Corepressor SMRT Specifically Represses the
Transcriptional Activity of Orphan Nuclear Receptor hB1F/hLRH-1

XU Ping-Long, 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        The orphan nuclear receptor hB1F (also known as NR5A2, LRH-1, FTF or
CPF) plays important roles in regulating the expression of several cellular and
viral genes actively involved in a wide range of biological processes such as
the bile acid biosynthesis, liver specific gene regulatory network and
hepatitis B virus replication. The activity of nuclear receptors is regulated
by multiple mechanisms, including coactivation and corepression. In this study,
it was found that the silencing mediator for retinoic acid receptor and thyroid
hormone receptor (SMRT) specifically represses the transcriptional activity of
hB1F, on either GAL4 dependent reporter system or the hB1F-responsive HBV
enhancer II/core promoter. The repression imposed by SMRT is observed in
different cell lines. Interestingly, hB1F couldn’t interact with SMRT directly, as demonstrated by mammalian
two-hybrid analysis or GST pull-down assay. Taken together, it can be concluded
for the first time that the transcriptional activity of hB1F is regulated
specifically by the corepressor SMRT via an indirect mechanism.

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

Human hepatitis
B virus enhancer II B1 binding factor (hB1F) 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). hB1F belongs to the
fushi tarazu factor I (FTZ-F1) subfamily of the nuclear receptor superfamily[1－4]. Members of the FTZ-F1 subfamily
possess a highly conserved FTZ-F1 box located downstream from the zinc fingers
in the DNA-binding domain and bind to their corresponding sites as monomer[5－7]. hB1F mainly expresses in the
pancreas and liver, and has also been found in ovary, intestine and
colon[1,2,8].

We reported previously that hB1F specifically
binds the B1 element of ENII and activates the enhancer to regulate the
expression of viral genes[1,9]. Recently, accumulating data on the biological
fuctions of hB1F in recent years have shown that it was an important
transcriptional activator in the bile acid and cholesterol homeostasis by
regulating the expression of key enzymes and transporters including cholesterol
7α-hydroxylase[2,8,10], sterol 12α-hydroxylase[11] and cholesteryl ester
transfer protein[12]. Additionally, hB1F also regulates the expression of
11β-hydroxylase[13], aromatase[14], scavenger receptor class B type I[15], and
several liver-enriched transcriptional factors such as HNF3β, HNF4α and
HNF1α[16].

So far, little
has been known about the molecular mechanism underlying hB1F-dependent promoter
activation. The transcription mediated by nuclear receptors frequently requires
the recruitment of specific coactivators and corepressors through interacting
domains on both the receptor and cofactor. Recruitment of coactivators such as
SRC-1 or CBP/p300 occurs mainly through direct or indirect interaction with
activation function modules of nuclear receptors, the ligand-dependent AF-2 and
N-terminal AF-1. While repression is achieved by recruitment of corepressors to
regulatory regions of nuclear receptor[17,18]. The silencing mediator for
retinoic acid receptor and thyroid hormone receptor (SMRT) and nuclear receptor
corepressor (NCoR) are two well-known corepressors, sharing a high homology in
the N-terminus[19,20]. Different nuclear receptors exhibit preference in
association with NCoR or SMRT[21]. SF-1, a FTZ-F1 related receptor, has been
shown to interact with the orphan nuclear receptor DAX-1 which in turn recruits
the corepressor NCoR[22,23].

In the present
study, the corepressor SMRT was found to specifically inhibit the
transcriptional activity of hB1F in a dose-dependent manner. However, mammalian
two-hybrid analysis and GST pull-down assay demonstrated that SMRT did not
directly interact with hB1F, suggesting that the corepressor SMRT exerts the
repression on the transcriptional activity of hB1F through an indirect
mechanism.

1    Materials
and Methods

1.1   Plasmids construction

The
hB1F-dependent reporter pENII/CpLuc with the enhancer II and the core promoter
(ENII/Cp) of HBV[1,9] was made from the pGL2basic (Promega). The pENIIm/CpLuc
reporter contains PCR-introduced point mutations (5′-GATCAACtACaGAtCTcGAG-3′, mutations in lowercase letters)
that disrupt the hB1F binding site in the B1 element of ENII. The GAL4
dependent reporter pG5Luc was made by replacing the CAT gene in pG5CAT
(Clontech) with the luciferase gene.

