INTRODUCTION

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Nuclear receptors comprise a large superfamily of relatively conserved transcription modulators that play a role in nearly every aspect of growth, differentiation, and development in organisms ranging from nematodes to humans (for reviews, see references 13, 58, 59, and 70). Family members are defined by the presence of two conserved functional domains. In the N-terminal portion of the protein there is a DNA binding domain (DBD) that contains two zinc fingers; in the C-terminal portion there is a large hydrophobic domain, termed the ligand binding domain (LBD), which is responsible for ligand binding, protein dimerization, and transcriptional activation. Recently, our understanding of the mechanism by which nuclear receptors modulate transcription was greatly enhanced by the finding that certain members interact in a ligand-dependent fashion with non-DNA binding coactivators from several different gene families, e.g., the p300 family, such as p300 and CBP (5, 28, 48), and the p160 family, such as GRIP1/TIF2 (38, 86) and SRC1/p160/ERAP160 (26, 48, 68) (reviewed in references 77 and 83). For several receptors, such as the thyroid hormone receptor, vitamin D receptor, retinoid (retinoic acid and retinoid X) receptors (RAR and RXR, respectively), and steroid (progesterone, estrogen, androgen, and glucocorticoid) receptors (PR, ER, AR, and GR, respectively), the binding of ligand is known to produce a conformational change in the protein which makes it more resistant to protease (1, 53, 54; see also reference 91 and references therein). Structural studies have shown that this change makes a small conserved region important for transactivation in the C-terminal end of the LBD, termed AF-2, more accessible to solvent, and therefore presumably to coactivators (3, 14, 73, 90). Coactivators in both the p300 and p160 families are known to bind receptors in a ligand-dependent fashion (5, 28, 37, 43, 60, 68, 84, 86). This interaction appears to be dependent upon nuclear receptor (NR) boxes in the coactivators which are comprised of LXXLL motifs (10, 32, 84, 85), and, at least in the p160 family, the AF-2 region of the receptor (37, 43, 60, 68, 86). Once tethered to the appropriate promoter by the receptor, the coactivators are thought to stimulate transcription by interacting with the basal transcription machinery (reviewed in reference 41) and/or by modulating the local nucleosome structure via histone acetylation (reviewed in references 39 and 92). The majority of the more than 150 different members of the nuclear receptor superfamily, however, have not yet been found to respond to ligands. The question then arises as to whether these so-called orphan receptors will also interact with coactivators and, if they do, whether the structural basis for that interaction is similar to that for receptors with known ligands.

Nuclear receptors, like many other proteins, are often subjected to alternative splicing which generates multiple isoforms of the receptors (23). Some isoforms are found more readily in cancerous than in noncancerous tissue (15, 21), while levels of others vary depending on the tissue or developmental state (34, 61, 69). Whereas the functional relevance of some of those variants is evident (e.g., producing alterations in DBDs or LBDs), the significance of other splicing variants is less clearly established, despite conservation among different species (17, 69). The question then arises as to whether some of these splicing variants differ in their interactions with different coactivators.

We wished to examine some of these issues with hepatocyte nuclear factor 4 (HNF4, NR2A1) (8), a highly conserved member of the superfamily. Three HNF4 genes HNF4, HNF4, and HNF4 (11, 35, 80), have been identified thus far in vertebrates, although most work has been done with the first cDNA cloned, HNF41 (80). HNF41 is directly linked to several human diseases: the coding region of HNF41 is mutated in maturity-onset diabetes of the young (MODY1) (4, 18, 27, 55, 62, 93), an HNF41 response element is mutated in hemophilia B Leyden (72), and HNF41 transcriptionally activates several hepatitis B viral genes (19, 24, 71). Deletion of the HNF41 gene from the mouse genome results in embryonic lethality at day 10 (7). HNF41 is known to activate a wide variety of genes involved in glucose, fatty acid, cholesterol, and amino acid metabolism in the liver, kidney, intestine, and pancreas (reviewed in reference 79). Whereas HNF41 is capable of activating transcription and binding DNA in the absence of exogenously added ligand (25, 44, 57, 80), there is a recent report of a potential ligand for HNF41 (33). However, many questions remain about the role of these compounds in HNF41 function.

