(Received for publication, February 8, 1996, and in revised form, September 26, 1996)
From the Department of Medicine and Biochemistry, Section of Molecular Genetics, Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118-2394 and the § Gene Regulation Laboratory, Hematology/Oncology Division, Department of Medicine, New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215
The hepatocyte nuclear factor 4 (HNF-4) is a
member of the nuclear receptor superfamily and participates in the
regulation of several genes involved in diverse metabolic pathways and
developmental processes. To date, the functional domains of this
nuclear receptor have not been identified, and it is not known whether
its transcriptional activity is regulated by a ligand or other signals.
In this report, we show that HNF-4 contains two transactivation
domains, designated AF-1 and AF-2, which activate transcription in a
cell type-independent manner. AF-1 consists of the extreme N-terminal
24 amino acids and functions as a constitutive autonomous activator of
transcription. This short transactivator belongs to the class of acidic
activators, and it is predicted to adopt an amphipathic -helical
structure. In contrast, the AF-2 transactivator is complex, spanning
the 128-366 region of HNF-4, and it cannot be further dissected
without impairing activity. The 360-366 region of HNF-4 contains a
motif that is highly conserved among transcriptionally active nuclear receptors, and it is essential for AF-2 activity, but it is not necessary for dimerization and DNA binding of HNF-4. Thus, HNF-4 deletion mutants lacking the 361-465 region bind efficiently to DNA as
homo- and heterodimers and behave as dominant negative mutants.
Remarkably, the full transactivation potential of AF-2 is inhibited by
the region spanning residues 371-465 (region F). The inhibitory effect
of region F on the HNF-4 AF-2 activity is a unique feature among
members of the nuclear receptor superfamily, and we propose that it
defines a distinct regulatory mechanism of transcriptional activation
by HNF-4.
The nuclear receptor superfamily comprises a large set of ligand-regulated transcription factors. This superfamily includes receptors for steroid hormones, retinoids, thyroid hormone, and vitamin D3, as well as a large number of structurally and functionally related transcription regulatory proteins whose natural ligands are not yet known, the so-called orphan receptors (reviewed in Refs. 1 and 2). Nuclear receptors exhibit a modular structure with six distinct regions (referred to as regions A-F), which correspond to functional domains. The N-terminal region A/B is highly variable among nuclear receptors and contains a ligand-independent transactivation function AF-1 (2). Region C contains a highly conserved DNA binding domain (DBD)1 composed of two zinc-coordinated modules and is responsible for specific binding to cognate response elements (Refs. 1 and 2 and references therein). The exact functions of regions D and F are not clear, although they appear to be well conserved for each receptor across species. Region D is postulated to function as a flexible hinge between the DBD and the ligand-binding domain (LBD), allowing rotational differences between these domains when dimeric receptors bind to direct, inverted, or palindromic repeats (2). Interestingly, the D regions of the thyroid hormone (TR) and retinoic acid receptors (RARs) interact with the co-repressor proteins N-CoR and SMRT, which mediate the ligand-independent transcriptional repression (3, 4).
Region E is functionally complex, since it contains the LBD, the
dimerization interface, and the ligand-dependent
transactivation function AF-2 (Ref. 2 and references therein). A short
activating domain has been identified in the C-terminal part of AF-2 in
many nuclear receptors, designated AF-2 AD, which is required for the ligand-dependent activity of AF-2 (5, 6, 7, 8). Mutagenesis of
the AF-2 AD can selectively abrogate AF-2 activity without affecting
nuclear receptor dimerization and DNA binding (2, 5, 6, 7, 8). The AF-2 AD
contains the highly conserved motif XE
(
being a hydrophobic amino acid, and X a nonconserved amino
acid), and it has been proposed to mediate interactions between the
receptors and transcription coactivators (2, 5, 6, 7, 8). Based on the
importance of this conserved region in ligand-dependent
transcriptional activation, it has been proposed that members of the
nuclear receptor superfamily that contain this motif may stimulate
transcription through a mechanism that involves a ligand (5). Recently,
the crystal structures of the unliganded human RXR
LBD (9) and the
liganded human RAR
(10) and rat TR
1 (11) LBDs revealed a novel
fold, termed the antiparallel
-helical sandwich, which consists of
12
-helices (H1-H12) packed in three layers and harboring an
internal hydrophobic ligand-binding core. The dimerization interface of
the RXR
LBD is formed primarily by helix H10 and to a lesser degree
H9 and a loop between H7 and H8 (9). Interestingly, the AF-2 AD
corresponds to the amphipathic
-helix H12, which in the unliganded
RXR
LBD extends into the solvent but in the liganded RAR
and
TR
1 LBDs is packed onto the body of the receptor, contributing to
the formation of the ligand-binding pocket and the surface that
interacts with putative transcription coactivators, such as TIF1,
TRIP1, SRC-1, or RIP140 (12, 13, 14, 15).
The hepatocyte nuclear factor 4 (HNF-4) belongs to the nuclear receptor superfamily, and it is expressed primarily in the adult liver, intestine, and kidney (16). The human and rat HNF-4 proteins are highly conserved, with an overall similarity of 96% (17). There is also a Drosophila homolog of HNF-4, which has lower similarity with the mammalian proteins, and it is expressed very early as maternal mRNA and during organogenesis (18). The mammalian HNF-4 has a distinctive F region among nuclear receptors whose function is unknown (16). Adult human and rat liver and kidney contain two isoforms of HNF-4, which differ by the presence or absence of a 10-amino acid segment in the middle of region F (17, 19). The longest of the isoforms, referred to as HNF-4A (17), is by far dominant in the liver, kidney, and the hepatoma cell line HepG2, whereas the shortest isoform (HNF-4B), which was cloned first, represents only a minor species in these tissues (17, 19). These isoforms are generated by differential splicing, but it is not known whether they have identical or different transactivation functions (20).
