INTRODUCTION

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Hepatocyte nuclear factor 4 (HNF4) constitutes transcription factor subfamily 2A (28), whose first member, HNF4 (NR2A1), has been identified as a factor interacting with promoter elements mediating liver-specific transcription (35). Based on the zinc finger motif of the DNA-binding domain and on a potential ligand-binding domain, HNF4 is classified as a member of the nuclear orphan receptor superfamily (33). Recently, it has been reported that acyl coenzyme A's (acyl-CoAs) are potential ligands of HNF4: acyl-CoAs containing fatty acids with 16 C residues or shorter act as agonists by increasing the DNA-binding potential of HNF4, whereas acyl-CoAs with 18 C residues or longer have antagonistic properties and inhibit DNA binding of HNF4 (11). HNF4 turned out to be present as well in nonhepatic cells such as kidney, intestine, stomach, and pancreas (23, 38, 45). The importance of HNF4 in gene control in tissues distinct from the liver has been documented by the fact that an inherited human disease is based on the expression of a mutated HNF4 gene in the  cells of the endocrine pancreas, leading to maturity-onset diabetes of the young (MODY1 [42]). Most interestingly, another MODY gene identified in humans represents the tissue-specific transcription factor HNF1 (43), known to be tightly regulated by HNF4 (17, 39, 44).

In addition to its role as a tissue-specific transcription factor, HNF4 is also a maternal component in the egg of Drosophila melanogaster (46) as well as of the amphibian species Xenopus laevis (12). In the mouse, an early embryonic function is implied by the fact that HNF4 transcripts are present in the primary endoderm at day 4.5 (6) and can be detected in totipotent embryonic stem cells (25). Disruption of the gene encoding HNF4 in the mouse established that HNF4 plays an essential role for the function of the visceral endoderm prior to gastrulation, leading to early embryonic death (2, 7). This embryonic lethality can be rescued by complementing the defective embryo with wild-type-derived visceral endoderm, leading to specification and early differentiation of the liver but to a dramatic failure in full hepatic differentiation (21).

In the Xenopus egg, we identified the HNF4 and the related HNF4 proteins as maternal transcription factors (13). Both proteins (12) are believed to contribute to the zygotic activation of the gene encoding HNF1, a distinct tissue-specific transcription factor of the homeodomain family. Consistent with this assumption, we have identified a functional HNF4 binding site in the HNF1 promoter (12, 13, 30). Furthermore, overexpression of HNF4 or HNF4 in Xenopus embryos leads to a dramatic increase in expression of the endogenous HNF1 gene as early as the late blastula stage (26). This demonstrates that the HNF1 promoter is accessible for artificial activation at these early embryonic stages. Surprisingly, in early Xenopus embryogenesis, HNF1 transcription is very low (1, 26), although the transcription factors HNF4 and HNF4 are abundantly present (12, 13). In addition, the HNF4 proteins are distributed in a gradient from the animal to the vegetal pole, with the highest concentration in the animal region that does not differentiate into tissues expressing the target gene HNF1 (12, 29). The discrepancy between the presence of the transcription factor HNF4 and the inactivity of its target gene HNF1 might now be explained by our finding that a novel component in the Xenopus embryo inhibits the DNA-binding activity of HNF4.

	MATERIALS AND METHODS

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Purification of HNF4 inhibitor from egg extracts. Xenopus eggs were dejellied in 2.5% cysteine hydrochloride (pH 7.8) and washed several times in H₂O. Eggs were homogenized in buffer A (20 mM Tris-HCl [pH 8], 10% glycerol, 0.1% EDTA) using 2.5 µl per egg, and the soluble components were recovered by centrifugation at 13,000 rpm. The supernatant was incubated for 10 min at 95°C, and the precipitated proteins were removed by high-speed centrifugation (50,000 rpm). The supernatant was bound to DEAE-Sepharose, step eluted with 50% buffer B (20 mM Tris-HCl [pH 8], 1 M NaCl, 10% glycerol, 0.1% EDTA) in buffer A and fractionated by anion-exchange chromatography on a MonoQ column (Pharmacia) using a gradient from 20% buffer B in buffer A to 100% buffer B. Fractions containing HNF4 inhibitor were identified in a gel retardation assay. Protein-free HNF4 inhibitor was prepared by digesting a MonoQ preparation with pronase (1 mg/ml) (Roche) according to the manufacturer's instructions, extracting the digested inhibitor with phenol-chloroform, and fractionating on a MonoQ column. This protein-free inhibitor preparation eluted at the same salt concentration as the undigested sample.