Aside from its physiological importance, HNF41 is of interest in that it possesses unique protein dimerization and DNA binding properties which define a distinct subfamily of nuclear receptors: HNF41 exists in solution and binds DNA response elements consisting of direct repeats exclusively as a homodimer (44). Furthermore, HNF41 is rather unique in its ability to activate transcription in the absence of exogenously added ligand in mammalian cells, in yeast cells, and in vitro (16, 25, 44, 57, 80). Finally, HNF41 possesses an unusually large region C terminal to the LBD (the F domain). Of the two splicing variants of HNF41 involving the F domain thus far identified (30, 50), one contains an additional 10 amino acids (aa) that have been inserted into the middle of the F domain (HNF42) (Fig. 1). Since deletion of the F domain has been shown to increase the activation of transcription by HNF41 in vivo (25), we wished to determine whether the F domain can modulate interaction with coactivators and whether the 10-aa splicing insert affects that modulation.

View larger version (41K): [in this window] [in a new window]   FIG. 1.   HNF4 constructs used in this study. Shown are naturally occurring rat HNF41 and HNF42 splicing variants and experimentally generated constructs containing the indicated amino acids. N1C268X is derived from a mutation found in a MODY1 patient (81, 97). The amino acid sequence of the conserved AF-2 region and the insertion in HNF42 are given in single-letter code. The underlined cysteine at position 409 is a serine in HNF41. The remaining residues shown are unique to the 2 insert; all others are identical between HNF41 and HNF42 (30). Conventional nomenclature for nuclear receptor domains (A to F) is given at the top. Numbers indicate amino acid residues. Zn++, zinc finger region; Rep, repressor region (aa 428 to 441 in HNF41).

In this study we used a variety of in vivo and in vitro assays, including transient transfections into mammalian cells, yeast two-hybrid and glutathione S-transferase (GST) pulldown assays, and protease digestion, to examine the interaction between HNF41 and HNF42 and coactivators GRIP1, SRC1a, p300, and CBP. The results show not only that HNF41 is capable of interacting physically and functionally with coactivators in vivo and in vitro in the absence of exogenously added ligand but that the F domain interferes with that interaction. We also show that the 10-aa insertion in HNF42 somewhat abrogates the interference by the F domain and propose a mechanism for that abrogation.

	MATERIALS AND METHODS

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Plasmids. All HNF4 constructs were derived from rat HNF41 (80) or HNF42 (30) and ligated into the vector pMT7 (46) via EcoRI adapters (Promega, Madison, Wis.), unless noted otherwise, for expression in vitro and in vivo. HNF42 cDNA was removed from HNF-4CL4 (kindly provided by S. Hata) by digestion with BamHI/EcoRI. HNF4.N1C374 and HNF4.N45.C455 were constructed by PCR amplification of HNF41 with previously described primers N1 (previously Npf7) and C374 or N45 and C455 (previously Cpf7), respectively (45, 46). N1C360 was constructed in a similar fashion with primers N1 and C360 (5'-GCGCTCGAGCTACAGGTTGTCAATCTTGGCCATC-3') but ligated into the BamHI/XhoI sites of pcDNA3.1(+) (Invitrogen, Carlsbad, Calif.). Construction of HNF4.N45.C374 and N1C268X in pMT7 has been previously described (46, 78). pSG5.GRIP1 contained full-length mouse GRIP1 coding region ligated into pSG5 (10). pRc/RSV-mCBP.HA.RK (kindly provided by R. Goodman) contained full-length mouse CBP with a C-terminal hemagglutinin (HA) tag driven by the Rous sarcoma virus promoter. pCMV.HA.p300 contained human p300 from nucleotides 1134 to 8329 with an HA tag fused to the NheI site at nucleotide 8329 driven by a cytomegalovirus promoter (12). The reporter construct pZLHIVAI-4 contained four HNF4 response elements (site A) from the human apolipoprotein AI gene (75) inserted at the BamHI site immediately upstream of positions 57 to +80 of the human immunodeficiency virus long terminal repeat (74) driving the firefly luciferase gene in pZLuc (78). Fusions of the Gal4 DBD to HNF1 (Gal4DBD-HNF4 constructs) for the yeast two-hybrid assay were prepared by inserting the appropriate PCR-amplified HNF41 coding regions into plasmid pGBT9 (Clontech, Palo Alto, Calif.) at the BamHI/SalI site for constructs HNF41.128-455 and HNF41.128-415 and at the EcoRI/SalI site for construct HNF41.128-370. Gal4 activation domain (Gal4AD)-GRIP1 and Gal4AD-SRC1a constructs have been previously described (10). GST.127.374 was constructed by inserting a PCR product containing amino acids 127 to 374 of rat HNF41 into the pGEX6P-1 vector (Pharmacia, Piscataway, N.J.), using EcoRI/XhoI sites. The fusion protein was expressed in Escherichia coli BL21(DE3)(pLysS) and bound to glutathione-agarose (Sigma, St. Louis, Mo.) by using standard protocols (2). A fragment containing residues 127 to 374 plus eight residues from the vector (N-Gly-Pro-Leu-Gly-Ser-Pro-Glu-Phe-C) was released from GST by cleavage with Precision Protease as directed by the manufacturer (Pharmacia) and verified by sequencing the N terminus via Edman degradation (UC Riverside peptide sequencing facility). The sequence of all PCR-derived products were verified by dideoxy sequencing.

