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Nucleic Acids Research, 2003, Vol. 31, No. 22 6640-6650
© 2003 Oxford University Press

Article

Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4{alpha} dimerisation and transcriptional activity

Jérôme Eeckhoute, Bénédicte Oxombre, Pierre Formstecher, Philippe Lefebvre and Bernard Laine*

INSERM U459, Faculté de Médecine Henri Warembourg, 1 Place de Verdun, F-59045 Lille Cedex, France

*To whom correspondence should be addressed. Tel: +33 03 20 62 69 51; Fax: +33 03 20 62 68 84; Email: blaine{at}lille.inserm.fr

Received June 18, 2003; Revised August 25, 2003;. Accepted September 23, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocyte Nuclear Factor 4{alpha} (HNF4{alpha}, NR2A1) is central to hepatocyte and pancreatic ß-cell functions. Along with retinoid X receptor {alpha} (RXR{alpha}), HNF4{alpha} belongs to the nuclear receptor subfamily 2 (NR2), characterised by a conserved arginyl residue and a glutamate residue insert in helix 7 (H7) of the ligand binding domain (LBD). Crystallographic studies indicate that R348 and E352 residues in RXR{alpha} H7 are involved in charge-driven interactions that improve dimerisation. Consistent with these findings, we showed that removing the charge of the corresponding residues in HNF4{alpha} H7, R258 and E262, impaired dimerisation in solution. Moreover, our results provide a new concept according to which helices of the HNF4{alpha} LBD dimerisation interface contribute differently to dimerisation required for DNA binding; unlike H9 and H10, H7 is not involved in DNA binding. Substitutions of E262 decreased the repression of HNF4{alpha} transcriptional activity by a dominant-negative HNF4{alpha} mutant, highlighting the importance of this residue for dimerisation in the cell context. The E262 insert is crucial for HNF4{alpha} function since its deletion abolished HNF4{alpha} transcriptional activity and coactivator recruitment. The glutamate residue insert and the conserved arginyl residue in H7 most probably represent a signature of the NR2 subfamily of nuclear receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatocyte Nuclear Factor 4 is a transcription factor encoded by two genes, HNF4{alpha} and HNF4{gamma}, leading to two subtypes of proteins: HNF4{alpha} (NR2A1) and HNF4{gamma} (NR2A2) (1). HNF4{alpha} is central to embryogenesis (2,3) and is required for the normal function of hepatocytes and pancreatic ß-cells (4,5). HNF4{alpha} occupies a key position in a complex transcription factor network and directly regulates the expression of genes involved in the transport and metabolism of various nutrients, as well as that of genes coding for serum proteins (1). HNF4{alpha} is linked to human diseases: mutations in HNF4{alpha} response elements in promoters of factors VII and IX are correlated with haemophilia, while mutations in the HNF4{alpha} gene have been found in patients carrying the syndrome of maturity onset diabetes of the young 1 (MODY1) (1,6).

Like other members of the nuclear receptor superfamily, HNF4{alpha} has a modular structure consisting of functional domains (7). Two of these domains, the DNA binding domain (DBD) and the ligand binding domain (LBD) are involved in dimerisation of HNF4{alpha}, which behaves as a homodimer (8). Dimerisation via the DBD is involved in HNF4{alpha} dimerisation on DNA, whereas dimerisation via the LBD is essential for dimerisation in solution and strongly stabilises the HNF4{alpha}–DNA complex (9,10). The LBD is also involved in other functions, including transcriptional activation and interaction with transcriptional partners. Both domains are well conserved in HNF4{gamma} (11). Crystal structures of the LBDs of HNF4{alpha} and HNF4{gamma} have recently been resolved (12,13). These LBDs adopt the canonical fold of {alpha}-helices (10 helices numbered H1–H12 to follow the conventional nomenclature) and ß-sheets arranged as an antiparallel {alpha}-helical ‘sandwich’ in a three-layer structure shared with the LBD of other nuclear receptors (14). The LBD structures of HNF4{alpha} and HNF4{gamma} are very similar and closely resemble that of RXR{alpha}, another member of the nuclear receptor subfamily 2 (NR2). Indeed, 194 core {alpha} carbons of the LBD of HNF4{alpha} and RXR{alpha} superimpose with a root mean square deviation (rmsd) of 1.26 Å and 223 core {alpha} carbons of the LBD of HNF4{gamma} and RXR{alpha} superimpose with a r.m.s.d. of 1.00 Å (12,13). In these structures, the LBD dimerisation interface is made of residues in helices 7 (H7), 9 (H9) and 10 (H10), the major part of the interface being made up of H10.

HNF4{alpha} and HNF4{gamma}, which activate transcription in the absence of exogenous ligands, are considered to be constitutive transcriptional activators (1,11,13,15,16). Their LBDs are tightly associated with endogenous fatty acids, which do not act as classical ligands but are likely required for the stability of the protein conformation (12,13). HNF4{alpha} transcriptional activity can be enhanced by other transcription factors, including COUP-TF (17), Smad3 and Smad4 (18), or by coactivators, such as members of the p160 family (19,20), CBP/p300 (20–22) and PGC-1 (23). The HNF4{alpha} transcriptional activity can also be repressed by negative transcriptional partners such as SHP (24), p53 (25) and SMRT (26).

