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Molecular Endocrinology 16 (6): 1367-1377
Copyright © 2002 by The Endocrine Society

Multiprotein Bridging Factor-1 (MBF-1) Is a Cofactor for Nuclear Receptors that Regulate Lipid Metabolism

Carole Brendel1, Laurent Gelman1 and Johan Auwerx

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre Nationale de la Recherche Scientifique/INSERM/Université Louis Pasteur, BP 163, 67404 Illkirch, France

Address all correspondence and reprint requests to: Dr. Johan Auwerx, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Parc d’Innovation, 1 rue Laurent Fries, 67404 Illkirch, France. E-mail: auwerx{at}igbmc.u-strasbg.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Multiprotein bridging factor (MBF-1) is a cofactor that was first described for its capacity to modulate the activity of fushi tarazu factor 1, a nuclear receptor originally implicated in Drosophila development. Recently, it has been shown that human MBF-1 stimulates the transcriptional activity of steroidogenic factor 1, a human homolog of fushi tarazu factor 1, which is implicated in steroidogenesis. Here we show that this cofactor enhances the transcriptional activity of several nonsteroid nuclear receptors that are implicated in lipid metabolism, i.e. the liver receptor homolog 1, the liver X receptor {alpha}, and PPAR{gamma}. MBF-1 interacts with distinct domains in these receptors, depending on whether the receptor binds DNA as a monomer or as a heterodimer with RXR. MBF-1 does not possess any of the classical histone modifying activities such as histone acetyl- or methyl transferase activities, linked to chromatin remodeling, but interacts in vitro with the transcription factor IID complex. MBF-1 seems therefore to act as a bridging factor enabling interactions of nuclear receptors with the transcription machinery.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MULTIPROTEIN BRIDGING FACTOR 1 (MBF-1) is a cofactor first identified in Bombyx mori (Bm) for its ability to interact with fushi tarazu factor 1 (Ftz-F1), a nuclear receptor that binds to DNA as a monomer (1). In vitro, BmMBF-1 can bind to the TATA box binding protein (TBP), suggesting that BmMBF-1 could recruit TBP at the promoters of Ftz-F1 target genes (2). Homologs of BmMBF-1 have subsequently been discovered. Yeast MBF-1 is a coactivator for the transcription factor GCN4 (3). In humans, human MBF-1 has been first identified as a factor implicated in endothelial differentiation (endothelial differentiation-related factor 1) (4). More recently human MBF-1 was also described as a transcriptional coactivator capable of interacting with the nuclear receptors SF-1/Ad4BP (steroidogenic factor-1/adrenal 4-binding protein), as well as with proteins of the cAMP response element-binding protein transcription factor family, such as activating transcription factor 1 (5).

To exert their regulatory activity on transcription, nuclear receptors recruit the transcriptional machinery at their target promoters. Some nuclear receptors are able to interact directly with elements of the core transcription machinery such as TBP or TBP-associated factors (TAFs). Nevertheless, cofactors seem to be necessary for activation to take place. Nuclear receptors implicated in lipid metabolism are no exception to this rule, and a better understanding of the mechanism underlying their transcriptional activity could be of particular interest in the prevention and treatment of metabolic diseases such as hyperlipidemia and atherosclerosis.

This study originated from an attempt to identify new cofactors for a well established nuclear receptor implicated in lipid metabolism, liver receptor homolog 1 (LRH-1). Based on the structural similarity between LRH-1 and SF-1, we first tested the interaction of MBF-1 with LRH-1 and then extended our studies to other nuclear receptors implicated in the control of metabolism, such as the liver X receptor {alpha} (LXR{alpha}) and PPAR{gamma}. We show here that MBF-1 is a coactivator not only for LRH-1, but also for LXR{alpha} and PPAR{gamma}. Although MBF-1 is known to bind to TBP, the precise mechanism by which it activates transcription remains unclear. We therefore tested whether MBF-1 was able to modify chromatin conformation through histone modification and/or through interaction with other components of the basal transcription machinery. We show here that MBF-1 has no enzymatic activity resulting in histone modification but interacts with the entire transcription factor IID (TFIID) complex.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MBF-1 Stimulates LRH-1, LXR{alpha}, and PPAR{gamma} Transcriptional Activities in Transfected Cells
Until now, very little information is available concerning LRH-1 cofactors (6, 7). Because MBF-1 has been described as a cofactor for SF-1, and because LRH-1 is a very similar receptor, we first addressed the question of whether MBF-1 could also enhance LRH-1-mediated gene activation on the promoter of the small heterodimer partner (SHP) gene, a well established LRH-1 target gene (8). RK13 cells were therefore cotransfected with expression vectors for LRH-1 and MBF-1 as well as a reporter construct where the SHP promoter drives the expression of the luciferase gene. As shown in Fig. 1AGo, LRH-1-mediated transactivation is stimulated in a dose-dependent manner by MBF-1 (Fig. 1AGo).