Transient transfection assays. Human embryonic kidney 293T cells and COS-7 cells were maintained at 37°C under 5% CO₂ in Dulbecco modified Eagle medium supplemented with penicillin-streptomycin and with 5% fetal calf and 10% bovine calf serum, respectively. Transient transfections into these cells using calcium phosphate precipitation were carried out essentially as previously described (78). All transfection results were normalized to the RSV.gal construct level; assays were performed at least twice in triplicate. Fold inductions were calculated relative to transfections lacking expression vectors. Activation of the reporter construct by coactivators in the absence of HNF4 was minimal compared to activation in the presence of HNF4 (not shown). Production of HNF4 protein was verified by expression in COS-7 cells by Western or gel shift analysis as previously described (44, 45). Production of HNF41 and HNF42 protein in 293T cells was verified by harvesting 3.0 × 10⁶ cells transfected with 25 µg of plasmid DNA (quantified by readings of optical density at 260 nm and analysis of ethidium bromide-stained agarose gels) 24 h after glycerol shock. The cells were lysed in 100 µl of 293T lysis buffer (50 mM HEPES [pH 7.9], 100 mM NaCl, 1.0 mM MgCl₂, 1 mM EDTA, 10% glycerol, 1% Triton X-100), gently agitated for 40 min at 4°C and centrifuged for 30 min at 12,000 × g to pellet the debris. The protein concentration of the supernatant was determined by the Bio-Rad assay, and equivalent amounts of total protein were analyzed by Western blot analysis as described in the figure legends.

GST pulldown assays. In vitro protein-protein interactions between various HNF4 constructs and GST-GRIP1 (aa 563 to 1121) were analyzed by a GST pulldown assay performed essentially as previously described (10). Briefly, 2 to 5 µl of in vitro-synthesized ³⁵S-labeled HNF4 (TNT kit; Promega) was incubated with 10 µl of packed conjugated glutathione-agarose beads (containing approximately 1 µg of GST fusion protein per µl) in 0.01% NETN in a total of 30 to 50 µl for approximately 1 h at 4°C with gentle agitation. The beads were spun in a Sorvall MC 12-V Microfuge at 2,000 rpm for 1 min and washed three times for 30 s each with 100 to 250 µl of 0.01% NETN. Finally, the beads were boiled in sodium dodecyl sulfate (SDS) loading buffer containing 10% -mercaptoethanol and pelleted. The resulting supernatants were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) on a 10% gel (2). The gels were either dried or transferred to polyvinylidene difluoride membrane (Millipore, Madison, Wis.) in 25 mM Tris base-0.19 M glycine and subjected to autoradiography. A PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.) was used for quantification. GST-GRIP1 NRmut was made by amplifying aa 563 to 1121 from a full-length GRIP1 construct containing two mutations in both NR box II (L693A and L694A) and NR box III (L748A and L749A) (10) and ligating it into the BamHI/EcoRI sites of pGEX-2TK (Pharmacia).

