The N-Terminal Domain of Transcription Factor IIB Is Required for Direct Interaction with the Vitamin D Receptor and Participates in Vitamin D-Mediated Transcription
Hisashi Masuyama,
Stephen C. Jefcoat, Jr. and
Paul N. MacDonald
St. Louis University Health Sciences Center Department of
Pharmacological and Physiological Science St. Louis, Missouri
63104
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ABSTRACT
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The interaction of the vitamin D receptor (VDR)
with transcription factor IIB (TFIIB) represents a potential physical
link between the VDR-DNA complex and the transcription preinitiation
complex. However, the functional relevance of the VDR-TFIIB interaction
in vitamin D-mediated transcription is not well understood. In the
present study, we used site-directed mutagenesis to demonstrate that
the structural integrity of the amino-terminal zinc finger of TFIIB is
essential for VDR-TFIIB complex formation. Altering the putative
zinc-coordinating residues (C15, C34, C37, or H18) to serines abolished
TFIIB interaction with the VDR as assessed in a yeast two-hybrid system
and by in vitro protein interaction assays. This
N-terminal, VDR-interactive domain functioned as a selective,
dominant-negative inhibitor of vitamin D-mediated transcription.
Expressing amino acids 1124 of human TFIIB (N-TFIIB) in COS-7
cells or in osteoblastic ROS17/2.8 cells effectively suppressed
1,25-dihydroxyvitamin D3
(1,25-(OH)2D3)-induced
transcription, but had no effect on basal or glucocorticoid-activated
transcription. A mutant N-terminal domain [N-TFIIB(C34S:C37S)] that
does not interact with VDR had no impact on
1,25-(OH)2D3-induced
transcription. Interestingly, both in vitro and in
vivo protein interaction assays showed that the VDR-TFIIB protein
complex was disrupted by the
1,25-(OH)2D3
ligand. Mechanistically, these data establish a functional role for the
N terminus of TFIIB in VDR-mediated transcription, and they allude to a
role for unliganded VDR in targeting TFIIB to the promoter regions of
vitamin D-responsive target genes.
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INTRODUCTION
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Vitamin D is essential for adequate intestinal absorption of
dietary calcium and for the normal development and maintenance of the
skeleton. In addition to mineral homeostasis, vitamin D also functions
in fundamental cellular processes such as regulating the proliferation
and differentiation of a variety of cell types (1, 2). The biologically
active metabolite of vitamin D3 is
1,25-dihydroxyvitamin D3
(1,25-(OH)2D3), and its cellular actions are
mediated through the vitamin D receptor (VDR), a member of the nuclear
receptor superfamily for steroid hormones, thyroid hormone, and
retinoids (3, 4). VDR is a ligand-activated transcription factor that
forms heterodimers with retinoid X receptor, binds to vitamin
D-responsive elements (VDRE) in the promoters of vitamin D-responsive
genes, and alters the rate of transcription of selected genes
(5, 6, 7).
The precise molecular details through which VDR affects the rate of RNA
polymerase II (RNAP II)-directed transcription are largely unknown. As
with other transcriptional regulatory proteins, one aspect of this
mechanism likely involves selective interaction of VDR with components
of the transcription preinitiation complex (PIC). These components
include the general transcription factors TFIIA, TFIIB, TFIID, TFIIE,
TFIIF, TFIIH, and RNAP II itself (8). The interaction of
transactivators with these general transcription factors may be direct
or it may occur indirectly through the action of bridging proteins such
as the TATA-binding protein (TBP)-associated factors or various
coactivator/corepressor proteins (8, 9, 10). One functional consequence of
the direct interaction of transactivators with the general
transcription factors is the recruitment of these basal components into
the PIC and subsequent enhancement of the overall transcriptional
process. For example, the transactivation domains of several proteins
are known to facilitate TFIID, TFIID/TFIIA, and TFIIB assembly into the
PIC (11, 12, 13, 14, 15). The prototypical example is Gal4-VP16, which interacts
directly with TFIIB through its acidic-rich activation domain and
recruits TFIIB into the PIC (14, 15). Importantly, mutants of TFIIB
that do not interact with GAL4-VP16 are able to function in basal
transcription, but they are not recruited to the PIC by GAL4-VP16, and
they are not functional in GAL4-VP16-activated transcription (16). This
strongly suggests that the interaction between an activator and TFIIB
is one crucial aspect of the transcriptional mechanism mediated by some
transactivating proteins.
TFIIB is also a central target for interaction with a variety of
steroid hormone receptors, including the estrogen receptor, thyroid
hormone receptor, progesterone receptor, and the VDR (17, 18, 19, 20, 21). A
precise functional role for TFIIB interaction with the nuclear
receptors in the mechanism of steroid-regulated transcription is
not well understood. Recent data from two groups indicate that the
thyroid hormone receptor (TR)-TFIIB interaction may be important for
transcriptional silencing mediated by the unliganded TR (18, 19). In
those studies, unliganded TR preferentially interacted with TFIIB and
interfered with the formation of a functional PIC. A separate study
suggested a positive role for TFIIB in vitamin D-mediated transcription
in which TFIIB expression was shown to augment VDR-activated
transcription of a reporter gene construct in P19 embryonal carcinoma
cells (20). However, that study did not examine whether direct
interactions between VDR and TFIIB were responsible for the observed
synergistic transcriptional response.