The expression
plasmid of GAL4-hB1F141－495 was made by inserting the PCR fragment of hB1F (aa141－495) digested with EcoRI and XbaI
into the C-terminus of the GAL4 DNA-binding domain (DBD) in the pM (Clontech).
The pVP16-SMRTc was made by inserting receptor-interacting domain (RID)[19,21]
of mouse SMRTα(residues 1975－2413), digested from pCMV-mSMRTα-FL (kindly provided by Prof. Ronald
M. Evans) with BglII and PstI, into the C-terminus of the activation domain
(AD) of VP16 in the pVP16 (Clontech). The prokaryotic expression plasmid of
pGST-hB1F186－495 was made
by inserting the PCR-amplified fragment of hB1F (aa186－495) into the SmaI site of the
pGEX-3X (Phamacia), in frame to the GST.

PCR
amplifications were performed with the high fidelity Pyrobest polymerase
(TaKaRa) with primers containing appropriate restriction enzyme sites to
facilitate cloning. All plasmids constructed with PCR fragments were verified
by sequencing.

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 at a 35 mm
dish with the standard calcium-phosphate precipitation method as previously
described[1] using 4 μg of total DNA, with 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
determined with luciferase assay system (Promega) and the β-galactosidase
activity was measured by a standard colorimetric method[24]. 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
2.5×105 cells per
dish, and transfected with 0.2 μg expression plasmid of GAL4-hB1F141－495 and 0.5 μg pCMV-lacZ. For the co-expression of SMRT, 1 μg expression plasmid of SMRT or
empty pcDNA3 vector was included in the transfection. After 48 h
post-transfection, cells were harvested and a small proportion of the cells was
kept for the measurement of the β-galactosidase activity 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)
using the rabbit anti-mouse Ig/HRP (DAKO) as a secondary antibody. Peroxidase
activity was detected by the ECL reaction with Western blot luminol reagent (Santa
CruZ).

1.4   Mammalian two-hybrid assay

Two-hybrid assay
of hB1F and SMRT was performed in Huh7 cells using mammalian MatchMarekr
two-hybrid assay kit (Clontech) according to the manufacturer’s instruction. 1 μg of vector for GAL4 or
GAL4-hB1F141－495 and 1 μg of vector for VP16 or VP16-SMRTc
(corresponding to residues 1975－2413) were cotransfected into Huh7 cells, along with 0.5 μg pG5Luc reporter and 0.5 μg pCMV-lacZ control. The luciferase
activity and the β-galactosidase activity were measured as described in part
1.2.

1.5   GST pull-down assay

GST and
GST-hB1F186－495 were
purified with the glutathione-Sepharose 4B beads (Amersham Pharmacia) from
lysates of BL21 (DE3) (Invitrogen) cultures containing appropriate expression
plasmids after inducing by 0.2 mmol/L IPTG for 2 h. Purified GST fusion
proteins were quantified by comparing with the BSA standard on Coomassic
stained SDS-PAGE.

Pull-down assay
was performed with purified GST fusion proteins and the full-length SMRT or
SRC-1 synthesized in vitro with TNT quick coupled transcription/translation
systems (Promega) in the presence of [35S] methionine (Amersham Pharmacia)
according to the manufacturer’s protocol.

Interaction was assessed as described in
reference[25]. 2 μg of GST
fusion proteins bound to glutathione-Sepharose 4B beads was incubated with 10 μL of [35S ]   labeled in vitro translated
protein in binding buffer containing 200 mmol/L KCl at 4 ℃ for 1.5 h. After extensive
washing, the mixture was boiled and resolved on 8% SDS-PAGE. Gels were fixed
and dried. Signals were detected and visualized by autoradiography.

2    Results

2.1   Corepressor SMRT specifically
represses the transactivation of hB1F

To investigate
the potential role of the corepressor SMRT in regulating the activity of hB1F,
the expression plasmid for the GAL4-hB1F141－495 fusion protein was cotransfected with different amount of the
expression plasmid of SMRT into human hepatocyte carcinoma Huh7 cells, along
with the GAL4 dependent pG5Luc reporter. As shown in Fig.1, GAL4-hB1F141－495 containing the complete hinge
region and LBD of hB1F was highly active in Huh7 cells; the coexpression of
SMRT did not affect the basal activity of the reporter; however, SMRT inhibited
the transactivation by hB1F in a dose-dependent manner.

Fig.1       SMRT
specifically inhibits the transactivation of hB1F

Huh7 cells were cotransfected with 200 ng of GAL4 or GAL4-hB1F141－495 expressing
vectors, different amount of vector for SMRT，1 μg pG5Luc reporter
and 0.5 μg internal control plasmid pCMV-lacZ.

