The Polymorphic N Terminus in Human Vitamin D Receptor Isoforms Influences Transcriptional Activity by Modulating Interaction with Transcription Factor IIB
Peter W. Jurutka,
Lenore S. Remus,
G. Kerr Whitfield,
Paul D. Thompson,
J.-C. Hsieh,
Heike Zitzer,
Poupak Tavakkoli,
Michael A. Galligan,
Hope T. L. Dang,
Carol A. Haussler and
Mark R. Haussler
Department of Biochemistry College of Medicine University
of Arizona Tucson, Arizona 85724
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ABSTRACT
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The human vitamin D receptor (hVDR) is a
ligand-regulated transcription factor that mediates the actions of the
1,25-dihydroxyvitamin D3 hormone to effect bone
mineral homeostasis. Employing mutational analysis, we characterized
Arg-18/Arg-22, hVDR residues immediately N-terminal of the first DNA
binding zinc finger, as vital for contact with human basal
transcription factor IIB (TFIIB). Alteration of either of these basic
amino acids to alanine also compromised hVDR transcriptional activity.
In contrast, an artificial hVDR truncation devoid of the first 12
residues displayed both enhanced interaction with TFIIB and
transactivation. Similarly, a natural polymorphic variant of hVDR,
termed F/M4 (missing a FokI restriction site), which lacks
only the first three amino acids (including Glu-2), interacted more
efficiently with TFIIB and also possessed elevated transcriptional
activity compared with the full-length (f/M1) receptor. It is concluded
that the functioning of positively charged Arg-18/Arg-22 as part of an
hVDR docking site for TFIIB is influenced by the composition of the
adjacent polymorphic N terminus. Increased transactivation by the F/M4
neomorphic hVDR is hypothesized to result from its demonstrated
enhanced association with TFIIB. This proposal is supported by the
observed conversion of f/M1 hVDR activity to that of F/M4 hVDR, either
by overexpression of TFIIB or neutralization of the acidic Glu-2 by
replacement with alanine in f/M1 hVDR. Because the f VDR genotype has
been associated with lower bone mineral density in diverse populations,
one factor contributing to a genetic predisposition to osteoporosis may
be the F/f polymorphism that dictates VDR isoforms with differential
TFIIB interaction.
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INTRODUCTION
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The traditional role of vitamin D, via its active hormonal
metabolite 1,25-dihydroxyvitamin D3
(1,25-(OH)2D3), is to
regulate calcium and phosphate metabolism to elicit normal bone
mineralization and remodeling.
1,25-(OH)2D3 also appears
to exert a number of nonclassical bioeffects in the immune, central
nervous, and endocrine systems, as well as in epithelial cell
differentiation (1). The generation of
1,25-(OH)2D3 from vitamin
D3, obtained initially from diet or derived from
sunlight-initiated photobiogenesis in skin, involves sequential
hydroxylations in liver and kidney (1).
1,25-(OH)2D3 ensures that
the proper ion product of calcium and phosphate exists in the blood for
optimal deposition of bone mineral by stimulating intestinal
absorption, bone resorption, and renal reabsorption of these ions. A
failure to achieve normal bone mineral accretion results in clinical
rachitic syndromes, such as nutritional rickets, which arises from the
simultaneous deprivation of sunlight exposure and dietary vitamin
D3, or hypocalcemic vitamin D-resistant rickets
(HVDRR), which can be a consequence of inadequate enzymatic
bioactivation of the vitamin. The molecular basis for a rare familial
form of HVDRR, in which there is tissue insensitivity to
1,25-(OH)2D3, has been
shown to reside in defects within the gene coding for the nuclear
vitamin D receptor (VDR) (2, 3). The phenotype of these latter HVDRR
patients, including hypocalcemia, secondary hyperparathyroidism, and
severe osteopenia, mimics classic nutritional rickets, thus implicating
VDR as the mediator of the bone mineral homeostatic actions of
1,25-(OH)2D3. Further
unequivocal evidence for the obligatory role of VDR in skeletal
maintenance is detailed in two recent reports describing the VDR null
mouse homozygote, which displays a phenotype similar to that of HVDRR
patients (4, 5).