Deletion analysis previously suggested that the amino-terminal domain
of TFIIB is important for in vivo interaction with the VDR
in the two-hybrid system (21). In contrast, the studies of Blanco
et al. (20) indicated that this region was not required for
VDR-TFIIB interaction in vitro. Therefore, in the present
study, site-directed mutagenesis of TFIIB was used to probe the
TFIIB-VDR interaction, and we establish that the structural integrity
of the N-terminal domain of TFIIB is indeed required for direct
interaction of TFIIB with the VDR. Moreover, expression of the TFIIB
amino-terminal domain resulted in selective, dominant-negative
inhibition of vitamin D-activated transcription in an osteoblast-like
cell line, suggesting that VDR interaction with the N-terminal domain
of TFIIB is one important aspect of the mechanism of vitamin D-mediated
gene expression in bone cells. We also demonstrate that the
1,25-(OH)2D3 ligand specifically disrupts the
formation of the VDR-TFIIB complex, thereby indicating a preferential
interaction of TFIIB with unliganded VDR. Taken together, these studies
establish the N terminus of TFIIB as a VDR-interactive domain that is
important for VDR-mediated transcriptional responses and suggest an
important role for VDR and the 1,25-(OH)2D3
ligand in the entry of TFIIB into the PIC on vitamin D/VDR target
genes.
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RESULTS
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Identification of Amino Acid Residues in the N Terminus of TFIIB
That Are Important for Interaction with VDR
As mentioned above, previous deletion analyses of TFIIB yielded
conflicting evidence for a role of the N-terminal domain of TFIIB in
mediating VDR-TFIIB complex formation (20, 21). Thus, to investigate
the importance of the amino-terminal domain of TFIIB as a VDR contact
site, several point mutations were introduced into this domain of
TFIIB, and their effects on VDR-TFIIB interactions were examined using
an in vivo two-hybrid assay and with in vitro
protein interaction assays. The N terminus of TFIIB contains a putative
zinc finger motif and a region of charged amino acids C-terminal to the
zinc finger (Fig. 1
, upper panel). Altering
any of the four residues that are putatively involved in coordinating
the zinc atom (C15, H18, C34, or C37) resulted in a marked impairment
of TFIIB to interact with VDR in the two-hybrid system. The VDR-TFIIB
interaction was reduced by approximately 7080% when each of these
four residues was changed to serine. In contrast, mutation of a number
of other residues within and immediately adjacent to this domain had
little effect on the interaction with VDR in this system (Fig. 1
, lower panel).

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Figure 1. Effects of Mutations in the Amino Terminus of TFIIB
on Its Interaction with VDR in the Two-Hybrid System
A schematic illustration of the putative zinc finger motif in TFIIB is
presented in the upper panel. Specific amino acid
substitutions were generated in this domain, and the effect of each
mutation on the interaction between VDR and TFIIB was examined in the
Hf7c strain of yeast using a two-hybrid assay. Relative growth on
histidine-deficient plates was assessed after 4 days at 30 C. VDR
interaction with each TFIIB mutant was quantitated in a
ß-galactosidase assay after overnight growth in a selection media
(SC-leu-trp) in the absence of 1,25-(OH)2D3.
Results are presented as the mean ± SD of triplicate
independent cultures.
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The effect of mutating the amino-terminal domain of TFIIB was also
examined with purified proteins using an in vitro protein
interaction assay. Wild type TFIIB and TFIIB(C34S:C37S) were expressed
as glutathione S-transferase (GST) fusion proteins and purified with
glutathione agarose and gel filtration chromatography. The purified
wild type and mutant GST-TFIIB fusions were compared for their relative
abilities to interact with increasing amounts of VDR (Fig. 2A
). Whereas strong interactions were observed between
VDR and wild type TFIIB (Fig. 2A
, lanes 13), the interaction between
VDR and the TFIIB(C34S:C37S) mutant was weak (Fig. 2A
, lanes 46).
When the relative stabilities of the VDR-TFIIB(WT) and the
VDR-TFIIB(C34S:C37S) complexes were examined, we observed that the
VDR-TFIIB(C34S:C37S) complex readily dissociated upon successive washes
of the complex (Fig. 2B
, lanes 46). This was in marked contrast to
the VDR-TFIIB(WT) complex, which was quite stable under these wash
conditions (Fig. 2B
, lanes 13). These data indicate that the
TFIIB(C34S:C37S) mutant exhibited a decreased relative affinity for the
VDR compared with TFIIB(WT). These in vitro results with
purified proteins agreed with the in vivo data generated in
the two-hybrid interaction assay (Fig. 1
). Thus, disrupting the
putative zinc finger motif in the amino terminus of TFIIB reduced its
ability to interact with VDR.