In the crystal structure of the RXR{alpha} LBD, H7 adopts an unusual {pi}-helical geometry that forces the glutamic acid residue in position 352 to bulge outward from the H7 axis (27). This structure gives rise to the formation of a series of intramolecular and intermolecular hydrogen bonds that improve RXR{alpha} LBD homodimerisation (27). In particular, residues E352 and R348 are directly involved in the dimerisation interface by forming charge-driven interactions. The {pi}-helical conformation near E352 in the RXR{alpha} homodimer and the resulting interactions were also observed in the PPAR{gamma}/RXR{alpha} LBD heterodimer (28). Sequence alignment indicates that a glutamic acid residue and an arginyl residue are also encountered at the equivalent positions in human and rat HNF4{alpha} (E262 and R258). Figure 1 shows that these residues are conserved in remote species such as Drosophila and in the Xenopus HNF4ß and are specifically encountered in members of the NR2 subfamily (27–29). Interestingly, in the crystal structure of the HNF4{alpha} LBD, a bulge near E262 was also observed in H7 (12). Gampe et al. described this glutamate as a single residue E insert in H7 and hypothesised that it may play a crucial role in the function of nuclear receptors belonging to the NR2 subfamily, including HNF4{alpha} (27,28). We investigated the role of the E262 and R258 amino acid residues in HNF4{alpha} function using biochemical and mutagenesis studies.



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  		Figure 1. Structure-based amino acid sequence alignment of H7 of nuclear receptor LBDs (adapted from references 27–29). Usual names of nuclear receptors are indicated on the left side whereas names proposed by the Nuclear Receptor Nomenclature Committee are indicated on the right side. The arrow and boxed R indicate the glutamate and arginyl residues specifically found in members of the NR2 subfamily. Positions of the functional domains, of the I-box and of activation function 2 (AF-2) are shown. *, position in isoform 2 of HNF4{alpha} (HNF4{alpha}2), which was used in this study.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DNA constructs
Plasmid pcDNA3 HNF4{alpha}2, described in Suaud et al. (30) was used as a template to create pcDNA3 HNF4{alpha}2-R258M, -E262A, -E262M, -E262K, -{Delta}E262, -{Delta}D261 and E327M constructs by site-directed mutagenesis using the QuickChangeTM kit from Stratagene according to the supplier’s recommendations. Vectors pcDNA3 HNF4{alpha}-{Delta}AF-2 (residues 1–358) and pSG5 HNF4{alpha}3 were described previously (31,32). Vector pcDNA3 HNF4{alpha}-{Delta}AF-2-E262A was generated by site-directed mutagenesis. Plasmid pGBKT7 HNF4{alpha}2, used to express HNF4{alpha}2 fused to a c-myc tag in vitro, was obtained by inserting a PCR fragment encompassing human HNF4{alpha}2 cDNA into the EcoRI and BamHI sites of pGBKT7 (Clontech). This vector was then used as a template to create pGBKT7 HNF4{alpha}2-E262A and -{Delta}E262 by site-directed mutagenesis. Plasmids pCMVß-NHA p300 and pGEX2TK p300(340–528) were kindly provided by S.R.Grossman. Plasmids pMT2 COUP-TFII, pTL1 myc-COUP-TFII {Delta}AB, pGEX5X2 PGC-1(36–797) and pGEX2TK SRC-1a(570–780) were gifts from S.K.Karathanasis, M.Leid, B.M.Spiegelman and M.Tsai, respectively. Plasmid pGEX2TK HNF4{alpha}2 was prepared by a strategy identical to that used for cloning pGEX2TK COUP-TFII (30) by inserting a PCR fragment encompassing the human HNF4{alpha}2 cDNA. This pGEX2TK HNF4{alpha}2 construct was then used as a template to generate pGEX2TK HNF4{alpha}2-E262A and -{Delta}E262 by site-directed mutagenesis. The human HNF1{alpha} promoter (–341/+183) cloned in pGL3 was a gift from G.Bell. All constructs were verified by DNA sequencing analysis.

Co-immunoprecipitation assays
Wild-type and mutated HNF4{alpha} were in vitro synthesised in reticulocyte lysates (Promega). In co-immunoprecipitation assays performed with an anti-c-myc tag antibody, 5 µl of non-radiolabelled wild-type or mutated c-myc-HNF4{alpha}2 was incubated with 5 µl of [35S]methionine-labelled wild-type or mutated HNF4{alpha}2 for 1 h at room temperature in 30 µl (final volume) of Ip buffer (50 mM Tris–HCl pH 7.0, 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.1% Nonidet P40, 0.2% bovine serum albumin, 0.5 mM phenylmethylsulphonyl fluoride and 0.1 mg/ml each of leupeptin, aprotinin and pepstatin). Then, 2 µg of anti-c-myc tag antibody (clone 9E10; Upstate Biotechnology) was added and incubation was continued for 1 h. An aliquot of 150 µl of Ip buffer containing 3 mg of hydrated protein A–Sepharose CL-4B beads (Sigma) was added and samples were incubated under constant agitation for an additional 1 h. After extensive washing of the beads with Ip buffer, bound proteins were resolved by SDS–PAGE. Co-immunoprecipitation assays performed with the {alpha}455 antiserum (33) were conducted similarly using 5 µl of non-radiolabelled wild-type or mutated HNF4{alpha}2, 5 µl of [35S]methionine-labelled HNF4{alpha}3 and 0.25 µl of {alpha}455 antiserum. Interactions were quantified using ImageQuant software on a PhosphorImager (Molecular Dynamics).

