{alpha}, and increasing amounts of SREBP-1c, and 24 h later, cell extracts were prepared and immunoprecipitated with either control IgG or HNF-4 antisera. The presence of PGC-1{alpha} or HNF-4 in the anti-HNF-4 immunoprecipitates was detected by Western blotting. Input indicates 5% of cell lysates used for each immunoprecipitation reaction. C and D, Increased SREBP-1c in the fed state is associated with a decrease in the association of endogenous PGC-1{alpha} with HNF-4 in mouse liver in vivo. Mice were fasted for 16 h (Fs) or fasted for 16 h and then fed for 5 h (Fd), and liver nuclear extracts were prepared. Analysis by CoIP using control IgG or HNF-4 antibody was as described in Materials and Methods. The presence of endogenous PGC-1{alpha} and SREBP-1c in the hepatic anti-HNF-4 immunoprecipitate was detected by Western blotting, and the positions of the proteins are indicated by arrows. Input indicates 5% of total nuclear extract used in each reaction.

 
We next asked whether endogenous PGC-1{alpha} in liver associates with endogenous HNF-4 in fasted mice and whether this association is decreased after refeeding, which results in increased nuclear SREBP-1c levels (Fig. 1BGo). Mice were fasted for 16 h or fasted for 16 h and then refed for 5 h. Nuclear SREBP-1c protein levels were substantially increased in liver nuclear extracts from refed mice, whereas PGC-1{alpha} levels were slightly decreased and HNF-4 levels were not markedly changed (Fig. 2CGo). Coimmunoprecipitation (CoIP) of PGC-1{alpha} with HNF-4 was markedly reduced by feeding of fasted mice (Fig. 2DGo). Conversely, CoIP of SREBP-1c with HNF-4 was not detected in fasted mice but, interestingly, was detected in the anti-HNF-4 immunoprecipitates after refeeding the mice (Fig. 2DGo). Consistent with the studies in cultured cells, these results suggest that HNF-4 also interacts with PGC-1{alpha} in vivo in mouse liver during fasting. The HNF-4/PGC-1{alpha} interaction is decreased, and conversely, the HNF-4/SREBP-1c interaction is increased upon feeding. These results are consistent with a hypothesis that SREBP-1c blocks the interaction between HNF-4 and PGC-1{alpha} upon feeding.

SREBP-1c Directly Binds to the Ligand-Binding Domain (LBD)/AF2 Domain of HNF-4
Next, we examined whether SREBP-1c directly interacts with HNF-4 using glutathione S-transferase (GST) pull-down assays. GST-SREBP-1c, which was bound to glutathione-Sepharose, was incubated with 35S-labeled full-length HNF-4 or with fragments of HNF-4 that contained the N-terminal DNA-binding domain (DBD), central LBD/AF2 domain, or C-terminal region containing the LBD/AF2 and negative regulatory domains (15, 35, 36, 37) (Fig. 3AGo). GST-SREBP-1c efficiently bound full-length HNF-4 and the fragments containing the central or C-terminal half of HNF-4 but not the N-terminal domain fragment (Fig. 3AGo). These results indicate that SREBP-1c interacts directly with HNF-4, and the interaction is mediated by the LBD/AF2 of HNF-4.


Figure 3
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  		Fig. 3. SREBP-1c Directly Interacts with HNF-4 in Vitro

GST pull-down assays were performed as described in Materials and Methods. Input indicates 20% of the total 35S-labeled proteins. A, A schematic diagram of full-length and deletion mutants of HNF-4 is shown to the left. NRD, Negative regulatory domain. N, M, and C, indicate HNF-4 mutants containing the N-terminal, central, and C-terminal regions, respectively. One microgram of GST or GST-SREBP-1c that had been bound to glutathione-Sepharose was incubated with [35S]HNF-4 full-length or mutants synthesized in vitro. B, A schematic diagram of nuclear forms of wild-type SREBP-1c and N-terminal transactivation (TA) domain-deleted SREBP-1c mutant is shown at the top. One microgram of GST or GST-HNF-4 that had been bound to glutathione-Sepharose was incubated with [35S]SREBP-1c wild type or N-terminal deleted mutant.

 
Whereas wild-type SREBP-1c efficiently interacts with HNF-4, a SREBP-1c mutant with the first 90 amino acids deleted did not bind to HNF-4 (Fig. 3BGo). These results indicate that the N-terminal transactivation domain of SREBP-1c is required for a direct interaction between SREBP-1c and HNF-4. It has been demonstrated that HNF-4 directly interacts with its coactivators including glucocorticoid receptor interacting protein-1 (GRIP-1), steroid receptor coactivator-1 (SRC-1), and PGC-1{alpha}, through the LBD/AF2 domain (21, 32, 35, 38). These previous studies, taken together with our GST pull-down and CoIP assays, suggest that SREBP-1c inhibits the interaction between HNF-4 and PGC-1{alpha} by directly binding to the LBD/AF2 domain of HNF-4.

SREBP-1c Inhibits HNF-4/ PGC-1{alpha} Transactivation of the CYP7A1 Promoter
To determine whether the effects of SREBP-1c on the interaction of HNF-4 with PGC-1{alpha} were functionally significant, we performed transfection reporter assays. To first determine whether the inhibition of HNF-4 transactivation by SREBP-1c requires the binding of HNF-4 to the direct repeat 1 site in the CYP7A1 promoter, CYP7A1-luc reporters containing wild-type or mutated HNF-4-binding sites were examined. In gel shift assays, binding of HNF-4 to the mutated promoter was completely abolished (data not shown). Although expression of SREBP-1c did not inhibit the activity of the pGL3 basic luciferase reporter (data not shown), cotransfection of increasing amounts of SREBP-1c plasmids inhibited the basal activity of the wild-type CYP7A1-luc promoter (Fig. 4AGo). In contrast, mutation of HNF-4-binding site in the promoter substantially decreased CYP7A1 promoter activity by more than 90%, and expression of SREBP-1c increased, rather than decreased, the mutated CYP7A1 promoter activity (Fig. 4AGo). These results indicate that an intact HNF-4-binding site is required for SREBP-1c inhibition of CYP7A1 promoter activity.