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Figure 2. The Integrity of the Putative Zinc Finger Motif of
TFIIB Is Essential for Interaction with VDR in an in
Vitro Protein Interaction Assay
A, Various amounts of baculovirus-expressed full-length VDR (0.2, 1, 5
µg) were incubated with 2.5 µg purified wild type (WT) GST-TFIIB
protein (lanes 13), or with identical concentrations of a purified
mutant GST-TFIIB (C34S:C37S) protein (lanes 46), or with GST (lanes
79). Protein-protein complexes were precipitated with
glutathione-agarose, washed extensively, and analyzed for VDR content
by Western immunoblot analysis. The relative amount of VDR precipitated
with GST-TFIIB represented 510% of the input VDR in this assay. B,
GST-TFIIB(C34S:C37S) dissociates more readily from VDR compared with
GST-TFIIB(WT). Baculovirus-expressed full-length VDR (1.3 µg) was
incubated with 5.0 µg GST-TFIIB(WT) (lanes 13) or with 5.0 µg
TFIIB(C34S:C37S) (lanes 46), and VDR-TFIIB complexes were analyzed by
Western analysis after the glutathione-agarose beads were washed one,
three, or five times.
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TFIIB folds into a complex structure through the formation of extensive
intramolecular contacts (i.e. the N terminus of TFIIB
interacts with the C terminus) (22). It is possible that mutations in
the zinc finger domain may disrupt the proper folding and drastically
alter the overall tertiary structure of TFIIB, thereby affecting a
distal site of TFIIB that is involved in VDR interaction. However, the
following data argue strongly against this possibility. A C-terminal
deletion mutant of TFIIB [TFIIB(
124-298), also called N-TFIIB]
interacted strongly with VDR. We observed approximately 3-fold higher
ß-galactosidase activity with this C-terminal deletion mutant
compared with wild type TFIIB (Fig. 3
). Thus, removing
the C terminus and eliminating intramolecular folding of TFIIB may
actually enhance the accessibility of VDR to this amino-terminal
domain. Importantly, introducing the C34S:C37S double mutant into the
TFIIB(
124-298) deletion construct virtually abolished the
interaction of this amino-terminal domain with VDR (Fig. 3
). Thus, in
both the presence and absence of the C terminus of TFIIB, the C34S:C37S
mutation disrupts TFIIB interaction with VDR, indicating a direct
interaction of VDR with this N-terminal domain.

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Figure 3. Disruption of VDR Interaction with the
TFIIB(C34S:C37S) Mutation Is Independent of the C-Terminal Domain of
TFIIB
TFIIB(WT), TFIIB( 124-298), TFIIB(C34S:C37S), and
TFIIB( 124-298:C34S:C37S) were examined for interaction with AS1-VDR
in the Hf7c strain of yeast in a two-hybrid interaction assay. The
TFIIB constructs were inserted into the pGAD.GH vector and are
illustrated schematically (left). Relative growth on
histidine-deficient plates was assessed after 4 days at 30 C, and
ß-galactosidase expression was quantitated after overnight growth in
SC(-leu-trp) medium. Results are presented as the mean ±
SD of triplicate independent cultures.
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The N-Terminal Domain of TFIIB Suppresses the
1,25-(OH)2D3/VDR-Induced
Transcription
To examine a functional role for VDR interaction with the amino
terminus of TFIIB, we tested the effect of coexpressing N-TFIIB
[TFIIB(
124-298)] in a vitamin D-responsive transient gene
expression system. The rationale was to overexpress that domain of
TFIIB that interacted with VDR and determine whether VDR-mediated
transcription was disrupted through the formation of nonproductive
VDR/N-TFIIB complexes. To test the approach, we expressed and purified
N-TFIIB and observed that increasing concentrations of the purified
amino-terminal domain of TFIIB effectively reduced the ability of
GST-TFIIB(WT) to interact with the VDR in the in vitro
protein interaction assay (Fig. 4
). Thus, N-TFIIB was
indeed an effective competitor for wild type TFIIB binding to the
VDR.

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Figure 4. The N Terminus of TFIIB Competes with Full-Length
TFIIB for Interaction with the VDR in Vitro
Wild type GST-TFIIB (1.5 µg) and baculovirus-expressed full-length
VDR (1.3 µg) were incubated without (lane 1) or with 0.5, 2.5, and
12.5 µg of purified N-TFIIB [TFIIB( 124-298)] (lanes 24) that
was expressed and purified as described in Materials and
Methods. VDR content was assessed as described in the legend to
Fig. 2 .
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Therefore, a VDR expression plasmid (SG5-VDR) and vitamin D-responsive
reporter gene constructs (VDRE4-TATA-GH and
VDRE4-TK-GH) were introduced into COS-7 cells in the
absence and presence of an expression vector that generates N-TFIIB
(Fig. 5A
). As predicted from the in vitro
experiment in Fig. 4
, expression of N-TFIIB in this transfection system
effectively suppressed
1,25-(OH)2D3/VDR-dependent transactivation of
both reporter gene constructs by more than 75% compared with similar
transfections with the parent expression vector (pcDNA3). Importantly,
expression of the amino-terminal mutation of TFIIB that does not
interact with VDR [N-TFIIB(C34S:C37S)] had no effect on VDR-mediated
reporter gene expression in this system. Moreover, the inhibitory
effect of N-TFIIB was selective for vitamin D-responsive promoter
activity since expression of the wild type amino terminus of TFIIB had
no impact on glucocorticoid-induced transactivation of
GRE2-TK-GH or on basal transcription of all of the reporter
constructs (Fig. 5A
). Under these conditions, N-TFIIB also did not
alter basal expression of the TK-GH and TATA-GH reporter constructs
that lack the hormone response elements (data not shown). The decrease
in vitamin D-activated transcription by N-TFIIB expression was not due
to differential expression of VDR in this transient expression assay
since Western analysis revealed equivalent levels of VDR in COS-7 cells
transfected with pcDNA3, pcDNA3-N-TFIIB, or pcDNA3-N-TFIIB(C34S:C37S)
(Fig. 5B
).

