(Received for publication, May 24, 1995; and in revised form, August 28, 1995)
From the
The key expression of the simian virus 40 (SV40) major late promoter could be repressed by the human TR4 orphan receptor via the +55 region of the SV40 major late promoter (nucleotide numbers 368-389, 5`-GTTAAGGTTCGTAGGTCATGGA-3`). Using the coupled in vitro transcribed and translated TR4 orphan receptor with a molecular mass of 67.3 kilodaltons, electrophoretic mobility shift assay showed specific binding with a dissociation constant of 1.09 nM between the TR4 orphan receptor and the SV40 +55 oligonucleotides. In addition, chloramphenicol acetyltransferase assay demonstrated that this SV40 +55 region can function as a repressor via the TR4 orphan receptor, suppressing the transcriptional activities of both SV40 early and late promoters. Together, our data suggest that the TR4 orphan receptor may play an important role for the suppression of the SV40 gene expression.
Steroid receptors regulate the transcription of complex gene
networks and subsequently control diverse aspects of growth,
development, and differentiation(1, 2) . The steroid
receptor superfamily includes receptors for steroid hormones, thyroid
hormones, retinoids, and a large number of orphan receptors whose
cognate ligands have not been identified. These steroid receptors
acting as transcriptional factors bind to specific DNA sequences,
hormone response elements (HREs), ()and thereby regulate
their target genes. In short, the HRE is composed of 6 base pairs
forming the core recognition motif. The palindromic half-site sequence
AGAACA is preferentially recognized by the receptors for androgen,
glucocorticoid, mineralocorticoid, and progesterone. In contrast,
estrogen, thyroid, retinoic acid, retinoid X, vitamin D, and many
orphan receptors preferentially bind to repeats of a half-site sequence
AGGTCA. However, a number of orphan receptors can be classified into a
third group of monomeric receptors which apparently bind as monomers to
a single half-site of AGGTCA preceded by a short AT-rich
sequence(2) .
We have isolated human and rat TR4 orphan receptor cDNAs from the hypothalamic supraoptic nucleus, prostate, and testis by degenerative polymerase chain reaction cloning(3) . The open reading frame of human TR4 orphan receptor cDNA encodes a polypeptide of 615 amino acids with a calculated molecular mass of 67.3 kilodaltons. In the 3`-untranslated region of the TR4 orphan receptor, an eukaryotic polyadenylation signal ATTAAA is present between the nucleotide numbers 2222 and 2227. Human TR4 orphan receptor shows a high degree of nucleotide sequence homology with the TR2-11 orphan receptor(4, 5) . The homology between these two orphan receptors in N-terminal, DNA-binding and C-terminal domains is 51, 81, and 65%, respectively (3) . The high homology between the TR2 and TR4 orphan receptors suggests that these two orphan receptors constitute an unique subfamily within the steroid receptor superfamily(3) . Recently, the TR4 orphan receptor has also been identified from human lymphoblastoma cells (named as TAK1), and the expression in a variety of tissues has been demonstrated by Northern blot analysis and in situ hybridization(6) . Most tissues contained TR4 transcripts at markedly variable levels. Thus, the TR4 orphan receptor may play some roles in the regulation of gene expression in specific cell types. Very recently, a DNA binding site for orphan receptors in the SV40 major late promoter (MLP) (7, 8, 9, 27) has been demonstrated to be a natural HRE for the TR2 orphan receptor(10) . Since both TR2 and TR4 orphan receptors are structurally related, we therefore set out to investigate the interaction between the transcriptional initiation site of the SV40-MLP and the TR4 orphan receptor. Various truncations of the TR4 orphan receptor were also generated to identify functional domains within the TR4 orphan receptor. Results from DNA binding studies and transfectional assays suggested that the TR4 orphan receptor can function as a suppressor for gene expression of the SV40. This may further expand the roles of the TR4 orphan receptor in the transcriptional regulation of viruses.
Figure 1:
Binding of the in vitro expressed TR4 orphan receptor to the +55 region of the
SV40-MLP. A, analysis of in vitro translated TR4
orphan receptor in SDS-12% polyacrylamide gel electrophoresis. Lane
1 displays C-methylated protein standards. The
mock-translated product and the TR4 orphan receptor expressed in a
coupled in vitro transcription-translation system are shown in lanes 2 and 3, respectively. The product of the TR4
orphan receptor is indicated on the right with an expected
molecular mass of 67.3 kilodaltons. The minor products probably arise
from internal initiation of translation or limited degradation. B, binding of the TR4 orphan receptor to the +55 region
of the SV40-MLP. EMSA was performed with the in vitro expressed TR4 orphan receptor and the
P-end-labeled
probe. Lane 1 shows the probe alone. Binding reaction mixtures
incubated with the probe and the in vitro synthesized TR4
orphan receptor (lane 2), in the presence of 100-fold molar
excesses of unlabeled wild-type (wt) oligonucleotides (lane 3) or mutant (mut) oligonucleotides (lane
4) are shown. Lane 5 displays binding reaction mixtures
incubated with mock-translated product and the probe. The retarded
complex is indicated by the arrowhead, whereas nonspecific
complexes appear between the retarded band and the free probe at the
bottom.