No ligand has been identified to date for HNF-4, which is classified as an orphan receptor. Nevertheless, it has been shown to play an important role in the regulation of several genes involved in diverse metabolic pathways. HNF-4 binds to its cognate elements as a dimer and is a positive regulator of several target genes, including the genes for apolipoprotein (apo) AI (21), apoB (22), apoCIII (22, 23), apoAII (22), apoAIV (24), medium chain acyl-CoA dehydrogenase (25), cellular retinol-binding protein II (26), the long terminal repeat of human immunodeficiency virus-1 (27), and the coagulation protease factor VII (28). Moreover, HNF-4 plays a key role in the transcriptional regulatory hierarchy of liver-specific gene expression, because it regulates the expression of HNF-1, a transcription factor important for the expression of several hepatic genes (29). Disruption of the murine HNF-4 gene, which is expressed in the visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos, indicating that HNF-4 is critical for early development (30). However, despite the recognition of the HNF-4 importance in developmental processes and the progress in identification of downstream targets of HNF-4, the functional domains of this nuclear receptor and the molecular mechanisms by which it regulates transcription are largely unknown.
As part of our ongoing studies on the mechanisms of transcriptional activation of apolipoprotein gene expression by HNF-4, we have performed a systematic analysis of the functional domains of HNF-4 that are involved in DNA binding, dimerization, and transactivation. We demonstrate here that HNF-4 contains two transactivation domains, designated AF-1 and AF-2, which are located in the A/B and D/E regions, respectively, and activate transcription in a cell type-independent manner. The AF-1 consists of the extreme N-terminal 24 amino acids and functions as an autonomous acidic transactivator. In contrast, the AF-2 transactivator is very complex, spanning the 128-366 region of HNF-4, and it cannot be further dissected without impairing activity. Unexpectedly, the full transactivation potential of the HNF-4 AF-2 is inhibited by sequences spanning region F. This is a unique feature among nuclear receptors, and it led us to propose that it may define a novel mechanism for regulation of the AF-2 activity.
All mutants of rat HNF-4 were
generated by polymerase chain reaction (PCR). To construct the N- and
C-terminal deletion mutants, the following oligonucleotides were
designed as the N-terminal primers: HNF-N,
5-GATATC
GCCGCCGCC
GACATGGCTGACTACAGTGCT3
; HNF-48F,
5
-TACTCGAG
GCCGCCGCC
AGCGCTCTGTGTGCCATCTGTGGCGATC-3
. The C-terminal primers were as follows: HNF-HF,
5
-GATGTCTGA
GGTGGACATCTGTCCATTGCT-3
; HNF-CD1, 5
-TCTAGA
GGCAGACCCTCCAAGCAGCATCTC-3
;
HNF-CD1b, 5
-GACTGA
CAGGTTGTCAATCTTGGCCAT-3
;
HNF-CD2, 5
-TCTAGA
GGTAATGCTCTGTAGAGTGGGCAGG-3
; HNF-CD3, 5
-TCTAGA
ATCTGGGTCAAAGAAGATGATGGCTTT-3
;
HNF-CD4, 5
-TCTAGA
GTCATTGCCTAGGAGCAGCACATCCT-3
;
HNF-CD5, 5
-TCTAGA
GCTGGCAATCCTCTTGGCCCGAAT-3
; HNF-CD6,
5
-TCTAGA
GATCCGATCCCGCTCATTTTGGAC-3
;
PCRHNF-C, 5
-TCTAGA
TCTGAGGGTGTGAGCCAGCAGAAGCCT-3
.
The underlined nucleotides indicate the cloning sites HindIII and BamHI as well as the initiator ATG and terminator TGA (TCA in reverse orientation) codons. The boldface letters indicate the Kozak sequence placed adjacent to the initiator ATG for optimal translation. The PCR products were digested with HindIII and BamHI and were cloned in the expression vector pcDNAI/Amp (Invitrogen) to generate constructs HNF-4B, HNF-HF, CD1 to CD6, D1HNF-4, and DCD1.
The HNF-4A isoform was constructed as follows. HNF-4 fragment A was
generated by PCR using the forward primer H-1
(5-GCCAGTGACGCGCCCCACGCCCACCA-3
) and the reverse primer H-3
(5
-
ACACATCTGTCCATTGCTGAGGT-3
). Similarly, HNF-4 fragment B was generated using the
forward primer H-2
(5
-GAGTGGCCCCGGCCCAGGGGGCAGGCAGCCACCCCTGAGACTCCACAGCCA-3
), which
contains the reverse complement of the underlined sequence of primer
H-3, and the reverse primer GAL-455R
(5
-TCTAGAGGATCCGGCGGCTTGCTAGATGGCTTCCT-3
). Aliquots containing 2% of
each of the amplified fragments A and B were mixed and used for another
round of PCR amplification in the presence of H-1 and GAL-455R primers.
The resulting PCR fragment was digested with HgaI and
BamHI, and it was used to replace the HgaI/BamHI fragment of HNF-4B cloned in the
plasmid pBXG1, containing the GAL4 DBD-(1-147) (31). Following
verification by sequencing, the resulting HNF-4A cDNA was
subcloned as an EcoRI/ BamHI fragment in the
pCDNAI/Amp vector.