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Figure 5. Suppression of
1,25-(OH)2D3/VDR-Induced Transactivation by the
N-Terminal Domain of TFIIB
A, The effect of N-TFIIB expression in a vitamin
D-responsive transient gene expression system in COS-7 cells. COS-7
cells were transfected with 2 µg of the VDRE4-TATA-GH or
the VDRE4-TK-GH reporter gene construct together with 10 ng
of the VDR expression plasmid. Another group of cells received the
glucocorticoid-responsive reporter gene construct termed
GRE2-TK-GH and 10 ng of the GR expression plasmid. These
three groups also received 1 µg of pcDNA3 parent expression plasmid
or pcDNA3 derivatives that express either N-TFIIB or
N-TFIIB(C34S:C37S). Sixteen hours after addition of the calcium
phosphate-DNA precipitate, the cells were washed, media were replaced,
and the cells were treated with ethanol vehicle, with 10-8
M 1,25-(OH)2D3, or with
10-7 M dexamethasone. GH secreted into the
media was quantitated 24 h following ligand addition using a RIA
kit. The results represent the mean ± SD of
triplicate determinations, and the number above each bar
represents fold activation relative to the ethanol-treated control
group. B, Relative level of expressed VDR in the presence and absence
of N-TFIIB expression. Cells were transfected with the indicated DNAs,
and expressed VDR was examined by Western blot analysis on extracts
from cells transfected with pSG5-VDR and pcDNA3 derivatives. C, The
effect of N-TFIIB expression in a vitamin D responsive osteoblast cell
line, ROS17/2.8. ROS17/2.8 cells were cotransfected with 5 µg of
VDRE4-TATA-GH, BGP(-1000)-GH, or BGP(-150)-GH reporter
gene constructs and 2 µg of the N-TFIIB expression plasmids described
in panel A. BGP(-1000)-GH contains approximately 1000 bp of
5'-flanking sequence of the native, vitamin D-responsive promoter from
the osteocalcin gene. BGP(-150)-GH contains only 150 bp of this
osteocalcin promoter and lacks the VDRE positioned around -450 bp. The
cells were treated and GH secretion was quantitated as described in
panel A. D, Concentration-dependent suppression of
1,25-(OH)2D3-induced transactivation with the
increasing amounts of N-TFIIB expression plasmids. Various amounts of
N-TFIIB expression vectors were transfected along with a constant
amount of SG5-VDR (10 ng) and VDRE4-TATA-GH reporter (2
µg) in COS-7 cells. ROS 17/2.8 cells received 5 µg
VDRE4-TATA-GH and the indicated concentrations of N-TFIIB
expression plasmid. The cells were treated with
1,25-(OH)2D3, and GH secretion was determined.
Suppression of 1,25-(OH)2D3-induced
transactivation was calculated by measuring secreted GH with N-TFIIB
relative to that of empty expression vector in the presence of
10-8 M 1,25-(OH)2D3.
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This same inhibitory effect of N-TFIIB on vitamin D-mediated
transcription was also observed in an authentic vitamin D-responsive
osteoblast-like target cell line, ROS 17/2.8 (Fig. 5C
). Vitamin
D-mediated expression of both the artificial VDRE4-TATA-GH
reporter and the reporter construct driven by the native osteocalcin
promoter [BGP(-1000)-GH] was inhibited approximately 60% by N-TFIIB
in ROS 17/2.8 cells. Again, this was not a generalized squelching
phenomena since N-TFIIB did not influence expression of the native
osteocalcin promoter deletion construct that lacks the VDRE
[BGP(-150)-GH]. VDR-mediated reporter gene expression in ROS 17/2.8
cells also was not affected by expressing N-TFIIB(C34S:C37S), in
agreement with the observation that this TFIIB mutant does not interact
with VDR in vitro or in vivo (Fig. 5C
). These
inhibitory effects were concentration-dependent with increasing amounts
of the N-TFIIB expression plasmid in both COS-7 cells and ROS 17/2.8
cells (Fig. 5D
) and were apparent with two distinct vectors in which
N-TFIIB expression was driven by either a cytomegalovirus promoter
(pcDNA3) or an SV40 promoter (pSG5). Significant effects were apparent
with 100 ng of either N-TFIIB expression plasmid. The excess of N-TFIIB
expression vector compared with VDR expression vector that was required
in these experiments may be related to the level of expression of each
protein from these vectors, the stabilities of the expressed proteins,
to the level of endogenous TFIIB that competes with N-TFIIB binding to
VDR, or to the relative affinity of the VDR-TFIIB complex.
These data suggest that N-TFIIB interfered selectively with vitamin
D-mediated transcription in a dominant negative fashion through the
formation of a nonproductive VDR/N-TFIIB complex, and this inhibited
the functional interaction of VDR with native TFIIB. Based on this
model, the inhibitory effect of N-TFIIB should be reversible with the
expression of additional VDR and/or additional TFIIB. The expression of
additional full-length VDR (Fig. 6A
) or additional,
full-length WT TFIIB (Fig. 6B
) reversed the negative impact of N-TFIIB
on vitamin D-activated transcription in COS-7 cells. Thus, VDR and
TFIIB were able to individually rescue the dominant-negative inhibition
of vitamin D-activated transcription by N-TFIIB.

