Characterization of a Strong Repression Domain
in the Hinge Region of Orphan Nuclear Receptor hB1F/hLRH-1

XU Ping-Long, SHAN Shi-Fang, KONG Yu-Ying, XIE You-Hua*, WANG Yuan*

( State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell  Biology, Shanghai
Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai
200031, China
)

Abstract        Human
hepatitis B virus enhancer II B1 binding factor (hB1F，also known as NR5A2, LRH-1, FTF or
CPF) is an orphan nuclear receptor and belongs to the fushi tarazu factor I (FTZ-F1)
subfamily. It plays important roles in the transcriptional regulation of a
number of genes involved in bile acid biosynthesis pathway, hepatitis B virus
(HBV) replication and liver specific regulatory network. Like other nuclear
receptors, hB1F is composed of modular functional domains. We characterized a
domain in its hinge region that imposes a strong repression on the
transcriptional activity of hB1F, which is important for the function of hB1F
on regulating the activity of HBV enhancer II/core promoter. Mutations of the
core residues in this domain abrogate the repression. Bioinformatic analysis
reveals that the amino acid sequence of this region is highly conserved only
among members of the FTZ-F1 subfamily. The repression is observed in five cell
lines tested, while the degree of the repression varies greatly, which does not
parallel with the expression level of the DEAD box protein of 130 kD (DP103), a
potential interacting protein of a homologous domain in the steroidogenic
factor 1 (SF-1). Moreover, the repression is not affected by the silencing
mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) and
steroid receptor coactivator 1 (SRC-1). Collectively, these data suggest a
novel regulatory mechanism for the transcriptional activity of hB1F.

Key
words     hB1F/hLRH-1;
orphan nuclear receptor；hinge region; repression domain; HBV enhancer II/core promoter

Human hepatitis
B virus enhancer II(ENⅡ) B1 binding factor (hB1F) is an orphan nuclear receptor and belongs
to the FTZ-F1 subfamily of the nuclear receptor superfamily. It has been
formally designated as NR5A2 and is also known as liver receptor
homologue-1(LRH-1), CYP7A promoter binding factor (CPF) and α-fetoprotein
transcription factor (FTF)[1－4]. hB1F can activate ENII by binding specifically to the B1
element, which in turn regulates the expression of viral genes[1,5]. Others
report that hB1F is an important activator in the bile acid and cholesterol
homeostasis by regulating the expression of key enzymes and transporters including
cholesterol 7α-hydroxylase (CYP7A)[3,6,7], sterol 12α-hydroxylase (CYP8B1)[8]
and cholesteryl ester transfer protein[9]. In addition, hB1F has also been
found to regulate the expression of 11 β-hydroxylase[10], aromatase
(CYP19)[11], scavenger receptor class B type I (SR-BI)[12] and a number of
liver-enriched transcriptional factors such as HNF3β, HNF4α and HNF1α[13].

We reported previously the cloning of hB1F
based on its interaction with the ENII of hepatitis B virus (HBV)[1]. Like
other nuclear receptors, hB1F contains several modular functional domains,
including an N-terminal A/B domain, a DNA-binding domain (DBD), a hinge region
whose function is not well defined, and a C-terminal ligand-binding domain
(LBD). Members of the FTZ-F1 subfamily also contain a particular FTZ-F1 box
located downstream from the zinc fingers in the DBD and bind to their
corresponding sites as monomer[14－16].

Despite
accumulating data on the biological function of hB1F, the functional domains of
hB1F and their implications in the molecular mechanism underlying
hB1F-dependent promoter activation are just being unveiled. The hinge region of
nuclear receptors was previously thought to merely connect the DBD and LBD.
This was an over-simplification and researchers have realized that actually the
hinge region can play a critical role in regulating the transcriptional
activity of nuclear receptors[17,18]. For FTZ-F1 subfamily, the hinge region
shows a highest variation in the whole sequence. Recently, a repression domain
was identified in the hinge region of the steroidogenic factor 1 (SF-1, NR5A1),
a homolog of hB1F, and the repression was found to be mediated by a DEAD-box
containing protein, DP103[19].