Plasmids ND1-ND5, containing internal deletions of amino acids
129-174 (ND1), 175-239 (ND2), 175-289 (ND3), 175-336 (ND4), and
175-369 (ND5) were generated as follows. The HNF-4 C-terminal fragments from residues 174, 240, 290, 337, and 370 to residue 455, were generated using as forward primer the oligonucleotides HNF-ND1
(5-
AGCATCACGGATGTGTGTGAGTCTA3
, HNF-ND2
(5
-
TACATCGTCCCTCGGCACTGTCCA-3
), HNF-ND3
(5
-
GCCAAGGGGCTGAGTGACCCAG-3
), HNF-ND4
(5
-
AGCATTACCTGGCAGATGATCGAGC-3
), and HNF-ND5
(5
-
GCCAGTGACGCGCCCCACGCC-3
), containing the indicated deletions, and as reverse primer the oligonucleotide PCRHNF-C. Similarly, the HNF-4 N-terminal fragments 1-28 (for ND1) and 1-174 (for ND2 to ND5) were generated by PCR using
as forward primer the oligonucleotide PCRHNF-N and as reverse primer
the oligonucleotides PCR-IDS1 (5
-GATCCGATCCCGCTCATTTTGGAC-3
) and
PCR-IDS2 (5
-GCTGGCAATCCTCTTGGCCCGAAT-3
). The PCR-IDS1 is the reverse
complement of the underlined sequence of oligonucleotide HNF-ND1, and
PCR-IDS2 is the reverse complement of the underlined sequence of
oligonucleotides HNF-ND2 to HNF-ND5. Aliquots containing 2% of each of
the amplified regions were mixed and used for another round of PCR
amplification in the presence of PCRHNF-N and PCRHNF-C primers. The
resulting PCR fragments were digested with BamHI and
HindIII and cloned in the pcDNAI/Amp vector to generate
constructs ND1-ND5.
The full-length and deletion mutants of GAL-HNF-4 chimeras were either obtained from pcDNA/HNF-4 plasmids by PCR amplification or generated by PCR using appropriate primers and were cloned in frame into plasmid pBXG1 at the EcoRI and BamHI sites. Mutants GAL-L366E and GAL-E363K were generated by PCR-mediated site-directed mutagenesis, using appropriate primers. All constructs were verified by DNA sequencing analysis.
In Vitro Transcription and TranslationpcDNAI/Amp constructs containing the wild type HNF-4 receptor and its deletion mutants were linearized with BamHI, transcribed in vitro with T7 RNA polymerase, and translated with rabbit reticulocyte lysates (Promega) in the presence of [35S]methionine, as recommended by the manufacturer.
Electrophoretic Mobility Shift Assay (EMSA)HNF-4 proteins produced by in vitro translation (2 µl) were incubated with 32P-labeled double-stranded oligonucleotide probes (10 fmol) for 15 min at 4 °C in the presence of 25 mM HEPES, pH 7.6, 40 mM KCl, 1 mM dithiothreitol, 5 mM MgCl2, and 0.6 µg of poly(dI-dC). Protein-DNA complexes were analyzed by electrophoresis in 5% nondenaturing gels, followed by autoradiography, as described previously (22, 27, 32).
Transient Transfection AssaysPlasmids were transfected
into HepG2, Caco-2, and HeLa cells and were assayed for their ability
to promote transcription of the chloramphenicol acetyltransferase (CAT)
gene constructs. All transient transfections were performed using the
calcium phosphate DNA coprecipitation method, as described previously
(22, 27, 32). The transfection mixture contained as a control the
RSV--gal plasmid. The
-galactosidase activity of the cell lysates
was determined as described previously (22, 27, 32), and the values
were used to normalize variabilities in the efficiency of transfection.
CAT activities were determined using 14C-chloramphenicol
and acetyl-CoA as described previously (22, 27, 32). The nonacetylated
and acetylated chloramphenicol forms were separated on IB2 silica gel
plates using chloroform/methanol (95:5) for development. The
radioactive spots, detected by autoradiography, were cut from the thin
layer plates and counted.
COS-1 cells were transfected with the
different GAL-HNF-4 chimeras. Protein extracts corresponding to
approximately 0.5-1 × 106 cells/lane were combined
with 5 × loading buffer (0.3 M Tris-HCl, pH 6.8, 10%
SDS, 50% glycerol, 25% -mercaptoethanol, and 0.05% bromphenol
blue) in a total volume of 70 µl and separated on 10 or 13%
SDS-polyacrylamide gels. Proteins were transferred to a nitrocellulose
membrane by electroblotting. Membranes were preincubated in PBS
containing 3% nonfat dry milk overnight at 4 °C. Subsequently, they
were incubated with the primary anti-GAL4 rabbit polyclonal antibody
(Upstate Biotechnology, Inc.) at a dilution of 1:500 in PBS containing
1% nonfat dry milk and 0.05% Tween 20 (PBST) for 1 h at
37 °C. Membranes were washed in the same buffer and incubated with
the secondary antibody, goat anti-rabbit IgG conjugated to alkaline
phosphatase (Kirkegaard & Perry Laboratories, Inc.) at a dilution
1:5,000 in PBST for 1 h at 37 °C. Membranes were washed in
PBST, and proteins were visualized by developing the alkaline
phosphatase color according to the manufacturer's specifications.
For indirect immunofluorescence analysis, wild type HNF-4 and mutant proteins CD1 and ND1, carrying a FLAG peptide (MDYKDDDDK) (Kodak, IBI) at their N termini, were cloned in pcDNAI/Amp vector and used to transfect COS-1 cells grown on coverslips (33). After transfection, cells on coverslips were fixed in 3.7% formaldehyde in PBS for 20 min at 20 °C, permeablized with 0.1% Nonidet P-40 in PBS for 10 min at 20 °C, and incubated with a specific monoclonal antibody M2 to the FLAG peptide, at a concentration of 9 µg/ml in PBS for 40 min at 37 °C. Cells were stained, using as a secondary antibody a rhodamine-labeled anti-mouse IgG (Kirkegaard & Perry Laboratories) at a 1:10 dilution (50 µg/ml), and incubated at 37 °C for 40 min, followed by extensive washes in PBS.
