(Received for publication, October 19, 1994)
From the
A DNA response element (TR2RE-SV40) for the TR2 orphan receptor, a member of the steroid-thyroid hormone receptor superfamily, has been identified in the simian virus 40 (SV40) +55 region (nucleotide numbers 368-389, 5`-GTTAAGGTTCGTAGGTCATGGA-3`). Electrophoretic mobility shift assay, using in vitro translated TR2 orphan receptor with a molecular mass of 67 kilodaltons, showed a specific binding with high affinity (dissociation constant = 9 nM) for this DNA sequence. DNA-swap experiments using chloramphenicol acetyltransferase assay demonstrated that androgen can suppress the transcriptional activities of SV40 early promoter via the interaction between this TR2RE-SV40 and the chimeric receptor AR/TR2/AR with the DNA-binding domain of the TR2 orphan receptor flanked by the N-terminal and androgen-binding domains of the androgen receptor. In addition, this TR2RE-SV40 can function as a repressor to suppress the transcriptional activities of both SV40 early and late promoters. Together, these data suggest the TR2RE-SV40 may represent the first identified natural DNA response element for the TR2 orphan receptor that may function as a repressor for the SV40 gene expression.
Several members of the steroid-thyroid hormone receptor
superfamily, including the androgen receptor (AR)(),
estrogen receptor, glucocorticoid receptor, progesterone receptor,
mineralocorticoid receptor, thyroid receptor (T
R),
1,25-dihydroxyvitamin D
receptor (VDR), and retinoid acid
receptor (RAR), play important roles in the control of vertebrate
differentiation and development(1) . These nuclear receptors
act as trans-regulators of transcription. Upon binding with
their cognate ligands, these hormone-receptor complexes interact with cis-acting DNA elements, termed hormone response elements
(HREs), to modulate the transcription of target genes. The functional
structure and organization of these receptors comprise a variable
N-terminal domain involved in the modulation of gene expression, a well
conserved DNA-binding domain with two zinc fingers, and a partially
conserved C-terminal ligand-binding domain(2) .
Human testicular receptor 2 (TR2) is one of the first orphan receptors identified that share structural homology with members of the steroid-thyroid hormone receptor superfamily(3, 4, 5, 6) . The TR2 orphan receptor cDNA was isolated from human prostate and testis cDNA libraries with a probe homologous to a highly conserved DNA-binding domain common to steroid hormone receptors. The TR2 orphan receptor cDNA encodes a protein of 603 amino acids with a calculated molecular mass of 67 kilodaltons(4, 5) . The expression of the TR2 orphan receptor has been detected in various cell lines and tissues, including the human prostatic carcinoma cell line LNCaP, testis, ventral prostate, and seminal vesical(7) .
The HREs
for steroid hormone receptors are structurally related but functionally
distinct(8) . Based on the finger model, the first zinc finger
in the DNA-binding domain of steroid hormone receptors may determine
target HRE specificity. The three amino acids at the C-terminal region
of the first zinc finger are categorized as the P (proximal) box, which
is important in base interaction. Consequently, HREs can be classified
into two main categories of repeat consensus sequences based on the P
box, the response element for glucocorticoid receptor and estrogen
receptor (ERE)(9) . The glucocorticoid response element group,
which includes glucocorticoid receptor, AR, progesterone receptor, and
mineralocorticoid receptor, recognizes AGAACA core consensus half-site.
On the other hand, the ERE group, which includes estrogen receptor,
TR, VDR, RAR, RXR, and many orphan receptors,
recognizes AGGTCA half-site. In addition, five amino acids localized in
the second zinc finger, referred to as D (distal) box, which is
important in dimerization contact forming, are involved in half-HRE
spacing functional discrimination. These two structural determinants (P
and D boxes) of target gene specificity may suggest a pathway for the
coevolution of receptor DNA-binding domains and regulatory gene
networks(8, 9) . On the basis of P and D box
sequences, the TR2 orphan receptor is assigned to the ERE subfamily and
predicted to bind to an AGGTCA repeat.
Recently, several transcriptional factors from HeLa cell nuclear extracts have been suggested to bind to the transcriptional initiation site of the simian virus 40 major late promoter (SV40-MLP). One of these factors turned out to be the hERR1 orphan receptor(6, 10) . One of the core sequences within the SV40-MLP was identified as AGGTCA, which could be a potential HRE for the TR2 orphan receptor. We, therefore, developed several strategies to test this hypothesis. Herein this paper is the summary of our results showing the TR2RE within SV40-MLP as the first identified natural HRE for the TR2 orphan receptor.