Elution of HNF4 inhibitor from SDS-polyacrylamide gels. Two microliters of a MonoQ inhibitor preparation was separated in a standard sodium dodecyl sulfate (SDS)-15% polyacrylamide gel, the lane was cut into slices, and the gel slices were shaken in 0.4 ml of H₂O for 2 h at 37°C. Eluted substances were precipitated with 1.6 ml of acetone (80°C), recovered by centrifugation, dried in a vacuum concentrator, and redissolved in 20 µl of H₂O. HNF4 inhibitor was localized by adding 1 µl of the fractions to gel retardation reactions.

Plasmid constructions. Expression vectors encoding HNF4 (44), Mt-HNF4 (19), and Mt-HNF4EF (R154X in reference 19) have been described. To construct HNF4AB, the region coding for amino acids 55 to 464 of rat HNF4 was amplified using the primer pair 5'-CACCAAGCTTCCACCATGGGTGTCAGTGCCC-3' (forward) and 5'-GGGGTACCTAGATGGCTTCCTGC-3' (reverse), introducing an HindIII site (italics). The PCR product was cloned into the SmaI site of pUC18, excised with HindIII, and cloned into the HindIII site of Rc/CMV.

Cell culture, transfection, and preparation of nuclear extracts. HEK 293 cells were cultured at 37°C in Dulbecco's modified Eagle's medium containing penicillin (100 U/ml), streptomycin (100 U/ml), L-glutamine (2 mM), and 10% heat-inactivated fetal calf serum (Biochrome). Cells were transfected with 30 µg of DNA per 10-cm cell culture dish using the DNA calcium phosphate coprecipitation method (9). High-salt nuclear extracts were obtained as described (5).

Gel retardation assays. Gel retardation assays were performed as described (16) using the following oligonucleotide probes: the HNF4 binding site of the Xenopus HNF1 promoter (44), the HNF1 binding site of the Xenopus albumin promoter (31), an AP1 binding site (37), an ATF/CREB binding site (37), an Sp1 site (22), and the HNF3 binding site of the transthyretin promoter (111/85 [18]).

Native gel electrophoresis and Western blotting. For native gel electrophoresis, 3 µl of nuclear extract of transfected HEK 293 cells and 1 µl of a MonoQ preparation of HNF4 inhibitor were incubated in GRBB (10 mM HEPES [pH 7.6], 60 mM KCl, 1 mM EDTA, 1 mM DTT, 4% Ficoll 400) for 15 min at room temperature. Probes were separated for 1.5 h at 100 V in a 4% polyacrylamide gel containing 0.25× TBE (90 mM Tris base, 90 mM boric acid, 1 mM EDTA). After electrophoresis, gel denaturation of proteins (see Fig. 6, lanes 5 to 8) was achieved by incubating the gel in a buffer containing 62.5 mM Tris (pH 6.7), 100 mM -mercaptoethanol, and 2% SDS for 20 min at 50°C.

	RESULTS

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Identification of the HNF4 inhibitor. In our previous experiments, we have shown by Western blotting that the transcription factor HNF4 is present in the fertilized egg as well as throughout the entire embryogenesis of Xenopus (12, 13). Surprisingly, using gel retardation assays, no HNF4 DNA-binding activity could be detected in embryonic extracts (data not shown).