Protease digestion assay. Protease digestion assays with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma) were carried out by the addition of the indicated amount of protease to in vitro-synthesized ³⁵S-labeled HNF4. One microliter of appropriately diluted trypsin was added to 2 µl of lysate diluted in 7 µl of 50 mM Tris (pH 7.0). Reactions were stopped after incubation at room temperature for 15 min by the addition of SDS loading buffer. Samples were subsequently analyzed as described above for the pulldown assays. Molecular weight (MW) markers (SigmaMarker, wide-MW range) included in a parallel lane in the gel were visualized by Coomassie blue staining of the membrane. Digestion with endoproteinase LysC (EndoLysC; Boehringer Mannheim, Indianapolis, Ind.) was carried out in a similar fashion except that the digestion buffer was 50 mM Tris (pH 8.6)-0.3 M NaCl-1 mM EDTA and incubation was for 1 to 2 h. Time courses of digestion with carboxypeptidase Y (Boehringer Mannheim) were carried out according to a previously published protocol (54). Briefly, lysate (10 µl) was incubated at room temperature with protease in a 210-µl reaction in 50 mM Tris-HCl (pH 6.7), and 21-µl aliquots were removed at the indicated times. Reactions were stopped by the addition of SDS loading buffer and 10 mM phenylmethylsulfonyl fluoride and placement on dry ice and were subsequently analyzed by SDS-PAGE and autoradiography. Proteolytic cleavage sites in Fig. 5E were determined by a comparison of observed MW measured from R_f values in SDS-PAGE to predicted MW based on amino acid composition, and by comparison of the bands to each other as explained in the text. When more than one residue could yield a fragment of a particular MW, the residue with the greatest surface probability according to Emini as determined by PEPTIDESTRUCTURE in the Genetics Computer Group package (20), was chosen as the potential cleavage site (e.g., R131 has a surface probability of 3.054 whereas R132 has one of 3.571). Efforts were made to minimize the number of differences between HNF41 and HNF42.

	RESULTS

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The F domain of HNF41 negatively regulates coactivator-mediated transcription in vivo. Full-length HNF41 and truncated forms lacking either or both of the N and C termini (HNF4.N1.C374, HNF4.N45.C455, and HNF4.N45.C374, respectively [Fig. 1]) were tested for responsiveness to coactivators GRIP1, p300, and CBP. HNF41 was cotransfected into 293T cells with the luciferase reporter construct pZLHIVAI-4 (Fig. 2A) and increasing amounts of GRIP1, p300, or CBP. All three coactivators were capable of enhancing transcriptional activation by full-length HNF41, although p300 and CBP yielded much greater enhancement than GRIP1 (Fig. 2B). The significance of the difference between the coactivators, if any, is not known. All three coactivators also enhanced HNF41-dependent transcription in other cell types, such as HeLa, and on other promoter constructs (data not shown).

View larger version (45K): [in this window] [in a new window]   FIG. 2.   The F domain of HNF41 inhibits transcriptional enhancement by coactivators. (A) Diagram of promoter region of reporter construct pZLHIVAI-4. HIV.LTR, human immunodeficiency virus long terminal repeat. (B to D) Transient cotransfections into 293T cells were performed as described in Materials and Methods with 2 µg of reporter construct, 0.5 µg of various HNF41 expression vectors as described in Fig. 1, and various amounts of GRIP1 (pSG5.GRIP1 full length), p300 (CMV.HA.p300 partial), or CBP (pRC.RSV.HA.CBP full length) expression vectors as indicated. Shown is the average fold induction of the relative light units normalized to a -Gal control (0.5 to 1.0 µg of RSV.gal) from one of several experiments. Error bars indicate the range of triplicate samples. Panel C and minus in panel D, no added coactivators. (D) One microgram GRIP1 and 5 µg of CBP expression vectors were used. Note the difference in scale of the y axes in panels C and D. (E) Electrophoretic mobility shift analysis of crude nuclear extracts from COS-7 cells transiently transfected with the various HNF4 expression vectors as indicated (HNF41 and HNF42, 25 µg of expression vector per 1.6 × 106 cells; N45C455, N1C374, and N45C374, 50 µg per 3.4 × 106 cells). DNA was introduced into the cells by standard calcium phosphate procedure, and the cells were harvested 40 h after glycerol shock. 32P-labeled APF1 oligonucleotide (0.5 ng per 7.5-µl reaction) was incubated with the protein extract (0.5 µg) in the presence of nonspecific DNA (0.5 µg of dI-dC, 0.5 µg of sonicated salmon sperm DNA) for 20 min at room temperature before the addition of antiserum (0.5 µl) as indicated. The incubation was continued for another 20 min before electrophoresis on a 6% native polyacrylamide gel. Details of procedures have been described previously (44, 78). Lanes: , no antiserum added; PI, preimmune antiserum; 445, antiserum to the C terminus of rat HNF41 (80); N1.14, antiserum raised in rabbit to a synthetic peptide corresponding to the first 14 aa of the N terminus of rat HNF41.