GST pull-down assays
GST pull-down assays were performed as described previously (30) using [35S]methionine-labelled, in vitro synthesised HNF4{alpha} and bacterially expressed GST fusion proteins. Interactions were quantified using ImageQuant software on a PhosphorImager (Molecular Dynamics).

Electrophoretic mobility shift assays (EMSA)
EMSA were performed using in vitro synthesised wild-type or mutated HNF4{alpha}2 and 32P-labelled oligonucleotides (0.2 ng) encompassing the HNF4{alpha} response element of the human HNF1{alpha} promoter (positions –66/–48) (1) or a mutated version of this response element (denoted HNF1 and HNF1 mt, respectively) or site B (position –67/–85) (1) of the human apolipoprotein CIII promoter (denoted CIIIB). Mutations in the HNF1 mt site are underlined in the following sequence, where half-sites are in upper case: tgaACTCCCaAGTTCAgtc. Complexes were formed in 20 µl (final volume) of binding buffer (100 mM KCl, 20 mM HEPES pH 7.9, 5 mM MgCl2, 0.1 mM EDTA, 20% glycerol, 1 mM DTT) in the presence of 1.25 µg of poly(dI·dC)–poly(dI·dC). Complexes were resolved by non-denaturing PAGE in 1x TEA. DNA binding was quantified using a PhosphorImager. In supershift assays, HNF4{alpha} proteins were incubated with 0.25 µl of the {alpha}455 HNF4{alpha} antiserum (33) for 15 min prior to adding the labelled probe.

Cell culture and transient transfection assays
Human embryonic kidney HEK 293 (1.5 x 105 cells per 24-well dishes), COS-1 (4 x 104 cells per 24-well dishes) and HeLa cells (5.5 x 104 cells per 24-well dishes) were grown and transfected as in Suaud et al. (31) with plasmid amounts indicated in the figure legends. Luciferase activities were measured using the Bright-Glo Luciferase assay system (Promega).

Western blot assays
Aliquots of 2.3 x 106 HeLa cells were transfected with 2 µg of wild-type or mutated HNF4{alpha}2 expression vector and whole-cell extracts were prepared as in Wang et al. (34). Western blot assays were carried out as in Suaud et al. (30).

Data analysis
Statistical analyses were based on Student’s t-test for unpaired data using Prism software. The statistical significance of differences between values obtained for mutant and wild-type HNF4{alpha} (P) is indicated in the legends to the figures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Residues E262 and R258 are important for HNF4{alpha}2 dimerisation in solution
Figure 1 depicts the amino acid sequence alignment of H7 in nuclear receptor LBDs. According to Gampe et al. (27,28), the inserted glutamate residue (indicated by the arrow in Fig. 1) may facilitate RXR{alpha} dimerisation (i) because of the {pi}-helix conformation its insertion generates and (ii) because of its carboxylic group, which forms a salt bridge and hydrogen bonds (27,28). A similar conformation was observed near the corresponding E262 residue in the HNF4{alpha} LBD (12). We investigated the role of this glutamate residue in HNF4{alpha}2 dimerisation. To this end, the HNF4{alpha}2 E262 residue was deleted in construct HNF4{alpha}2-{Delta}E262 and its charge was inverted in construct HNF4{alpha}2-E262K and removed in constructs HNF4{alpha}2-E262A and HNF4{alpha}2-E262M, the bulk of the residue being conserved in the latter mutation.

We analysed whether these mutations affected the ability of labelled HNF4{alpha}2 mutants to form dimers in solution with wild-type HNF4{alpha}2 (HNF4{alpha}2 WT). Dimerisation was studied by either co-immunoprecipitation assays using c-myc-HNF4{alpha}2 WT or GST pull-down assays using GST-HNF4{alpha}2 WT. As expected, HNF4{alpha}2 WT efficiently bound to both c-myc-HNF4{alpha}2 WT (Fig. 2A) and GST-HNF4{alpha}2 WT (Fig. 2B). Conversely, all mutants failed to bind efficiently to c-myc-HNF4{alpha}2 WT (Fig. 2A) or GST-HNF4{alpha}2 WT (Fig. 2B). Next, we analysed the effects of mutations on dimerisation of a given mutant with itself. For these assays, we focused on mutants E262A and {Delta}E262. In co-immunoprecipitation assays, no dimer was detectable with either E262A or {Delta}E262 (Fig. 2C). This result was not due to a lower expression of mutated c-myc-HNF4{alpha}2 (Fig. 2C, insert). Impaired dimerisation of mutants was confirmed by pull-down experiments performed in increasingly stringent conditions. HNF4{alpha}2 WT dimerisation was unaltered at 300 mM KCl and decreased by only 25% at 600 mM KCl (Fig. 2D). In sharp contrast, dimerisation dropped dramatically at 300 and 600 mM KCl for both mutants (Fig. 2D). Thus, removing the carboxylic group at position 262 is sufficient to strongly impair HNF4{alpha}2 dimerisation in solution.