In this study, we characterize the
corresponding homologous domain in the hinge region of hB1F. We find that this
domain, like its counterpart in SF-1, strongly inhibits the transcriptional
activity of hB1F. Some point mutations within this domain significantly enhance
hB1F-dependent promoter activation. This repression is observed in several cell
lines. However, the repression effect varies greatly in different cell lines,
which does not parallel with the expression level of DP103. Moreover, the
repression is not affected by the corepressor SMRT and p160 family coactivator
SRC-1 which regulate the transcriptional activity of hB1F (to be published
elsewhere).

1    Materials and Methods

1.1   Plasmids construction

PCR reactions
were performed with the high-fidelity Pyrobest polymerase (TaKaRa). The plasmids
constructed using PCR-amplified fragments have been verified by sequencing.
Sequences of the primers used in the routine plasmid construction are not
presented in this paper, but are available from the corresponding authors.

The hB1F-responsive luciferase reporter
pENII/CpLuc was made by inserting the PCR-amplified enhancer II/core promoter
(ENII/Cp) fragment of HBV (subtype adr1)[1,5] into the pGL2basic (Promega). The
GAL4-dependent pG5Luc was made by replacing the CAT gene in the pG5CAT (Clontech)
with the luciferase gene.

The pcDNA3-hB1F
was made by inserting the full-length hB1F cDNA from pGAD-16[1] into the EcoRI
and XbaI sites of the pcDNA3 (Invitrogen). Using the pcDNA3-hB1F as the
template, point mutations in the hinge region were introduced with GeneEditor
in vitro Site-directed mutagenesis system (Promega) according to the
manufacturer’s instruction. The following oligonucleotides were used in the
mutagenesis (mutated nucleotides are in lowercase letters). KS225AA, 5′-CTAGCCGGGCCATCgctgCTGAGTACCCAGACC-3′; Y227A, 5′-TCAAGTCTGAGgctCCAGACCCC-3′. The pCR3.1-hSRC-1α plasmid was a
gift from Dr. Ming-Jer Tsai (Baylor College of Medicine, USA) and the
pCMX-mSMRTα-FL plasmid was kindly provided by Dr. Ronald M. Evans (Salk
Institute for Biological Studies, USA).

The plasmid
coding for the GAL4-hB1F141－495 fusion protein was generated by inserting the PCR-amplified
fragments of hB1F (encoding aa141－495, aa219－495 and aa228－495) digested with EcoRI and XbaI to the C-terminus of the GAL4
DNA-binding domain in the pM (Clontech). The plasmids coding for the GAL4-DBD
fused mutant hB1F141－495 (KS225AA and Y227A) were made in the same way.

1.2   Transfection and luciferase assays

COS, HeLa, HepG2
and Huh7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal calf serum (FCS). Y1 cells were grown in F12 medium
supplemented with 10% FCS. Transient transfection was carried out with the
standard calcium-phosphate precipitation method[20], using 4 μg total DNA, which included 0.5 μg of pCMV-lacZ to normalize the
transfection efficiency. 48 h after transfection, cells were harvested and
lysed in 1× reporter
lysis buffer (Promega). The luciferase activity was assayed with luciferase
assay system (Promega) and the β-galactosidase activity was measured by a
standard colorimetric method[20]. Luciferase activities from different
transfections were normalized by the β-galactosidase activities. Each
transfection was performed in duplicate dishes and repeated at least three
times.

1.3   Western blotting

Huh7 cells were
seeded in 35 mm dishes at a density of 2.5×105 cells, and were transfected with 1 μg expression plasmids coding for
the wild type and mutant GAL4-hB1F141－495, and 0.5 μg pCMV-lacZ. 48 h after transfection, cells were harvested and a
small proportion of the cells was removed for the measurement of the
β-galactosidase activity, which was used to normalize the transfection
efficiency. Adjusted amount of the whole cell extracts were subjected to 10%
Tris-glycine SDS-PAGE and transferred to nitrocellulose membrane (Protran,
S&S). Immunoblotting was carried out with an anti-GAL4-DBD monoclonal
antibody SC-510 (Santa CruZ),
followed by the rabbit anti-mouse Ig/HRP (DAKO) as the secondary antibody.
Peroxidase activity was detected by the ECL reaction with Western blot luminol
reagent (Santa CruZ).