Figure 1:
Analysis of in vitro expression of the TR2 orphan receptor. A, schematic
structure of a high level in vitro expression plasmid
pSPUTK-TR2. The full-length coding sequence of the TR2 orphan receptor
cDNA was constructed into pSPUTK vector under the control of the SP6
promoter with a 5`-untranslated region of the Xenopus -globin gene and the consensus Kozak translational initiation
site (UTK). B, analysis of in vitro translated TR2 orphan receptor by SDS-12% polyacrylamide gel
electrophoresis. Lane1 displays
C-methylated protein standards. The mock-translated
product and the TR2 orphan receptor expressed in a coupled in vitro transcription-translation system are shown in lanes2 and 3, respectively. The expected molecular mass of 67
kilodaltons for the TR2 orphan receptor is indicated on the right.
Figure 2:
Binding of the TR2 orphan receptor to
+55 region of the SV40-MLP. EMSA was performed with in vitro expressed TR2 orphan receptor and the P-labeled probe
corresponding to SV40 nucleotides 368-389 (see
``Experimental Procedures''). Lane1 shows
the probe alone. Binding reaction mixtures incubated with in vitro synthesized TR2 orphan receptor and the probe (lane2), in the presence of 100-fold molar excesses of
unlabeled wild-type (wt) oligonucleotides (lane3) or mutant (mut) oligonucleotides (lane4), are shown. Lane5 displays binding
reaction mixtures incubated with in vitro expressed TR3 orphan
receptor and the probe. The retarded complex is indicated by the arrowhead, while nonspecific complexes appear between the
retarded complex and the free probe at the
bottom.
Figure 3: Schematic structure of the TR2RE among the transcriptional initiation site of the SV40-MLP. The arrow indicates the position of transcriptional initiation site of the SV40-MLP. The sequence of either the wild-type or mutant TR2RE, an imperfect direct repeat of the AGGTCA half-site, is boxed. The TR2RE is located at the +55 region of the major late transcript. Numbers under the sequence show SV40 nucleotide residues(19) .
Figure 4:
Binding affinity of the TR2 orphan
receptor to the TR2RE. Constant amounts of in vitro expressed
TR2 orphan receptor were used in a series of EMSAs with varying
concentrations of P-labeled TR2RE. The specific
DNA-protein complex and the free probe were quantified by
PhosphorImager (Molecular Dynamics). We plotted the ratio between
TR2-bound (nM) and free DNA with respect to TR2-bound DNA. The
dissociation constant (K
) value was
determined from the minus reciprocal of the slope of the line generated
from the experimental data. A Scatchard plot of the results is
shown.
Figure 5:
Confirmation of the repressor function of
the TR2RE by chimeric TR2 orphan receptor. A, scheme of the
expression plasmid AR/TR2/AR chimera. Chimeric AR/TR2/AR plasmid
contains the DNA-binding domain of the TR2 orphan receptor in the
middle flanked by the N-terminal and androgen-binding domains of AR at
both ends in the pSG5 vector(14) . Thus, the DNA-binding domain
of the AR is replaced with that of the TR2 orphan receptor (shadedbox). H, HpaI; R, EcoRI; S, SacII; and X, XhoI. B, induction of CAT activity in HeLa cells cotransfected with
chimeric AR/TR2/AR plasmid and different reporter plasmids in the
presence or absence of 10 nM synthetic androgen (R1881). Both
pSV55wt1 and pSV55mut1 reporter plasmids are described in Fig. 7A. All CAT assays were normalized for the level
of -galactosidase activity. Each value represents the mean
± S.D. of three independent experiments. The relative folds of
CAT conversion from the cotransfection with chimera plasmid (lanes1-4) or positive control pCMV-AR (lanes5 and 6) and CAT reporter plasmids pSV55wt1 (lanes1 and 2), pSV55 mut1 (lanes3 and 4), or pMSG-CAT (lanes5 and 6) in the presence of 10 nM R1881 (lanes2, 4, and 6) are shown. All experiments
were done in CTS during cell culture.
Figure 7:
TR2RE may function as a repressor in
vitro. A, schematic structure of the reporter plasmids
containing either the wild-type or mutant TR2RE in parent pCAT-promoter
vector. One copy of the wild-type and mutant TR2RE with the same
direction as the promoter and CAT gene were cloned into the
pCAT-promoter vector (a) at the blunted BglII site,
termed pSV55wt1 (b) and pSV55mut1 (c), respectively. B, induction of CAT activity in HeLa cells cotransfected with
the expression plasmid pSG5-TR2 and different reporter plasmids in the
presence of normal FBS or CTS. All CAT assays were normalized for the
level of -galactosidase activity. Each value represents the mean
± S.D. of three independent experiments. The relative folds of
CAT conversion from the cotransfection with pSG5-TR2 and parent
pCAT-promoter vector (a, lanes1 and 2), pSV55wt1 (b, lanes3 and 4), or pSV55mut1 (c, lanes5 and 6) in the presence of FBS (lanes2, 4, and 6) or CTS (lanes1, 3, and 5) are indicated.