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  		Figure 2. E262 and R258 are involved in HNF4{alpha}2 dimerisation in solution. (A and B) Analyses by co-immunoprecipitation assays and GST pull-down assays, respectively, of dimerisation between immobilised wild-type HNF4{alpha}2 fused to c-myc or GST (c-myc-HNF4{alpha}2 WT or GST-HNF4{alpha}2 WT) and wild-type or mutated [35S]methionine-labelled HNF4{alpha}2. Graphs in (A) and (B) indicate means ± SE of HNF4{alpha}2 mutant binding relative to that of the wild-type protein from three independent experiments. Inputs were taken into account for binding quantifications. (C) Dimerisation, analysed by co-immunoprecipitation assays of HNF4{alpha}2 WT, -{Delta}E262 or -E262A. For each assay, [35S]methionine-labelled HNF4{alpha}2 was incubated with the same protein fused to the c-myc tag. Control of synthesis of c-myc-HNF4{alpha}2 WT and mutated proteins is shown in the insert. (D) Dimerisation, analysed by GST pull-down assays of HNF4{alpha}2 WT, -{Delta}E262 and -E262A. For each assay, [35S]methionine-labelled HNF4{alpha}2 was incubated with the same protein fused to GST. Pull-down assays were performed in the indicated ionic strength conditions. The graph indicates means ± SE of HNF4{alpha} binding at 300 or 600 mM KCl relative to binding at 100 mM KCl (set to 100%) from three independent experiments. (E) Dimerisation, analysed by co-immunoprecipitation assays, between immobilised c-myc-HNF4{alpha}2 WT and [35S]methionine-labelled HNF4{alpha}2 WT or -R258M. The graph indicates mean ± SE of HNF4{alpha}2-R258M binding relative to that of the wild-type protein from four independent experiments. Inputs were taken into account for binding quantifications.

 
In RXR{alpha}, R348 is also involved in dimerisation (27,28). Interestingly, this arginyl residue is specifically conserved in all members of the NR2 subfamily (Fig. 1). This led us to investigate the role of the corresponding R258 residue of HNF4{alpha}2 in dimerisation. When the positive charge of this residue was removed in the R258M mutant, HNF4{alpha} dimerisation dropped (Fig. 2E), although less markedly than when elicited by E262 mutations (compare decrease in dimerisation in Fig. 2A and E). Together, these results show the involvement of the charged groups of residues E262 and R258 in the stabilisation of HNF4{alpha}2 dimerisation in solution.

Mutations of residues E262 and R258 do not impair DNA binding
Since HNF4{alpha} is known to bind DNA as a homodimer (8), we analysed the effect of E262 and R258 mutations on HNF4{alpha}2 DNA binding. Surprisingly, none of the mutations altered HNF4{alpha}2 binding to the HNF4{alpha} response element of the HNF1{alpha} promoter (HNF1 site, Fig. 3A). The specificity of the band shift was ascertained by supershifting it with HNF4{alpha} antiserum {alpha}455 (33) (Fig. 3B). Similar results were obtained on another HNF4 response element, site B of the apoCIII promoter (CIIIB site), which is also a direct repeat 1 (DR1) (Fig. 3E, results in the absence of competitor). Results obtained with increasing amounts of labelled probe exclude that lack of detection of DNA binding impairment by E262A mutation was due to saturated binding (Fig. 3C). Similar results were obtained with other mutants (data not shown). Mutated and wild-type HNF4{alpha} yielded a retarded band of identical intensity and electrophoretic mobility. More specifically, we did not observe a band of higher mobility corresponding to a HNF4{alpha} monomer bound to DNA as observed for the RAR monomer (15). In addition, to rule out the possibility that mutants bound to DNA as two adjacent monomers, we performed EMSA with the HNF1 mt site containing one mutated half-site and obtained no binding of the mutants (Fig. 3D). Thus, HNF4{alpha} mutants, like wild-type HNF4{alpha}, bind DNA as homodimers. To evidence possibly diminished interactions with DNA, we performed EMSA in less-favorable conditions. First, we increased the ionic strength (300 versus 100 mM KCl in Fig. 3A) in EMSA but found no difference in DNA binding between wild-type and mutated HNF4{alpha} (data not shown). Second, we carried out competition experiments with COUP-TFII, which binds to several HNF4{alpha} response elements. The rationale here was to check whether this nuclear receptor impinges on DNA binding of HNF4{alpha}2-E262A more efficiently than on wild-type HNF4{alpha}2. In these assays, the truncated COUP-TFII {Delta}AB was used to distinguish complexes formed with either HNF4{alpha}2 or COUP-TFII. EMSA was performed on the CIIIB site since COUP-TFII does not bind to the HNF1 site (17,30). COUP-TFII {Delta}AB similarly competed for HNF4{alpha}2 WT and -E262A DNA binding (Fig. 3E). Also, unlabelled DNA competed equally for DNA binding of both proteins (data not shown). In addition, we observed that mutations did not alter binding to a direct repeat 2 (DR2), to which HNF4{alpha} binds less efficiently than to a DR1 (9) (data not shown). It appears therefore that the E262 residue does not play a significant role in HNF4{alpha}2 DNA binding.