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Figure 6. Rescue of N-TFIIB Effects on Vitamin D/VDR-Mediated
Transcription
A, Rescue of the suppressed transactivation by VDR. COS-7 cells were
cotransfected with 100 ng VDR together with 2 µg reporter construct
(VDRE4-TATA-GH) and 100 ng N-TFIIB. The cells were treated
and GH secretion was quantitated as described in Fig. 4 . B, Rescue of
the suppressed transactivation by full-length TFIIB. COS-7 cells were
cotransfected with 100 ng full-length TFIIB together with 2 µg
reporter construct (VDRE4-TATA-GH) and 100 ng N-TFIIB. The
cells were treated and GH secretion was quantitated as described in
Fig. 5 .
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The Effect of the
1,25-(OH)2D3
Ligand on the VDR-TFIIB Interactions
Having demonstrated the importance of the N terminus of
TFIIB in VDR interaction in vitro and in vivo and
the potential impact of that interaction on vitamin D-mediated
transcription, the role of the 1,25-(OH)2D3
ligand in this process was examined. First, we investigated the effect
of 1,25-(OH)2D3 on the interaction between VDR
and TFIIB in the yeast two-hybrid protein interaction assay.
Surprisingly, the interaction between VDR and TFIIB was decreased in a
concentration-dependent fashion with increasing amounts of
1,25-(OH)2D3 (Fig. 7A
).
Significant effects were observed between
10-10-10-9 M
1,25-(OH)2D3, and the effect was maximal at
10-7 M 1,25-(OH)2D3.
At this latter level of 1,25-(OH)2D3, VDR-TFIIB
interaction was approximately 20% of control values in the absence of
ligand as determined by a ß-galactosidase assay (Fig. 7A
). This
ligand-dependent decrease in VDR-TFIIB interaction was also apparent in
the in vitro assay with purified components. Here, VDR
interaction with GST-TFIIB was markedly diminished with increasing
amounts of 1,25-(OH)2D3 (Fig. 7B
, lanes 14).
Nonspecific interaction of VDR with GST was not observed in the absence
or presence of 1,25-(OH)2D3 in this assay
(lanes 57).

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Figure 7. Concentration-Dependent Inhibition of VDR
Interaction with TFIIB by the 1,25-(OH)2D3
Ligand
A, Ligand disrupts in vivo interaction between VDR and
TFIIB in the two-hybrid assay. Yeast expressing the AS1-VDR and
GAD.GH-TFIIB two-hybrid plasmids were grown for 24 h at 30 C in
the absence and presence of increasing concentrations of
1,25-(OH)2D3. VDR-TFIIB interaction was
assessed in a ß-galactosidase assay. Results are presented as the
mean ± SD of triplicate independent cultures. B,
Ligand disrupts interactions between purified proteins in
vitro. Baculovirus-expressed full-length VDR (1.3 µg) was
incubated with 2.5 µg GST-TFIIB or GST in the absence or presence of
10-10, 10-8, or 10-6
M 1,25-(OH)2D3. Protein-protein
complexes were precipitated with glutathione-agarose, washed five
times, and analyzed for VDR by Western immunoblot analysis.
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The inhibition by 1,25-(OH)2D3 on VDR
interaction with TFIIB showed the appropriate ligand selectivity (Fig. 8
). Only in the presence of 10-7
M 1,25-(OH)2D3 was a decrease in
VDR-TFIIB interaction observed. Other vitamin D3
metabolites, such as 24,25-dihydroxyvitamin D3 (24,
25-(OH)2D3), 25-hydroxyvitamin
D3 (25OHD3), and other
ligands for nuclear receptors, such as 9-cis-retinoic acid
(9-cis-RA) and dexamethasone, did not affect the interaction between
VDR and TFIIB as assessed by the in vivo two-hybrid protein
interaction assay and the in vitro GST fusion protein assay
(Fig. 8
, A and B). Thus, the two different systems that examined
VDR-TFIIB interaction in vivo and in vitro
indicated that the 1,25-(OH)2D3 ligand
selectively disrupts VDR interaction with TFIIB.

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Figure 8. Specific Effect of
1,25-(OH)2D3 on Disrupting VDR Interaction with
TFIIB
A, The in vivo two-hybrid protein interaction
assay. ß-Galactosidase expression was quantified in liquid culture
after overnight culture in the absence or presence of 10-7
M 1,25-(OH)2D3,
24,25-(OH)2D3, 25OHD3,
dexamethasone, or 9-cis-RA. Results are presented as the
mean ± SD of triplicate independent cultures. B, The
in vitro GST fusion protein interaction assay.
Baculovirus-expressed full-length VDR (1.3 µg) was incubated with 2.5
µg GST-TFIIB in the absence or presence of 10-7
M 1,25-(OH)2D3,
24,25-(OH)2D3, 25OHD3,
dexamethasone, or 9-cis-RA. The protein-protein complex
were precipitated with glutathione-agarose, washed five times, and
analyzed for VDR by Western immunoblot analysis.