View larger version (82K): [in this window] [in a new window]   FIG. 1.   Identification of an HNF4 inhibitor in Xenopus eggs that gradually disappears during development. (A) Xenopus liver extract (3 µl) was incubated with the labeled oligonucleotide containing the HNF4 binding site of the human apolipoprotein B promoter and analyzed in a gel retardation assay. Monoclonal antibody H4/55, specific for HNF4 (32), is in lane 2 (ab). Lanes 3 to 8 contain in addition 1 µl of embryonic extracts from the developmental stages given (27). The HNF4-DNA complex is indicated, and the supershifted complex containing the antibody is marked with an open triangle. (B) Gel retardation assay essentially as in panel A, but using the HNF1 binding site of the albumin promoter in combination with monoclonal antibody XAD5, specific for HNF1 (1). (C) Gel retardation assay using rat liver nuclear extract and a CREB binding site (lanes 1 to 3), an AP1 site (lanes 4 to 6), an HNF3 site (lanes 7 to 10), and an Sp1 site (lanes 11 to 14). Protein-free HNF4 inhibitor preparation (1 µl) was added to the reactions as indicated. We used this protein-free inhibitor preparation to avoid a change in protein concentration that may alter the DNA binding in the gel retardation assays. To identify protein-DNA complexes, antibodies (ab) were added as indicated: lane 2, 2 µg of antibody CREB-1 (Santa Cruz; C-21) directed against the DNA-binding domain of CREB; lanes 5 to 6, 2 µg of antibody against c-Fos (Santa Cruz; K-25). The complex supershifted by antibody to c-Fos (K-25) is marked by an open triangle in lane 6. In lanes 8 and 12, a 100-fold excess of unlabeled specific competitor (s) was added (HNF3 site and Sp1 site, respectively). In lanes 9 and 13, a 100-fold excess of unlabeled nonspecific competitor (n) was included in the reaction (Sp1 and HNF3 site, respectively). HNF3, -, and - protein-DNA complexes are indicated in lane 10 (18), and the Sp1 protein-DNA complex is marked by an arrowhead in lane 14 (22).

Purification of the HNF4 inhibitor from egg extracts. Xenopus eggs were homogenized in a low-salt buffer, and the soluble components were recovered by centrifugation. As the activity of the HNF4 inhibitor is heat resistant (data not shown), we incubated the supernatant at 95°C for 10 min and removed the precipitated proteins by high-speed centrifugation. The supernatant was bound to DEAE-cellulose, step eluted with 0.5 M salt, and fractionated by anion-exchange chromatography on a MonoQ column (Fig. 2A). Individual fractions were tested for the presence of HNF4 inhibitor activity in gel retardation assays containing hepatic HNF4. As summarized in Fig. 2A, the inhibitory activity coelutes in MonoQ fractions 8 to 10 with a major protein with an apparent molecular mass of 25 kDa. Further purifications on Superose, Mono-S, phenyl-Sepharose, and octyl-Sepharose revealed copurification of the 25-kDa protein with the HNF4 inhibitor (data not shown).

View larger version (36K): [in this window] [in a new window]   FIG. 2.   Purification and characterization of the HNF4 inhibitor. (A) Elution profile of a heat-treated Xenopus egg extract on a MonoQ column using a salt gradient from 0.2 to 1.0 M NaCl. An aliquot of 0.1 µl of each column fraction was added to a gel retardation assay using rat liver extract and the HNF4 binding site of the Xenopus HNF1 promoter (44). The results are given for fractions 6 to 12 only, as the other fractions did not contain any inhibitory activity. In the lower panel, a Coomassie blue-stained SDS-15% polyacrylamide gel of the same frac- tions is shown using 5 µl of each fraction. The major protein of 25 kDa is indicated, and the minor bands of between 60 and 120 kDa are marked by arrowheads. (B) MonoQ-purified inhibitor preparation (2 µl) was separated on an SDS-15% polyacrylamide gel and stained with Coomassie blue. The migration of molecular size markers is shown. An adjacent lane containing the same amount of inhibitor was cut into individual fractions as indicated. An aliquot representing 5% of each fraction was added to a gel retardation assay using the HNF4 binding site, as given in Fig. 1.

Inhibitory function is protease resistant. The HNF4 inhibitor purified by MonoQ-Sepharose chromatography as given in Fig. 2A was digested with pronase. To monitor protease activity, bovine serum albumin (BSA) was added as an internal control to the HNF4 inhibitor. SDS-polyacrylamide gel electrophoresis revealed that BSA and the 25-kDa protein were completely digested (Fig. 3A, compare lanes 1 and 2). After digestion, the protease was inactivated by adding a protease inhibitor cocktail, and the mixture was analyzed for HNF4 inhibitor activity in a gel retardation assay using hepatic extract. Figure 3B illustrates that the protease-digested HNF4 inhibitor (lane 5) retained its inhibitory function compared to untreated inhibitor sample (lane 4). In a control experiment involving pronase-digested BSA, no inhibition was observed (lane 3), indicating that no pronase activity remained in the gel retardation assay. Similar results were obtained using trypsin and proteinase K digestion (data not shown).