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  		Figure 3. Mutations of E262 and R258 residues do not impair HNF4{alpha}2 DNA binding. (A) DNA binding of HNF4{alpha}2 mutants to the 32P-labelled HNF4{alpha} response element of the HNF1{alpha} promoter (HNF1 site). Control of in vitro synthesis of wild-type and mutated HNF4{alpha}2, used in EMSA, is shown in the insert (values on the right end indicate molecular size markers). The graph indicates means ± SE of mutated HNF4{alpha}2 DNA binding relative to that of the wild-type protein from three independent experiments. (B) Specificity of binding. Unprogrammed reticulocyte lysate (mock) yielded no shifted band. Supershifting was performed in the presence of the specific {alpha}455 HNF4{alpha} antiserum. (C) EMSA performed with a constant amount of HNF4{alpha}2 WT or HNF4{alpha}2-E262A and increasing amounts of labelled HNF1 probe. (D) HNF4{alpha}2-E262A did not bind as a monomer to the half-site of the HNF4{alpha} response element (HNF1 mt). (E) Competition experiments with COUP-TFII {Delta}AB. EMSA were performed on the HNF4 response element of the apoCIII promoter (CIIIB site) using in vitro synthesised HNF4{alpha}2 WT or HNF4{alpha}2-E262A and increasing amounts of the competitor COUP-TFII {Delta}AB. The amount of reticulocyte lysate in each lane was held constant by the appropriate addition of unprogrammed lysate. The positions of HNF4{alpha}2 and COUP-TFII {Delta}AB homodimers bound to DNA are indicated.

 
Differential effects on dimerisation in solution and DNA binding of mutations in H7 and H10 of HNF4{alpha} LBD
Within the LBD, the I-box has been shown to constitute a dimerisation interface that mediates cooperative binding to DNA of nuclear receptors (35). The I-box almost perfectly overlaps H9 and H10 that form the major portion of the dimer interface in RXR{alpha} and HNF4{alpha} homodimers (12,13,27,36). Mutagenesis studies also showed that the HNF4{alpha} I-box is an important interaction interface for homodimerisation in solution (37) and is likely involved in DNA binding (15). We therefore compared the effects of mutations in H7 and H10 on HNF4{alpha} dimerisation in solution and DNA binding. Until now, the role of the HNF4{alpha} I-box has been studied using multiple (double, triple or quadruple) mutations (15,37). For example, the K300E-E327K HNF4{alpha} double mutant was used to show the involvement of the salt bridge between these residues in the exclusive homodimerisation of HNF4{alpha} (15). For our comparative study, we chose a single mutation located in H10 E327M. In co-immunoprecipitation assays with c-myc-HNF4{alpha}2 WT, we observed that the E327M mutation decreased HNF4{alpha}2 dimerisation in solution by 25% (Fig. 4A). The extent of this decrease was similar to that observed for the R258M mutation (Fig. 2E). A similar drop in HNF4{alpha}2 dimerisation in solution with E327M and R258M mutations was also observed in GST pull-down assays (Fig. 4B). Note that the impairment of dimerisation by the E262 mutations was stronger, as observed in both co-immunoprecipitation and GST pull-down assays (Fig. 2A and B). The stronger effect of the E262A mutation on HNF4{alpha}2 dimerisation in solution was confirmed by a different co-immunoprecipitation assay, where unlabelled HNF4{alpha}2 WT, -R258M, -E262A or -E327M and the {alpha}455 antiserum raised against their common C-terminus were used to co-immunoprecipitate labelled HNF4{alpha}3, which contains a different C-terminus not recognised by this antiserum. R258M, E262A and E327M mutations strongly impaired the ability of HNF4{alpha}2 to interact with HNF4{alpha}3 (Fig. 4C), thus confirming the involvement of these three residues in HNF4{alpha} dimerisation. Quantification again indicated that the E262A mutation resulted in the strongest impairment of HNF4{alpha}2 dimerisation in solution (Fig. 4C).



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  		Figure 4. Differential effects on dimerisation in solution and DNA binding of mutations in H7 and H10 of the HNF4{alpha} LBD. (A) Dimerisation, analysed by co-immunoprecipitation assays, between immobilised c-myc-HNF4{alpha}2 WT and [35S]methionine-labelled HNF4{alpha}2 WT or -E327M. The graph indicates mean ± SE of HNF4{alpha}2-E327M binding relative to that of the wild-type protein from three independent experiments. Inputs were taken into account for binding quantifications. (B) Dimerisation, analysed by GST pull-down assays, between immobilised GST-HNF4{alpha}2 WT and [35S]methionine-labelled HNF4{alpha}2 WT, -R258M and -E327M. Pull-down assays were performed in various ionic strength conditions as in Figure 2D. The graph indicates means ± SE of mutant binding relative to that of the wild-type HNF4{alpha} from three independent experiments. Inputs were taken into account for binding quantifications. (C) Dimerisation, analysed by co-immunoprecipitation assays using the {alpha}455 antiserum, between immobilised HNF4{alpha}2 WT, -R258M, -E262A or -E327M and [35S]methionine-labelled HNF4{alpha}3, which is not recognised by the {alpha}455 antiserum. Control of HNF4{alpha}2 protein synthesis is shown in the insert. The graph indicates means ± SE of HNF4{alpha}3 retention by HNF4{alpha}2 mutants relative to HNF4{alpha}3 retention by HNF4{alpha}2 WT from four independent experiments. Control of protein synthesis was taken into account for binding quantifications. (D) DNA binding of HNF4{alpha}2-E327M on the HNF1 site, analysed by EMSA performed as in Figure 3A. Supershifts were obtained in the presence of the {alpha}455 HNF4{alpha} antiserum as indicated. Control of protein synthesis is shown in the insert. The graph indicates mean ± SE of HNF4{alpha}2-E327M DNA binding relative to that of the wild-type protein from three independent experiments.

