(Received for publication, September 16, 1994; and in revised form, November 10, 1994)
From the
The vitamin D receptor (VDR) heterodimerizes with retinoid X receptors (RXR) on many vitamin D-responsive
promoter elements, suggesting that this complex is the active factor in
vitamin D-mediated transcription. However, the mechanism of
transcriptional regulation following VDRRXR binding to
DNA is not well characterized. Using a yeast two-hybrid protein
interaction assay, we demonstrate that VDR forms specific
protein:protein contacts with the basal transcription factor TFIIB.
Deletion analysis indicated that the carboxyl-terminal ligand binding
domain of VDR interacted with a 43-residue amino-terminal domain in
TFIIB. The interaction with TFIIB showed selectivity for the ligand
binding domain of VDR as similar regions of RXR
or of
retinoic acid receptor
did not couple with TFIIB. Binding assays
with purified proteins showed a direct interaction between VDR and
TFIIB in vitro. These data suggest a mechanism for
VDR-dependent transcription in which protein contacts between VDR and
TFIIB may impart regulatory information to the transcription
preinitiation complex.
The biological effects of 1,25-dihydroxyvitamin D (1,25(OH)
D
) (
)are mediated
through a soluble protein termed the vitamin D receptor, or VDR. The
VDR is a member of the superfamily of nuclear receptors for steroid
hormones, thyroid hormone, and retinoids. As such a member, the VDR
acts as a ligand-induced transcription factor that binds to specific
DNA elements in vitamin D-responsive genes and ultimately influences
the rate of transcription(1) . Purified VDR does not bind to
authentic vitamin D-responsive elements with high affinity unless an
additional nuclear protein, designated NAF or RAF (for nuclear or
receptor auxiliary factor) is
present(2, 3, 4) . RAF may be related to
retinoid X receptors (RXRs) since RXRs mimic
RAF activity (5, 6, 7) and highly purified
RAF contains RXR immunoreactivity(7) . Involvement of
RXR in vitamin D function is supported by the observations
that vitamin D-dependent transcription is augmented by exogenous
RXR in transient expression systems (5, 7) and that numerous VDR mutants that do not
interact with RXR also fail to activate transcription in
vivo(8) . Thus, in one current hypothesis,
1,25(OH)
D
-dependent gene expression is mediated
by heterodimeric binding of VDR and RXR to the vitamin
D-responsive element with the heterodimer serving as the functional
transcription factor.
The mechanisms that link receptor:response
element binding to alterations in RNA polymerase II transcription are
not well understood but are likely to involve protein:protein
interactions between the receptor and the transcription preinitiation
complex. Transcription factors IIB and IID (TFIIB and TFIID) are key
targets for transactivator interactions, and, in many cases, additional
adapter proteins function to mediate contacts between the
transactivator and these basal factors. In this regard,
TAF110 links transcription factor Sp1 to
TFIID(9, 10) , TAF
40 is involved in
VP16/TFIIB interactions(11) , and a putative coactivator also
links phosphorylated cAMP response element binding protein to
TFIIB(12) . Alternatively, transactivators may contact
components of the basal transcription machinery directly. Both the
acidic activation domain of VP16 and the glutamine-rich activation
domain of the Ftz protein interact directly with
TFIIB(13, 14, 15) . Furthermore, progesterone
receptor, estrogen receptor, thyroid hormone receptor, and chicken
ovalbumin upstream promoter transcription factor interact with TFIIB,
suggesting that steroid receptor function is mediated through TFIIB
interactions(16, 17) . Presently, the mechanisms
linking VDR
RXR heterodimers to the transcription
preinitiation complex are not known. In the current study, we
demonstrate that VDR forms a highly specific, direct protein:protein
contact with TFIIB. The carboxyl-terminal portion of VDR contacts
TFIIB, whereas similar regions of RXR or of retinoic acid
receptor (RAR) do not associate with TFIIB.
Figure 1:
Specific interactions of VDR with
RXR and with TFIIB in a two-hybrid system. A,
the two-hybrid protein interaction assay. The Gal4 DNA binding domain (DBD)-VDR hybrid protein and the Gal4 activation domain (ACT)-RXR
hybrid protein are illustrated
schematically. The interaction of the VDR and RXR domains
results in Gal4-dependent transcription of the lacZ and HIS3 reporter sequences that are integrated into the HF7c
yeast genome. B, growth characteristics of various two-hybrid
combinations on His-deficient media. Individual colonies were streaked
on media containing histidine (leftplate) or lacking
histidine (rightplate) to identify plasmid
combinations that resulted in expression of the HIS3 gene. Area1, AS1-VDR(93-427) + GAD-SNF4; area2, AS1-SNF1 +
GAD-RXR
(223-462); area3,
AS1-SNF1 + GAD-SNF4; area4,
AS1-VDR(93-427) + GAD-RXR
(223-462); area5, AS1-SNF1 + GAD-TFIIB; area6, AS1-VDR(93-427) + GAD-TFIIB. Specific
interaction is observed only with the following hybrid pairs: yeast
SNF1 and SNF4 (area3), VDR and RXR (area4), and VDR and TFIIB (area6).