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  		Figure 1. MBF-1 Stimulates the Transcriptional Activity of LRH-1, LXR{alpha}, and PPAR{gamma} in Transfected Cells

A, RK13 cells were cotransfected with the pGL3-hSHP(569)-Luc reporter construct (1 µg/well), with an expression vector for LRH-1 (0.5 µg/well), and increasing amounts of an expression vector for MBF-1 (0, 2, 6 µg/well). B, RK13 cells were cotransfected with the pGL3-(LXRE)5TK-Luc reporter construct (400 ng/well), with an expression vector for LXR{alpha} (0.8 µg/well), and increasing amounts of an expression vector for MBF-1 (0, 240, 480 ng/well). Cells were then grown during 24 h in the presence or absence of 10-5 M 22(R)-hydroxycholesterol (22(R)-HC). C, RK13 cells were cotransfected with the pGL3-(Jwt)3TK-Luc reporter construct (400 ng/well), with an expression vector for PPAR{gamma} (0.8 µg/well), and increasing amounts of an expression vector for MBF-1 (0, 240, 800 ng/well). Cells were then grown during 24 h in the presence or absence of 10-7 M rosiglitazone (Rosi) or 10-5 M 15-deoxy-{Delta}12,14-PGJ2 (15PGJ2).

 
In many cases LRH-1 is able to bind to and to activate the same target genes as LXR{alpha}, another nuclear receptor implicated in lipid metabolism (9). Moreover, LRH-1 has been shown to act as a competence factor for LXR{alpha} in certain circumstances (6, 10). We therefore tested whether MBF-1 could also coactivate LXR{alpha} in transient transfection assays, using a luciferase reporter gene driven by multimerized response elements for LXR, in the absence or presence of 22(R)-hydroxycholesterol, a LXR ligand (Fig. 1BGo). LXR{alpha} activity was stimulated in a dose-dependent manner by MBF-1 both in the presence or absence of 22(R)-hydroxycholesterol. The enhancement of the expression of the reporter gene by MBF-1 when no exogenous LXR{alpha} is transfected can be explained by the presence of a small amount of endogenous LXR{alpha} or -ß in the cells used for transfection.

In contrast to LRH-1, LXR{alpha} belongs to a subgroup of nuclear receptors that require heterodimerization with RXRs to transactivate their target genes. We next tested whether MBF-1 could also activate another member of this family, also implicated in lipid metabolism, i.e. PPAR{gamma} (11). A peroxisomal proliferator response element-driven reporter construct pGL3-(Jwt)3TKLuc was therefore transfected together with an expression vector for PPAR{gamma}, and increasing amounts of MBF-1, in the presence or absence of a synthetic (rosiglitazone) or a natural (15-deoxy-{Delta}12,14-PGJ2) PPAR{gamma} ligand (Fig. 1CGo). PPAR{gamma} transcriptional activity was also stimulated in a dose-dependent manner by MBF-1. The effect of MBF-1 on PPAR{gamma} activity was again evident both in the presence or absence of agonists.

MBF-1 could act as a coactivator in vivo only if coexpressed with these receptors. MBF-1 has already been shown to be ubiquitously expressed (4, 5), with high levels in liver and colon, two tissues in which LRH-1, LXR{alpha}, or PPAR{gamma} are expressed and have important functions. We performed complementary Northern blot studies and confirmed that MBF-1 is broadly expressed in almost all cell lines or tissues analyzed (Fig. 2Go). Most notable was the demonstration that MBF-1 mRNA is expressed in many cells and tissues in which LRH-1, LXR{alpha}, and PPAR{gamma} are active, e.g. cells of hepatic origin (HepG2 and Hep3B cells, Fig. 2AGo, lanes 8 and 9) and liver (Fig. 2BGo, lane 1), cells of intestinal origin (CaCo2 cells, Fig. 2AGo, lanes 3 and 4), and intestine (Fig. 2BGo, lane 13), as well as adipose tissue (Fig. 2BGo, lane 2). MBF-1 expression in adipose tissue is noteworthy because it has not yet been established whether MBF-1 is expressed there.



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  		Figure 2. MBF-1 Is Expressed in the Same Tissues as LRH-1, LXR{alpha}, and PPAR{gamma}

RNA was isolated from different cell lines (A) or rat tissues (B) and analyzed by Northern blot hybridization with a MBF-1 labeled cDNA probe. Equivalent amounts of loaded RNA in each lane (20 µg) were confirmed by hybridization to control 36B4 probe. B, 1: liver; 2: adipose tissue; 3: muscle; 4: heart; 5: adrenals male; 6: adrenals female; 7: brain; 8: spleen; 9: kidney; 10: testis; 11: ovary; 12: lung; 13: intestine. Ndiff., Nondifferentiated; diff., differentiated.