Figure 4
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  		Fig. 4. SREBP-1c Inhibits HNF-4/PGC-1{alpha} Transactivation of the CYP7A1 Promoter

A, HepG2 cells were cotransfected with 200 ng –1887 human CYP7A1-luc (black bars) or a mutant –1887 human CYP7A1-luc (white bars) in which the HNF-4 site was mutated, 300 ng CMV ß-gal plasmid, and increasing amounts of pcDNA3.1-SREBP-1c. The values for firefly luciferase activity were normalized by dividing by values for ß-galactosidase activity. The SEM was calculated from triplicate assays. B and C, HepG2 cells were cotransfected with 200 ng of either –371-human CYP7A1-luc (B) or –371-mouse Cyp7a1-luc (C), 50 ng CMV-HNF-4, 100 ng pcDNA3 PGC-1{alpha}, and increasing amounts of pcDNA3.1-SREBP-1c wild type or the N-terminal transactivation (TA) domain-deleted mutant as indicated. The values for firefly luciferase activity were normalized by dividing by values for ß-galactosidase activity. The SEM was calculated from triplicate assays. D, Cos-1 cells were cotransfected with 200 ng (Gal4)5-TK-luc, 25 ng Gal4DBD or Gal4DBD-HNF-4 (130–368), 50 ng pcDNA3 PGC-1{alpha}, and increasing amounts of pcDNA3.1SREBP-1c wild type or the N-terminal TA domain deleted mutant as indicated. The values for firefly luciferase activity were normalized by dividing by those for ß-galactosidase activity.

 
Because the N-terminal domain of SREBP-1c is required for a direct interaction between SREBP-1c and HNF-4 (Fig. 3BGo), we asked whether this N-terminal region was required for the inhibition of HNF-4 activity. Expression of increasing amounts of wild-type SREBP-1c, but not of the SREBP-1c mutant with N-terminal domain deleted, progressively inhibited HNF-4/PGC-1{alpha} transactivation of both the human CYP7A1 (Fig. 4BGo) and mouse Cyp7a1 (Fig. 4CGo) promoters in HepG2 cells. These results indicate that SREBP-1c functionally inhibits HNF-4/PGC-1{alpha} transactivation of the CYP7A1 gene and that inhibition requires the N-terminal domain of SREBP-1c, which is also required for direct interaction with HNF-4.

To determine whether SREBP-1c inhibits HNF-4/PGC-1{alpha} directly without binding to DNA, we used a Gal4 reporter system. Expression of Gal4-HNF-4 increased the activity of a promoter containing Gal4-binding sites several-fold, and expression of PGC-1{alpha} dramatically enhanced the HNF-4 transactivation (Fig. 4DGo, lanes 1–3). Increasing amounts of wild-type SREBP-1c progressively suppressed the HNF-4/PGC-1{alpha} activation (Fig. 4DGo, lanes 4–6), whereas the SREBP-1c mutant lacking the HNF-4 interacting domain did not inhibit the HNF-4/PGC-1{alpha} activation (lanes 7–9). Comparable levels of wild-type and the mutant proteins were detected in transfected cells (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). If SREBP-1c acts by inhibiting the interaction of PGC-1{alpha} with HNF-4, then expression of increasing amounts of PGC-1{alpha} should be able to reverse the SREBP-1c-mediated inhibition. Transfection of increasing amounts of PGC-1{alpha} expression plasmid reversed the inhibition of the HNF-4/PGC-1{alpha} transactivation by the wild-type SREBP-1c (Fig. 4DGo, lanes 6 and 10–12). These transfection studies, along with protein-protein interaction studies (Figs. 2Go and 3Go), suggest that SREBP-1c inhibits HNF-4 transactivation in the regulation of CYP7A1 by blocking functional interaction of HNF-4 with PGC-1{alpha} without binding to DNA. Consistent with these results, in vitro gel mobility shift assays revealed that SREBP-1c bound efficiently to a known SREBP-1 site but did not bind the CYP7A1 promoter region containing the HNF-4 site (supplemental Fig. 2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).

SREBP-1c Inhibits PGC-1{alpha}-Enhanced Transactivation of Estrogen Receptor (ER), CAR, PXR, and Farnesoid X Receptor (FXR)
Because our data suggest that SREBP-1c directly interacts with the LBD/AF2 domain of the HNF-4 and inhibits HNF-4 transactivation (Figs. 3AGo and 4Go), SREBP-1c could inhibit the activities of other nuclear receptors that also contain the BD/AF2 domain. To test this possibility, we carried out functional assays using the Gal4 reporter system. The expression plasmid for PGC-1{alpha} was expressed with expression plasmids for Gal4-chimeric proteins containing the LBD of each of the nuclear receptors ER{alpha}, CAR, PXR, and FXR. Expression of PGC-1{alpha} substantially increased the transactivation by each of the ligand-activated nuclear receptors (Fig. 5Go, A–D). Interestingly, increasing amounts of SREBP-1c substantially suppressed the transactivation of all of these nuclear receptors (Fig. 5Go, A–D). These results suggest that, like inhibition of HNF-4 by SREBP-1c through the direct interaction with the LBD/AF2 domain, SREBP-1c may more generally block the interaction of coactivators with the LBD/AF2 domains of other nuclear receptors.