To
investigate the functional properties of HNF-4 we cloned HNF-4A,
HNF-4B, and a number of N-terminal, C-terminal, and in-frame internal
deletion mutants in the mammalian expression vector pcDNAI/Amp (Fig. 1A). In vitro translated
HNF-4A, HNF-4B, and various deletion mutants were tested for DNA
binding in an EMSA, using the CIIIB element of the apolipoprotein CIII
promoter as a probe, which is a high affinity binding site for HNF-4
(22, 34). This analysis showed that HNF-4A and HNF-4B bound strongly to
DNA (Fig. 1B, lanes 1 and 4), in
agreement with previous studies (16, 17, 22). Mutants HNF-HF, CD1, and
CD1b, lacking part or all of the F region also bound strongly to DNA,
(Fig. 1B, lanes 2, 3, 5), indicating that region F is not required for DNA binding. In contrast, mutant CD2 lacking residues 340-465 lost its ability to bind to element CIIIB (Fig. 1B, lane 6), indicating that
sequences spanning the region 340-360 are required for efficient DNA
binding. Similarly, mutants CD3 and CD4, harboring progressive
C-terminal deletions within region E, lost their ability to bind to
element CIIIB (Fig. 1B, lanes 7 and
8). This inability to bind DNA was not due to inefficient
synthesis of these proteins, because the integrity and amounts of the
in vitro translated proteins were monitored by
SDS-polyacrylamide gel electrophoresis and 35S-sensitive
autoradiography prior to EMSA analysis (Fig. 1D). However,
additional C-terminal deletions that removed either the entire region E
(mutant CD5) or both regions E and D (mutant CD6) restored DNA binding,
although with reduced affinity (Fig. 1B, lanes 9 and 10). Mutant DCD1, lacking both regions A/B and F, also
bound strongly to DNA (Fig. 1B, lane 11),
indicating that A/B is also not required for DNA binding. Taken
together, these results map the HNF-4 DBD between residues 48-128,
which corresponds to the well-studied DBD of the nuclear receptors that
contains two zinc finger modules (2). In addition, these results
indicate that, in the absence of the 340-465 region, DNA binding is
inhibited by sequences carboxyl to residue 174, and at least a portion
of these sequences is located in the 175-239 region.
In a similar series of experiments, we also tested the DNA binding properties of the internal deletion mutants ND1-ND5 produced by in vitro translation. Mutant ND1 lacking most of D domain, bound strongly to CIIIB probe (Fig. 1B, lane 12), indicating that the 129-174 region is not required for DNA binding. Similarly, mutant ND2 lacking residues 175-240, also bound to DNA (Fig. 1B, lane 13). Interestingly, mutants ND3 and ND4 lacking the regions 175-289 and 175-336, respectively, lost their ability to interact efficiently with element CIIIB (Fig. 1B, lanes 14 and 15). In contrast, deletion of the entire E region in mutant ND5 restored DNA binding, and two weak protein-DNA complexes were observed reproducibly (Fig. 1B, lane 16). These complexes were not due to proteolytic degradation of ND5 protein, as shown in Fig. 1D. Comparison of the protein-DNA complexes formed by CD5 and ND5, suggests that the presence of region F in ND5 does not destroy DNA binding but leads to the formation of two complexes that have weaker intensities than the CD5-DNA complex. Taken together, these results indicate that the region between residues 175-240 is not required for DNA binding; nevertheless, it is necessary for the formation of a tight protein-DNA complex. Moreover, in the absence of this region, DNA binding is inhibited by sequences carboxyl to residue 241, and at least a portion of these sequences is located in the 337-369 region.
It was previously shown that HNF-4 binds to DNA as a dimer (16, 27, 35). To identify the dimerization domain(s) of HNF-4, we employed EMSA analysis to monitor the formation of heterodimers between wild type HNF-4B and its C-terminal deletion mutants CD1-CD6. When equal amounts of in vitro translated HNF-4B and CD1 were mixed and then tested for binding to CIIIB probe, in addition to complexes that corresponded to HNF-4B and CD1, a complex with intermediate electrophoretic mobility (heterodimer) was also formed (Fig. 1C, lanes 1 and 2), indicating that one molecule each of HNF-4B and CD1 bound concomitantly to element CIIIB. In contrast, heterodimers were not observed between HNF-4B and any of the mutants CD2-CD6 (Fig. 1C, lanes 3-7), indicating that sequences within regions D and E are required for formation of dimers capable of DNA binding and that at least a portion of these sequences is located in the 340-360 region. Because mutants CD5 and CD6 retained their ability to bind to DNA but did not dimerize with HNF-4B, we conclude that they bind to DNA as monomers. Moreover, mutant DCD1 also formed dimers with HNF-4B that bound to DNA (Fig. 1C, lane 8), indicating that the A/B domain is not required for dimerization and that mutant DCD1 contains an intact dimerization interface. Similar results were obtained when HNF-4A and CD1b were used in a similar assay (Fig. 1C, lanes 9-11), indicating that sequences carboxyl to residue 360 are not required for efficient dimerization of HNF-4.
To map more precisely the dimerization domain of HNF-4, we tested mutants ND1-ND5 for their ability to dimerize with DCD1 in a similar series of EMSA experiments. The mutant DCD1 was used in these experiments, because its size (considerably smaller than HNF-4) facilitated the resolution of heterodimers in the dimerization assay. Mutant ND1 formed heterodimers with DCD1 that bound to DNA (Fig. 1C, lane 12), indicating that the region spanning residues 129-174 is not required for dimerization. In contrast, mutants ND2-ND5 failed to dimerize with DCD1 (Fig. 1C, lanes 13-16), indicating that sequences within E are required for formation of dimers capable of DNA binding and that at least a portion of these sequences is located in the 175-240 region. The inability of ND2 to dimerize with HNF-4 suggests that it binds to DNA as a monomer. Furthermore, since the region spanning residues 175-240 is required for efficient dimerization but not DNA binding, it is possible that the lack of this region in mutant ND2 may induce conformational changes in ND2 and explain, at least in part, the abnormal appearance of the ND2-DNA complex. Taken together, these data indicate that the dimerization domain of HNF-4 is located in the 175-360 region.