Initial characterization of the two-hybrid system is presented in Fig. 1B. The specificity of the VDR/RXR interaction was examined using SNF1 and SNF4, authentic interacting yeast proteins. The AS1-SNF1 and GAD-SNF4 constructs expressed functional proteins since yeast transformed with this combination grew well on His-deficient media (right plate, area 3). In contrast, yeast expressing either the VDR and SNF4 combination (area 1) or the SNF1 and RXR combination (area 2) did not grow, indicating that neither the VDR nor the RXR fusion proteins interacted with the SNF proteins in this system. Importantly, specific interactions between VDR and RXR were evident as yeast expressing AS1-VDR(93-427) and GAD-RXR(223-462) grew well on histidine-deficient media (area 4).
Figure 2:
The ligand binding domain of VDR interacts
with the amino-terminal domain of TFIIB. A, amino- and
carboxyl-terminal deletion mutants of AS1-VDR were tested against
GAD-TFIIB in the two-hybrid system. Relative growth on His-deficient
plates was assessed after 4 days, and -galactosidase expression
was quantitated in liquid cultures. Results are presented as the mean
(± standard deviation) of triplicate cultures. The DNA box in
the figure represents the DNA binding domain of VDR. B,
amino-terminal and internal deletion mutants of TFIIB were constructed
in the GAD.GH vector. Each TFIIB construct was examined for interaction
with AS1-VDR by monitoring expression of the HIS3 and lacZ reporter genes in the two-hybrid system. Openboxes in the illustration represent the imperfect direct repeats in the
amino acid sequence of TFIIB.
A similar analysis of TFIIB is presented in Fig. 2B. Elimination of most of the carboxyl terminus
of TFIIB (125-297 and
66-294) did not interfere
with VDR-TFIIB interactions. In fact, these mutants appeared to
interact with VDR better than full-length TFIIB, possibly due to the
removal of interfering domains in TFIIB (23) . However,
deleting the amino-terminal regions (
1-42 and
1-123) eliminated
-galactosidase expression and growth
on His-deficient media. Importantly, addition of the most extreme
amino-terminal 43 residues to the
1-123 mutant partially
restored activity, suggesting that this amino-terminal domain is
sufficient to mediate TFIIB-VDR interactions.
Figure 3:
Specificity of the interaction between VDR
and TFIIB. HF7c yeast were transformed with AS1-VDR (leftplate), AS1-RXR (middleplate), or AS1-RAR
(rightplate)
together with the indicated GAD.GH constructs. Individual colonies were
streaked on His-deficient plates to identify plasmid combinations that
result in HIS3 gene expression. Specific interactions were
noted with AS1-VDR/GAD-RXR, AS1-VDR/GAD-TFIIB,
AS1-RXR/GAD-VDR, and
AS1-RAR/GAD-RXR.
Figure 4:
Interaction of VDR with TFIIB and with
RXR in vitro. A, ligand-dependent
interactions. Baculovirus-expressed full-length VDR (1.3 µg) was
incubated with 15 µg of GST (lanes4 and 5), 7.5 µg of GST-TFIIB (lanes6 and 7), or 7.5 µg of GST-RXR (lanes8 and 9) in the absence or presence of 0.8
10
M 1,25(OH)
D
. Protein:protein complexes were
precipitated with glutathione-agarose, washed extensively, and analyzed
for human VDR by Western immunoblot analysis. Lanes1 and 2 represent 2.5% of the total baculovirus-expressed
human VDR present in the interaction assay. B, role of the
TFIIB amino terminus in VDR-TFIIB interactions. Wild-type GST-TFIIB (lane 1), GST-TFIIB(
45-124) (lane 2), and
GST-TFIIB(
1-124) (lane 3) were examined for their
ability to interact with VDR as described
above.
Fig. 4B illustrates
the effect of several TFIIB deletion mutants in the in vitro VDR interaction assay. In agreement with the results of the
two-hybrid system, the TFIIB(1-124) mutation did not
interact appreciably with VDR, while addition of amino acids 1-43
to this construct (lane2) partially restored
interaction with VDR compared with wild-type TFIIB (lane1). Thus, these data indicate that the amino terminus of
TFIIB mediates interactions with the VDR both in vivo and in vitro.