Figure 5
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  		Fig. 5. SREBP-1c Inhibits PGC-1{alpha}-Enhanced Transactivation of Other Nuclear Receptors Such As ER, CAR, PXR, and FXR

A–D, Cos-1cells were cotransfected with 200 ng (Gal4)5-TATA-luc, 300 ng CMV ß-gal plasmid, 25 ng of Gal4DBD-ER, Gal4DBD-CAR, Gal4DBD-human PXR, or Gal4DBD-FXR, 100 ng of PGC-1{alpha}, and indicated amounts of pcDNA3.1-SREBP-1c. Cells were treated overnight with 10 nM estradiol, 10 µM 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), 10 µM rifampicin, or 1 µM GW4064 for ER, CAR, PXR, or FXR, respectively, and harvested for reporter assays. E, Cos-1 cells were cotransfected with 200 ng (Gal4)5-TATA-luc, 25 ng Gal4-HNF-4, and expression plasmids for 100 ng SRC-1, SRC-2 (GRIP-1), or SRC-3, and cells were harvested for reporter assays. The values for firefly luciferase activity were normalized by dividing by values for ß-galactosidase activity. The SEM was calculated from triplicate assays.

 
SREBP-1c Specifically Inhibits PGC-1{alpha}- or GRIP-1-Mediated Transactivation of HNF-4
To determine whether the observed SREBP-1c inhibitory effects on HNF-4 are specific to PGC-1{alpha}, we performed functional assays using the Gal4 reporter system. Transfection of SRC-1, SRC-2 (GRIP-1), or SRC-3 increased HNF-4-mediated transactivation of the reporter (Fig. 5EGo). Although increasing amounts of SREBP-1c did not inhibit the SRC-1 or SRC-3 coactivation of HNF-4, SREBP-1c markedly reduced the GRIP-1-mediated enhancement of HNF-4 transactivation. Together with our functional studies, these results suggest that SREBP-1c specifically inhibits PGC-1{alpha}- or GRIP-1-mediated transactivation of HNF-4.

Effects of Feeding and Insulin Treatment on the Association of HNF-4, PGC-1{alpha}, and SREBP-1c with the CYP7A1 Promoter
To determine whether the inhibition of HNF-4/PGC-1{alpha} transactivation by SREBP-1c observed in cultured cells is physiologically relevant in vivo, we examined the association of these proteins with the mouse Cyp7a1 promoter under different nutritional conditions. Mice were fasted for 16 h or refed for 6 h after fasting, and livers were collected for chromatin immunoprecipitation (ChIP) and real-time RT-PCR assays. We initially postulated that fasting and feeding may alter DNA binding of HNF-4 because it had been shown that HNF-4 binding to the L-type pyruvate kinase promoter was inhibited by protein kinase A in the fasting state (39). However, our ChIP studies consistently showed that HNF-4 binding to the Cyp7a1 promoter was not altered in fasting and feeding states (Fig. 6AGo). In contrast, association of PGC-1{alpha} with the Cyp7a1 promoter was markedly decreased, whereas recruitment of SREBP-1c was increased after feeding (Fig. 6AGo). Because SREBP-1c does not interact with PGC-1{alpha} (40), it is unlikely that recruited SREBP-1c interacts directly with PGC-1{alpha} and masks the epitopes from the antibody.


Figure 6
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  		Fig. 6. Effects of Feeding the Mice or Insulin Treatment in HepG2 Cells on the Association of Proteins and Acetylated H3 Levels at the Native Cyp7a1 Promoter

A–C, Mice were fasted (Fs) for 16 h or fasted for 16 h and then fed (Fd) for 6 h. Liver extracts were prepared and ChIP (A and B) and real-time RT-PCR (C) assays carried out as described in Materials and Methods. Reproducible results for association with the native Cyp7a1 promoter for endogenous proteins in vivo in mice were obtained in multiple ChIP assays. D and E, HepG2 cells were treated with insulin or vehicle for 6 h and further subjected to ChIP assays (C) and quantitative RT-PCR (D). C and E, In real-time RT-PCR assays, the amounts of PCR product were normalized by dividing by the amount of 36B4 PCR product. The SEM was calculated from three independent assays, and differences between experimental groups were analyzed by the Student’s t test. *, P < 0.05; NS, not statistically significant. F, HepG2 cells were transiently transfected with pcDNA3.1 SREBP-1c (+) or pcDNA3.1 (–) and then further subjected to ChIP assays as described in Materials and Methods.

 
Interestingly, histone deacetylase (HDAC) 2 was also recruited, and acetylated histone H3 was reduced after feeding the mice (Fig. 6BGo). In contrast, no association of these proteins with the control GAPDH coding region was detected. These results indicate that PGC-1{alpha} dissociates from the Cyp7a1 promoter after feeding concomitant with recruitment of SREBP-1c. The dissociation of PGC-1{alpha} from the promoter is consistent with the results above, indicating that SREBP-1c competes with PGC-1{alpha} for binding to HNF-4. The recruitment of HDAC2 and decreased histone acetylation would be consistent with a suppressor role for SREBP-1c.

We also examined the effects of fasting and feeding on the mRNA levels of Cyp7a1 and SREBP-1c. Hepatic Cyp7a1 mRNA levels were decreased, whereas SREBP-1c mRNA levels were increased by refeeding of fasted mice as expected (Fig. 6CGo). Because an orphan nuclear receptor, small heterodimer partner (SHP), is a well-known transcriptional corepressor of Cyp7a1 (3, 4, 41, 42), it was possible that feeding induces SHP, which then mediates the inhibition of Cyp7a1 expression. However, SHP mRNA levels were significantly decreased by 70% after feeding, so that Cyp7a1 suppression in the fed state does not result from increased SHP levels (Fig. 6CGo).

Because increased levels of insulin after feeding induces expression of SREBP-1c (29, 43), we also examined the effect of insulin treatment of HepG2 cells on the mRNA levels and association with the CYP7A1 promoter of HNF-4, SREBP-1c, and PGC-1{alpha}. Although association of HNF-4 with the promoter was little changed, SREBP-1c was recruited and PGC-1{alpha} was dissociated at the promoter after insulin treatment (Fig. 6DGo). In real-time RT-PCR assays, insulin treatment reduced CYP7A1 mRNA levels by 60% and increased SREBP-1c mRNA levels by 2.5-fold, whereas the SHP mRNA levels did not significantly change (Fig. 6EGo). The results from insulin treatment of HepG2 cells, therefore, were similar to those observed in mice in vivo after feeding.