The Transactivation Potential of HNF-4 Is Negatively Regulated by Region FTo study the transcription activation domain(s) of HNF-4
independently of its DNA binding, dimerization, and nuclear
localization properties, we constructed a series of plasmids containing
the full-length HNF-4 and its deletion mutants fused in-frame to the yeast GAL4 DBD-(1-147) (36). The GAL4 DBD-(1-147) in addition to
specific DNA binding activity contains signals for dimerization and
nuclear localization (36). The various GAL-HNF-4 chimeric constructs
were cotransfected into HepG2 cells with the reporter plasmid
pG5CAT, which contains five GAL4 DNA-binding sites upstream of the -globin promoter and the CAT gene (37). The expression levels
and integrity of the wild type HNF-4 and its deletion mutants fused to
GAL4 DBD-(1-147) in transfected COS-1 cells was monitored by Western
blot analysis using an anti-GAL4 antibody (Fig. 2). In
general, the transfected cells were shown to express wild type and
mutant proteins in comparable amounts, with insufficient variations to
account for the observed differences in their transcriptional activation potential (Fig. 2). Cotransfection with GAL-HNF-4A and
GAL-HNF-4B activated the transcription of reporter pG5CAT by approximately 10-fold as compared with the activation obtained by
pBXG1, containing the GAL4 DBD-(1-147) alone (Fig. 3).
This finding indicates that the full-length HNF-4A is a transcriptional activator, comparable with HNF-4B. Surprisingly, cotransfection with
mutants GAL-HNF-HF and GAL-CD1 resulted in 45- and 65-fold increase of
the pG5CAT activity, respectively (Fig. 3). This
enhancement was not due to differences in expression or stability of
the fusion proteins, as was demonstrated by Western blot analysis (Fig.
2). Therefore, we conclude that region F has an inhibitory function on
the transcriptional activation potential of both HNF-4 isoforms, and it
has been designated the negative regulatory domain. The negative effect
of region F was unexpected, because no similar function has been
described to date for this region in other members of the nuclear
receptor superfamily. In contrast, it was previously suggested that
region F might be an activator of transcription because of its high
content in proline residues (16). However, our results clearly indicate
that the activation potential of HNF-4 resides outside region F, and in
fact, its activity is inhibited by F.
A Stretch of 24 Amino Acids at the Extreme N-terminal Domain of HNF-4 Is an Autonomous Transactivator
To identify the activation domain(s) of mutant CD1, we tested a series of HNF-4 C-terminal deletion mutants fused to GAL4 DBD-(1-147) for their potential to transactivate the pG5CAT reporter construct in HepG2 cells. Cotransfection with mutant GAL-CD2 resulted in loss of transcriptional activation of pG5CAT (Fig. 3). The dramatic decrease in the transactivation potential of GAL-CD2 indicates that the region 340-370 is necessary for high levels of transcriptional activation by GAL-CD1.
Low levels of pG5CAT activity were maintained upon
cotransfection with expression plasmids GAL-CD3 and GAL-CD4, which lack regions 290-465 and 240-465, respectively (Fig. 3). However, deletion of the residues 175-465 in expression plasmid GAL-CD5 resulted in a
dramatic increase of the pG5CAT transcriptional activity (Fig. 3). These results indicate that an activation function(s) of
HNF-4 is located in the region 1-174, which includes domains A/B, C,
and D. To examine the influence of region F on this activation function(s), we tested the transactivation potential of construct GAL-ND5. As shown in Fig. 3, the activity of GAL-ND5 was only 2-fold
lower than that of GAL-CD5, indicating that the negative regulatory
domain only slightly affects the transcriptional activity elicited by
domains A/B, C, and D. Furthermore, to identify which of these domains
was responsible for the observed transactivation potential of GAL-CD5,
we tested constructs GAL-CD6 to GAL-CD10, which contain deletions of
regions A/B, C, and D in various combinations. Transactivation was
obtained with GAL-CD6 but not with GAL-CD7, GAL-CD8, or GAL-CD9,
suggesting the presence of an activating domain in region A/B (Fig. 3).
This was confirmed by the high transactivation elicited by GAL-CD10,
demonstrating the presence of a potent activator in the region spanning
residues 1-48. Interestingly, Chou-Fasman (38) and Garnier-Robson (39)
algorithms predicted that the region spanning residues 1-24 has the
potential to adopt an -helical structure, whereas the region
spanning residues 25-48 is unstructured. Based on these structural
predictions, we asked whether the transactivation potential could be
further sublocalized in either of these two regions, by testing mutants
GAL-CD11 and GAL-CD12 for their potential to activate the reporter
pG5CAT. The results showed that GAL-CD11 retained full
transactivation potential, whereas GAL-CD12 had no activity above
background levels (Fig. 3). Therefore, we conclude that the small
region spanning residues 1-24 contains a potent transactivation
domain, designated AF-1. This transactivator has an estimated
isoelectric point of 3.04 (DNASTAR package), and it is therefore
classified as an acidic activator (40). These results suggest that
although AF-1 is a powerful autonomous transactivator, in the context
of the intact GAL-HNF-4 chimera elicits a weaker transactivation than
when it is separated from the rest of the HNF-4 sequences. It also
appears that in the context of GAL-CD2 to GAL-CD4 chimeric constructs, the activity of AF-1 is inhibited by sequences in region E, and at
least a portion of these sequences is located in the 175-239 region.
To investigate the possibility that HNF-4
contains a second transactivation domain, we generated N-terminal
deletions of HNF-4 fused to GAL4 DBD-(1-147). Deletion of the AF-1
domain resulted in 5-fold reduction of transcriptional activity of the
otherwise intact HNF-4B (construct GAL-D1HNF-4 in Fig.
4). Remarkably, deletion of region F resulted in strong
transactivation (construct GAL-D1CD1 in Fig. 4), indicating the
existence of a second activation domain spanning residues 48-370,
whose activity is inhibited by F. Additional cotransfection experiments
using a series of N- and C-terminal deletion mutants of HNF-4 fused to
GAL4 DBD-(1-147), revealed that the regions D and E contain a powerful
transactivator, designated AF-2 (Fig. 4). Comparison of the activities
of constructs GAL-D2HNF-4 and GAL-D2CD1 indicates that the activation
potential of AF-2 is strongly inhibited by F.