To detect the
effect of the coexpression of SMRT on expression level of GAL4-hB1F141－495, Western blot was performed on lysates
of cells transfected with the expression plasmid of GAL4-hB1F141－495 with or without the
coexpression of SMRT, using an anti-GAL4-DBD monoclonal antibody. As shown in
Fig.2, the coexpression of SMRT did not affect the expression of the hB1F141－495 fusion protein, indicating that
SMRT specifically repressed the activity of hB1F.

The corepression by SMRT was also examined
in other cell lines, including COS, HeLa, HepG2, and Y1 cells. As shown in
Fig.3, repression by SMRT was apparent in COS, HepG2, Huh7
and Y1 cells, while relative weak in HeLa cells.

Fig.2       Western
blot assay of the GAL4-hB1F141－495 fusion protein

Huh7 cells were cotransfected
the 200 ng GAL4-hB1F141－495 expressing vector, 0.5 μg internal control plasmid
pCMV-lacZ and 1 μg SMRT expressing vector or empty vector of pcDNA3.

Fig.3       Varied
effect of SMRT on the activity of hB1F in different cell lines

Five different cell lines including COS, HeLa, HepG2, Huh7 and Y1 cells
were cotransfected with 200 ng of GAL4 or GAL4-hB1F141－495 expressing
vectors, 2 μg expression vector of SMRT, 0.5 μg internal control plasmid
pCMV-lacZ and 1 μg pG5Luc reporter.

2.2   SMRT represses the activity of HBV
enhancer II/core promoter via hB1F

A cotransfection
assay with hB1F-responsive pENII/CpLuc reporter was performed in Huh7 cells. As
shown in Fig.4, exogenously expressed hB1F (200 ng) activated the reporter
about 5.5-fold. While the coexpression of SMRT (2 μg) reduced the reporter activity
significantly. SMRT also repressed the reporter activity in the absence of the
exogenously expressed hB1F, which probably occurred via the endogenous hB1F or
via other transcriptional factors interacting with the ENII/Cp. To distinguish
these two possibilities, the pENIIm/CpLuc reporter was constructed, in which
the functionally critical hB1F-binding site in the B1 element of ENII was
mutated[1,9]. As expected, hB1F could barely activate the pENIIm/CpLuc
reporter. Coexpression of SMRT obviously did not inhibit the reporter (Fig.4),
indicating that SMRT inhibited the activity of ENII/Cp primarily via hB1F.
These data strongly suggested that there is a functional correlation between
hB1F and SMRT.

Fig.4       SMRT
represses the activity of HBV enhancer II/core promoter via hB1F

(A) Schematic representation of the reporters pENII/CpLuc and
pENIIm/CpLuc. Solid boxes in HBV enhancer II indicate two potential binding
sites of hB1F[1,9]. In pENIIm/CpLuc reporter, the primary hB1F binding site was
mutated (see Material and Methods). (B) The repression of hB1F by SMRT was
examined on the pENII/CpLuc or pENIIm/CpLuc reporter in Huh7 cells. 1 μg of pENII/CpLuc
or pENIIm/CpLuc reporter plasmid, 0.5 μg internal control plasmid
pCMV-lacZ, 200 ng of the expression plasmid for hB1F and 2 μg of the
expression plasmid for SMRT or the empty pcDNA3 vector were transfected in each
dish.

2.3   hB1F does not interact with SMRT
physically

The functional correlation of SMRT and hB1F
implicates a potentially physical interaction of these two factors. To explore
this possibility, a mammalian cell two-hybrid assay was performed. Expression
plasmids for GAL4-hB1F141－495 or VP16-SMRTc containing the RID of SMRT were cotransfected into
Huh7 cells along with the reporter pG5Luc respectively. The RID of SMRT
[Fig.5(A)] has been reported extensively to mediate the interaction between
SMRT and nuclear receptors[26]. As shown in Fig.5(B), coexpression of GAL4 and
VP16-SMRTc did not affect the reporter activity, while coexpression of VP16 and
GAL4-hB1F141－495
stimulated the reporter. However, further activation was not observed when
GAL4-hB1F141－495 and
VP16-SMRTc were coexpressed, suggesting that hB1F did not interact with the RID
of SMRT in Huh7 cells.

Fig.5       hB1F
does not interact with SMRT in mammalian two-hybrid assay

(A) Schematic representation of domains of SMRT, depicted are the
repression domain, the nuclear receptor interacting domain (RID), CBF1/Su(H)
interacting domain[18], and the fragment used in construction of VP16-SMRTc
plasmid. (B) Represents mammalian two-hybrid assay of the interaction between
SMRT and hB1F performed in Huh7 cells as described in “Material and
Methods”. The average values with standard deviations of three independent
duplicate experiments are shown.