To test whether increased nuclear levels of SREBP-1c resulted in dissociation of PGC-1{alpha} from the CYP7A1 promoter, the nuclear form of SREBP-1c was exogenously expressed by transfection of HepG2 cells. HNF-4 association with the promoter was little changed upon exogenous expression of SREBP-1c, but association of PGC-1{alpha} was decreased, whereas SREBP-1c recruitment was increased (Fig. 6FGo). These changes are similar to those observed in vivo after feeding and in HepG2 cells treated with insulin (Fig. 6Go, A and D), conditions under which SREBP-1c levels are increased. The results are consistent with increased SREBP-1c mediating the dissociation of PGC-1{alpha} from the CYP7A1 promoter after feeding or insulin treatment. Combined with in vitro binding studies, the results are consistent with the increased SREBP-1c competing with PGC-1{alpha} for binding to HNF-4 at the native CYP7A1 promoter.

Effect of the SREBP-1c Dominant-Negative (DN) Mutant on Functional Interaction of HNF-4 and PGC-1{alpha}
To directly test effects of SREBP-1c on the nutritional regulation of CYP7A1 in animals in vivo, we used adenovirus-mediated expression of a dominant-negative (DN) mutant of SREBP-1c. Because this DN mutant abolishes DNA binding but efficiently dimerizes with wild-type SREBP-1c, the DN mutant has been used to inhibit the activity of endogenous SREBP-1c (31, 44, 45). To first determine whether the DN mutant inhibited SREBP-1c activity by mechanisms other than inhibition of DNA binding, we first tested whether the SREBP-1c DN blocks the direct interaction between SREBP-1c wild type and HNF-4 using competition GST pull-down assays. Increasing amounts of reticulocyte lysates containing the SREBP-1c DN, but not control lysates, inhibited the HNF-4/SREBP-1c interaction in a dose-dependent manner, indicating that the DN mutant can directly inhibit wild type SREBP-1c suppression of HNF-4 independent of effects on DNA binding (Fig. 7AGo).


Figure 7
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  		Fig. 7. Functional Characterization of the SREBP-1c DN Mutant

A, Competition GST pull-down assays. One microgram of GST or GST-HNF-4 that had been bound to glutathione-Sepharose was incubated with [35S]SREBP-1c wild type (2 µl), and increasing amounts (2, 4, and 6 µl) of cold SREBP-1c DN synthesized using the in vitro TNT system or control lysates (control) were added in the binding reactions. B, Cos-1 cells were cotransfected with expression plasmids for HNF-4, PGC-1{alpha}, SREBP-1c wild type, and increasing amounts of the SREBP-1c DN mutant, cell extracts were prepared, and CoIP assays were done as described in Materials and Methods. C, Cos-1 cells were cotransfected with Gal4-TK-luc, Gal4DBD or Gal4DBD-HNF-4, pcDNA3 PGC-1{alpha}, and increasing amounts of SREBP-1c wild type or the DN mutant as indicated. The values for firefly luciferase activity were normalized by dividing by those for ß-galactosidase activity. D, HepG2 cells were cotransfected with –371-hCYP7A1-luc, CMV-HNF-4, pcDNA3 PGC-1{alpha}, and increasing amounts of SREBP-1c wild type, or the DN mutant as indicated. The values for firefly luciferase activity were normalized by dividing by values for ß-galactosidase activity. The SEM was calculated from triplicate assays.

 
Next, we examined whether the SREBP-1c DN can reverse the SREBP-1c inhibition of the HNF-4/PGC-1{alpha} interaction in cells assessed by CoIP. Cos-1 cells were transfected with expression plasmids for HNF-4, PGC-1{alpha}, and SREBP-1c with increasing amounts of SREBP-1c DN. Expression of SREBP-1c inhibited association of PGC-1{alpha} with HNF-4, and expression of increasing amounts of the SREBP-1c DN progressively reversed the inhibition (Fig. 7BGo).

We also tested whether the SREBP-1c DN blocks the SREBP-1c-mediated inhibition of HNF-4/PGC-1{alpha} transactivation. In the Gal4 reporter system, expression of Gal4-HNF-4 increased promoter activity, which was dramatically enhanced by expression of PGC-1{alpha} (Fig. 7CGo, lanes 1–4). Increasing amounts of SREBP-1c wild type progressively suppressed the HNF-4/PGC-1{alpha} activation (Fig. 7CGo, lanes 4–7), whereas the DN mutant abolished the inhibition mediated by SREBP-1c wild type (lanes 7–10). Similar effects were observed when the CYP7A1 promoter-luc was used (Fig. 7DGo). These results demonstrate that the SREBP-1c DN interferes with the interaction between wild-type SREBP-1c and HNF-4 and thereby effectively blocks the inhibitory action of wild-type SREBP-1c on CYP7A1 regulation.

Adenovirus-Mediated Expression of the SREBP-1c-DN Mutant in Mouse Liver Partially Reversed the Inhibition of Cyp7a1 Gene in the Fed State
Finally, we examined the effects of adenovirus-mediated hepatic expression of SREBP-1c-DN on Cyp7a1 regulation in response to feeding in mice in vivo. Tail vein injection of adenoviruses producing transcriptional factors and cofactors has been shown to result in expression in the liver, whereas marked expression was not detected in other tissues, such as muscle, pancreas, and white adipose tissue (30, 46). Using this approach, the function of transcription factors and cofactors, such as HNF-4, PGC-1{alpha}, forkhead box O1 (Foxo-1), and forkhead box A2 (Foxa-2), in the regulation of liver metabolism has been successfully studied (21, 23, 47, 48).