The importance of region 360-366, which contains the conserved motif
XE
, on the AF-2 activity was assessed with
mutants GAL-D2CD1a and GAL-D2CD1b. Deletion of residues 367-370
(mutant GAL-D2CD1a) did not affect the transactivation potential of
AF-2. However, further deletion of residues 361-366 (mutant
GAL-D2CD1b) resulted in abolishment of transactivation, indicating that
this region contains an activation domain critical for AF-2 activity, designated AF-2 AD. To assess the contribution of the conserved glutamic acid Glu363 in the transactivation potential of
HNF-4 AF-2, we changed it to a lysine (mutant GAL-E363K). This mutant
was transcriptionally inactive, indicating that Glu363 is
critical for the HNF-4 AF-2 activity. Notably, although HNF-4 is a
strong transactivator, it has a leucine at position 366, similar to
nuclear receptors ARP-1, EAR-2, and EAR-3, which are negative
regulators of transcription, and in contrast to the RAR, RXR, and TR
families of receptors, which contain a glutamic acid residue in the
analogous position critical for transactivation (Fig.
5). We therefore addressed the importance of the leucine Leu366 in the transactivation potential of HNF-4, by
changing it to glutamic acid (mutant L366E). Surprisingly, the AF-2
activity was abolished (Fig. 4), indicating that Leu366 is
critical for the HNF-4 AF-2 activity. Taken together, these observations imply that the HNF-4 AF-2 AD may interact with a coactivator(s) different from those that interact with RARs, RXRs, and
TRs.
To further delineate the N-terminal boundaries of AF-2, we analyzed mutants GAL-D3CD1 to GAL-D6CD1, harboring progressive 10-amino acid deletions in region D (Fig. 4). The AF-2 activity decreased gradually in GAL-D3CD1, GAL-D4CD1, and GAL-D5CD1, and it was abruptly abolished in GAL-D6CD1, indicating that the region 128-175 is critical for HNF-4 AF-2 activity. However, this region did not function as an autonomous transactivation domain in construct GAL-CD9 (Fig. 3). Our attempts to further dissect the AF-2 domain using a series of C-terminal and internal deletions of the D/E regions fused to GAL4 DBD-(1-147) were unsuccessful (Fig. 4), leading us to conclude that AF-2 is a complex domain, requiring the presence of the entire 128-366 region for full activity. Interestingly, construct GAL-D7 had no transactivation potential, indicating that the HNF-4 AF-2 AD cannot function as an autonomous transactivator, in contrast to AF-2 ADs of other nuclear receptors, which can activate transcription autonomously (2, 5, 6, 7, 8). Moreover, construct GAL-D10 had no transactivation potential (Fig. 4), consistent with our previous results that region F is not an activation domain.
The AF-1 and AF-2 Domains of HNF-4 Can Activate Transcription in a Cell Type-independent MannerBecause the expression of HNF-4 is
restricted primarily in hepatic, intestinal, and renal cells, we
addressed the question whether the activator and inhibitory domains of
HNF-4 can function exclusively in cell types where HNF-4 is expressed
or in other cell types as well. To investigate this question, we
performed cotransfection experiments with the above described
GAL4-HNF-4 chimeric constructs in Caco-2 and HeLa cells. These
experiments showed that the pG5CAT transactivation pattern
in Caco-2 and HeLa cells was remarkably similar to that observed in
HepG2 cells (Fig. 6). Taken together, these results
suggest that AF-1 and AF-2 domains can activate transcription through
communication with ubiquitous rather than cell-specific basal
transcription factors and coactivators.
Effects of HNF-4 Deletion Mutants on ApoB and ApoCIII Gene Transcription
We showed previously that HNF-4 binds with high
affinity to regulatory elements BA1 (41) and CIIIB (34) of the apoB and apoCIII promoters, respectively, and activates the transcription of
these promoters (22). Having identified the HNF-4 functional domains
with the GAL-HNF-4 chimeric constructs, we then wished to study the
effects of these domains on the transcription of native apoB and
apoCIII promoters. For this purpose, we cotransfected selected HNF-4
mutants (shown in Fig. 7A) with the reporter
constructs apoB-1800CAT (22), (BA1)5CAT (42), and apoCIII-890CAT (34) in HepG2 cells. To verify the nuclear localization of these mutants, we
performed immunofluorescence analysis in COS-1. These HNF-4 mutants
(carrying a FLAG peptide at their N termini) were transiently transfected in COS-1 cells, and the expressed proteins were detected with an anti-FLAG M2 monoclonal antibody. This analysis showed that
HNF-4B and its deletion mutants CD1, CD2, CD3, CD4, CD6, and ND1 were
localized in the nucleus (Fig. 8 and data not shown). As
seen in Fig. 7B, HNF-4A and HNF-4B slightly reduced the
activity of the apoB-1800CAT construct, in agreement with previous
studies (22). Mutants HNF-HF and CD1 increased the activity of
apoB-1800CAT by approximately 2-fold (Fig. 7B), consistent
with the results obtained from the GAL-HNF-4 chimeras, indicating that
region F functions as a negative regulatory domain in the context of
the native apoB promoter. In contrast, cotransfection of CD1b resulted in dramatic loss of the transcriptional activity of apoB-1800CAT. Since
CD1b binds to DNA as a dimer, it is possible that this reduction in
activity results from the heterodimerization of CD1b with wild type
endogenous HNF-4, suggesting that CD1b may act as a dominant negative
regulator. Moreover, mutant D1HNF-4 was only 10% less active than
HNF-4B, indicating that AF-1 has a small contribution to the overall
HNF-4 activity in the native apoB promoter (Fig. 7B).
Surprisingly, mutant DCD1 was over 60% less active than CD1 (Fig.