GST pull-down
assays were also performed to further investigate the property of the
interaction between SMRT and hB1F. The soluble fusion protein GST-hB1F186－495 containing most of the hinge
region and the complete LBD was employed in the pull-down assay. As shown in
Fig.6, the immobilized GST-hB1F186－495 interacted specifically with [35S]-labeled in vitro translated
coactivator SRC-1. However, it could only very weakly interact with the labeled
full-length SMRT. The results indicated that SMRT was unlike to interact
physically with hB1F.

3    Discussion

Transcriptional repression
has been recognized as a critical function of some nuclear receptors and is
thought to be mediated by association of nuclear receptors with corepressors,
such as SMRT and NcoR[17,18]. Corepressors are considered to silent
transcription by promoting a closed chromatin structure through  histone deacetyltaion[27]. But the
precise mechanism for the repression of nuclear receptors is not well
understood, especially for orphan nuclear receptors. Dynamic regulation of the
activity of hB1F may be essential for the diverse spatial and temporal
functions of hB1F on a wide array of target genes, and protein cofactors are
likely to play a central role in modulating the activity of hB1F. The results
presented herein demonstrate that the corepressor SMRT can repress the
transcriptional activity of hB1F specifically, but unlikely via a physical
interaction. The repression effect is specific to hB1F, since SMRT does not
repress the activity of a mutated enhancer II/core promoter, in which the
binding site of hB1F was altered.

Fig.6       In
vitro GST pull-down assay of the interaction between hB1F and SMRT

(A) E. coli expressed GST or GST-hB1F186－495 was incubated with
[35S]-labeled in vitro translated SMRT and SRC-1. The lane on the left contains
one-tenth the amount of SMRT or SRC-1 protein used in the incubation
respectively. (B) Purified GST and GST-hB1F186－495 proteins were used in
pull-down assays.

Functional
interaction of hB1F and SMRT implicates potential physiological significance.
mRNA level of SMRT varies from one cell type to another or during
differentiation[28,29]. The physiological level of SMRT likely influences the
activity of hB1F and hence the expression of its target genes. For instance,
SMRT transcripts are entirely absent in the liver for a transitory period at
birth when the liver must take charge of the functions necessary to ensure the
metabolism of the newborn independent of maternal circulation. The absence of
SMRT expression likely facilitates the transcriptional activity of hB1F, thus a
number of genes involved mainly in lipid and glucose metabolism, which are
targets of hB1F or hB1F regulated transcriptional factors such as HNF4 and
HNF1[16,30], may be activated within a short time frame.

The corepressor SMRT and NCoR contain two
receptor interacting domains (RID1 and RID2) that mediate interaction with
nuclear receptors. Nuclear receptors exhib it preference to corepressors and RIDs[21].
SF-1, a close homolog of hB1F, has been shown to directly interact with DAX-1,
which in turn recruits NcoR[23]. Furthermore, phosphorylation of S203 residue
in its hinge region seems to facilitate SF-1 to interact with SMRT in pull-down
analysis in vitro, whereas in vivo interaction and functional correlation has
not been demonstrated[31]. Our results suggest that despite an apparent
functional correlation between hB1F and SMRT, these two factors are unlikely to
interact directly, either in cells or in vitro. Nevertheless, the data of
mammalian two-hybrid interaction assay could not exclude the possibility that
hB1F might interact with domains of SMRT other than RID, though such an
interaction pattern between SMRT and nuclear receptors has not been reported.
On the other hand, it can be postulated that other uncharacterized factors
might bridge the repression function of SMRT on hB1F. It is noteworthy that the
repression effect of SMRT is not apparent in HeLa cells compared with other
cells, which may implicate an absence of the bridging factor in this cell line.

In conclusion, our data demonstrated that
corepressor SMRT specifically represses the transcriptional activity of hB1F,
thus inhibits the function of hB1F-responsive promoter. This functional correlation
is probably not owing to a physical interaction between hB1F and SMRT, but
possibly bridged by some uncharacterized coregulators, which suggests a novel
regulatory mechanism for the activity of hB1F.

Acknowledgment         The authors are grateful to Dr. Ronald M. Evans of Salk Institute
for Biological Studies for the pCMV-mSMRTα-FL and Dr. Ming-Jer Tsai of Baylor
College of Medicine for kindly providing the pCR3.1-hSRC-1α.

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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 Technology 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