The VDR is a member of the nuclear receptor superfamily of
proteins that contain amino acid homologies within two separate
functional domains (6, 7, 8). The N-terminal region of VDR is configured
into two zinc-coordinated fingers responsible for DNA recognition and
binding, whereas the C-terminal segment binds the
1,25-(OH)2D3 hormone (9).
This common modular structure reflects the similar molecular actions
employed by the members of the nuclear receptor superfamily in
translating a hormonal signal into a transcriptional response. Upon
binding 1,25-(OH)2D3, VDR
regulates specific gene transcription by binding as a heterodimer with
the retinoid X receptor (RXR) (10, 11, 12, 13) to a DNA enhancer sequence,
termed the vitamin D-responsive element (VDRE), that is present within
the promoter region of vitamin D-controlled genes (14, 15, 16). Thus, VDR
belongs to the same subgroup of nuclear receptors that includes the
thyroid hormone receptor (TR) and retinoic acid receptor (RAR), which
also heterodimerize with RXR on their respective DNA-responsive
elements (17).
In addition to its interaction with RXR, the VDR has been shown to
associate with several additional proteins to form the active
transcriptional complex required for gene regulation (1). These
molecules, termed coactivators, include proteins of the p160 class that
possess histone acetyl transferase (HAT) activity such as SRC-1 (18),
GRIP1 (19), and ACTR (20). Other coactivators postulated to stimulate
VDR-mediated transactivation are TIF1 (21), NCoA-62 (22), p65 (23),
DRIP205/TRAP220 (24, 25), and components of the transforming growth
factor-ß (TGF-ß) signaling pathway, including Smad3 (26, 27).
Moreover, VDR has been reported to interact directly with components of
the basal transcription machinery, such as TATA-binding protein
associated factors TAFII135 (28) and
TAFII55 (29), with concomitant enhancement in
ligand-stimulated transcription. Finally, the basal transcription
factor IIB (TFIIB) has been shown by several laboratories to interact
both physically and functionally with this receptor (30, 31, 32, 33), and one
of the regions required for TFIIB association is localized within the
C-terminal hormone-binding domain of the VDR (30, 31, 34).
The VDR gene harbors several polymorphisms, both in the coding and
noncoding portions of the gene (1, 35). However, only one of these
polymorphisms results in an actual change in the VDR primary sequence.
This polymorphism occurs within the first ATG start codon of human VDR
(hVDR) and contains a FokI restriction endonuclease site
(designated f). Absence of the FokI site (denoted F)
indicates that the first codon is ACG, resulting in translational
initiation at an in-frame ATG three codons downstream (36, 37).
Therefore, the FokI polymorphism produces either a 424 (F)
or a 427 (f) amino acid hVDR protein. These two isoforms are thus
structurally distinct, unlike those hVDRs that contain polymorphisms
present in the 3'-portion of the gene that are either silent codon
changes or are found in introns or in the 3'-untranslated region
(1).
Because of the central role of VDR in calcium and phosphate homeostasis
to ensure the deposition of bone mineral, the FokI
polymorphism has been studied in the context of its potential influence
on bone mineral density (BMD). In several different populations,
including American and Japanese premenopausal women, as well as
Mexican-American and Italian postmenopausal women, an association
between enhanced BMD and the F allele has been reported (36, 37, 38, 39, 40, 41), but
no mechanism for this relationship has been proposed.
In the present study, we identify a novel region in the VDR
N-terminal segment required for functional interaction with TFIIB and
define specific residues that participate in transcriptional
stimulation mediated by
1,25-(OH)2D3 via contact
with TFIIB. Thus, similar to the estrogen and glucocorticoid receptors
(42, 43), VDR appears to possess an activation function 1-like domain
(AF-1) N-terminal of the zinc fingers. We also provide unique evidence
that, as a direct result of differential interaction of the two
receptor isoforms with TFIIB at this N-terminal region, the F hVDR
possesses more potent transcriptional activity. This observation may
provide the mechanistic basis for the enhanced BMD associated with the
FF vs. ff genotype, and intimates that, unlike the
inactivating mutations which generate the severe HVDRR phenotype,
smaller differences in VDR activity over a lifetime could significantly
impact the risk of bone fractures and osteoporosis.