View larger version (47K): [in this window] [in a new window]   FIG. 3.   Protease digestion of the HNF4 inhibitor. (A) MonoQ-purified inhibitor preparation (2 µl) was combined with 20 µg of BSA and either separated directly on an SDS-15% polyacrylamide gel (lane 1) or digested with pronase prior to electrophoresis (lane 2). (B) Gel retardation assay using rat liver nuclear extract and the labeled HNF4 binding site as oligonucleotide is given in lane 1 and supplemented in lane 2 with the polyclonal antibody (ab) H4/66 specific for HNF4 (32). In lanes 3, 4, and 5, pronase-digested BSA, untreated HNF4 inhibitor, and pronase-digested inhibitor were added, respectively. (C) Untreated and trypsin-digested HNF4 inhibitor was separated in adjacent lanes of an SDS-15% polyacrylamide gel. The gel was cut into slices, and the HNF4 inhibitor was eluted and assayed in a gel retardation assay as given in Fig. 2B. (D) Aliquots of the indicated gel fractions of the untreated and trypsin-digested lanes of panel C were assayed in a gel retardation assay using the labeled oligonucleotide representing either the HNF4 (H4) or the HNF1 (HP1) binding site. The DNA-protein complexes containing HNF1 and HNF4 are indicated.

HNF4 inhibitor is distinct from acyl-CoAs, the potential ligands of HNF4. Recently, Hertz et al. (11) reported that acyl-CoAs are potential ligands of HNF4 and that acyl-CoAs containing long-chain fatty acids inhibit DNA binding of HNF4. To investigate whether the HNF4 inhibitor has similar properties, we compared the effect of acyl-CoAs on the DNA binding of HNF4 in the gel retardation assays used to identify the HNF4 inhibitor. Figure 4 illustrates that with increasing amounts, palmitoyl (C16:0)-CoA inhibits HNF4 binding to DNA (lanes 5 to 8). However, this effect is not specific, as a very similar inhibition is seen on the DNA binding of HNF1 (lanes 9 to 12). In contrast, a serial dilution of the HNF4 inhibitor specifically inhibits HNF4 DNA binding (lanes 13 to 16), but has no effect on the DNA-binding activity of HNF1 (lanes 17 to 20). Stearoyl (C18:0)-CoA also shows a nonspecific effect, because HNF4 and HNF1 are equally affected by this compound (lanes 21 to 24). In contrast, myristoyl (C14:0)-CoA has no effect on the DNA binding of either of these two transcription factors (lanes 25 to 28). From these experiments, we conclude that the HNF4 inhibitor is more specific than the acyl-CoAs tested and thus represents a distinct effector molecule.

View larger version (64K): [in this window] [in a new window]   FIG. 4.   Comparison of the action of the HNF4 inhibitor with the effect of acyl-CoAs. Gel retardation assays using the labeled oligonucleotide representing either the HNF4 (H4) or the HNF1 (HP1) binding site were performed in the presence of various concentrations of palmitoyl-CoA (lanes 5 to 12), stearoyl-CoA (lanes 21 to 24), and myristoyl-CoA (lanes 25 to 28). The DNA-protein complexes containing HNF1 and HNF4 are indicated, and the reaction with the corresponding antibodies (ab) is given in lanes 2 and 4. For comparison, the effect of the addition of HNF4 inhibitor is shown in lanes 14 to 20.

Ligand-binding domain of HNF4 is not required for mediating the inhibitory effect. To test whether the HNF4 inhibitor acts via ligand-binding domain E of HNF4, extending from amino acids 172 to 377, we used the deletion construct Mt-HNF4EF containing the N-terminal part of HNF4 (amino acids 1 to 153), as given in Fig. 5A. This construct and the wild-type protein used as a control (Mt-HNF4) contain a Myc tag at the N terminus that allows efficient identification of the protein in transfected HEK 293 cells. Since binding of the Mt-HNF4EF protein to DNA is weak and can only be detected in the presence of the Myc tag-specific antibody (lane 8 in Fig. 5B) (19), HNF4 inhibitor was added in the presence of the Myc tag-specific antibody. As Fig. 5B illustrates, the truncated protein Mt-HNF4EF and the full-length protein Mt-HNF4 are inhibited in complex formation by the same HNF4 inhibitor concentrations (compare lanes 9 to 12 with lanes 3 to 6). Therefore, the ligand-binding domain is not necessary to convey inhibitor sensitivity to HNF4.