 
MBF-1 Interacts in Vitro with LRH-1, LXR{alpha}, PPAR{gamma}, and RXR{alpha}
To test whether MBF-1 interacts directly with LRH-1, LXR{alpha}, and PPAR{gamma}, pull-down experiments with purified proteins were carried out. The different nuclear receptors were in vitro translated and incubated with either glutathione-S-transferase (GST)-MBF-1 or GST-p300Nt fusion proteins, the latter being used as a positive control (12). We previously established that LRH-1 and LXR{alpha} interacted in vitro with p300 (C. Brendel, unpublished results). LRH-1, LXR{alpha}, and PPAR{gamma} all interacted with MBF-1 in GST pull-down assays (Fig. 3Go, A–C). No interaction was detected with the GST protein alone. In addition, RXR{alpha}, the obligate heterodimerization partner for LXR{alpha} and PPAR{gamma}, could also interact with MBF-1 (Fig. 3DGo). The interaction between PPAR{gamma} and MBF-1 was not affected by the presence of the synthetic PPAR{gamma} ligand rosiglitazone (Fig. 3CGo). This was in contrast to the interaction between PPAR{gamma} and p300, which was stimulated by rosiglitazone.



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  		Figure 3. MBF-1 Interacts in Vitro with LRH-1, LXR{alpha}, and PPAR{gamma}

35S-Radiolabeled, in vitro translated LRH-1 (A), LXR{alpha} (B), PPAR{gamma} (C), or RXR{alpha} (D) proteins were incubated with GST, GST-MBF-1, or GST-p300Nt fusion proteins bound to glutathione-Q Sepharose beads, in the absence or presence of 10-5 M rosiglitazone (Rosi) in the case of PPAR{gamma}. The beads were then washed and the samples separated on a 12% SDS-polyacrylamide gel. Proteins were then detected by autoradiography.

 
MBF-1 Interacts with Distinct Domains in Nuclear Receptors, Depending on Their DNA-Binding Mode
Nuclear receptors are in general composed of distinct domains: an A/B domain, which harbors the ligand-independent activating function 1 (AF-1), a C domain for DNA binding, a D domain often referred to as the hinge region, and an E domain, which harbors the ligand-binding domain and the ligand-dependent activating function 2 (AF-2) (13). To determine which of these domains is implicated in the interaction with MBF-1, deletion mutants of these receptors were either produced in vitro or purified from bacteria and incubated with GST-MBF-1. As expected, the Ftz-F1 box of LRH-1 was necessary for the interaction of LRH-1 with MBF-1, which is consistent with what was previously published for other receptors of the Ftz-F1 subfamily (2, 5). Interestingly, the DE domain was fully dispensable for the interaction between LRH-1 and MBF-1 (Fig. 4AGo). The DE domains of both LXR{alpha} and PPAR{gamma}, however, were necessary and sufficient for the interaction with MBF-1 (Fig. 4Go, B and C). As observed in the transfection assays (Fig. 1Go), the interaction between LXR{alpha}DE and MBF-1 was neither altered by the addition of the natural LXR{alpha} ligand 22(R)-hydroxycholesterol (Fig. 4BGo) nor by the addition of a synthetic ligand (T0901317) (data not shown). Similarly, the interaction between PPAR{gamma}DE and MBF-1 was not altered by the addition of rosiglitazone, whereas the interaction between PPAR{gamma} and p300Nt was enhanced by the presence of a ligand (Figs. 3CGo and 4CGo).



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  		Figure 4. MBF-1 Interacts with Distinct Domains in Nuclear Receptors that Bind DNA as Monomers vs. Heterodimers

A and B, Different 35S-radiolabeled, in vitro translated LRH-1 (A) or LXR{alpha} (B) domains, which are schematically represented, were incubated with GST or the GST-MBF-1 fusion protein bound to glutathione-Q Sepharose beads. When indicated 22(R)-hydroxycholesterol (22(R)-HC) was added at 10-4 M. The beads were then washed and the samples separated on a 4–20% gradient or 15% SDS-polyacrylamide gel. Proteins were detected by autoradiography. C, In vitro translated PPAR{gamma}ABC and purified PPAR{gamma}DE proteins, which are schematically represented, were incubated with GST or the GST-MBF-1 fusion protein bound to glutathione-Q Sepharose beads. When indicated rosiglitazone (Rosi) was added at 10-5 M. The beads were then washed and the samples separated on a 15% SDS-polyacrylamide gel. Proteins were detected by Western blot using antibodies directed against the AB or DE domains of PPAR{gamma}.

 
Next we mapped the regions in MBF-1 that contact the three receptors. Pull-down experiments were performed using in vitro translated LRH-1, LXR{alpha}, and PPAR{gamma} and different regions of MBF-1 fused to the GST protein (Fig. 5Go). MBF-1 interacts with all the above nuclear receptors principally through its central region (amino acids 37–113). This interaction domain coincides with the domain previously shown to be required for the interaction with SF-1 (5). It seems therefore that the interaction domain in MBF-1 necessary for the binding with the different nuclear receptors is the same regardless of the DNA-binding modes of these receptors.



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  		Figure 5. The Central Domain of MBF-1 Is Necessary and Sufficient for Interaction with LRH-1, LXR{alpha}, and PPAR{gamma}

A, Schematic representation depicting the MBF-1 domain structure and the various constructs used in the GST pull-down assays. B, 35S-radiolabeled, in vitro translated LRH-1, LXR{alpha}, or PPAR{gamma} proteins were incubated with GST or the GST-MBF-1 deletion mutants bound to glutathione-Q Sepharose beads. The beads were then washed and the samples separated on a 12% SDS-polyacrylamide gel. Proteins were detected by autoradiography.