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RESULTS
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hVDR Mutants Used to Probe Interactions with TFIIB
Previous reports by us and our collaborators (30) and others
(31, 32) have demonstrated that VDR interacts both physically and
functionally with TFIIB in vitro, in transfected mammalian
cells, and in the yeast two-hybrid system. Furthermore, one region of
hVDR that appears to be important for contacting TFIIB occurs within
the C-terminal hormone binding domain (30, 34). In the present
study, we endeavored to determine whether additional regions in hVDR
are important for mediating the interaction between the receptor and
TFIIB. A series of hVDR mutants was constructed as depicted in Fig. 1
. These mutants included truncations of
the hormone-binding (
161427,
134427,
115427,
84427)
or DNA-binding (
188) domains, as well as smaller internal
deletions in the N-terminal region of VDR (
513,
1423,
1517, and
1821). Specific point mutants were also synthesized
that resulted in the loss of a positively charged arginine residue by
replacement with alanine (R18A and R22A) or that contained a
conservative change from arginine to lysine (R22K), thus maintaining a
positive charge at this position. All of these mutants were constructed
from the cDNA encoding a polymorphic form of hVDR known as F/M4, so
denoted to indicate that this hVDR lacks the first three amino acids
and thus translation begins from the fourth residue, a Kozak consensus
methionine, in the amino acid sequence numbering convention described
previously (44). Another polymorphic variant, termed f/M1, represents
the full-length hVDR (amino acids 1427) with translation commencing
from the first residue, also a Kozak consensus methionine. The f/M1
receptor was created by mutagenesis using the F/M4 cDNA as a template
and inserting the naturally occurring codons present in the f/M1
receptor [coding for the first three amino acids (MEA-) Fig. 1
].
Finally, a point mutant within the context of the f/M1 polymorphic VDR
was generated by converting glutamate to alanine at position 2.

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Figure 1. Construction of hVDR Mutants Used to Probe
Interactions with TFIIB
The central portion of the figure depicts a schematic
representation of full-length hVDR (427 amino acids) containing an
N-terminal DBD and C-terminal ligand binding region. Illustrated
directly above the central hVDR molecule are the two
polymorphic designations for the receptor: f/M1 encoding the
full-length hVDR and F/M4, which codes for a shorter 424-amino acid
protein isoform. Shown above the F/M4 hVDR are C- and
N-terminal truncations that were generated in this receptor isoform by
site-directed mutagenesis. The bottom portion of the
panel depicts smaller internal deletions and point mutations
(arrows) that were generated in the F/M4 hVDR isoform in
this study. The numbers after each delta symbol
represent the amino acids deleted in each case [using the Baker
et al. (44 ) numbering system]. For point mutations
(E2A, R18A, R22A, and R22K), the number of the mutated residue is
designated along with the corresponding amino acid substitution.
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Mapping Regions of hVDR Required for Interaction with hTFIIB
To define regions of hVDR that associate with TFIIB, we employed
an in vitro coprecipitation or pull-down assay using
glutathione-S-transferase (GST) fusion protein methodology
and in vitro transcribed and translated (IVTT) receptor
proteins. Figure 2A
depicts the basic
interaction between wild-type (WT) hVDR and a GST-TFIIB fusion protein
bound to Sepharose beads (lane 4), with no interaction occurring when
only GST-Sepharose (GST-S) is used (lane 3) or when the IVTT reaction
contains only the pSG5 parent expression vector (lanes 1 and 2). The
association between RXR
and TFIIB (lane 6) is much weaker than
between VDR and TFIIB (lane 4), and the amount of RXR
that is
coprecipitated does not increase in the presence of unliganded VDR
(compare lanes 6 and 7). Importantly, inclusion of
1,25-(OH)2D3 markedly
stimulates association of RXR
-VDR and TFIIB (lane 8). These
observations indicate that the RXR-VDR heterodimer, which forms in
solution in the presence of
1,25-(OH)2D3 and represents
the functionally relevant molecular species in mediating the activation
of VDRE-controlled genes, can readily interact with TFIIB in a presumed
ternary complex.