View larger version (49K): [in this window] [in a new window]   FIG. 5.   Effect of the HNF4 inhibitor on various HNF4 deletion constructs in gel retardation assays. (A) Schematic drawing of the deletion constructs used. Domains C and E represent the DNA-binding and the potential ligand-binding domains of HNF4, respectively. (B) Nuclear extracts from HEK 293 cells transfected with the Myc-tagged full-length HNF4 (lanes 1 to 6) or with the C-terminal deletion construct Mt-HNF4EF (lanes 7 to 12) were analyzed in gel retardation assays using the labeled HNF4 binding site. The addition of the monoclonal Myc tag-specific antibody 9E10 is shown (ab). A protein-free inhibitor preparation was added (1, 0.3, 0.1, or 0.03 µl) in lanes 3 to 6 and 9 to 12, respectively. The arrowhead marks the HNF4-DNA complex, and the open triangles indicate the complex supershifted by the antibody. (C) Nuclear extracts from HEK 293 cells transfected with the full-length HNF4 (lanes 1 to 5) or with the N-terminal deletion construct HNF4AB were analyzed in gel retardation assays using the labeled HNF4 binding site. The polyclonal antibody XH4 specific for HNF4 (13) was added in lanes 2 and 7 (ab). The protein-free inhibitor preparation was used at 1, 0.3, or 0.1 µl in lanes 3 to 5 and 8 to 10, respectively. The arrowhead marks the HNF4-DNA complex, and the open triangle marks the complex supershifted by the antibody. The unusually broad protein-DNA complex containing HNF4AB is marked by an arrow. Lanes 11 and 12 show the abundance of the transfected HNF4 proteins in 3 µl of HEK 293 cell extract in a Western blot of an SDS-12% polyacrylamide gel using monoclonal antibody H4/39f (32).

HNF4 inhibitor alters the electrophoretic mobility of HNF4. To elucidate whether the activity of the HNF4 inhibitor requires DNA binding, we separated nuclear extracts from HEK 293 cells transfected with Myc-tagged HNF4 constructs on native polyacrylamide gels and located the HNF4 protein by Western blotting using the Myc tag-specific antibody. Figure 6 illustrates that HNF4 migrates faster than the same protein that has been incubated with the HNF4 inhibitor (compare lanes 1 and 2). In contrast, the mobility of the Myc-tagged HNF1 protein that migrates as a doublet is not affected by addition of the HNF4 inhibitor (lanes 3 and 4). Notably, a weak signal was consistently seen in the Western blot for the untreated HNF4 protein (lane 1) compared to the inhibitor-treated sample (lane 2). In contrast, when the gel was denatured in SDS prior to transfer to the membrane, similar intensities for the HNF4 protein were obtained (Fig. 6, lanes 5 and 6). From this observation, we conclude that properties of HNF4 are altered by addition of the HNF4 inhibitor. This alteration leads to decreased mobility as well as to an increase in accessibility to the Myc tag-specific antibody. This effect was only seen on native polyacrylamide gels, whereas an equal mobility of HNF4 with or without HNF4 inhibitor incubation was observed on SDS gels (Fig. 6, lanes 13 and 14).

View larger version (64K): [in this window] [in a new window]   FIG. 6.   Effect of the HNF4 inhibitor on the electrophoretic mobility of various HNF4 deletion constructs. Nuclear extracts (3 µl) from HEK 293 cells transfected with an expression vector encoding either Myc-tagged HNF4 (lanes 1, 2, 5, and 6), Myc-tagged HNF1 (lanes 3, 4, 7, and 8), Myc-tagged C-terminal deletion construct Mt-HNF4EF (lanes 9 and 10), or the N-terminal deletion construct HNF4AB (lanes 11 and 12) were subjected to electrophoresis on a native 4% polyacrylamide gel. Prior to electrophoresis, the extracts were mixed with 1 µl of MonoQ-purified HNF4 inhibitor as indicated. The gels were blotted and probed with the Myc-specific antibody 9E10 (lanes 1 to 10) or the HNF4-specific monoclonal antibody H4/39f (32) (lanes 11 and 12). The gel with lanes 5 to 8 was denatured before blotting. Lanes 13 and 14 are blotted from an SDS-15% polyacrylamide gel containing Myc-tagged transfected HNF4 and probed with the Myc tag-specific antibody 9E10.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have characterized a novel component that specifically inhibits the DNA-binding activity of HNF4. DNA binding of unrelated transcription factors, namely the POU/homeobox protein HNF1 (Fig. 1B), the leucine zipper proteins CREB and AP1, and the forkhead/winged helix protein HNF3 (Fig. 1C), is clearly not affected by HNF4 inhibitor. In contrast, DNA binding of Sp1 seems to be weakened by HNF4 inhibitor to some extent (Fig. 1C). Although this effect is much less obvious than the inhibition of HNF4 DNA binding, it could point to a partial action of HNF4 inhibitor on other zinc finger proteins as well. The inhibitor also acts on the progesterone receptor as well as on the retinoic acid RAR/RXR, but does not affect the estrogen receptor (data not shown). Therefore, not all members of the nuclear receptor superfamily are affected.