 
MBF-1 Interacts with LXR{alpha} and PPAR{gamma} in Mammalian Cells and Stimulates Specifically Their Ligand-Dependent Transcriptional Activity
Because our pull-down experiments pointed out a new mode of interaction between MBF-1 and nuclear receptors of the LXR{alpha} and PPAR{gamma} family, we wanted to confirm this interaction mechanism in a more physiological environment by testing the association of MBF-1 and the DE domains of LXR{alpha} and PPAR{gamma} in a mammalian two-hybrid system. We used, on the one hand, expression vectors for chimeric proteins composed of the DE domains of LXR{alpha} or PPAR{gamma} fused to the Gal4 DNA binding domain and, on the other hand, an expression vector for a fusion protein of MBF-1 and the activation domain of the VP16 transcription factor. The luciferase reporter construct comprises five tandem repeats of the Gal4 UAS cloned upstream of the thymidine kinase promoter and the luciferase reporter gene. The ectopic expression of VP16-MBF-1, compared with VP16 alone, resulted in a 2.5-fold and 10-fold induction of the luciferase activity when DNA-binding domain (DBD)-Gal4-LXR{alpha}DE and DBD-Gal4-PPAR{gamma}DE, respectively, were present (Fig. 6Go, B and C). These results confirm our in vitro data and show that the DE domains of LXR{alpha} and PPAR{gamma} are sufficient to recruit MBF-1 to promoters in vivo. However, in the case of LXR (but not PPAR{gamma}), ligand had to be added to demonstrate the interaction in the mammalian two-hybrid assay (Fig. 6Go, A and B). In contrast, ligand was not necessary for the interaction between MBF-1 and LXR in transfection and GST pull-down assays (Figs. 1Go, 3Go, and 4Go). In the mammalian two-hybrid experiments, it is possible that the LXR DE domain is not folded in the same fashion due to its fusion to the Gal4 DBD domain. Alternatively, LXR could be bound to a corepressor that must be displaced by the binding of a ligand before MBF-1 can be recruited. To distinguish between these hypothesis, a GST pull-down experiment with GST-MBF-1 and DBD-Gal4-LXRDE was performed. No significant interaction between in vitro translated DBD-Gal4-LXRDE and GST-MBF-1 was detected either in the absence or in the presence of a ligand (data not shown). These results suggest that the chimeric DBD-Gal4-LXRDE protein is probably folded differently compared with the nonchimeric protein that interacts in the presence and absence of a ligand (Fig. 4Go). In the mammalian two-hybrid system, under normal cellular conditions (i.e. pH and ionic strength), this defect is overcome in the presence of a ligand, and DBD-Gal4-LXRDE and MBF-1 are then able to interact (Fig. 6BGo).



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  		Figure 6. MBF-1 Interacts with LXR{alpha} and PPAR{gamma} in a Mammalian Two-Hybrid System

A schematic representation of the experiments is shown at the bottom of the figures. DE, Ligand binding domain of LXR{alpha} or PPAR{gamma}. A and B, RK-13 cells were cotransfected with expression vectors for the DBD-Gal4 or DBD-Gal4-LXR{alpha}DE fusion proteins (0, 2 µg/well), and expression vectors encoding VP16 or VP16-MBF-1 (1 µg/well) and the reporter construct pGl3-(UAS)5TK-Luc (1 µg/well). Cells were grown 24 h in the absence (A) or presence (B) of 10-5 M 22(R)-hydroxycholesterol. The histogram represents the transcriptional activity of the DBD-Gal4 or DBD-Gal4-LXR{alpha}DE fusion proteins in the presence of cotransfected pCMX-VP16-MBF-1 compared with their activity in the presence of the cotransfected pCMX-VP16 vector, which was set arbitrarily to 1. C, An experiment similar to that in panel A was performed using DBD-Gal4-PPAR{gamma}DE fusion protein in the absence of ligand. D, RK13 cells were cotransfected with expression vectors for the DBD-Gal4, or DBD-Gal4-LXR{alpha}DE fusion proteins (80 ng/well), an expression vector for MBF-1 (480 ng/well), and the pGL3-(UAS)5TKLuc reporter construct (400 ng/well). Cells were then grown 24 h in the presence of 10-5 M 22(R)-hydroxycholesterol. The histogram represents the transcriptional activity of the DBD-Gal4 or DBD-Gal4-LXR{alpha}DE fusion proteins in the presence of cotransfected pCMX-MBF-1 compared with their activity in the presence of the cotransfected pCMX empty vector, which was set arbitrarily to 1. E, An experiment similar to that in panel D was performed using the DBD-Gal4-PPAR{gamma}DE fusion protein in the presence of 10-6 M rosiglitazone.