Mice were infected with adenoviral vectors expressing the SREBP-1c-DN mutant (Ad-SREBP-1c-DN) or control (Ad-empty) via tail vein injection. After 3 d, the injected mice were either fasted for 16 h or fed for 8 h after fasting, and livers were collected for further analyses. In ChIP assays, binding of HNF-4 to the native Cyp7a1 promoter was similar in both fasted and refed mice, and expression of SREBP-1c DN had little effect on HNF-4 binding (Fig. 8AGo, lanes 4–6). The decrease in the association of PGC-1{alpha} with the Cyp7a1 promoter observed after feeding of fasted mice was markedly, but partially, reversed in mice infected with SREBP-1c DN (lanes 7–9). Consistent with these results, SREBP-1c association was decreased in livers of refed mice infected with the SREBP-1c-DN (Fig. 8AGo, lanes 10–12). These studies provide direct evidence that SREBP-1c mediates the decreased association of PGC-1{alpha} with the native Cyp7a1 promoter in the liver after feeding of fasted mice.


Figure 8
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  		Fig. 8. Expression of the SREBP-1c-DN Mutant in Mouse Liver Partially Reversed the Inhibition of Cyp7a1 Gene by SREBP-1c in Fed State

Mice were injected with about 1 x 109 active viral particles of control Ad-empty or Ad-SREBP-1c-DN in 200 µl PBS via tail veins, and 3 d after infection, mice were fasted overnight or fasted overnight and then fed for 8 h. Livers were collected for ChIP assays (A) and real-time RT-PCR (B–E). A, Effects of expression of the SREBP-1c-DN on the association of endogenous HNF-4, SREBP-1c, and PGC-1{alpha} with the Cyp7a1 promoter in mouse liver. Livers were pooled from three mice from each group, and association of proteins with the native Cyp7a1 promoter was detected by ChIP assay as described in Materials and Methods. Fs, Fd, E, and DN, indicate fasting, feeding, Ad-empty, and Ad-SREBP-1c DN, respectively. B–E, Livers were collected for real-time RT-PCR to measure mRNA levels of FAS (B), Cyp7a1 (C), Cyp8b1 (D), and SHP (E). Total RNA was isolated from livers for real-time RT-PCR, and the amount of PCR product was divided by the amount of 36B4 PCR product. The SEM was calculated from four to six mice, and differences between Ad-empty and Ad-SREBP-1c-DN samples were analyzed by the Student’s t test. *, P < 0.05; NS, statistically not significant.

 
To ensure that the SREBP-1c DN expressed in the liver inhibits wild-type SREBP-1c activity, we measured mRNA levels of the fatty acid synthase (FAS) gene, a well-known SREBP-1c target involved in lipogenesis (49, 50). As expected, the mRNA levels of the FAS gene in mice infected with control Ad-empty were substantially increased in the refed mice, and the increase was largely inhibited in mice infected with Ad-SREBP-1c DN (Fig. 8BGo), indicating that the SREBP-1c DN mutant efficiently blocks endogenous SREBP-1c activity.

We next evaluated the in vivo role of SREBP-1c on nutritional regulation of the Cyp7a1 mRNA levels. The mRNA levels of Cyp7a1 gene were decreased upon feeding by 80% as expected, and importantly, the decrease was partially, but significantly, reversed by expression of Ad-SREBP-1c DN (Fig. 8CGo).

HNF-4 has been shown to transactivate the Cyp8b1 gene, which is involved in the neutral pathway of bile acid biosynthesis (20, 51). Therefore, we tested whether SREBP-1c DN also affects expression of this gene. The Cyp8b1 mRNA levels were decreased in the fed state, but the levels were partially, but significantly, increased in mice infused with Ad-SREBP-1c DN (Fig. 8DGo). These results suggest that the expression of Cyp7a1 and Cyp8b1 is regulated by SREBP-1c, but the incomplete reversal of these genes by SREBP-1c DN further suggests that SREBP-1c-independent pathways are also involved in the regulation of these genes in response to feeding.

We finally tested whether SHP expression is altered by SREBP-1c DN in the fed state. The SHP mRNA levels were decreased in fed animals, and blocking endogenous SREBP-1c activity by the SREBP-1c DN did not result in marked changes in the SHP mRNA levels (Fig. 8EGo). These results suggest that the inhibitory effects of SREBP-1c on the expression of Cyp7a1 are not mediated by induction of SHP expression.

These in vivo experiments, along with in vitro and cell studies, suggest that SREBP-1c functions as a non-DNA-binding transcriptional inhibitor and interferes with functional interaction of HNF-4 and PGC-1{alpha} in CYP7A1 regulation, which mediates, at least in part, the suppression of this key gene involved in hepatic bile acid biosynthesis in the fed state.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We provide evidence that a key lipogenic activator, SREBP-1c, functions as a non-DNA-binding inhibitor and mediates, in part, the repression of CYP7A1 upon feeding. GST pull-down, CoIP, and functional studies indicated that SREBP-1c directly interacts with the LBD/AF2 domain of HNF-4 and inhibits the interaction between HNF-4 and its coactivator PGC-1{alpha}, which inhibits HNF-4 transactivation of CYP7A1. In ChIP assays, although HNF-4 binding was not altered, PGC-1{alpha} was dissociated and SREBP-1c was recruited at the Cyp7a1 promoter after feeding mice and insulin treatment in HepG2 cells. Interestingly, HDAC2 was recruited and acetylated histone H3 levels were decreased at the promoter upon feeding of mice. Similar to feeding and insulin effects, overexpression of SREBP-1c in cells also resulted in dissociation of PGC-1{alpha} from the CYP7A1 promoter. Inhibition of wild-type SREBP-1c activity in mice livers by adenovirus-mediated overexpression of the SREBP-1c DN mutant partially, but significantly, reversed suppression of Cyp7a1 expression that was observed upon feeding, implicating SREBP-1c as a physiological regulator of bile acid biosynthesis.