The HNF4 inhibitor is hydrophilic and resistant to protease (Fig. 3B), RNase, DNase, and N-glycosidase (data not shown). As the activity of the HNF4 inhibitor is protease resistant, the activity resides in a nonprotein component. On the other hand, the inhibitor contains a peptide bond, as the electrophoretic mobility is increased upon protease digestion (Fig. 3C). In fact, we assume that there are two distinct peptide bonds, because the inhibitory activity is sensitive to trypsin as well as pronase digestion, as assayed on a denaturing polyacrylamide gel, but passes through a 5-kDa membrane only after pronase treatment (data not shown). In conclusion, our data support a model in which the inhibitor consists of a protease-resistant core carrying the inhibitory activity and a distinct protease-sensitive region.

Although the chemical identity of the HNF4 inhibitor is not yet known, it is most unlikely that it represents a member of the acyl-CoAs, which have recently been proposed to be ligands for HNF4 (11). In fact, the HNF4 inhibitor is more specific, as it does not inhibit the transcription factor HNF1, a member of the homeodomain proteins, whereas various acyl-CoAs were found to inhibit DNA binding of HNF1 as well (Fig. 4). More importantly, we could show that the HNF4 inhibitor does not require the potential ligand-binding domain of HNF4 to exert its action (Fig. 5B and Fig. 6, lanes 9 and 10).

The inhibitor is a relatively small molecule whose protease-resistant core of about 5 kDa retains full inhibitory function and thus resembles the relatively small size of the well-known ligands of the nuclear receptor superfamily. However, the inhibitor would be a novel type of ligand, as it acts on an HNF4 derivative lacking the ligand-binding domain. Based on our finding that the inhibitor interferes with the DNA-binding property of HNF4, it acts as an antagonist and may thus be similar to androstane, which inhibits the constitutive androstane receptor (8). However, such an interpretation is quite speculative, because at present it remains unclear whether the inhibitor binds to HNF4 or just induces the observed changes without binding. In fact, several different attempts made to isolate the inhibitor using the HNF4 protein as a bait failed, and thus we favor a model in which the inhibitor binds only transiently to the HNF4 protein.

It is well established that HNF4 is a phosphoprotein and that the extent of phosphorylation is modulated (14, 40). However, we exclude an inhibitor-induced change in phosphorylation because the mobility of HNF4 in SDS-polyacrylamide gels is known to be influenced by phosphorylation (14), but we could not see any change in the mobility of HNF4 on SDS-polyacrylamide gels upon incubation with the inhibitor (Fig. 6, lanes 13 and 14).

The action of the HNF4 inhibitor can be traced in three distinct ways. In the first, the DNA-binding activity of HNF4 is inhibited in the gel retardation assay and is therefore measured in the presence of the HNF4 DNA-binding site (Fig. 1 to 5). However, the inhibitor also affects HNF4 in the absence of the DNA-binding site by altering the mobility in a native polyacrylamide gel (Fig. 6). In these native polyacrylamide gels, HNF4 inhibitory action is evident in a third way, because inhibitor treatment leads to an altered accessibility of HNF4 to the antibody in the Western blot. This is most evident using the Myc tag-specific antibody that reacts to the artificial N-terminal tag of HNF4, but also occurs using a monoclonal antibody raised against the ABCD domains of HNF4 (36).