In order to
determine the DNA protein binding affinity between the TR4 orphan
receptor and the SV40 +55 region in more detail, we performed
Scatchard binding analysis by the EMSA (Fig. 2). Constant
amounts of in vitro expressed TR4 orphan receptor (60 ng) were
incubated with different concentrations of the SV40 +55 probe
(0.025-12.8 ng). DNA-protein complexes were resolved in the EMSA (Fig. 2A). Scatchard plot analysis revealed a single
binding component for the specific DNA-protein complex with a
dissociation constant (K) of 1.09 nM and B
of 0.06 nM (Fig. 2B).
These results basically fit the range of K
for
steroid receptors and their HREs(15) .
Figure 2:
Binding affinity of the TR4 orphan
receptor to the SV40 +55 region. A, binding of the in
vitro expressed TR4 orphan receptor to various concentrations of
the probe in the EMSA. Constant amounts of in vitro expressed
TR4 orphan receptor (60 ng) were incubated with different
concentrations of the probe (0.025-12.8 ng). The specific
DNA-protein complex (indicated by the arrowhead) and the free
probe were quantified by PhosphorImager (Molecular Dynamics). Six
points of experimental data are shown here. B, Scatchard plot
analysis. The ratio between specific DNA-protein binding (bound,
nM) and free DNA probe with respect to specific DNA-protein
binding (bound/free) was plotted. The dissociation constant (K) and B
values
were generated from Ebda program (Biosoft).
Figure 3:
Domain feature of the TR4 orphan
receptor in the recognition of the SV40 +55 region. A,
schematic structure of various truncations of the TR4 orphan receptor.
Plasmids pET14b-TR4C and pET14b-TR4S represents N- and C-terminal
truncations of the TR4 orphan receptor, respectively. The DNA-binding
domain (DBD) is included in these constructs. Each number
shows the number of the amino acid residue within the TR4 orphan
receptor(3) . Molecular masses of the intact TR4 orphan
receptor; N- and C-terminal truncations of the TR4 orphan receptors are
also indicated. B, analysis of the in vitro expressed
TR4 orphan receptor and its variants in SDS-12% polyacrylamide gel
electrophoresis. Lane 1 shows C-methylated
protein standards. Lanes 2-4 display the intact TR4
orphan receptor, N- and C-terminal truncations, respectively. C, binding of the TR4 orphan receptor and its variants to the
SV40 +55 region. Lane 1 shows the probe alone. Lane 2 displays binding reaction mixtures incubated with mock-translated
product and the probe. Binding reaction mixtures incubated with the
probe and the intact TR4 orphan receptor (lanes 3-7 and 16-23), the N-terminal truncated TR4 orphan receptor (lanes 8-11 and 16-19), or the
C-terminal truncated TR4 orphan receptor (lanes 12-15 and 20-23), in the presence of 100-fold molar
excesses of unlabeled wild-type (wt) oligonucleotides (lanes 4, 9, 13, 17 and 21), mutant (mut) oligonucleotides (lanes 5, 10, 14, 18 and 22), monoclonal
anti-TR4 orphan receptor antibody (mAb) (lanes 6, 11, 15, 19 and 23), or RPMI medium
1640 (lane 7) are shown. The retarded complexes are indicated
by small, medium, and large arrowheads for the DNA N-
or C-terminal truncated TR4 orphan receptor complex, the DNA-intact TR4
orphan receptor complex, and DNA-protein-antibody complex,
respectively.
Figure 4:
Repression of CAT activity of the SV40-MLP
via the +55 region. HeLa cells were co-transfected with the
expression plasmid pCMX-TR4 and two different reporter plasmids,
pBL-SVL-CAT and pBL-SVLRE-CAT. All CAT assays were normalized for the
level of -galactosidase activity. Each value represents the mean
± S.D. of three different
experiments.