Physical interaction of HNF41 with coactivators GRIP1 and SRC1a is inhibited by the F domain. To determine whether HNF41 interacts physically with coactivators, a yeast two-hybrid assay was performed (Fig. 3A). When the entire hinge region plus LBD and F domain were fused to the Gal4 DBD (HNF41.128-455), a small but significant amount of -galactosidase (-Gal) activity was produced in the absence of coactivators, indicating that the HNF41 fragment contains a transcriptional activation domain active in yeast. Since no GRIP1 or SRC1 homologs have been found in Saccharomyces cerevisiae, the HNF4 construct must interact with either some other coactivator in yeast or the basal transcription machinery directly. Interestingly, when the Gal4-HNF4 fusion construct was truncated at amino acid 415 (HNF41.128-415) or amino acid 370 (HNF41.128-370), the basal level of -Gal activity in the absence of any exogenously added coactivator was greatly increased, as observed in mammalian cells (Fig. 2C). The presence of the GRIP1-Gal4AD fusion protein significantly enhanced the -Gal activity further but only with those GRIP1 constructs containing three NR boxes (full-length and 320 to 1121). The increase in -Gal activity indicates a physical interaction in the yeast two-hybrid system. Similar results were obtained with the related coactivator SRC1a (Fig. 3A). These results indicate that, like other nuclear receptors, HNF41 interacts with GRIP1 and SRC1. What has not been shown for other receptors, however, is the inhibition of interaction between a nuclear receptor and coactivators by portions of the F domain.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Interaction between HNF41 and coactivators in vivo and in vitro is inhibited by the presence of the F domain. (A) Interactions between HNF41 and coactivators GRIP1 and SRC1a were examined in the yeast two-hybrid assay (Clontech) with various Gal4DBD-HNF4 (pGBT9) and Gal4AD-GRIP1 or -SRC1a (pGAD424) constructs as described in Materials and Methods and previously (10). Shown is one representative experiment of two or more independent transformations into S. cerevisiae SFY526 containing an integrated -Gal reporter construct. Standard deviations are from four independent clones from a single transformation. Numbers indicate amino acid sequence encoded in the various constructs. NR boxes, nuclear receptor interaction motifs as previously described (10). (B) In vitro pulldown assays between the GST control and GST-GRIP1 (aa 563 to 1121) and in vitro-translated 35S-HNF41 constructs as indicated were performed as described in Materials and Methods. Shown is the phosphorimage after SDS-PAGE of eluted material as well as percent binding of input protein (10% input is shown). One of several experiments is shown. Positions of 14C-labeled MW markers are shown at the left. Negative controls for the pulldown assays shown in panel B, using in vitro-translated 35S-C/EBP and 35S-luciferase, are not shown. (C) As for panel B. wt, GST-GRIP1 as in panel B; GRIP1 NRmut, as wt GRIP1 except with mutations in NR boxes II and III. The presence of the F domain and AF-2 is indicated for each HNF4 construct.