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DISCUSSION
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We and others recently demonstrated that VDR interacts with TFIIB
(20, 21), a key component of the general transcription machinery
and a pivotal target for transactivator interaction with the PIC. This
interaction represents a potential physical link between VDR bound at a
distal enhancer sequence (i.e. the VDRE) and RNAP II
activity at the core promoter element(s). However, a precise functional
role for VDR interaction with TFIIB in vitamin D-mediated transcription
is not well understood. In the current study, we developed a
transcriptional interference assay to examine the potential importance
of VDR-TFIIB interactions in vitamin D-activated transcription in
mammalian cells. Toward this end, site-directed mutagenesis was used to
firmly establish the requirement of the amino-terminal domain of TFIIB
in the formation of the TFIIB-VDR complex in vitro and
in vivo. Based on these data, a TFIIB derivative (N-TFIIB)
was developed that contained the N-terminal, VDR-interactive domain,
but lacked other regions of TFIIB that are required for general
transcription. When N-TFIIB was expressed in mammalian cell lines to
disrupt interactions between VDR and native TFIIB, this derivative
interfered with VDR/vitamin D-activated transcription. These studies
support a functional role for the interaction between VDR and the N
terminus of TFIIB in the mechanism through which VDR alters target gene
transcription in mammalian cells.
TFIIB is a single polypeptide that contains two functionally important
regions, one in the N terminus and one in the C terminus (23, 24, 25). The
crystal and solution structures for the C-terminal region of human
TFIIB (residues 106316) have been solved (26, 27). The C-terminal
core region is required for interactions with the TFIID(TBP)-TATA
element complex, making direct contacts with the C-terminal stirrup of
TBP and with the phosphoribosyl backbone of the DNA both upstream and
downstream of the TATA element (26, 27, 28, 29). Structural data on the
amino-terminal region of TFIIB have not been reported. However, a
putative zinc (Zn2+)-binding sequence
(CysX2HisX17CysX2Cys) is located in
this N-terminal region. Limited proteolysis and epitope mapping studies
both indicate that the N terminus of the native molecule is exposed to
solvent and readily accessible for macromolecular interactions (23, 30). This is supported by functional studies indicating that the N
terminus is required for incorporation of TFIIF and RNAP II into the
PIC, probably through interaction with the RAP30 subunit of TFIIF
(23, 24, 25). However, this is not the only role that the amino-terminal
domain plays. For example, Colgan et al. (31, 32)
established the importance of the amino terminus of TFIIB in contacting
the Ftz transactivator protein in vivo. Our current studies
using the yeast two-hybrid system and in vitro interaction
assays with purified proteins also establish the importance of this
region in VDR-TFIIB interactions. Disrupting the structural integrity
of the N terminus by mutating those residues putatively involved in
coordinating the Zn2+ atom (C15, H18, C34, and C37 of human
TFIIB) dramatically reduced TFIIB interaction with VDR in
vivo and in vitro. The fact that these two diverse
transactivators (Ftz and VDR) interact with the same region of TFIIB
further support the hypothesis put forth by Colgan et al.
(31, 32) that the N terminus of TFIIB, although important for RNAP
II/TFIIF recruitment, may also have an important regulatory role in
transcription mediated by activator proteins.
The transcriptional interference assay with N-TFIIB provides support
for a role of the TFIIB amino terminus in regulated transcription by
VDR. Under the conditions of the assay, N-TFIIB did not affect basal
transcription of several different promoter constructs in both COS-7
cells and ROS 17/2.8 cells. It is likely that the relative level of
N-TFIIB in these experiments was not sufficiently great enough to
compromise RNAP II/TFIIF activity on basal promoters with comparatively
low transcriptional throughput. Moreover, expression of N-TFIIB in this
assay had no effect on glucocorticoid-activated transcription. This
indicates that N-TFIIB did not limit the participation of RNAP II/TFIIF
in higher order transcription elicited by all transactivating proteins,
but of the two nuclear receptors examined, N-TFIIB was selective for
VDR-activated transcription. The transcriptional interference was most
likely due to N-TFIIB interaction with VDR because a mutant N-TFIIB
that does not interact with VDR [N-TFIIB(C34S:C37S)] showed no
ability to interfere with VDR-mediated transcription, and the effects
of N-TFIIB were rescued by the expression of additional wild type VDR
or TFIIB. Moreover, the in vitro competition assay clearly
showed the ability of N-TFIIB to compete with wild type TFIIB for
binding to the VDR. Taken together, these studies indicate that N-TFIIB
interferes with vitamin D-activated transcription by disrupting the
interaction between VDR and native TFIIB. As indicated by the
contrasting studies of our group and Ozatos group (20), it is
possible that VDR interacts with multiple domains of TFIIB, one in the
N terminus and another in the C terminus. However, our current data
would clearly support a functional importance for VDR interaction with
the N terminus of TFIIB in the mechanism of vitamin D-activated
transcription.
What are the possible steps in the vitamin D transactivation cascade?