1.4   RT-PCR

Semi-quantitative
RT-PCR was performed to determine the relative expression levels of DP103 in
different cell lines. Total RNA was isolated from COS,
HeLa, HepG2, Huh7 and Y1 cells with the Trizol reagent (Gibco BRL). 2 μg of each RNA was used in the
reverse transcription reaction using SuperScriptTM II (Gibco BRL). The primers
for the DP103 were designed to amplify a 285 bp region conserved among the
human, mouse and rat cDNAs (sense: 5′-TTCCTGTGAAAAGCCACTCAGA-3′; antisense: 5′-CCACAGGTTTCTCTAACCCCTC-3′). Meanwhile, the primers for the housekeeper gene GAPDH were
included in the same PCR reactions, which yielded a 145 bp fragment (sense: 5′-CCATGACAACTTTG GTATCGTG-3′; anti-sense: 5′-GCCAGTAGAGGC-AGGGATGA-3′). PCR reactions were carried out
with the following condition: 94 ℃ for 1 min, 50 ℃ for 1 min and 72 ℃ for 30 s, total 28 cycles. The amplified products were separated on
2% agarose gels and visualized by ethidium bromide staining.

2    Results

2.1   Identification of a strong
repression domain in the hinge region of hB1F

In the present
study, we analyzed the functional domains in the hinge region of hB1F (Fig.1)
with transient transfection and reporter assays, using truncated hB1F proteins
fused to the GAL4-DBD. In human hepatocyte carcinoma Huh7 cells, the
GAL4-hB1F141－495 that
contained the hinge region and LBD of hB1F was highly active. The truncation to
residue 219 showed an apparently diminished activity. However, further truncation
to residue 228 resulted in a dramatically increased activity, which was 30-fold
higher than the GAL4-hB1F219－495 (Fig.2). Similar results were obtained from reporter assays in
HeLa cells (data not shown). The data suggested that a strong repression domain
might be located around aa219–227.

Fig.1       Schematic
structure of hB1F

The DBD, FTZ-F1 box, hinge region,
conserved region II and region III in LBD, and AF-2 are depicted. The amino
acid residues at the boundary of these domains are indicated. Point mutations
in the repression domain were shown with mutated residues in lowercase letters.

To confirm the
above results, point mutations were introduced into the putative repression
domain in the GAL4-hB1F141－495 (Fig.1) and the transient transfection of Huh7 cells was
performed. As shown in Fig. 2, the Y227A mutant retained a similar activity as
the wild type GAL4-hB1F141－495 while the KS225AA mutant displayed a much stronger activity (～35-fold).

Fig.2       Reporter
gene analysis of mutants in repression domain

Mutants depicted in Fig.1 and
truncations of hB1F fused to the GAL4-DBD were transfected (0.5 μg) along with the
pG5Luc (1 μg) into Huh7 cells.

Western blot experiment using the
anti-GAL4-DBD monoclonal antibody was performed to rule out the possibility that
the difference in the transcriptional activities between the wild type and the
KS225AA mutant was resulted from the different expression levels of these
proteins. As shown in Fig.3, similar protein levels were observed between the
wild type and mutants, thus indicated that it was the mutation in the KS225AA
mutant that caused the enhanced transcriptional activity.

Fig.3       Western
blot analysis of GAL4-hB1F141－495 expression level in Huh7 cells

Wild type (WT) or mutated
GAL4-hB1F141－495 expressing plasmids (1 μg) were transfected in Huh7
cells, and detected with an anti-GAL4-DBD monoclonal antibody.

Transient
transfection assays were also performed with the natural hB1F-responsive
pENII/CpLuc reporter (Fig.4). No obvious difference in the activity was
observed between the wild type full-length hB1F and the Y227A mutant. However,
the KS225AA mutant showed an increased transcriptional activity, which was
2.5-fold higher than the wild type hB1F. Taken together, a strong repression
domain was located in the aa219－227 of the hinge region.

Fig.4       Reporter
gene assays of hB1F mutants on hB1F-responsive HBV ENII/Cp reporter

Wild type (WT) or mutants of hB1F
depicted in Fig. 1 (0.5 μg) were transfected with the pENII/CpLuc reporter (1
μg) into Huh7 cells.