Using various deletion constructs, it is clear that domain E, the potential ligand-binding domain of HNF4, is not required to get the DNA-binding inhibition in the gel retardation assay (Fig. 5B). In contrast, the HNF4 deletion construct HNF4AB, lacking the A/B domains, is apparently not affected in its binding affinity by the inhibitor. However, this construct has a low binding activity compared to the full-length protein, implying some effect of the A/B domain on the DNA-binding domain. Surprisingly, this N-terminal truncation of HNF4 leads to an unusual broad DNA-protein band in the gel retardation assay (Fig. 5C, lane 6). As this diffuse band is sharpened by the addition of HNF4 inhibitor (Fig. 5, lane 8), we conclude that the inhibitor is still able to act upon the truncated HNF4 derivative lacking the A/B domain. This interpretation agrees with our finding that the same construct is affected in its mobility in native gel electrophoresis in the absence of the DNA-binding site (Fig. 6, lanes 11 and 12).

The addition of the inhibitor in the absence of DNA leads to decreased mobility of HNF4 in the native polyacrylamide gel, implying either some conformational change, multimerization, or a decrease in negative charge (Fig. 6, lanes 1 and 2). Whereas decreased mobility was also obtained for the deletion construct HNF4AB lacking the A/B domain (Fig. 6, lanes 11 and 12), the mobility of construct Mt-HNF4EF lacking the ligand-binding domain is increased (Fig. 6, lanes 9 and 10). This opposite effect of the HNF4 inhibitor on the electrophoretic mobility of HNF4 derivatives is quite unexpected. In this context, it is noteworthy that Mt-HNF4EF migrates much more slowly than the full-length Mt-HNF4 in a native polyacrylamide gel, although the theoretic isoelectric point of Mt-HNF4EF is lower, 4.5 compared to 4.9 for full-length Mt-HNF4, and thus should migrate faster at the pH used for electrophoresis. Possibly during electrophoresis under native conditions, HNF4 interacts with other components of the extract that influence the electrophoretic migration. Therefore, not only a change in the charge or in the conformation of HNF4 upon inhibitor addition has to be considered, but an influence on the interaction with other components as well. Indeed, it is well established that HNF4 interacts with a series of other factors involved in transcriptional regulation (10, 15, 20, 34, 41).

The presence of an HNF4 inhibitor in the Xenopus embryo gives a most attractive explanation for our finding that the HNF4 target gene HNF1 is only marginally expressed in the early embryo, although maternal HNF4 proteins  and  are abundantly present (12, 13). From injections of RNAs encoding HNF4 into fertilized Xenopus eggs, we know that the endogenous HNF1 gene is competent for activation and can be activated as early as the blastula stage by overexpression of HNF4 (26). Indeed, under these conditions, overexpressed HNF4 can be found in embryonic extracts in gel retardation assays (data not shown), implying that the HNF4 inhibitor is titrated out by the introduced HNF4 protein. Therefore, we assume that the HNF4 inhibitor inactivates the maternal HNF4 transcription factor and that this inhibition is gradually relieved during development. This release of suppression during development is supported by our finding that the inhibitory activity is lost in the process of development (Fig. 1). During this time period of development, the amount of HNF4  and  proteins does not change significantly (12, 13), but the expression of the HNF1 gene increases dramatically (1, 26). Thus, we propose that the decrease in the HNF4 inhibitor is responsible for the activation of HNF4-dependent target genes during Xenopus development. The balance of positive and negative regulators crucial for the concerted activation of the developmental program is a well-established phenomenon during embryogenesis (3, 4, 24). We further speculate that the distribution of the inhibitor within the embryo is a crucial determinant in the patterning of the HNF4 response. Clearly, firm conclusions concerning the regulatory role of the HNF4 inhibitor awaits its chemical identification, which will allow us to determine its distribution within the embryo and to test its function in injected embryos.

The identification of an HNF4 inhibitor in Xenopus embryos opens up a potential way to manipulate the activity of HNF4, a gene product mutated in MODY1 patients. Recent analysis of the mutated HNF4 factors found in MODY patients have shown that these naturally occurring mutations impair the function of the transcription factor to greatly varying degrees (19 and references therein), but that most of these mutants retain the C and D domain that is responsive to the HNF4 inhibitor. Therefore, compounds with properties of the HNF4 inhibitor may constitute a novel approach to interfering with HNF4 function.