The F domain of HNF41 obscures an AF-2-independent binding site for GRIP1. Since the F domain begins immediately following the AF-2 region, we hypothesized that it might inhibit transcription by obscuring the AF-2 region and thereby impeding access to coactivators. To test this, we first needed to verify that the AF-2 region of HNF41 is required for interaction with GRIP1. Since the AF-2 regions of other receptors have been found to interact with the NR boxes of coactivators, we also examined the role of GRIP1 NR boxes II and III in HNF41 binding in vitro. The results (Fig. 3C) were rather surprising. As expected, full-length HNF41 did not significantly bind a GST-GRIP1 construct mutated in two of the three NR boxes (NRmut). However, when the F domain was deleted, there was appreciable binding to the GRIP1 mutant as well as to the wild-type (wt) GRIP1 (N1C374). Perhaps even more surprising, when HNF41 was further truncated to remove the AF-2 region, significant binding to both wt and mutant GRIP1 was again observed (N1C360). A similar result was observed with even further truncation of HNF41 (N1C268X).

HNF42 preferentially activates transcription and responds to coactivators in vivo and in vitro. Since the results presented above indicated that the F domain of HNF41 modulated the interaction with coactivators and since there is a prevalent splicing variant of HNF41 that contains a modified F domain (HNF42 [Fig. 1]), we determined the effect of this splicing variant on HNF4 transactivation function. Transient transfection analysis comparing levels of activation by HNF41 and HNF42 showed not only that HNF42 activated transcription approximately fourfold better than HNF41 in the absence of added coactivator but also that GRIP1 and CBP each stimulated transcription by HNF42 approximately sevenfold more than they stimulated transcription by HNF41 (Fig. 4A). The greater effect of both coactivators on HNF42 was also seen over a range of DNA concentrations (Fig. 4B), although at larger amounts of DNA the effect of the coactivators was somewhat less. Western blot analysis verified that the HNF41 and HNF42 proteins were expressed to similar levels in the 293T cells used for the transfections and in COS-7 cells (Fig. 4C).

View larger version (32K): [in this window] [in a new window]   FIG. 4.   HNF42-mediated transcription is preferentially enhanced by coactivators GRIP1 and CBP. (A) Transient cotransfections were performed as for Fig. 2 with 0.1 µg of HNF41 or HNF42 and 5 µg of GRIP1 or CBP expression vectors as indicated. Error bars indicate range of the fold induction between triplicate samples. Plotted on the y axis is the fold induction compared to the reporter alone. Numbers in the plot represent fold induction of HNF42 relative to HNF41 under similar conditions. (B) As for panel A except with increasing amounts of expression vectors as shown. Error bars not shown for 0, 1, and 5 µg of GRIP1 were all less than 10%. Note difference in scale in y axis in between the GRIP1 and CBP panels. The difference between HNF41 and HNF42 in the presence and absence of coactivators was seen in at least three independent experiments, all done in triplicate. (C) Western blot analysis of HNF41 and HNF42 proteins transiently expressed in 293T and COS-7 cells, using chemiluminescence (Pierce, Rockford, Ill.). Twenty-five micrograms of total protein of 293T extracts (lanes 1 and 2) and 10 µg (lanes 3 and 4) or 25 µg (lanes 5 and 6) of nuclear extracts of COS cells were analyzed with a 1:5,000 dilution of 445 (see the legend to Fig. 2). Extracts from nontransfected cells showed no bands in this region of the gel (not shown). (D) GST pulldown experiments were performed as for Fig. 3B with 35S-HNF41 and 35S-HNF42 and GST control (GST) or GST-GRIP1 (aa 563 to 1121) (GRIP1). Shown are 2 of 13 representative experiments with percent binding of input (In, 10%) normalized to GST control beads and a graph of the ratio of percent bound HNF42 to percent bound HNF41 (% Bd 2/% Bd 1) (each controlled for binding GST beads) for all 13 experiments (two of which are the average of duplicate samples). The dashed line indicates a ratio of 1.0, which one would expect if there were no difference between HNF41 and HNF42. The average ratio for the 13 experiments was 1.6. A nonparametric paired t test (Wilcoxon signed-rank test) performed by the STATView program yielded a P value of 0.0024 for the 13 experiments, indicating that the difference noted between HNF41 and HNF42 is statistically significant.