 
The functional relevance of the interaction between the DE domains of LXR{alpha} and PPAR{gamma} was then addressed by studying specifically the effects of MBF-1 expression on LXR{alpha} and PPAR{gamma} transcriptional activities. We used a modified mammalian two-hybrid system in which MBF-1 was no longer fused to an artificial transactivating domain but was expressed as the full-length, wild-type protein. Addition of MBF-1 increased the transcriptional activities of the chimeric DBD-Gal4-LXR{alpha}DE and DBD-Gal4-PPAR{gamma}DE proteins in the presence of a ligand (Fig. 6Go, D and E), supporting the idea that MBF-1 coactivates the AF-2 function of LXR{alpha} and PPAR{gamma} in vivo. Little enhancement of transcription can be seen in the absence of ligand (data not shown). Together with the transfection data shown in Fig. 1Go, B and C, the data in Fig. 6Go suggest that the interaction between the DE domains of LXR{alpha} and PPAR{gamma}, on the one hand, and MBF-1, on the other hand, occur when the DE domains of the receptors are docked to DNA.

MBF-1 Has No Intrinsic Transcriptional Activity and Has Neither Histone Acetyltransferase nor Methyltransferase Activity in Vitro
In an attempt to determine the molecular mechanisms underlying coactivation by MBF-1, we first tested whether this cofactor could on its own induce transcription when tethered to a promoter. We constructed a mammalian expression vector for a chimeric protein composed of the Gal4 DBD and full-length MBF-1. Expression of DBD-Gal4-MBF-1 did not result in activation of transcription on a Gal4-responsive reporter construct, whereas the DBD-Gal4-PPAR{gamma}DE fusion protein did so in the presence of a synthetic PPAR{gamma} ligand (Fig. 7Go). We conclude from this experiment that MBF-1 is not endowed with any activating domain.



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  		Figure 7. MBF-1 Has No Intrinsic Transcriptional Activity

RK13 cells were transfected with increasing amounts of an expression vector for the DBD-Gal4-MBF-1 fusion protein (2, 10, 40, 100 ng/well) or with an expression vector for DBD-Gal4-PPAR{gamma}DE (40 ng/well), in the presence of the pGL3-TK-Luc or pGL3-(UAS)5TK-Luc reporter constructs (200 ng/well). Cells were then grown 24 h before harvesting for luciferase determination. For DBD-Gal4-PPAR{gamma}DE, 10-6 M rosiglitazone was added when indicated. A schematic representation of the experiment is shown at the bottom of the graph.

 
As several coactivators affect the structure of chromatin through histone modifications, we tested whether MBF-1 has histone acetyltransferase or methyltransferase activity. For the histone acetylase assay, core histones were incubated with 14C acetyl-coenzyme A (CoA) and either MBF-1 or p300, which was used as a positive control, or both proteins together. Although p300 was able to acetylate histones, no acetylation could be detected in the presence of MBF-1 alone (Fig. 8AGo). The degree of histone acetylation achieved by p300 was furthermore not altered by the addition of MBF-1. A similar experiment was done to analyze whether MBF-1 has any methyltransferase activity. The coactivator-associated arginine (R) methyltransferase-1 (CARM-1) was used as a positive control in our assay (Fig. 8BGo). We could not detect any methylase activity for MBF-1 or any change in CARM-1 activity upon addition of MBF-1.



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  		Figure 8. MBF-1 Has Neither Histone Acetyl Transferase nor Methyl Transferase Activity in Vitro

A, Core histones (0.17 µg) were incubated 1 h at 30 C with 14C-acetyl-CoA, p300, MBF-1 (250–500 ng), or both proteins together. Samples were then boiled in 2x sample buffer and separated on a 15% SDS polyacrylamide gel. The amount of histones was visualized by Coomassie staining, and histone acetylation was visualized by autoradiography. B, Core histones (1.3 µg) were incubated 1 h at 30 C with 3H-S-adenosyl-L-methionine and CARM-1 or MBF-1. Samples were then boiled in 2x sample buffer and separated on a 15% SDS polyacrylamide gel. The amount of histones was visualized by Coomassie staining, and histone methylation was visualized by autoradiography.

 
MBF-1 Interacts with TFIID in Vitro
Another explanation for MBF-1 action on transcription would be that MBF-1 acts as a bridging factor between nuclear receptors and the basal transcription machinery. It has been shown previously that MBF-1 can interact with TBP (1, 2). Nevertheless, TBP occurs in vivo within distinct protein complexes, the most notable being TFIID, in which the numerous TAFs surrounding TBP could preclude its interaction with MBF-1. We therefore performed pull-down experiments using the purified TFIID complex and the GST-MBF-1 protein. GST-MBF-1 interacts both with the TBP protein alone as well as the TFIID complex, whereas the GST protein alone interacts with none of them (Fig. 9Go).