SREBP-1c binds to SREs or E-boxes in the promoters of target genes and stimulates the program of hepatic fatty acid metabolism in response to increased insulin and glucose in the fed state (25, 26, 44). However, recent studies also showed that SREBP-1c can act as a transcriptional inhibitor by competing with other transcription factors for binding to DNA. For instance, SREBP-1c suppresses transcription of the insulin receptor substrate-2 gene by competing with forkhead transcription factors for binding to DNA at the promoter, resulting in reduction of IRS-2 expression and increased insulin resistance in the fed state (52).

In contrast, our data suggest that the SREBP-1c does not inhibit the HNF-4/PGC-1{alpha} transactivation of CYP7A1 by competing for DNA binding of HNF-4. The competition GST pull-down experiments (Fig. 7AGo) showed that the DN mutant blocked the binding of wild-type SREBP-1c with HNF (in the absence of DNA), resulting in impaired interaction of SREBP-1c with HNF-4. Second, in the Gal4 transcription system, the DN blocked the inhibition by SREBP-1c, and in this case, there is no DNA-binding site for SREBP-1c (Fig. 7CGo). These data support the idea that SREBP-1c is acting as a non-DNA-binding inhibitor and directly interacts with the LBD/AF2 domains of HNF-4, which mediate the interaction of HNF-4 with coactivators like PGC-1{alpha} and GRIP-1 (32, 35). The conclusion that SREBP-1c competes with coactivators such as PGC-1{alpha} and GRIP-1 for binding to HNF-4 is consistent with our observations from ChIP assays that PGC-1{alpha} was dissociated and SREBP-1c was recruited upon feeding of mice.

Binding of SREBP-1c to the LBD/AF2 region of HNF-4 may also mask the surface of the ligand binding pocket so that binding of endogenous ligands as well as coactivators to HNF-4 may be inhibited. Although long-chain fatty acids, such as palmitate, have been suggested as endogenous ligands for the orphan nuclear receptor HNF-4 (53), whether the long-chain fatty acids are physiological ligands for HNF-4 is not clear, and the molecular basis of modulation of HNF-4 activity by these ligands is unknown.

Interestingly, SREBP-1c also inhibited PGC-1{alpha}-enhanced transactivation of ER, CAR, PXR, and FXR as well as HNF-4 (Fig. 5Go). Because SREBP-1c directly interacts with the LBD/AF2 domain of HNF-4 and inhibits the HNF-4 activity by blocking functional interaction with PGC-1{alpha} and GRIP-1, other nuclear receptors could be similarly inhibited by SREBP-1c via competition for binding of coactivators to the LBD/AF2 domains.

Our studies demonstrate that increased SREBP-1c activity after feeding suppresses CYP7A1 transcription but does not rule out additional mechanisms that might contribute to the suppression. The inability to completely reverse the suppression of CYP7A1 after feeding by expression of SREBP-1c DN suggests other mechanisms might be operating in vivo. For example, insulin-activated protein kinase B (PKB)/Akt has been shown to be involved in the functional interaction between Foxo-1 and PGC-1{alpha} in the regulation of hepatic gluconeogenesis (54). Such a mechanism could also contribute to decreased interaction between HNF-4 and PGC-1{alpha} in the nutritional regulation of the Cyp7a1 gene. Interestingly, a potential PKB motif, RXRXXS/T (amino acids 125–130, RDRIST), which is conserved from human to Xenopus, is located in the DBD of HNF-4. However, the chimeric protein GST-HNF-4 was not phosphorylated by PKB in vitro (37), nor have we observed phosphorylation of HNF-4 in anti-HNF-4 immunoprecipitates by PKB in Cos-1 cells (our unpublished data). Recent studies also demonstrated that DNA binding, protein stability, and nuclear localization of HNF-4 are substantially altered by phosphorylation of HNF-4 mediated by tyrosine phosphorylation or protein kinase C (55, 56). Therefore, increased insulin in the fed state may result in phosphorylation of HNF-4, which could also contribute to CYP7A1 repression. Whether HNF-4 is phosphorylated in response to insulin in hepatic cells in vivo and whether the functional interaction between HNF-4/PGC-1{alpha} is modulated by the insulin-activated kinase pathway are important questions for future studies.

The orphan nuclear receptor SHP is a well-known repressor of CYP7A1 expression in the negative-feedback regulation of bile acid synthesis (3, 4). We recently reported that SHP inhibits transcription of CYP7A1 by coordinately recruiting chromatin-modifying enzymes, including mSin3A/HDACs corepressor complex, G9a histone lysine methyltransferase (41), and the Swi/Snf-Brm chromatin remodeling complex (42). A recent study showed that adenovirus-mediated overexpression of SREBP-1c in HepG2 cells induces expression of the human SHP gene by binding to the SREBP-responsive element in the promoter, which is not conserved in mouse SHP promoter, suggesting the existence of species differences in the induction of SHP by SREBP-1c (57). Based on these previous reports and considering that SHP is a key corepressor of CYP7A1 gene expression, our initial hypothesis was that SHP is the key mediator of the suppression of CYP7A1 in the fed state. However, our studies, in which a fasting/feeding protocol and insulin treatment were used, instead of overexpressing SREBP-1c, consistently showed that SHP mRNA levels increased during fasting by about 3- to 4-fold, and SHP mRNA levels rapidly decreased upon feeding of fasted animals. Also, the SHP mRNA levels were not increased but were slightly decreased in insulin-treated HepG2 cells. Our observations are, therefore, the opposite of what would be expected if SHP mediates suppression of Cyp7a1 expression after feeding. Consistent with the changes in mRNA levels, association of SHP with the Cyp7a1 promoter was not detected by ChIP assay after feeding (our unpublished data). Instead, we consistently observed that SHP and SHP-associated mSin3A corepressor were associated with the SREBP-1c promoter in the fasted state, and these proteins were dissociated from the promoter after feeding (our unpublished data). These results, taken together, suggest that the decrease in SHP expression after feeding may result in de-repression of SREBP-1c expression and, thus, indirectly contribute to suppression of CYP7A1 expression by SREBP-1c.