The presence of the 2 insert alters the protease sensitivity of HNF4. To determine whether there are any structural differences between HNF41 and HNF42 that could explain the enhanced binding and responsiveness to GRIP1 (and CBP) we performed a series of protease digestion experiments using the ³⁵S-labeled HNF4 constructs. First, a time course of digestion of HNF41 and HNF42 was performed with carboxypeptidase Y, an exopeptidase that sequentially cleaves amino acids in a C- to N-terminal fashion. The results indicate that there is indeed a difference between HNF41 and HNF42. Not only did the full-length HNF42 begin to disappear faster than the full-length HNF41 (Fig. 5A; compare lanes 8 to 10 to lanes 1 to 3), but HNF42 yielded a protected fragment that was significantly more pronounced than a similar fragment in HNF41 (compare lanes 9 to 13 to lanes 2 to 6). Since the protected fragment migrates slightly slower than uncleaved N1C374 (lane 15), this finding suggests that there might be a structural difference between HNF41 and HNF42 C terminal to aa 374 (i.e., in the F domain).

View larger version (50K): [in this window] [in a new window]   FIG. 5.   The presence of the 2 insert alters the protease sensitivity of HNF4. (A) Autoradiograph after SDS-PAGE (10% gel) of a time course (in minutes) of proteolytic digestion of 35S-labeled HNF41 and HNF42 with carboxypeptidase Y (80 ng/µl, final concentration) which cleaves sequentially from the C terminus (see Materials and Methods for details). Uncleaved N1C374 serves as an MW marker in lane 15. Arrows point to protected fragments mentioned in the text. (B) As for panel A except that HNF41 and HNF42 were digested with increasing amounts of EndoLysC as indicated. a, a', b, and b', arbitrary labeling of fragments referred to in the text. a* and b* represent cleavage in the very C terminus only (most likely K439 in HNF41 and K449 in HNF42) since cleavage at the first N-terminal lysine residue, K61, would yield a band migrating much faster. (C) As for panel B except digestion of HNF41 and HNF42 was compared to digestion of N1C374. Tryp, trypsin; LysC, EndoLysC. Labeling of EndoLysC fragments is the same as in panel B. MW, MW markers (positions are indicated in kilodaltons). (D) As for panel C except that N127.374, an engineered fragment of 28.6 kDa containing residues 127 to 374 of HNF41 plus an additional eight residues (described in Materials and Methods), was added to the 35S-N1C374 trypsin digestion right before loading on the gel. Coomassie blue-stained blots before and after cutting out the engineered fragment (double-edged arrow) are shown on the top, and the corresponding autoradiographs (Autorad) are shown on the bottom. M, MW markers; c, same as in panel C. The arrow in the after-cutting blots shows how the band corresponding to the engineered fragment was moved. (E) Map of potential cleavage sites for trypsin (Lys-X or Arg-X) in rat HNF41 and HNF42. Tick marks indicate either a lysine (K) or an arginine (R) residue. To simplify the presentation, only those residues that are thought to be cleaved (plus K356) are indicated by a residue number. Note the complete absence of the Arg and Lys residues in the A/B domain. Also shown are the receptor domains (A to F), the AF-2 region (aa 360 to 368), a previously identified repressor region (aa 428 to 441) (40) (see Discussion), and the amino acid sequence of the insert in HNF42 (aa 410 to 419) and its predicted secondary structure as determined by the Chou-Fasman algorithm in PEPTIDESTRUCTURE in the Genetics Computer Group package (20). T, strong probability of a beta turn; h, possible alpha helix; , no predicted structure. (F) Schematic representation of the trypsin digestion products shown in panels C and D and observed (Obs) and calculated MW (Calc) MW (in thousands) of each fragment in descending order. As discussed in the text, HNF42 appears to have a tryptic cleavage site near R168 that is not present in HNF41. The large number of K and R residues in domain C results in some ambiguity in the determination of the N-terminal cleavage sites. The ambiguity was resolved by relying on predictions of MW and surface probability as explained in the text. When two potential cleavage sites were close together and exhibited similar surface probabilities, such as R413 and R415 in HNF42, the internal most site was used (e.g., R413). Band c of N1C374 is predicted to represent cleavage only in the N terminus since carboxypeptidase Y data indicated that its C terminus is rather resistant to digestion (data not shown). 1, HNF41; 2, HNF42, 374, HNF4.N1C374. Numbers indicate amino acid residues.