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  		Figure 9. MBF-1 Interacts with TFIID in Vitro

The TFIID complex (A) or in vitro translated TBP (B) was incubated with GST or the GST-MBF-1 fusion protein bound to glutathione-Q Sepharose beads. The beads were then washed and the samples separated on a 7.5% SDS-polyacrylamide gel. Proteins were detected by Western blot using an antibody directed against TBP.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors are one of the largest families of transcription factors. Some unliganded receptors can actively repress transcription by interacting with corepressors. After ligand binding, conformation of these receptors changes, allowing the recruitment of coactivators and consequent activation of transcription. Cofactors can modify the structure of chromatin. These modifications can lead either to a condensation of chromatin, which represses transcription, or to a decondensation, which facilitates the recruitment of the basal transcription machinery. Certain cofactors also favor the formation of preinitiation complexes by making direct interactions with components of the basal transcription machinery. MBF-1 is a cofactor first identified in Bombyx mori for its ability to induce the activity of the Ftz-F1 nuclear receptor (1). Subsequently, it was shown that the yeast homolog of MBF-1 interacts with the GCN4 transcription factor (3), whereas the human homolog was shown to interact with a monomeric nuclear receptor, SF-1, and other transcription factors such as activating transcription factor 1 (5).

We first show here that MBF-1 enhances the transcriptional activity of three nuclear receptors involved in lipid metabolism, i.e. LRH-1, LXR{alpha}, and PPAR{gamma} (11, 14, 15). Hence, MBF-1 could be itself a new key component of lipid homeostasis as it could potentially coordinate the action of these nuclear receptors. All the nuclear receptors and transcription factors that were reported to bind MBF-1 so far interact with MBF-1 via a basic region. Therefore, it was first inferred that this basic motif was the hallmark of all MBF-1 partners (5). LRH-1 also contains such a motif, and its interaction with MBF-1, which implicates this motif, was somehow expected. However, we clearly demonstrate here that MBF-1 is also a cofactor for LXR{alpha} and PPAR{gamma}, which do not contain a motif resembling the previously identified basic region. MBF-1 interacts with the DE domains of LXR{alpha} and PPAR{gamma}, thereby coactivating their ligand-dependent transcriptional activity. In the absence of ligand, MBF-1 interacted with both LXR and PPAR in transfection and GST pull-down assays (Figs. 1Go, 3Go, and 4Go). The ligand-independent interaction between MBF-1 and PPAR{gamma} could also be seen in the mammalian two-hybrid system (Fig. 6CGo). The interaction between VP16-MBF-1 and DBD-Gal4-LXRDE, however, could only be detected in the mammalian two-hybrid system in the presence of a ligand (Fig. 6BGo). In transfections, the promoter context might facilitate the ligand-independent recruitment of MBF-1 by LXR. In addition, these cells probably contain other factors, such as endogenous oxysterol ligands, that could influence interactions between MBF-1 and LXR.

Interestingly, MBF-1 does not possess a characteristic LxxLL or IxxII motif, a hallmark of many cofactors that is implicated in the interaction with transcription factors, and seems to contact all nuclear receptors through the same central domain (amino acids 37–113). The different nuclear receptors apparently display different interaction interfaces for MBF-1, depending on their mode of interaction with DNA. In fact, it appears that nuclear receptors of the nuclear receptor 5 subfamily that preferentially bind DNA as monomers interact with MBF-1 through their basic Ftz-F1 box (belonging to the C domain), whereas receptors that heterodimerize with RXR and bind DNA as heterodimers seem to interact with MBF-1 through their DE domain. Although several nuclear receptors are shown here to interact with MBF-1, MBF-1 interaction is not a general feature of all nuclear receptors. In fact, retinoid orphan receptor {alpha}, which binds DNA as a monomer (16) but which belongs to the nuclear receptor 1 subfamily, does not interact with MBF-1 in vitro (data not shown). This result hence indicates that MBF-1 might be a specific cofactor for a subset of nuclear receptors.

MBF-1 does not contain an intrinsic activation domain and can hence not induce transcription by itself when tethered to a promoter (Fig. 7Go). Consistent with this, the interaction of MBF-1 with the receptors as well as its coactivating function could only be detected when the AF2 function of the receptors is activated by previous binding of a ligand (Fig. 6Go, D and E). Although MBF-1 acts as a coactivator, its exact mechanism of function is still unclear. MBF-1 does not seem to possess any of the histone modifying activities such as histone acetyl transferase or methylase activities, which have been associated with modulation of the transcriptional response. It had been shown previously that MBF-1 could interact with TBP in vitro (1, 2). Nevertheless, TBP participates in vivo in the formation of multiple complexes, and the factors surrounding TBP could preclude its interaction with MBF-1. Interestingly, we show here that MBF-1 can also interact directly with the TFIID complex, which consists of the TBP protein and TAFs (17). This interaction most probably occurs through a direct association between MBF-1 and TBP, the interaction surface of which must still be accessible in the TFIID complex, as supported by three-dimensional structure studies (18, 19). We cannot with certainty rule out, however, that MBF-1 also contacts some TAFs.