Insight into potentially novel aspects of the mechanism of VDR-mediated
transcription was realized with the remarkably potent, specific
interference of the 1,25-(OH)2D3 ligand on
VDR-TFIIB complex formation. Recently, Yen et al. (33)
showed that unliganded VDR suppressed transcription of DR-3 and
osteopontin VDRE-driven reporter gene constructs, suggesting that
unliganded VDR may function as a transcriptional repressor, much like
unliganded thyroid hormone receptor (TR). As suggested for thyroid
hormone in disrupting TR-TFIIB interactions (18, 19), it is possible
that part of the mechanism for VDR may involve unliganded VDR
interaction with TFIIB to negatively influence the efficient formation
of the PIC, resulting in a repressed transcriptional state. However,
using osteocalcin VDRE-driven reporter constructs in both COS-7 cells
and CV-1 cells, we do not observe repressed transcription by unliganded
VDR, suggesting that the repressive effect may be response element or
promoter specific. An alternative mechanism suggested by our data
involves TFIIB recruitment to the immediate vicinity of vitamin
D-responsive promoters through TFIIB interaction with unliganded VDR
bound to the VDRE. This creates a "primed" state on that promoter
in which limiting transcription factors such as TFIIB are more readily
available for higher order transcription elicited by the hormone. The
1,25-(OH)2D3 ligand binds to VDR and releases
TFIIB for assembly into the PIC forming on that particular vitamin
D-responsive promoter. Here, the apoVDR-TFIIB interaction serves to
sequester TFIIB (and possibly other limiting factors) around or near a
particular vitamin D-responsive promoter area. Thus, it is not simply
the formation of the VDR-TFIIB complex that leads to vitamin
D-activated transcription, but VDR-directed targeting of TFIIB to
vitamin D-responsive promoters and then a ligand-induced release of the
sequestered TFIIB to the assembling PIC. In preliminary studies
examining PIC assembly on a vitamin D-responsive promoter, we have
observed a VDR-dependent and
1,25-(OH)2D3-dependent increase in the relative
amount of TFIIB that assembles into the PIC (H. Masuyama and P.
MacDonald, unpublished observation). This indicates that although
1,25-(OH)2D3 negatively impacts VDR-TFIIB
interaction, the ligand has an overall positive influence on the
relative level of TFIIB in the PIC. Such an observation is compatible
with the mechanism discussed above, and it also supports previous
observations by Ozatos group showing synergistic activation of
VDR-mediated transcription by exogenous TFIIB (20).
Although we have shown that VDR interaction with the N terminus of
TFIIB is involved in the mechanism of vitamin D-mediated gene
expression, it is clearly only a part of the transcriptional cascade.
The nuclear receptors are known to interact with a variety of putative
coactivator and corepressor proteins that also function in the
mechanism of steroid-regulated gene transcription (34, 35, 36, 37, 38). For
example, steroid receptor coactivator-1 (SRC-1), a putative coactivator
for many of the steroid hormone receptors, is thought to mediate
interactions between liganded receptors and the PIC (36). The nuclear
receptor corepressor (N-CoR) and the silencing mediator for retinoid
and thyroid hormone receptors (SMART) factors are nuclear corepressor
proteins that interact with unliganded TRs and retinoid receptors to
repress transcription of responsive genes in the absence of hormone
(34, 35). Horlein et al. (35) proposed that certain nuclear
receptor-regulated gene transcription may consist of a combination of
hormone-dependent derepression mediated by corepressor proteins such as
N-CoR and transactivation mediated through coactivators such as SRC-1.
With specific regard to vitamin D-mediated transcription, the role of
the known coactivator/corepressor proteins has not been established.
Apparently, VDR does not interact with N-CoR (35), and its interaction
with SRC-1 was not investigated (36). VDR has been reported to interact
with thyroid hormone receptor interacting protein-1 (TRIP-1) (37), a
putative cofactor that interacts in a ligand-dependent fashion with the
AF-2 region of several nuclear receptors (37, 38). Indeed, our
laboratory has isolated TRIP-1 as a ligand-dependent, VDR-interactive
clone in a two-hybrid screen of an osteoblast cDNA library (H. Masuyama
and P. MacDonald, unpublished observation). However, the precise role
of TRIP-1 in steroid hormone-mediated transcription or its role as a
nuclear receptor coactivator protein is still unresolved. Based on
these observations, it is possible that VDR couples to the PIC through
direct interactions with the N terminus of TFIIB as well as through
interactions with novel coactivator/corepressor proteins that remain to
be identified. Understanding the complex interplay of the receptor with
TFIIB and with the various coactivator/corepressor proteins and the
precise means through which these interactions result in efficient
ligand-mediated transcription will be the focus of future research
efforts.
 |
MATERIALS AND METHODS
|
---|
Preparation of Two-Hybrid Expression Vectors
All two-hybrid plasmid constructs used the pAS1 and pGAD.GH
yeast expression vectors (39, 40). The AS1-VDR construct, which
contained the Gal4 DNA-binding domain (amino acids 1147) and the
carboxyl-terminal region of VDR (amino acids 93427), was previously
described (21). Point mutations were introduced into the human TFIIB
cDNA with oligonucleotide-directed mutagenesis (41, 42). The N-terminal
domain of TFIIB (N-TFIIB) was an NcoI/BglII
deletion construct, termed TFIIB(
124-296) (21). A mutated version of
TFIIB(
124-296) was also generated in which cysteine residues at
amino acid positions 34 and 37 were altered to serine residues by
site-directed mutagenesis. This construct was designated
TFIIB(
124-296;C34S:C37S) or more simply as N-TFIIB(C34S:C37S). All
point mutants were confirmed by DNA-sequencing reactions, and each
mutant was subcloned into the pGAD.GH vector to examine in the
two-hybrid assay.