Figure 6:
Suppression of CAT activity of the
SV40-MLP via the TR2RE. A, scheme of two CAT reporter
plasmids. While pBL-SVL-CAT plasmid (a) consists of the SV40
late promoter without the TR2RE, pBL-SVLRE-CAT plasmid (b)
contains the SV40 late promoter with the TR2RE and intron of major
capsid protein VP1 followed by the CAT gene. B, induction of
CAT activity in HeLa cells cotransfected with the expression plasmid
pSG5-TR2 and two different reporter plasmids. All CAT assays were
normalized for the level of -galactosidase activity. Each value
represents the mean ± S.D. of three independent experiments. The
relative folds of CAT conversion from the cotransfection with pSG5-TR2
and either pBL-SVL-CAT (lane1) or pBL-SVLRE-CAT (lane2) are shown.
It has been previously shown that some orphan receptors may
affect the expression of viruses. For example, chicken ovalbumin
upstream promoter transcription factor can bind to a negative
regulatory region in the human immunodeficiency virus type 1 long
terminal repeat(20) . Hepatitis B virus enhancer I contains
response elements of enhancer binding factor to polyoma C, hepatocyte
nuclear factor 4, and retinoid X receptor
(RXR)(21) . The TR3 orphan receptor
and nuclear factor I can induce transcription of the mouse mammary
tumor virus long terminal repeat(14, 22) . Recently,
hERR1 and other orphan receptors have also been suggested to bind to
the transcriptional initiation site of the SV40-MLP that may be
involved in the early-to-late shift in the SV40 gene
expression(10) . Our finding that the TR2 orphan receptor can
bind to a negative response element in the transcriptional initiation
site of the SV40-MLP will further expand the knowledge of the influence
of orphan receptors on the gene expression of viruses.
The AGGTCA
motif has been proposed to be the primary target element for the
members of the ERE subfamily in the steroid-thyroid hormone receptor
superfamily(8, 9) . Many family members, including the
TR2 orphan receptor identified in the present study, can bind to this
core sequence. However, it is not known yet how distinct sets of target
genes are regulated by these nuclear receptors. In addition to the
primary sequence, recent evidence has suggested that the orientation
and spacing of repeat core elements determine the specificity of
hormonal regulation of these receptors ((23) ; also, see (24) for review). More recently, a novel cooperative dimer
interaction within the DNA-binding domains of family members has been
proposed(25, 26) . A region in the first zinc finger
of the DNA-binding domain of the TR or RAR can interact
with the second zinc finger in the DNA-binding domain of the
RXR to promote selective DNA binding to direct repeats spaced
by 4 and 5 nucleotides, respectively. The resulting polarity
established by this protein-protein interaction places RXR in
the 5`-position (5`-RXR-T
R-3` or
5`-RXR-RAR-3`) of the direct repeats(25) . In
addition, the formation of either 5`-T
R-VDR-3` or
5`VDR-T
R-3` heterodimeric complex can be controlled by the
ligand for the downstream receptor. Thus, polarity is an important
regulatory property of heterodimeric nuclear receptor
complexes(26) .
The TR3 orphan receptor, another orphan receptor identified initially in our laboratory, was used here as a negative control in the EMSA (Fig. 2). The TR3 orphan receptor, a human homologue of rat NGFI-B and mouse nur77, is also a member of the steroid-thyroid hormone receptor superfamily(27, 28, 29) . It has been demonstrated that rat NGFI-B can bind to a response element (NBRE), 5`-AAAGGTCA-3`, which contains only one AGGTCA half-site(30) . Thus, NGFI-B has been grouped into a special monomer binding subfamily. Herein, we have shown that the TR3 orphan receptor cannot bind to the TR2RE, an imperfect direct repeat sequence. This finding is in good agreement with the data from NGFI-B(30) .
Based on the binding affinity between
some members of the ERE subfamily and the TR2RE, we found that the TR2
orphan receptor prefers TR2RE to other natural HREs, such as
TRE, RARE
, and NBRE. (
)The binding affinity
between the TR2 orphan receptor and the TR2RE is high and specific.
Interestingly, sequence analysis of the TR2RE in the GenBank data base
indicated that human hepatitis B virus and human papillomavirus type 33
may also contain binding sites for the TR2 orphan receptor. Whether the
TR2 orphan receptor has any function in these viral gene expressions
could be an interesting question to ask.
Lydon et al. (31) showed that the TR2 orphan receptor is not constitutively active. The chimera receptor (PR/PR/TR2), which replaces the ligand-binding domain of progesterone receptor with that of the TR2 orphan receptor, can be activated by dopamine, a neurotransmitter. Our previous report (28) showed that chimeric receptor TR3/AR/TR3 can be constitutively activated in both COS1 and prostate PC-3 cells but not TR2/AR/TR2. These two independent data all suggested that the TR2 orphan receptor may need its cognate ligand for the activation.
In summary, our data indicated that this TR2RE is indeed the first identified natural HRE for the TR2 orphan receptor. Moreover, our results showing that TR2RE can function as a repressor for the SV40 gene expression may suggest that several potential HREs for the TR2 orphan receptor could exist in other viruses. This prediction may open a new chapter for the study of interaction between the viral expression and the TR2 orphan receptor.