In Bombyx mori, MBF-1 functions in association with a partner, MBF-2, which is able to interact with TFIIA (20). Because no mammalian homolog of MBF-2 has yet been reported, it would be interesting to determine whether such a homolog exists and whether it fulfils the same function. Primary database searches and attempts to clone a putative human MBF-2 with the gene trapper cDNA-positive selection system (Life Technologies, Inc., Gaithersburg, MD) were unsuccessful (Brendel, C., unpublished data). It is therefore probable that MBF-1 does not require MBF-2 for its activity in higher species, or alternatively that the human counterpart of BmMBF-2 is very distant in protein and gene structure.

Due to the high conservation of MBF-1 between eukaryotes and the existence of an ortholog in several archea, MBF-1 has been considered as a putative basal transcription factor itself, which could be associated with the core transcription initiation complex (21). This hypothesis is further reinforced by our current data showing its interaction with both TBP and TFIID and by the fact that MBF-1 contains a helix-turn-helix structure, which is also found in TFIIB and TFIIE (21).

In conclusion, MBF-1 seems to function as a bridging factor between certain nuclear receptors and TFIID, hence increasing the transcriptional activity of these receptors. The sharing of a cofactor between LRH-1, LXR{alpha}, and PPAR{gamma} may be a way to coordinate and integrate signals derived from different transduction pathways in the cell and highlights a potential role for MBF-1 in lipid homeostasis. It is noteworthy that MBF-1 is the first positive cofactor described for human LRH-1. It will be interesting in the future to determine which other elements of the transcription machinery also interact with MBF-1 and how these interactions are regulated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
22(R)-Hydroxycholesterol was purchased from Sigma (St. Louis, MO). 15-Deoxy-{Delta}12,14-PGJ2 (15PGJ2) was purchased at BIOMOL Research Laboratories, Inc. (Plymouth, PA). LG100268 and rosiglitazone were kind gifts of R. Heyman (X-ceptor Therapeutics, San Diego, CA). The antibodies directed against the AB domain of PPAR{gamma} (PPAR{gamma}AB) were described before (22), the antibodies directed against the DE domain of PPAR{gamma} (PPAR{gamma}DE) were a kind gift of J. Berger (Merck \|[amp ]\| Co., Inc., Rahway, NJ), and the antibodies against TBP were a gift of L. Tora (Institut de Génétique et de Biologie Moléculaire et Cellulaire). Protease inhibitor cocktail was purchased at ICN Biochemicals, Inc. (Orsay, France).

Cell Culture and Transient Transfection Assays
RK-13 cells (ATCC, Manassas, VA) were maintained at 37 C, 5% CO2 and grown in MEM supplemented with 10% FCS, L-glutamine, and antibiotics [penicillin-streptomycin–seromed A2213 (Life Technologies, Cergy Pontoise, France)]. Cells were transfected by the calcium phosphate-DNA coprecipitation technique as described previously (23). Empty expression vectors were used to maintain equivalent amounts of DNA in the transfections. pCMX-MBF-1 is an expression vector containing the full-length human MBF-1 cDNA. pCMX-LRH-1 was produced by insertion of a PCR product, corresponding to the human LRH-1 cDNA, into the pCMX vector using EcoRI and XmaI restriction sites. The human LRH-1 PCR product and pCMX-LXR{alpha} were gifts. pSG5-hPPAR{gamma}2 was described elsewhere (22). The pGL3-(LXRE)5TK-Luc reporter construct contains five tandem repeats of the DR-4 LXR response element (5'-GCGGTTCCCAGGGTTTAAATAAGTTCATCTAGAT) cloned upstream of the herpes simplex virus thymidine kinase (TK) promoter and the luciferase (Luc) reporter gene. The pGL3-hSHP (569)-Luc and pGL3-(Jwt)3TK-Luc reporter constructs were described elsewhere (6). For mammalian two-hybrid assays, the full-length MBF-1 cDNA was cloned downstream of the VP16 activation domain to obtain pCMX-VP16-MBF-1. pcDNA3-DBD-Gal4-LXR{alpha}DE and pcDNA3-DBD-Gal4-hPPAR{gamma}DE are constructs where the ligand binding domain of LXR{alpha} and PPAR{gamma}, respectively, have been cloned downstream of the Gal4 DBD. The luciferase reporter construct pGL3-(UAS)5TK-Luc comprises five tandem repeats of the Gal4 upstream activating sequence (UAS) cloned upstream of the thymidine kinase promoter. To test the transcriptional activity of MBF-1, the full-length MBF-1 cDNA was cloned downstream of the Gal4 DBD to obtain pcDNA3-DBD-Gal4-MBF-1. The pCMV-ßGal vector was used as a control of transfection efficiency.

RNA Analysis
RNA extraction and Northern blot analysis of RNA were performed as described (23). MBF-1 mRNA was analyzed using a full-length human MBF-1 cDNA probe. A human acidic ribosomal phosphoprotein 36C4 cDNA clone was used as control (24). All probes were labeled by random priming (Roche Molecular Biochemicals, Mannheim, Germany).