 
In contrast to E262A and R258M mutations, the E327M mutation moderately but significantly decreased HNF4{alpha}2 DNA binding to the HNF1 site (Fig. 4D). The specificity of the band shift was ascertained by supershifting it with the {alpha}455 antiserum (Fig. 4D). Interestingly, the intensities of the supershifted bands obtained with wild-type and E327M HNF4{alpha} were similar. It has previously been shown that the impaired DNA binding of HNF4{alpha} mutants (i.e. K300E-E327K and R154X) due to decreased dimerisation could be rescued in the presence of antibodies, which facilitate dimerisation. This phenomenon is likely due to the bivalency of antigen recognition by antibodies (15,32). Conversely, when decreased DNA binding is not due to a loss of dimerisation, DNA binding is not recovered in the presence of antibodies, as observed for the D126Y and D126H HNF4{alpha} mutants (38). Therefore, the decreased DNA binding and impaired dimerisation of the E327M mutant are directly correlated. E327M mutation also impaired HNF4{alpha}2 DNA binding to the CIIIB site and to the synthetic DR2 site (data not shown). Our results not only confirm that H10 and H9 constitute a motif of the LBD dimerisation interface required for efficient DNA binding of HNF4{alpha} (15), but also show that another region of the LBD dimerisation interface, namely H7, is not required for strong DNA binding of this protein.

Deletion of the E262 residue dramatically impairs HNF4{alpha}2 transcriptional activity
Next we investigated the effects of mutations of E262 and R258 residues on HNF4{alpha}2 transcriptional activity. The HNF4{alpha}2-mediated activation of the HNF1{alpha} promoter was not altered by any of the substitution mutations in HeLa cells (Fig. 5A). However, HNF4{alpha} transcriptional activity was abolished by E262 deletion (Fig. 5A). In HEK 293 and COS-1 cells, transcriptional activity was unaffected by substitution mutations but was dramatically impaired by the deletion mutation {Delta}E262 (Fig. 5B and C, where only data with wild-type, E262A and {Delta}E262 are shown). The loss of activation by HNF4{alpha}2-{Delta}E262 was neither due to a lower protein expression, as controlled by western blotting (Fig. 6D), nor to an unfolding of the protein, as assessed by limited protease mapping assays using chymotrypsin or trypsin (data not shown). Deletion of the neighbouring residue, {Delta}D261, also abolished HNF4{alpha} transcriptional activity (Fig. 5A, last bar). The dramatic drop in HNF4{alpha} transcriptional activity caused by the E262 deletion prompted us to determine whether this mutant exhibits a dominant-negative effect on the wild-type protein. However, HNF4{alpha}2-{Delta}E262 was unable to repress the transcriptional activity of wild-type HNF4{alpha} (Fig. 5E).



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  		Figure 5. Deletion of E262 strongly affects HNF4{alpha}2 transcriptional activity. HeLa (A), HEK 293 (B) and COS-1 cells (C) were transiently transfected with 12.5 ng of expression vector for wild-type or mutated HNF4{alpha}2 or the corresponding empty vector (–) together with 250 ng of HNF1{alpha} promoter construct. Fold induction refers to the activity with no HNF4{alpha}2 derivative (–), which was set to 1. Results are means ± SE of three independent experiments performed in triplicate. **, P = 0.0015, 0.0060 and 0.0018 for the {Delta}E262 mutant in (A–C), respectively; ***, P < 0.0001 for the {Delta}D261 mutant in (A). (D) Western blotting of HeLa cell extracts. (E) HNF4{alpha}-{Delta}E262 does not exhibit a dominant-negative activity on wild-type HNF4{alpha}. COS-1 cells were transfected as in (C), except that equal amounts of wild-type HNF4{alpha} and HNF4{alpha}-{Delta}E262 or control vector (–) were co-transfected. (F and G) Effects of substitution mutations on the dominant-negative activity of HNF4{alpha}-{Delta}AF-2. COS-1 cells were transfected as in (C), except that in (F) plasmids expressing wild-type, E262A or E262K HNF4{alpha} were co-transfected with an equal amount of vector expressing HNF4{alpha}-{Delta}AF-2 or the control vector (–), whereas in (G) pcDNA3 HNF4{alpha}2 WT was co-transfected with an equal amount of vectors expressing HNF4{alpha}-{Delta}AF-2 or HNF4{alpha}-{Delta}AF-2-E262A or the control vector (–). Activation of the HNF1{alpha} promoter is expressed relative to that obtained when only full-length proteins were expressed. Results are means ± SE of three independent experiments performed in triplicate. **, P = 0.0040 in (F); ***, P < 0.0001 in (F); *, P = 0.0278 in (G).