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Figure 2. Human TFIIB Interacts with hVDR and RXR in a
1,25-(OH)2D3-Dependent Ternary Complex
A, Plasmids (1.0 µg) expressing either hVDR or hRXR were used as
templates in IVTT reactions (see Materials and Methods)
to generate [35S]methionine-labeled hVDR and hRXR
proteins. Negative control IVTT reactions (lanes 1 and 2) employed the
pSG5 template without a receptor cDNA insert. All reactions were then
incubated with 10-6 M
1,25-(OH)2D3 (lane 8) or ethanol vehicle (lanes
17) for 1 h at 22 C, followed by incubation with either 20 µl
of GST-hTFIIB fusion protein bound to Sepharose beads (GST-hTFIIB-S;
lanes 2, 4, and 68) or 20 µl GST alone bound to Sepharose (GST-S;
lanes 1, 3, and 5) for 1 h at 4 C. In lanes 7 and 8, equal
portions of hVDR and hRXR protein were mixed before incubation with
1,25-(OH)2D3. The beads were then washed
extensively, and the amount of coprecipitated hVDR and hRXR was
detected by electrophoresis of denatured bead samples followed by
autoradiography. B, The amount of extract analyzed as input (panel B)
was 5% of the amount used in the coprecipitation reactions (panel A).
The arrows indicate the migration position of hVDR and
hRXR .
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We next evaluated the association of the WT hVDR and several C-terminal
truncation mutants in the GST pull-down assay. Figure 3A
reveals that removal of the
hormone-binding domain results in a significantly attenuated
interaction of hVDR with TFIIB (compare lanes 2 and 10), consistent
with earlier evidence for a TFIIB contact site residing in the
hormone-binding domain of hVDR, approximately between amino acids 257
and 355 (30). However, further truncation of the receptor enhances this
level of association (lanes 6 and 8), with the most pronounced
interaction occurring with the smallest truncation, namely
84, that
possesses only the first 83 amino acids in hVDR (lane 4). These results
suggest that an additional segment within the N-terminal portion of
hVDR may be required for full TFIIB association. Indeed, removal of the
first 88 amino acids (
188) in the receptor results in a dramatic
decrease in the level of interaction with TFIIB (Fig. 3B
, lane 4, and
Fig. 4A
, lane 6) as
compared with that observed with the
84 truncation (Fig. 3A
, lane
4), while the association of the C-terminal truncations (Fig. 3B
, lanes
6, 8, and 10) is similar to that observed in Fig. 3A
. Nonspecific
binding to GST-S alone (odd-numbered lanes) is minimal. In addition,
all of the truncations were expressed, based on analysis of 5% of the
sample size employed in the coprecipitation reaction (input panels),
although the level of expression for the truncations was generally less
than that of the WT control. Taken together, these results strongly
implicate the VDR DNA-binding domain (DBD), and/or its N-terminal
extension, as important for TFIIB interaction, in vitro.

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Figure 4. Analysis of Internal Deletants and Point Mutants in
the N-Terminal Region of hVDR and Identification of Residues Needed for
Association with TFIIB
A, WT, N-terminally truncated ( 188), and internally deleted
( 513 and 1423) hVDR proteins were synthesized in the IVTT
system and incubated with either 20 µl of GST-TFIIB-S (even
numbered lanes) or 20 µl GST-S (odd numbered
lanes) for 1 h at 4 C. Lanes 1 and 2 contain an IVTT
reaction that employed pSG5 template without the hVDR cDNA insert as a
negative control. The beads were then washed and analyzed as described
in Fig. 2 . B, An additional set of internally deleted hVDRs ( 1517
and 1821; lanes 710) was generated and analyzed in parallel with
WT, 513, and 1423 hVDRs (lanes 16) for interaction with GST-TFIIB-S or GST-S. C, WT and two
point mutant hVDRs (R18A and R22A) were generated in the IVTT system. A
control IVTT reaction (lane 1) employed the pSG5 template without the
hVDR cDNA insert. Reactions were then incubated with 10-6
M 1,25-(OH)2D3 (even
numbered lanes) or ethanol vehicle (odd numbered
lanes) for 1 h at 22 C followed by incubation with either
20 µl GST-TFIIB-S (lanes 1 and 38) or 20 µl GST-S (lane 2) for
1 h at 4 C. The beads were then washed and analyzed as described
in Fig. 2 . D, Extracts from cells transfected with either TFIIB alone
(lane 1), WT hVDR alone (lane 2), or TFIIB and WT or mutant hVDRs
(lanes 35) were immunoprecipitated with a TFIIB antibody-protein
A/G-Sepharose complex. The immunoprecipitates were subjected to 520%
SDS/PAGE followed by immunoblotting with an anti-VDR monoclonal
antibody (9A7 ) to detect the level of hVDR interaction with TFIIB in
a cellular context. The amount of extract analyzed as input in each
panel was 5% of the amount used in the coprecipitation reactions. The
arrows indicate the migration position of the hVDR
proteins. The bands below the migration position of
full-length hVDR represent proteolytic products in each reaction. The
results are representative of three independent trials for panel C and
two independent trials each for panels A, B, and D.