Production of Proteins and Pull-Down Experiments
For GST pull-down assays, deletion mutants of LRH-1, LXR{alpha}, and PPAR{gamma} were subcloned downstream of the GST cDNA in the pGex-4T1 vector (Pharmacia Biotech, Orsay, France). The MBF-1-GST fusion proteins were expressed in Escherichia coli and purified on a glutathione affinity matrix (Pharmacia, St. Louis, MO).

TFIID was purified from nuclear extracts. Nuclear extracts were loaded on a heparin column (Heparine ultrogel A4R, IBF Biotechnics, Villeneuve-la-Garenne, France) equilibrated in buffer BC100 (Tris, pH 7.4, 20 mM; EDTA 0.2 mM; glycerol 20%; KCl 100 mM). The column was washed with BC100, and a TFIID-containing fraction was eluted by rinsing the column with BC500 (same buffer as BC100 but with 500 mM KCl). This fraction was dialyzed against BC100 and applied to a diethylaminoethyl-Sephacel column (Pharmacia Biotech). The diethylaminoethyl column was then washed with BC190 and a TFIID-containing fraction was eluted by rinsing the column with BC550. The TFIID in this fraction was then immunoprecipitated with the TBP-specific 5TF-2C1 monoclonal antibody coated on protein G-Sepharose beads (25). The beads were then washed with BC500, and the complex was eluted by adding the peptide used to raise the antibody at the concentration of 2 mg/ml in BC100.

In vitro 35S-radiolabeled translated proteins (TNT T7 Quick Rabbit Reticulocyte, Promega Corp., Madison, WI) or purified proteins (PPAR{gamma}DE, TFIID) were incubated 1 h at 25 C in pull-down buffer (PBS 1x, glycerol 10%, Nonidet P-40 0.5%, protease inhibitor cocktail) with either the GST protein alone or the different GST fusion proteins bound on glutathione-Q Sepharose beads. 22(R)-Hydroxycholesterol (10-4 M) or rosiglitazone (10-5 M) were also added when indicated. The beads were then washed five times in pull-down buffer and boiled in 2x sample buffer (12.5 mM Tris-HCl, 20% glycerol, 0.002% bromophenol blue, 5% ß-mercaptoethanol). The samples were separated on 12% SDS polyacrylamide gels, which were either dried when radiolabeled proteins were used or transferred to nitrocellulose membranes for Western blotting. Blots were developed with antibodies directed against PPAR{gamma}AB, PPAR{gamma}DE, or TBP.

Histone Acetyl Transferase and Methyl Transferase Assays
For the histone acetyl transferase assay, core histones (Roche, Meylan, France) (0.17 µg), were incubated 1 h at 30 C with 14C-acetyl CoA and p300 or MBF-1 (250–500 ng) in HAT buffer (Tris 50 mM, glycerol 10%, EDTA 0.1 mM, KCl 50 mM, sodium butyrate 20 mM, DTT 1 mM, and protease inhibitor cocktail). p300 was produced in baculovirus-infected-SF9 cells and purified on a nickel column as described elsewhere (26). For the methyl transferase assay, core histones (1.3 µg), were incubated 1 h at 30 C with 3H-S-adenosyl-L-methionine (Sigma) (3.35 µCi) and GST-CARM or GST-MBF-1 in MET buffer (Tris 20 mM, NaCl 1 M, and EDTA 4 mM). Samples were then boiled in 2x sample buffer and separated on a 15% SDS polyacrylamide gel. All histones were first stained with a Coomassie blue solution, after which the gel was dried, and acetylated or methylated histones were visualized by autoradiography.


   ACKNOWLEDGMENTS
 
We thank J. Berger, P. Chambon, J.-M. Egly, R. Heyman, J. Kadonaga, D. Mangelsdorf, O. Morand, K. Schoonjans, M. Stallcup, and L. Tora for the gift of materials and/or helpful discussions.


   FOOTNOTES
 
This work was supported by grants from the Centre Nationale de la Recherche Scientifique, INSERM, Hôpitaux Universitaires de Strasbourg, the Juvenile Diabetes Foundation, Association pour la Recherche contre le Cancer, the National Institute of Health, the Human Frontier Science Program, and the European Union. C.B. was supported by grants from the Société de Nutrition et de Diététique de Langue Française and Nestlé. L.G. was supported by grants from Laboratoires Fournier and the Association pour la Recherche contre le Cancer.

1 These authors contributed equally to this work. Back

Abbreviations: Bm, Bombyx mori; CARM-1, coactivator-associated arginine (R) methyltransferase-1; CoA, coenzyme A; DBD, DNA-binding domain; Ftz-F1, fushi tarazu factor 1; GST, glutathione-S-transferase; LRH-1, liver receptor homolog 1; LXR{alpha}, liver X receptor-{alpha}; MBF-1, multiprotein bridging factor 1; SF-1, steroidogenic factor 1; SHP, small heterodimeric partner; TAF, TBP-associated factor; TBP, TATA box binding protein; TFIID, transcription factor IID.

Received for publication September 26, 2001. Accepted for publication February 5, 2002.


   REFERENCES
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 DISCUSSION
 MATERIALS AND METHODS
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