2.2   Bioinformatic analysis of the repression
domain

In an attempt to
investigate the sequence and structure features of this repression domain, the
amino acid sequence of hB1F was submitted to the PredictProtein server (Columbia University Bioinformatics Center). As analyzed by the PHD software[21,22],
no apparent secondary structure was detected near residues 219－227. Nevertheless, this region is
highly conserved among 25 known members of the FTZ-F1 subfamily as shown by the
ProDom analysis[23] (Fig.5). Moreover, this repression domain is only found in
the FTZ-F1 subfamily but not in other proteins in the Swiss-Prot and TrEMBL
sequence databases provided by ProDom tool, suggesting a unique regulatory
mechanism

for the transcriptional activity adopted by
members of the FTZ-F1 subfamily.

Fig.5       ProDom
analysis of the repression domain

Schematic diagram depicting the result of
ProDom analysis indicated the high conserved sequence of repression domain was
only existed among members of FTZ-F1 subfamily (NR5). Left panel stands for the
ID of proteins containing the homologue domain with hB1F and their sequences
employed in alignment analysis. Remarkably, all these proteins belong to nuclear
receptor FTZ-F1 subfamily. Consensus sequences are marked with deep color.
Thereinto, the identity residues are labeled as capital, and the homologue
residues are labeled as lowercase letter in “consensus”. #, stands for acidic
residues; %, stands for aromatic residues.

2.3   Repression effect varies in
different cell lines

To determine
whether the activity of the repression domain was dependent on the cell
context, we examined the repression by cotransfection analysis in five cell
lines derived from different tissues. Although wild type GAL4-hB1F141－495 showed different activities,
increased activities were observed with the KS225AA mutant in all these cell
lines. Interestingly, the effects varied in different cell lines [Fig.6(A)].
Dramatically increased activities were observed in COS and Huh7 cells, while moderate increased activities were seen in HeLa and HepG2
cells. In Y1 cells, only a marginal increase in the transcriptional activity
was observed. As expected, The Y227A mutant did not display increased
activities in all cell lines tested. These data implicated a potential cell
type-related repression by the repression domain. Some cofactors, which
expressed differently in different cell lines, may be required.

Fig.6 Repression effect varied in
different cell lines

(A) Transfection assays with wild type and mutant GAL4-hB1F141－495 were performed
in five different cell lines: COS, HeLa, HepG2, Huh7 and
Y1 cells. 0.5 μg of each expression plasmid and 1 μg of the pG5Luc reporter were
transfected in each transfection assay. (B) Semi-quantitative RT-PCR analysis
of the expression levels of DP103 in different cell lines. Amplification of
GAPDH served as internal controls for total amount of mRNA and the
amplification reaction.

Since a novel
protein, DP103 is reported to interact with the homologous repression domain in
the hinge region of SF-1 and represses the transcriptional activity of SF-1 in
a dose-dependent manner[19], we examined the expression levels of DP103 in
these cells by semi-quantitative RT-PCR. As shown in Fig.6(B), DP103 was
present in all five cell lines. Relatively high amount of DP103 was detected in
COS and HeLa cells while a little lower amount in HepG2 and Y1 cells. The
lowest amount was observed in Huh7 cells. Obviously, the expression level of
DP103 did not correlate with the degree of the repression observed in different
cell lines, suggesting that other protein(s) be involved in the

repression by
the repression domain on the transcriptional activity of hB1F.

2.4   Repression is not influenced by the
corepressor SMRT or coactivator SRC-1

Finally we
investigated whether the corepressor SMRT[24] or coactivator SRC-1[25] might influence
the repression by the repression domain. Both factors regulated the
transcriptional activity of hB1F (to be published elsewhere). As shown in Fig.7,
SMRT repressed the transcriptional activities of both the wild type and the
KS225AA mutant similarly. On the other hand, SRC-1 also potentiated the
transcriptional activities of the wild type and the   KS225AA mutant with similar proportion. Therefore, the
repression by the repression domain and the regulations by SMRT or SRC-1 are
mutually independent processes.

Fig.7       Repression
is not influenced by the corepressor SMRT or coactivator SRC-1

Cotransfection assays of SMRT or SRC-1 with the wild type or KS225AA
mutant of GAL4-hB1F141－495 were performed in COS cells. 200 ng of the expression plasmids of
GAL4-hBF141－495, 2 μg of the expression plasmids of SMRT or SRC-1, and 1 μg of the pG5Luc
reporter were used in each transfection.