	DISCUSSION

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The results of this study show that HNF41 responds to transcriptional coactivators GRIP1, p300, and CBP in vivo (Fig. 2 and 4). They also show a direct physical interaction between HNF41 and GRIP1 and SRC1a which appears to involve at least some of the NR boxes of GRIP1 and SRC1a (Fig. 3). Unlike most other nuclear receptors, however, addition of an exogenously added ligand was not required for the in vivo or in vitro effects of coactivators on HNF41. While this work was in progress, others observed similar interactions between HNF41 and CBP and between SRC1 and GRIP1 (9, 22, 64, 87, 94). Our results, however, show for the first time that the F domain of a nuclear receptor can partially block interaction with a coactivator (Fig. 2 and 3) and that the blockage is abrogated by a 10-aa insertion in the F domain generated by naturally occurring alternative splicing (HNF42 [Fig. 4]). They also show that the insertion induces structural changes in the F domain and elsewhere in the protein which could explain the altered blockage (Fig. 5).

There are a few reports of splicing variants of coactivators and corepressors acting differentially with nuclear receptors (31, 47, 76). There is also one report of nuclear receptor splicing variants differentially responding to corepressors (36). However, to our knowledge, this is the first published report showing that a naturally occurring splicing variant in a nuclear receptor interacts differentially with a coactivator. This is an important finding since nearly every nuclear receptor gene exhibits some degree of alternative splicing, although the functional significance of that splicing is not always known. As is seen in Fig. 4, interaction with coactivators can accentuate the difference between transcription factor isoforms that are otherwise difficult to detect, especially in transient transfection systems in which the factors tend to be expressed far beyond physiological concentrations. Furthermore, this phenomenon of splicing variants interacting differentially with coactivators might be more generally applicable to other transcription factors systems for which alternative splicing has also been shown to play a role in the control of gene expression (81).

Proposed mechanism for inhibitory action of the HNF4 F domain. Iyemere et al. (40) recently reported a repressor region from aa 428 to 441 in rat HNF41 and observed that it showed a significant degree of similarity to a previously defined repressor region in the C-terminal extension of the LBD of PR (49, 91) (Fig. 6A). Shortly thereafter, the three-dimensional crystal structure of the LBD of PR was solved and showed that the repressor region forms a beta strand which is tightly fixed in position by an antiparallel beta-sheet interaction with another beta strand between helix 8 and helix 9 in the LBD (90). We have incorporated these findings and our current results into a model to explain the mechanism by which the F domain of HNF41 inhibits transcription (Fig. 6B). We propose that the F domain of HNF41 inhibits transcription by virtue of the repressor region, and possibly other regions, contacting another portion of the protein, most likely the LBD as in PR. This contact might obscure, at least partially, an activation region(s) such as AF-2 and thereby limit access to coactivators (Fig. 3 to 5). In HNF42, the predicted structure of the region suggests that the 10-aa insert introduces a turn in the F domain (Fig. 5E), which might cause a partial displacement of the repressor region(s), thereby exposing a protease cleavage site (Fig. 5) and the activation region(s). The net result is that the activation region(s) is somewhat more accessible to coactivators and that HNF42 activates transcription more efficiently than HNF41 (Fig. 4). In HNF4.N1.C374 and HNF4.N45.C374 (HNF4374), there is no F domain to obscure the activation region(s), resulting in maximal physical contact with coactivators and hence transactivation (Fig. 2 and 3).

View larger version (30K): [in this window] [in a new window]   FIG. 6.   Model for the inhibition of transactivation and coactivator binding by the F domain of HNF41 and HNF42. (A) Shown is an alignment of the repressor regions in rat HNF41 (rHNF41) and human PR (hPR) to a region in the C-terminal extension of human GR, AR, and MR (hGR, hAR, and hMR) and the distance to the AF-2 which is located at the C-terminal end of the LBD. Residues that differ from the consensus are shown. Identical residues are indicated by dashes. Numbers indicate amino acid residues, which are given in single-letter code. , hydrophobic residue; x, any residue. (B) Schematic diagram (not drawn to scale) of proposed interactions between the F domain and the remainder of the receptor of HNF41, HNF42, and HNF4.N1.C374 or HNF4.N45.C374 (HNF4374). Relative transcriptional activities from Fig. 2 and 4 are indicated. Specific regions shown are defined in the key at the bottom. The activation region(s) includes the AF-2 region, and possibly other regions, as discussed in the text.