A central question in eukaryotic transcription concerns the mechanism by which site-specific activators stimulate the transcriptional process. One current hypothesis suggests that activators function by contacting components of the RNA polymerase II preinitiation complex. The basal transcription factor TFIIB is important in this regard. TFIIB assembly into the preinitiation complex is a rate-limiting step that is increased by direct interaction with a transactivator such as VP16(13, 14) . Furthermore, a TFIIB mutant that does not interact with VP16 still functions in basal transcription but not in VP16-activated transcription(24) . Thus, interaction between an acidic activator and TFIIB is necessary for transcriptional activation in some systems. In the present study, we demonstrate that TFIIB forms a highly specific protein:protein contact with the VDR. The interaction was apparent both in vivo in the yeast two-hybrid interaction system and in vitro in protein interaction assays with purified TFIIB and VDR. Thus, TFIIB may be a pivotal link in the communication between VDR and the transcription preinitiation complex during vitamin Dmediated transcription. However, functional analysis of VDR and/or TFIIB mutants will be required to test this hypothesis.
The carboxyl-terminal
region of TFIIB contains two imperfect repeats of approximately 75
amino acids with a cluster of basic residues in the first repeat that
may fold into an amphipathic -helix(25, 26) . A
conserved cysteine-rich sequence is present in the amino terminus that
may form a zinc binding motif(27) . Limited proteolysis and
deletion analysis reveal that these two domains are functionally
distinct(28, 29) . The amino terminus may be required
for recruitment of RNA polymerase II and/or TFIIF, while the
carboxyl-terminal region interacts with the TATA binding protein-DNA
complex. The bipartite nature of TFIIB is also evident in its
interactions with VDR in that the carboxyl-terminal domain of TFIIB is
dispensable for VDR coupling while the amino terminus is absolutely
required. In contrast, VP16 couples to the putative amphipathic
-helix of TFIIB(24) , and this region is not involved in
VDR-TFIIB interactions. Thus, the activation domains of different
classes of transcription factors may ultimately influence gene
expression by interacting with distinct domains on TFIIB.
Several
other steroid-thyroid receptors interact with TFIIB, and different
regions of each receptor mediate this
interaction(16, 17) . TFIIB interacts with the
carboxyl-terminal ligand binding domain of the estrogen
receptor(17) , with two distinct domains of the thyroid hormone
receptor (16) and with the DNA binding domain of the
progesterone receptor(17) . In comparison, the amino-terminal
DNA binding domain of VDR was not crucial for VDR-TFIIB interactions (Fig. 2A). Multiple requisite domains in the carboxyl
terminus of VDR were essential. Removal of either
Leu-Ser
or
Leu
-Ser
completely abolished
VDR-TFIIB interactions, and neither region on its own was sufficient to
promote strong interactions with TFIIB. Both regions are rich in
hydrophobic and charged amino acid residues, and, specifically, the
region between Leu
and Tyr
has the
propensity to form an amphipathic
-helix that may be potentially
important in VDR-TFIIB interactions.
Recent mutagenesis of VDR
suggests that several widely spaced regions of the ligand binding
domain also mediate VDR-RXR and VDR-RAF
interactions(8) . The extreme carboxyl-terminal residues of the
VDR are also critical for VDR-RXR interactions, indicating
that TFIIB and RXR may interact with a common domain in VDR.
However, one aspect of the present study suggests that distinct domains
of VDR interact with TFIIB and RXR since VDR-RXR
interaction was enhanced by 1,25(OH)D
, whereas
the VDR-TFIIB interaction was ligand independent (Fig. 4). It is
conceivable that a ligand-induced conformational change exposes a
domain of the VDR involved in heterodimerization with RXR, and
this domain is not directly involved in VDR interactions with TFIIB.
More refined mutagenesis and binding studies are required to test this
hypothesis.
With regard to specificity, we noted a clear preference
of TFIIB for the carboxyl terminus of VDR. TFIIB did not interact to
the same extent with the carboxyl terminus of RXR (Fig. 3) nor did GST-TFIIB interact appreciably with murine
RXR in our in vitro assay (data not shown). Our
studies do not rule out the possibility that TFIIB interacts with other
isoforms or with more amino-terminal domains of RARs or RXRs.
However, a nearly full-length RXR
construct
(AS1-RXR
(Glu
-Thr
))
showed no interaction with the TFIIB fusion in the two-hybrid system
(data not shown), supporting the concept that RXR does not
interact appreciably with TFIIB under these conditions. It is an
intriguing possibility that the RXR portion of the heterodimer
may contact the preinitiation complex through a distinct protein, such
as another general transcription factor or a bridging protein.
Regardless, these data indicate that fundamental differences exist in
the mechanisms leading to transcriptional regulation by the nuclear
receptors for retinoids and vitamin D.