3    Discussion

Activity of
nuclear receptors is frequently regulated by multiple mechanisms, including
activation and repression, dependent on the different ligands, specific
promoters, cell type, signal cross-talk and development phases[26,27]. In the
present study, we characterized a repression domain in the hinge region of
hB1F. Point mutations of the internal K224S225 residues abolished the strong
repression on the transcriptional activity of hB1F. Although a homologous
repression domain in the hinge region of SF-1 has recently been reported, our
results demonstrate for the first time that the repression domain is capable of
regulating the activity of a natural promoter of FTZ-F1 related receptors.
Since activation domains or repression domains of nuclear receptors frequently
exhibit cell type- or promoter-specific functions, our data indicate that this
repression domain is active at least on the enhancer II/core promoter of HBV.

The hinge region
of nuclear receptors has been viewed as a flexible unstructured region between
the DBD and LBD. Recently, several regulatory elements in the hinge region of
nuclear receptors have been reported. For example, a domain in the hinge region
of the androgen receptor inhibits the activity of the AF-2 in the LBD[18].
Besides the repression domain, phosphorylation of the S203 residue in the hinge
region of SF-1 by MAPK signal pathway results in maximized activity[17]. These
results and our data suggest that the hinge region is more than a simple
flexible connector but participates in specifically regulating the function of
nuclear receptors.

The result of
ProDom analysis was interesting. Although the hinge region is the least
conserved part between SF-1 and hB1F with a identity of only 25%,residues
around aa219－227 are
highly conserved, even among other members of the FTZ-F1 subfamily. Moreover,
this conserved domain has only been found in members of the FTZ-F1 subfamily.
Therefore, it is reasonably to speculate that this repression domain was unique
to and pivotal for the function of FTZ-F1 related receptors, including hB1F.
However, why such a repression domain is required for the function of FTZ-F1
related factors is elusive.

Transcription
mediated by nuclear receptors requires the recruitment of multiple specific
coactivators and corepressors. These proteins are recruited to the target
promoters mainly through direct interaction with specific domains of nuclear
receptors. In SF-1, the repression domain has been reported to interact with a
DEAD-box containing protein DP103[19]. Owing to the high homology of
therepression domain between SF-1 and hB1F, it is likely that DP103 is also
involved in the interaction with the repression domain of hB1F and regulates
its transcriptional activity. However, our data suggest the presence of other hB1F-specific
cofactors that interact with the repression domain. It has been reported that
DP103 is expressed abundantly in the testis and in relatively lower amount in
the steroid-producing ovary and adrenal gland, as well as in the placenta,
brain, and kidney by Northern blot analysis of mouse tissues[19]. This
expression pattern parallels SF-1 expression, but does not correlate with that
of hB1F, which is mainly expressed in the pancreas and liver[1,4]. Our results
of RT-PCR demonstrated that the expression level of DP103 in different cell
lines did not correlate with the effect of repression on the transcriptional
activity of hB1F in these cell lines. Therefore, it is possible that DP103
might not be the cofactor interacting with the repression domain of hB1F.
Another unidentified protein, such as a homolog of DP103, may be involved in
the repression of the activity of hB1F. The identification of this cofactor
will be critical for the understanding of the repression mechanism by the
repression domain of hB1F.

In conclusion,
our data provide evidences for a novel regulatory domain in the hinge region of
hB1F that represses the transcriptional activity of hB1F.

Acknowledgements            Authors
are grateful to Dr. Ming-Jer Tsai of Baylor college of Medicine for kindly providing the
pCR3.1-hSRC-1α expression plasmid and Prof. Ronald M. Evans of Salk Institute
for Biological Studies for the pCMV-mSMRTα-FL expression plasmid.

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_____________________________________

Received: June
17, 2003        Accepted:
July 9, 2003

This work was supported by the grants from
the National Natural Science Foundation of China (No. 30100088), the National
High Tech-nology R&D Program (2001AA221261), Basic Research Program from
Ministry of Science and Technology (G1999054105), and the Qi Ming Xing Project
from Shanghai Science and Technology Committee (01QA14046)

*Corresponding authors:

WANG Yuan: Tel, 86-21-54921103; Fax,
86-21-54921011; e-mail, [email protected]

XIE You-Hua: Tel, 86-21-54921105; Fax,
86-21-54921011; e-mail, [email protected]

Updated at: 2003-10-09