 


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  		Figure 6. Deletion of E262 markedly decreases recruitment of transcriptional partners. (A) GST pull-down assays were performed using GST-SRC-1a (570–780), GST-p300 (340–528), GST-PGC-1 (36–797) or GST-COUP-TFII and [35S]methionine-labelled WT or mutated HNF4{alpha}2. Inputs correspond to 5 or 2% (for the experiment with GST-SRC-1a) of amounts of labelled proteins used in the assays. (B and C) Effects of mutations of the E262 residue on the enhancement of HNF4{alpha}2 transcriptional activity by p300 and COUP-TFII, respectively. HeLa cells were transiently transfected with 12.5 ng of wild-type or mutated HNF4{alpha}2 expression vector, 250 ng of HNF1{alpha} promoter construct and 250 ng of empty control vector (white bars) or expression vectors (black bars) for p300 or COUP-TFII. Shown are per cent enhancements of wild-type and mutated HNF4{alpha} activities. Results are means ± SE of three independent experiments performed in triplicate. *, P = 0.0109; **, P = 0.0037.

 
To gain further insight into the consequences of altered dimerisation of HNF4{alpha} by substitution mutations, we took advantage of the fact that a truncated HNF4{alpha} lacking the AF-2 activation function module (HNF4{alpha}-{Delta}AF-2, left part of Fig. 5F) exhibits a dominant-negative effect through its ability to dimerise with the wild-type protein (7). In a first set of experiments, we compared HNF4{alpha}-{Delta}AF-2 repression on the transcriptional activities of wild-type HNF4{alpha} and of the two mutants E262A and E262K. The rationale was that the dominant-negative mutant would exhibit a weaker repression on mutant HNF4{alpha}, with impaired dimerisation, than on wild-type HNF4{alpha}. HNF4{alpha}-{Delta}AF-2 reduced the activity of wild-type HNF4{alpha} by 50% while it reduced that of E262A and E262K mutants by 29 and 26%, respectively (Fig. 5F). Accordingly, compared to HNF4{alpha}-{Delta}AF-2, HNF4{alpha}-{Delta}AF-2 bearing the E262A mutation (HNF4{alpha}-{Delta}AF-2-E262A) exhibited a weaker repressive activity on the wild-type protein. Indeed, HNF4{alpha}-{Delta}AF-2 reduced the wild-type HNF4{alpha}-mediated activation of transcription by 50% while HNF4{alpha}-{Delta}AF-2-E262A reduced it by 30% (Fig. 5G). By demonstrating that substitution mutations of E262 affect repression of HNF4{alpha}-mediated activation of transcription, our results highlight the importance of this residue for dimerisation in a cell context.

The E262 insert is required for efficient recruitment of coactivators by HNF4{alpha}
Next, we investigated the effects of the mutations on the physical interaction between HNF4{alpha}2 and its transcriptional coactivators. In this study we included interaction with COUP-TFII, which acts as an HNF4{alpha} transcriptional partner on the HNF1{alpha} promoter (17). GST pull-down assays showed that E262 deletion markedly impaired HNF4{alpha}2 interaction with SRC-1a, p300, PGC-1 and COUP-TFII, whereas substitution mutations of E262 did not alter these interactions (Fig. 6A). In line with these data, enhancement of HNF4{alpha}2 transcriptional activity on the HNF1{alpha} promoter by p300 or COUP-TFII was not altered by substitution mutations but was abolished by the E262 deletion, as evidenced by transient transfection assays (Fig. 6B and C). Deletion of the E262 residue also disrupted the functional cooperation between HNF4{alpha}2 and SRC-1a (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We observed that two charged residues located in H7 of the HNF4{alpha} LBD, i.e. R258 and E262, are involved in HNF4{alpha} dimerisation in solution. We failed to detect significant impairment of DNA binding of their substitution mutants. At first glance, this may seem striking, since dimerisation via the LBD is thought to be crucial for strong HNF4{alpha} DNA binding. However, the accuracy of our findings is verified by the fact that (i) results obtained by various pull-down and co-immunoprecipitation assays all converge and (ii) the same methods and EMSA allowed us to detect the expected impairments due to the E327M mutation located in H10. In addition, our results are in agreement with recent crystallographic studies of the HNF4{alpha} LBD, which indicate that H7, together with H9 and H10, constitute the dimerisation interface of this nuclear receptor (12,13). The interest of our work was to describe the critical role of the charge of R258 and E262 residues in dimerisation in solution of HNF4{alpha}, a role suggested by Gampe et al. from the crystal structure of RXR{alpha} (27,28). Mutants of nuclear receptors that are deficient in dimerisation in solution after in vitro analysis but still able to efficiently bind DNA have already been described (39–41). To explain the behaviour of several of these mutants, it was suggested that dimers were stabilised on DNA via the DBD dimerisation interface. However, such a hypothesis is unlikely here since the E327M mutation impaired DNA binding in spite of its lower effect on dimerisation in solution than E262 mutations. A more probable explanation of the efficient DNA binding of H7 mutants is that the HNF4{alpha} dimerisation interface is improved by a DNA binding-induced conformational change of the LBD. An allosteric effect of DNA, which modulated HNF4{alpha} recruitment of co-repressors, has recently been documented (42). We also observed an allosteric effect of DNA that most likely occurs in the LBD and modulates coactivator recruitment (data not shown).