Our data from molecular, cellular, and in vivo studies together indicate that increased SREBP-1c in the fed state mediates, in part, the trans-repression of the CYP7A1 gene by interfering with the functional interaction of HNF-4 with its coactivator PGC-1{alpha}. Bile acid-induced SHP has also been shown to inhibit SREBP-1c transcription in mice in vivo (58). Therefore, our studies along with these reports suggest the existence of functional inhibitory cross-talk between hepatic lipogenesis and bile acid biosynthesis mediated by SREBP-1c. It has been well established that SREBP-1c is a DNA-binding transcriptional activator of many genes involved in the hepatic lipogenic pathway in response to increased insulin and glucose upon feeding (24, 25, 26). Our current studies show that SREBP-1c also functions as a non-DNA-binding transcriptional inhibitor and interferes with functional interaction of HNF-4 and PGC-1{alpha} in CYP7A1 regulation. This mechanism may be relevant to the known suppression of many other HNF-4/PGC-1{alpha} target genes involved in hepatic gluconeogenesis, fatty acid ß-oxidation, as well as bile acid synthesis in response to nutritional feeding.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Construction
To construct the adenoviral vector encoding SREBP-1c DN, the EcoR1 fragment encoding SREBP-1c from pBabe-SREBP-1c/ADD1 DN (44) was first inserted into the EcoR1 site of pcDNA3. Then, the HindIII/EcoRV fragment encoding SREBP-1c from pcDNA3-SREBP-1c DN was inserted into HindIII/EcoRV-digested Ad-CMV-Track vector (59). Ad-empty and Ad-SREBP-1c DN adenoviral vectors were amplified in Ad-293 cells and purified as described (59), and the number of active viral particles was determined by infection of Ad-293 cells or Cos-1 cells seeded on coverslips with increasing amounts of the viral vectors followed by confocal microscopy as described (6, 60). A mouse genomic DNA fragment containing the HNF-4-binding site in the Cyp7a1 promoter (–371 to + 24) was amplified from genomic DNA from livers by PCR using pfx polymerase enzyme (Invitrogen, Inc., Carlsbad, CA). After digestion with Kpn1 and Xho1, the DNA fragment was inserted between Kpn1/Xho1 sites of the pGL3 basic luciferase reporter vector (Promega, Inc., Madison, WI). The positive clone was confirmed by DNA sequencing.

Cell Culture
Human hepatoma cells (HepG2, ATCC HB8065; American Type Culture Collection, Rockville, MD) were maintained in phenol-red-free DMEM/F12 (1:1) media. Monkey kidney Cos-1 cells (from American Type Culture Collection) and Ad-293 cells derived from HEK cells (Cell Biolabs, Inc., San Diego, CA) were maintained in DMEM. Media were supplemented with 100 U/ml penicillin G-streptomycin sulfate and 10% heat-inactivated fetal bovine serum. For treatment with insulin, HepG2 cells were incubated in serum-free media containing 0.1% BSA overnight and then incubated for an additional 6 h after addition of insulin to 100 nM or vehicle (PBS).

Mouse in Vivo Experiments and Tail Vein Injection of Adenoviral Vectors
BALB/c male mice (8–12 wk old) were maintained on a 12-h light, 12-h dark cycle. Mice were randomly divided into groups and fasted for 16 h or fasted for 16 h and then fed chow (Harlan Teklad Co., Madison, WI) containing high carbohydrate (77% carbohydrate, 18% casein, 1% vitamin mix, 4% minerals) for indicated times using the fasting/refeeding protocol as previously described (29, 39, 50, 52). Feeding a fat-free and high-carbohydrate diet after fasting has been used in in vivo experiments as a potent insulin-stimulating protocol in animal experiments (29, 39, 50, 52). Because the expression of CYP7A1 is regulated by a diurnal rhythm (61), feeding was always started at 1700 h to reduce experimental variation. For experiments determining the time course of mRNA changes, mice were fasted for 0, 1, 3, 6, or 24 h or fasted for 24 h and then fed for 1, 3, or 6 h, and mRNA levels were determined by quantitative real-time RT-PCR.

For expression of SREBP-1c-DN in the liver, mice were injected with about 1 x 109 active viral particles of Ad-empty or Ad-SREBP-1c-DN in 200 µl PBS via tail veins as described (21, 23, 47, 48). Three days after infection, mice were fasted overnight or fasted overnight and then fed for 6–8 h. The mice were killed, and livers were collected for further analyses. All the animal use and adenoviral protocols were approved by the Institutional Use and Care of Animals and Biosafety Committees at University of Illinois and were in accordance with National Institutes of Health guidelines.

Real-Time and Semiquantitative RT-PCR
Total RNA was isolated from mouse liver or HepG2 cells using Trizol reagent, and cDNA was synthesized using a reverse transcriptase kit (Promega). Real-time RT-PCR was performed with an iCycler iQ (Bio-Rad, Inc., Hercules, CA) following the manufacturer’s instructions. The amount of PCR product for each mRNA was normalized by dividing by the amount of ß-actin or 36B4 PCR mRNA. Sequences of the primers for real-time and semiquantitative RT-PCR are available upon request.

CoIP Assays
Cos-1 cells in six-well plates or 10-cm plates were transfected with expression plasmid DNA for HNF-4 and PGC-1{alpha} and increasing amounts of plasmid DNA for SREBP-1c. After 24 h, cell lysates were prepared, and CoIP was carried out as described (6, 41, 42). Briefly, cells were harvested and resuspended in 250 µl for six-well plates or 500 µl for 10-cm plates of lysis buffer [20 mM K+-HEPES (pH 8.0), 0.2 mM EDTA, 5% glycerol, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM dithiothreitol, and protease inhibitors). After incubation on ice for 10 min followed by brief sonication and centrifugation, 1–3 µg of either control IgG or HNF-4 antibody (sc-8987; Santa Cruz Biotechnology, Santa Cruz, CA) was added, and the samples were incubated for 4 h to overnight at 4 C. Immune complexes were collected by incubation with 25 µl 25% protein A-Sepharose slurry for 2 h followed by centrifugation. Immunoprecipitates were washed four times with lysis buffer (supplemented with NaCl up to 250 mM). The presence of PGC-1{alpha} and SREBP-1c in the immunoprecipitates was detected by Western blotting using antisera against PGC-1{alpha} (sc-13067) and SREBP-1c (sc-8984). For in vivo studies, mice were either fasted for 16 h or fasted for 16 h and then fed for 5–6 h. Liver nuclei were isolated by homogenization followed by centrifugation through a sucrose cushion, and nuclear extracts were prepared. About 600–800 µg nuclear extracts were used for each CoIP reaction using control IgG or HNF-4 antibody as described above.