7B), suggesting that in the absence of region F, domains AF-1 and AF-2 act synergistically to activate the apoB promoter (Fig.
7B). Finally, cotransfection with ND1 resulted in moderate loss of apoB-1800CAT transcriptional activity, indicating that residues
128-175 may be involved in the apoB transactivation by HNF-4.
The potential of the HNF-4 functional domains to activate or repress transcription of a basal promoter was also tested using the homopolymeric promoter construct (BA1)5CAT, which contains five copies of the regulatory element BA1 in front of the apoB TATA box and the CAT gene (42). As seen in Fig. 7C, HNF-4A and HNF-4B increased the transcription of (BA1)5CAT by approximately 10-12-fold, in agreement with previous studies (22). Again, HNF-HF and CD1 elicited 2- and 4-fold higher transactivation than HNF-4A, respectively, confirming the inhibitory effect of region F on HNF-4 function. In contrast, CD1b and ND1 drastically reduced the basal (BA1)5CAT activity, suggesting that they act as dominant negative mutants, possibly through heterodimerization with wild type endogenous HNF-4 molecules. Furthermore, cotransfections of a constant amount of HNF-4B with reporter (BA1)5CAT and increasing amounts of CD1b in HepG2 cells, resulted in a dose-dependent decrease of the transactivation obtained by HNF-4B (Fig. 7D). These results demonstrate directly that CD1b is a dominant negative mutant of HNF-4.
The effects of HNF-4 deletion mutants on the activity of apoCIII-890CAT reporter were also tested in a similar series of experiments. As shown in Fig. 7E, HNF-4A, HNF-4B, and HNF-HF activated the apoCIII promoter by 7-, 9-, and 11-fold, respectively. Furthermore, a 60-fold enhancement in transactivation was obtained by CD1, demonstrating that region F has a negative effect on HNF-4 activity in the context of native promoters (Fig. 7E). In contrast, cotransfections with CD1b and ND1 resulted in loss of apoCIII promoter transactivation, similar to that observed with the apoB reporter constructs. Interestingly, cotransfections with D1HNF-4 resulted in 40% reduction of transactivation, indicating that the contribution of AF-1 to the overall HNF-4 activity may depend on promoter context. Moreover, CD1b reversed the HNF-4B-dependent transactivation of apoCIII-890CAT in a dose-dependent manner (Fig. 7F), demonstrating that it is a potent dominant negative mutant of HNF-4 on a native promoter.
In this report, we have performed a systematic
structural-functional analysis of the orphan nuclear receptor HNF-4. We
have mapped the HNF-4 DBD between residues 48-128, which corresponds to the well-studied DBD of members of the nuclear receptor superfamily (2). This domain consists of a 66-residue highly conserved core
containing two zinc finger modules (region C), followed by a 9-residue
carboxyl-terminal extension. Crystallographic studies of the
RXR-TR
heterodimer bound on a direct repeat DR4 response element
have revealed a polar head-to-tail assembly of the two DBDs on the two
repeats (43). The two monomers occupy adjacent major grooves on one
side of the DNA double helix, with the carboxyl-terminal extension
making extensive minor groove contacts. Since HNF-4 also binds to
direct repeats as a dimer, it is reasonable to expect that the HNF-4 DBDs may have a similar structural
arrangement on the cognate response elements.
Our results also show that both HNF-4A and HNF-4B isoforms bind to DNA
as dimers and that the dimerization domain spans residues 175-360 in
region E. Recently, the crystal structure of the unliganded RXR LBD
homodimer has revealed a novel antiparallel
-helical fold consisting
of 12
-helices (H1-H12) (9). The breakpoints between the
-helices are conserved throughout the nuclear receptor superfamily,
and this structure evidently represents a prototypic fold of nuclear
receptor LBDs (9, 10, 11, 44). The dimerization interface is formed
primarily by
-helix H10 and to a lesser degree by H9 and a loop
between H7 and H8 (9). Based on the high degree of similarity between
the RXR
and HNF-4 LBDs and on the predicted high
-helical content
of the HNF-4 LBD (data not shown), we have assigned the putative
-helices H3-H12 in the region E of HNF-4 (Fig. 9).
According to this analysis, C-terminal deletions up to residue 360 have
no effect on dimerization of HNF-4 because they lie carboxyl to H10 and
leave the dimerization interface intact. However, the additional
deletion of residues 340-360 in mutant CD2 results in abolishment of
dimerization. We attribute this loss of dimerization primarily to the
removal of tryptophan 340 in H10, because the residue in the
corresponding position in the RXR
LBD (Leu430)
participates in hydrophobic van der Waals contacts (contacts H10-H10)
and is critical for dimerization (9). Interestingly, deletion of
residues 175-240 in mutant ND2 removes the
-helices H3, H4, H5, and
the
-strand s1, resulting also in loss of dimerization. Although the
corresponding region in the RXR
LBD does not participate directly in
the formation of the dimerization interface, its effect on HNF-4
dimerization may be attributed to the requirement of the secondary
elements encompassed in this region for the correct architectural fold
of the HNF-4 LBD.
The loss of DNA binding activity in mutants CD2, CD3, and CD4 and the
recovery of this activity in CD5 and CD6 is surprising because all of
these mutants contain an intact DBD. These results indicate that, in
the absence of the 340-465 region, sequences located carboxyl to
residue 174 inhibit efficient DNA binding of HNF-4 monomers. Although
disruption of the -helices that compose the dimerization interface
can account for the loss of dimerization, it cannot explain why mutants
CD2-CD4 do not bind to DNA as monomers. A similar inhibitory effect of
C-terminal sequences on the DBD activity was described previously for
the nuclear receptor ARP-1, and a model was proposed to explain the DNA
binding and dimerization behavior of this receptor (45). In this model,
the DBD is masked by a region in the C-terminal domain of the receptor,
thus inhibiting efficient DNA binding of receptor monomers. Upon
dimerization, the DBD is unmasked and binds to DNA. It is therefore
possible that a similar masking of the DBD by sequences in the E region may also be operational in HNF-4. Dimerization of HNF-4, an event that
requires the 334-360 region, will then result in unmasking of the DBD
and strong binding to DNA.