Like the substitution mutants in H7, several missense HNF4{alpha} mutants, including naturally occurring mutants associated with diabetes (6,30,38), also exhibit unaltered or slightly impaired transcriptional activities in transient transfection assays. Failure to detect a subtle loss of HNF4{alpha} function in these assays where the protein is overexpressed does not exclude that these mutations may have greater consequences on HNF4{alpha} function when the protein is expressed at normal levels. Interestingly, a significant effect of substitution mutants in H7 on HNF4{alpha} transcriptional activity could be detected by analysing the repression of this activity with an HNF4{alpha} mutant exhibiting a dominant-negative activity. These results demonstrate the importance of E262 for HNF4{alpha} dimerisation in a cell context. Moreover, it should be kept in mind that the activity of nuclear receptors can be repressed by proteins that prevent their dimerisation (43,44). HNF4{alpha} is a target of AMP-activated protein kinase, which inhibits HNF4{alpha} activity by decreasing its dimerisation (45). The ability of HNF4{alpha} to form a highly stable homodimer in solution (8) is likely crucial in this type of repressive mechanism that does not exclusively occur on DNA. Other pathways that do not necessarily require DNA binding include cross-regulatory mechanisms in which nuclear receptors are frequently involved. In these mechanisms, dimerisation can be of major importance (46). Since HNF4{alpha} is known to interact with numerous other transcription factors, including Smad proteins (18), it likely participates in cross-regulatory pathways. Therefore, involvement of H7 residues in dimerisation in solution may be crucial in HNF4{alpha} biological activities.

Results obtained with the deletion mutant HNF4{alpha}-{Delta}E262 indicate that the E insert is required for HNF4{alpha} transcriptional activity and recruitment of transcriptional partners. Note that, probably due in part to its impaired dimerisation, HNF4{alpha}-{Delta}E262 did not exhibit a dominant-negative activity. None of the MODY1-associated HNF4{alpha} mutants can repress wild-type HNF4{alpha} activity (6). Even single or double mutations in the AF-2 module, which is crucial for HNF4{alpha} transcriptional activity, do not result in a dominant-negative effect (J.Eeckhoute, unpublished results). Interestingly, HNF4{alpha}-{Delta}D261, a mutant having a deletion of the neighbouring residue, exhibited an abolished transcriptional activity but an unaltered DNA binding (EMSA data not shown), a behaviour that is very similar to that of the {Delta}E262 mutant. These findings may be explained by the loss of the special conformation (the {pi}-helix) in H7, which was hypothesised to result from the presence of an additional residue, the ‘E insert’ (28). The D261 and E262 deletions most probably modify the orientation of the side chain of residues in H7. Several residues in H7 point towards the HNF4{alpha} ligand binding pocket and probably participate in fatty acid binding, which is required for the stability and function of HNF4{alpha} and HNF4{gamma} (12,13). The marked decrease in HNF4{alpha} transcriptional activity and recruitment of transcriptional partners caused by the E262 deletion may reflect the alteration in fatty acid binding secondary to the loss of the {pi}-helix conformation generated by this glutamate residue.

From a mechanistic point of view, our results unravel distinct contributions of H7, H9 and H10 to HNF4{alpha} dimerisation and DNA binding. H9 and H10 constitute the core of the LBD dimerisation interface of several non-steroid nuclear receptors (27,36,47,48). This core is required for efficient DNA binding (35). In HNF4{alpha}, this core is also involved in dimerisation required for DNA binding, as evidenced using a double mutation in H9 and H10 (15). We observed that a single mutation in H10, E327M, was sufficient to alter HNF4{alpha} dimerisation and DNA binding. The role of H7 in nuclear receptors has been less extensively studied and only recent crystallographic data indicate that its contribution to dimerisation varies according to the receptor and, for RXR{alpha}, with its homodimeric or heterodimeric state (47). Concerning HNF4{alpha}, our results clearly show that H7 has a crucial role in dimerisation in solution but does not contribute to the dimerisation activity required for DNA binding.

In conclusion, our results demonstrate the key role of H7 in HNF4{alpha} dimerisation in solution and provide a new concept by which helices of the HNF4{alpha} LBD dimerisation interface contribute differently to the dimerisation required for DNA binding: whereas H9 and H10 are required for DNA binding, H7 is involved solely in dimerisation in solution. Our data also show the key role of the E262 insert that causes a special conformation in H7 observed in both RXR{alpha} and HNF4{alpha}. The key role of this glutamate is likely shared by other members of the NR2 subfamily, where it is specifically encountered, and most probably the E insert corresponds to a signature of this subfamily.


   ACKNOWLEDGEMENTS
 
The authors are indebted to Drs S. R. Grossman, S. K. Karathanasis, B. M. Spiegelman, M. Leid and M. Tsai for the generous gift of plasmids. They acknowledge Dr P. Lefebvre’s collaborators for helpful discussions, I. Briche for technical assistance and L. Hauffman-Touzet for proofreading.


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 DISCUSSION
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