GST Pull-Down Assays
GST and GST fusion proteins, GST-HNF-4, GST-SREBP-1c wild type, and GST-SREBP-1c mutants, were expressed in Escherichia coli BL21-RIL [DE3 (pLys)] and purified by binding to glutathione-Sepharose (Pharmacia, Piscataway, NJ). 35S-labeled HNF-4 (37) and SREBP-1c (32) proteins were synthesized by in vitro transcription and translation (TNT kit; Promega) according to manufacturer’s instructions. One microgram of GST, GST-HNF-4, or GST-SREBP-1c wild type and mutants was incubated at 4 C for 2 h with 2–3 µl reticulocyte lysate containing the labeled proteins in 100 µl binding buffer [25 mM HEPES-KOH (pH 7.6), 100 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 1% Nonidet P-40) in the presence of protease inhibitors. After the incubation, the Sepharose beads were extensively washed, proteins were eluted with SDS-PAGE buffer and separated by SDS-PAGE, and radioactivity was visualized by autoradiography.

Transient Transfection and Reporter Assay
HepG2 or Cos-1 cells were cotransfected with plasmid DNA of –371 hCYP7A1-luc (62), –1887 hCYP7A1-luc (62), –1887 hCYP7A1 HNF-4 site-mutated-luc (5), –371 mouse Cyp7a1-luc, Gal4-TK-luc reporter (63), G4DBD, G4DBD-HNF-4 (130–368) (64), G4DBD-ER, G4DBD-CAR, G4DBD-human PXR (6), Gal4 DBD-FXR (65), pCMV-HNF-4, pcDNA3-PGC-1{alpha} (6), pcDNA3.1-SREBP-1c, and the SREBP-1c mutant, which express constitutive nuclear mature forms of SREBP-1c (32), as indicated in the figure legends. Empty vector DNA was added as needed so that the same amounts of CMV expression vector DNA were present in each transfection. Transfection was carried out using Lipofectamine 2000 in 24-well plates. Twenty-four hours after transfection, cells were collected, and luciferase and ß-galactosidase activities were determined. Firefly luciferase activities were divided by ß-galactosidase activities to normalize for transfection efficiency. Consistent results were observed in at least two independent triplicate transfection assays in each experiment.

ChIP Assays
ChIP assays with chromatin isolated from HepG2 cells or in mouse liver were essentially carried out as described (5, 6, 41, 42). Details of mouse liver ChIP methodology are in supplemental Materials and Methods (published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). For HepG2 cells, cells were incubated in serum-free media containing 0.1% BSA overnight and then treated with 100 nM insulin (Sigma Chemical Co., St. Louis, MO) in the presence of BSA for 6 h. For exogenous expression of SREBP-1c, HepG2 cells in 15-cm plates (about 1–2 x 107 cells per plate) were transfected by electroporation with pcDNA3.1-SREBP-1c, or pcDNA3.1 empty vector as a control, and incubated for 24 h. Chromatin was isolated from the cells and incubated with 2–3 µg antibodies or normal serum. The immune complex was collected by centrifugation and extensively washed, and the bound chromatin was eluted as described (5, 6, 42). Genomic DNA was purified and used as a template for semiquantitative PCR using primer sets for CYP7A1 and GAPDH as a control. The amounts of genomic DNA and PCR cycles used were determined to be within a linear range of amplification. Primer sequences are available upon request. Each ChIP experiment was repeated two to four times with reproducible results.


   ACKNOWLEDGMENTS
 
We thank Drs. Jae Bum Kim, H. Shimano, B. Katzenellenbogen, R. Sato, I. Talianidis, A. Fukamizu, and J. Chiang for kindly providing plasmids. We thank Dr. T. Willson for providing GW4064. We thank Dr. B. Kemper for critically reading the manuscript.


   FOOTNOTES
 
This work was supported by the National Institutes of Health Grant DK62777 and American Heart Association Grant 557772 (to J.K.K.).

Disclosure Statement: B.P., S. F., and J.K.K. have nothing to declare.

First Published Online July 17, 2007

Abbreviations: Ad, Adenovirus; CAR, constitutive androstane receptor; ChIP, chromatin immunoprecipitation; CoIP, coimmunoprecipitation; CYP, cytochromes p450; DBD, DNA-binding domain; DN, dominant-negative; ER, estrogen receptor; FAS, fatty acid synthase; FXR, farnesoid X receptor; GRIP-1, glucocorticoid receptor interacting protein-1; GST, glutathione S-transferase; HDAC, histone deacetylase; HNF-4, hepatic nuclear factor-4; LBD, ligand-binding domain; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1{alpha}, peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha}; PKB, protein kinase B; PXR, pregnane X receptor; SHP, small heterodimer partner; SRC-1, steroid receptor coactivator-1; SREBP, sterol-responsive element-binding protein.

Received for publication April 19, 2007. Accepted for publication July 11, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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NURSA Molecule Pages Link:

Nuclear Receptors:   SHP  |  FXRα  |  PXR  |  CAR  |  HNF4α  |  ERα
Coregulators:   PGC-1  |  SRC-1  |  GRIP1  |  AIB1
Ligands:   Rifampicin  |  GW4064  |  17β-Estradiol  |  TCPOBOP



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