Yeast Transformation and ß-Galactosidase Assays
The pGAD-TFIIB wild type and mutant constructs were
cotransformed with pAS1-VDR (93427) into the yeast strain Hf7c, which
was made competent with lithium acetate (43). Transformants were plated
on media lacking leucine and tryptophan (SC-leu-trp) and were grown for
4 days at 30 C to select for yeast that had acquired both plasmids.
Triplicate independent colonies from each plate were grown overnight in
2 ml of SC (-leu-trp). These samples were diluted to 0.02
OD600 units and were incubated overnight with or without
the indicated concentrations of 1,25-(OH)2D3 or
other hormones. Cells were harvested and assayed for ß-galactosidase
activity as described (44).
Production and Purification of Glutathione S-Transferase (GST)
Fusion Proteins and the in Vitro Protein Interaction
Assay
The cDNAs for wild type TFIIB, TFIIB(C34S:C37S) and
N-TFIIB [TFIIB(
124-296)], were subcloned into pGEX-KT (45).
GST-fusion proteins of wild type TFIIB, TFIIB(C34S:C37S), were
expressed in the DH5
strain of bacteria and were purified by
glutathione-agarose and Sephadex G-75 size exclusion chromatography.
The N-TFIIB protein was purified by gluthatione-agarose chromatography
and was separated from the GST moiety by thrombin cleavage (45).
Proteins were analyzed by SDS-PAGE and stained with Coomassie blue to
confirm their purity. Full-length VDR was expressed in a baculovirus
expression system (46). The GST-TFIIB fusion proteins were incubated
with 0.5 µg VDR in 100 µl of GBB buffer (20 mM Tris-Cl,
pH 7.6, 200 mM NaCl, 0.1% Nonidet P-40, 1 mM
dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 50 µg/ml
pepstatin A, and 0.1 mM phenylmethylsulfonyl fluoride) for
1 h at 4 C. After the addition of 20 µl of a 50% slurry of
glutathione agarose, the proteins were incubated for 30 min at 4 C. The
glutathione-agarose and associated protein complexes were washed five
times in 200 µl of GBB buffer, solubilized in SDS sample buffer, and
analyzed for VDR content by Western blot analysis using monoclonal
antibody 9A7
as described previously (21).
Transient Transfection Studies
The VDRE4-TATA-GH GH reporter plasmid contained four
copies of the rat osteocalcin VDRE adjacent to the rat osteocalcin
promoter fragment (-40 to +32) linked to a human GH reporter sequence
in p0GH (47). VDRE4-TK-GH, BGP(-1000)-GH, and
BGP(-150)-GH were described previously (46, 48).
GRE2-TK-GH contained two copies of a consensus
glucocorticoid-responsive element (GRE) upstream of the viral thymidine
kinase promoter in the pTK-GH vector (47). N-TFIIB
[TFIIB(
124-296)] and the mutant N-TFIIB
[TFIIB(
124-296);C34S:C37S] were subcloned into the pcDNA3
expression vector (Invitrogen Co. San Diego CA). Full-length TFIIB was
subcloned into the pSG5 expression vector (Stratagene, San Diego, CA).
The pSG5-VDR and pSG5-GR expression plasmids were described previously
(46, 49). COS-7 cells were cotransfected with reporter gene constructs
(VDRE4-TATA-GH, VDRE4-TK-GH, or
GRE2-TK-GH), receptor expression vectors (pSG5-VDR or
pSG5-GR), and TFIIB expression vectors (N-TFIIB, mutant N-TFIIB,
full-length TFIIB, or expression vector alone). ROS 17/2.8 cells were
also cotransfected with reporter gene constructs
[VDRE4-TATA-GH, BGP(-1000)-GH or BGP(-150)-GH), and
TFIIB expression vectors (N-TFIIB, mutant N-TFIIB, or expression vector
alone). In all transfections, the amount of total DNA was kept constant
at 10 µg by adding pTZ18U (U.S. Biochemical, Cleveland, OH) as a
carrier plasmid, and the cells were transfected by standard calcium
phosphate coprecipitation procedures as described previously (46).
Transfected cells were treated with 10-8 M
1,25-(OH)2D3, 10-7 M
dexamethasone, or ethanol vehicle for 24 h, and the amount of
secreted GH was determined with a RIA kit (Nichols Institute, San Juan
Capistrano, CA).
 |
ACKNOWLEDGMENTS
|
---|
We gratefully acknowledge Dr. Peter Jurutka for the
GRE2-TK-GH plasmid and Dr. Mark Haussler for his continued
support and encouragement. We wish to thank Dr. Milan R. Uskokovic
(Hoffmann-LaRoche, Nutley, NJ) for providing the
1,25-(OH)2D3 and Dr. Alex Brown for other
vitamin D compounds. We also acknowledge Dr. Diane R. Dowd for
experimental suggestions and for a critical reading of the
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Paul N. MacDonald, Ph.D., St. Louis University Health Sciences Center, Department of Pharmacological and Physiological Science, 1402 South Grand Boulevard, St. Louis, Missouri 63104.
This work was supported in part by NIH Grants R29DK-47293 and
R01DK-50348 to P.N.M.
Received for publication June 3, 1996.
Revision received October 18, 1996.
Accepted for publication November 5, 1996.
 |
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