Several different types of activation domains have been identified and
classified according to the frequency of certain amino acids, including
acidic, proline-rich, glutamine-rich, and serine/threonine-rich activators (46). The HNF-4 AF-1 is located in the first 24 amino acids
of the receptor, and it is classified as an acidic activator based on
the net charge of its amino acid composition. Although it has the
potential to adopt an amphipathic -helical structure, its exact
structure within the context of the intact receptor or during its
interaction with other proteins is unknown. Interestingly, it has been
reported recently that the AF-1 of RAR
2 is also an acidic activator
(47), raising the possibility that the AF-1s of HNF-4 and RAR
2 may
transduce related signal pathways and may function by similar
mechanisms.
In contrast to the small size of AF-1, the HNF-4 AF-2 spans residues
128-366 and is not amenable to further dissection. AF-2 is very
complex, and it cannot be classified in any of the conventional groups
of transactivators. The inability to further dissect the AF-2 without
impairing its activity may be attributed to the essential role of the
encompassed subdomains in the formation of the final activation
surface, which eventually interacts with coactivators or other basal
transcription factors. Our results also corroborate previous studies on
the importance of the AF-2 AD (region 360-366), which contains the
conserved motif XE
in the AF-2 transactivation potential. The crystal structures of the unliganded RXR
and the liganded RAR
and TR
1 LBDs reveal that the AF-2 AD corresponds to
the amphipathic
-helix H12, which in the unliganded receptor extends
into the solvent but in the liganded receptors is packed onto the body
of the receptor, contributing to the formation of the hydrophobic
ligand-binding pocket (10, 11). These structural studies suggest a
mechanistic model for ligand-induced transactivation, in which
ligand-binding by the receptor induces conformational changes that
reposition the
-helix H12 so that it folds back toward the body of
the receptor, sealing the ligand-binding pocket and generating a
surface that allows efficient interactions between AF-2 and putative
coactivators. It is noteworthy that in the RAR
LBD crystal
structure, the H12 is stabilized in this position by a crucial salt
bridge between lysine 264 in H4 and glutamic acid 414 in H12.
Importantly, the corresponding positions in the HNF-4 LBD are occupied
by arginine 212 in H4 and glutamic acid 363 in H12 (Fig. 9). It is
therefore likely that a similar salt bridge exists in the HNF-4 LBD
between Arg212 and Glu363 that stabilizes the
packing of H12 onto the body of the receptor in a RAR
-like fold. In
this conformation, glutamic acid 363 of the HNF-4 AF-2 AD extends into
the solvent, allowing interactions with putative coactivators. In this
context, the critical importance of leucine 366 for HNF-4 AF-2 activity
is intriguing, because other ligand-activated receptors, such as the
RAR, RXR, and TR families of receptors, contain a glutamic acid in the
corresponding position (Fig. 5), which is essential for AF-2 function
(2, 5, 6, 7, 8). For example, in the RAR
crystal structure, the
corresponding residue (Glu417) faces outward, and it is
thought to participate in interactions with putative coactivators. Our
results, therefore, suggest that the AF-2 AD of HNF-4 may interact with
a different coactivator(s) than those that interact with RARs, RXRs,
and TRs.
Unlike other members of the nuclear receptor superfamily, HNF-4 possesses a distinctive F region with unknown function. Its high content (23%) in proline residues led to the early suggestion that it might be an activator of transcription (16). In contrast, our work definitively shows for the first time that region F has a negative effect on the AF-2 activity. This is a novel feature in the nuclear receptor superfamily, and it may define a new level of regulation of the HNF-4 activity. Although the precise mechanism for this inhibition is unclear, it is conceivable that region F may mask critical residues of the AF-2 transactivation surface, either by itself or through interaction with another protein(s). An attractive target for such masking is the AF-2 AD because of its absolute requirement for AF-2 function and its proximity to region F. Since the AF-2 AD is essential for AF-2 function, even partial masking of this domain would be sufficient to diminish AF-2 activity. The significant inhibition of the AF-2 activity by the first half of region F in mutant HNF-HF is consistent with a model involving masking of the surface close to AF-2 AD by sequences within the segment 371-410. Notably, deletion of region F in mutant CD1 results in an apparent synergistic transactivation of the apoB promoter by AF-1 and AF-2 domains (compare activities of constructs HNF-4B, D1HNF-4, CD1, and DCD1 in Fig. 7B). Interestingly, evidence has been presented recently for a ligand-dependent functional interaction between AF-1 and AF-2 in the estrogen receptor (48). It is therefore possible that a similar mechanism is operational in HNF-4, where region F may play a role in modulating the degree of interaction between AF-1 and AF-2, thus controlling the transcriptional synergism between these domains. Conceivably, alleviation of the F-mediated inhibition in vivo could be brought about by disruption of its interaction with the protein(s) that causes masking, proteolytic cleavage of F, or conformational changes induced by posttranslational modification(s) or upon binding to a putative ligand. Although the physiologic significance of the inhibitory function of region F on the HNF-4 activity has not yet been established, we envision that this may define a novel regulatory mechanism that may modulate the transactivation potential of HNF-4 in response to certain signals.
In conclusion, we have delineated the boundaries of the DBD,
dimerization, and transactivation domains AF-1 and AF-2 of the orphan nuclear receptor HNF-4, and we have identified a novel negative
regulatory function within region F (Fig. 10). Finally, we have discovered a potent dominant negative HNF-4 mutant that can be
a useful tool for studying the HNF-4 role during embryonic development
and adult physiology.
This paper is dedicated to the loving memory of our friend, husband, and colleague Christos Cladaras, who initiated this work.
We thank Dr. Dimitris Thanos for providing the constructs pBXG1 and pG5CAT and for valuable comments and discussions.