Because our data demonstrated that the TR4 orphan receptor may suppress the SV40 gene expression, we tried to determine if the TR4 orphan receptor needs activator(s) for the activation of its repression. In order to test this hypothesis, we used regular fetal bovine serum (FBS) versus charcoal-treated FBS (CTS) in the transient transfection experiments. As shown in Fig. 5, the repressor function of the TR4 orphan receptor was dependent on the presence of a potential activator(s) in the serum. In contrast, charcoal can eliminate such a repressor(s) by the removal of the potential TR4 activator(s) from the serum. These results further indicated that this activator-dependent repression of the SV40 gene expression may rely on the interaction between the TR4 orphan receptor and the +55 region. As expected, the mutant +55 region had no influence on the repression of the SV40 gene expression in the presence of either FBS or CTS.
Figure 5:
The SV40 +55 region may function as a
repressor in vitro. HeLa cells were co-transfected with the
expression plasmid pCMX-TR4 and three different reporter plasmids, pCAT
promoter, pSV55wt1, and pSV55 mut1, in the presence of either FBS or
CTS. Plasmids pSV55wt1 and pSV55 mut1 contain one copy of the SV40
+55 oligonucleotides and the mutant SV40 +55 oligonucleotides
with the same orientation as the SV40 early promoter in the parent pCAT
promoter vector (Promega), respectively. All CAT assays were normalized
for the level of -galactosidase activity. Each value represents
the mean ± S.D. of three different
experiments.
In the present study, we have demonstrated that the human TR4
orphan receptor may suppress gene expression of the SV40-MLP. Some
orphan receptors have been shown to affect gene expression of viruses.
For example, we have previously identified that human TR2 orphan
receptor, a closely related subclass member to the TR4 orphan receptor,
can bind and repress transcriptional initiation from the +55
region of the SV40-MLP(10) . Another orphan receptor (TR3)
identified in our laboratory was also able to induce the
transcriptional activity of the mouse mammary tumor virus long terminal
repeat(14, 17, 18) . Evidence suggests that
other orphan receptors, for instance, chicken ovalbumin upstream
promoter transcription factor, retinoid X receptor , human
estrogen-related receptor 1, and hepatocyte nuclear receptor 4 may
influence viral expression(7, 19, 20) . These
results support the possibility that several orphan receptors acting as
repressors in the transcriptional regulation may be able to serve as
antiviral mediators. This antiviral or tumor-suppressed mediator may
further expand the potential physiological function played by some
orphan receptors in a wide variety of animal species.
Both TR4 and
TR2 orphan receptors can recognize imperfect direct repeats of a
half-site sequence AGGTCA within the +55 region of the SV40-MLP.
Nucleotide sequence comparison shows a high degree of homology between
these two orphan receptors(3, 21) . It is not
surprising that both orphan receptors can bind to the same DNA response
element with relatively similar binding affinity (1 versus 9
nM of K) based on structure-function
relationship. However, it is not clear how distinct sets of target gene
are regulated by these two orphan receptors. In addition to the primary
sequence of the core recognition motif, orientation, spacing, and
sequences outside the core motif are also important in controlling the
selectivity of nuclear receptors for their HREs. In terms of nuclear
protein, non-zinc finger regions (e.g. T and A boxes in
addition to P and D boxes), transcriptional inactivation (Ti) domain,
and heptad repeats at the C-terminal region, polarity properties, and
ligand specificity of nuclear receptors contribute to the recognition
of DNA response elements(2) . Moreover, nuclear accessory
factor and even nuclear matrix three-dimensional structure also
influence these DNA-protein interactions(2, 22) .
Monomeric, homodimeric, and heterodimeric modes of DNA binding result from receptor-specific differences in the DNA binding and dimerization domains(2) . It has been documented that the C-terminal heptad repeats which are structurally similar to the leucine zipper dimerization domains are involved in the dimerization of thyroid hormone and retinoic acid receptors(23, 24, 25, 26) . However, we were unable to demonstrate homo- or heterodimer formation using the intact and truncated TR4 orphan receptors by EMSA in the present study. Whether the TR4 orphan receptor has the ability to form monomeric, dimeric, or both modes of DNA binding may remain an interesting puzzle to be solved.
Most recent evidence suggests that both ligands and phosphorylation play important roles in activation of steroid receptors(16) . Many orphan receptors have been discovered by cross-hybridization with known steroid receptor cDNAs. Consequently, these orphan receptors have unknown ligands (or do not need ligands to be activated) and usually unknown physiological function(16) . Therefore, identification of ligands for these orphan receptors remains a key to understand the possible roles of these transcriptional regulators. In this current study, we found that TR4 orphan receptor requires a potential activator(s) in the serum to be activated. This activator-dependent suppression of the SV40 gene expression by TR4 orphan receptor can be eliminated in the presence of CTS. Further characterization of this activator(s) will be the next important step to follow.