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To define a smaller region in hVDR that participates in contacting
TFIIB, a series of short internal deletants was constructed within the
N-terminal extension of the DBD. Surprisingly, the
513 mutant
displayed a striking enhancement in association with TFIIB (Fig. 4A
, lane 8, and Fig. 4B
, lane 4) compared with the WT receptor (Fig. 4A
, lane 4, and Fig. 4B
, lane 2), while the
1423 deletant was
attenuated in its interaction with TFIIB (Fig. 4A
, lane 10, and Fig. 4B
, lane 6). An additional pair of even shorter deletants revealed a
similar pattern, with
1517 displaying enhanced (Fig. 4B
, lane 8)
and
1821 (lane 10) showing a reduced association with TFIIB. These
results suggest that residues between amino acids 18 and 23 within the
N-terminal domain of hVDR are mediators of TFIIB association, and that
the apparent affinity of this interaction can be influenced by the
composition of amino acids N-terminal of Arg-18.
Charged amino acids within the residue 1823 segment of hVDR were
selected for conversion to alanine by site-directed mutagenesis. Two
such mutants, designated R18A and R22A, were evaluated in the GST
pull-down assay. The results from these experiments, shown in Fig. 4C
, indicate a significant loss in the ability of these two mutant hVDRs to
interact with TFIIB (compare lanes 3 and 4 to lanes 58). No
interactions occur when only GST-Sepharose is used (lane 2) or when the
cDNA template in the IVTT reaction does not contain the hVDR insert
(lane 1). In addition, the presence of the
1,25-(OH)2D3 hormone does
not significantly influence the association with TFIIB (compare lanes 3
and 4, 5 and 6, and 7 and 8), suggesting that contact between TFIIB and
the residues in the N-terminal domain of hVDR is independent of
hormonal ligand.
Because the results described above were obtained with the pure
protein-protein interaction system of IVTT GST pull-down, we also
evaluated WT and mutant hVDR association with TFIIB in the context of
cellular extracts employing a complementary coimmunoprecipitation
protocol utilizing TFIIB antibody. In the experiment depicted in Fig. 4D
, both overexpressed WT hVDR (lane 3) and the
513 mutant (lane
4) coimmunoprecipitate with TFIIB under these conditions, whereas R22A
hVDR does not exhibit detectable binding to TFIIB (lane 5).
Interestingly, when the level of TFIIB association with
513 is
normalized to differences in expression of this mutant (input panel,
right),
513 interacts more efficiently with TFIIB than
WT hVDR, approximating the enhanced level of association observed when
employing the IVTT system (Fig. 4
, A and B). Thus, similar results for
VDR-TFIIB interaction to those observed with the IVTT GST pull-down
assay are obtained with an independent, and perhaps more
physiologically relevant, methodology.
Transcriptional Activity of N-Terminal hVDR Mutants Is Related to
the Magnitude of Receptor-TFIIB Interaction
Having demonstrated a requirement for the presence of the N
terminus of hVDR to bind TFIIB optimally, in vitro, we next
probed the functional significance of this domain in mediating
1,25-(OH)2D3-stimulated
transcription of a reporter gene under the control of the rat
osteocalcin VDRE (four tandem copies of the VDRE linked to GH gene,
[CT4]4-TKGH) in a variety of transfected
mammalian cells. Figure 5A
illustrates
that, in this cotransfection system,
1,25-(OH)2D3
(10-8 M) treatment of
COS-7 cells overexpressing WT hVDR results in an approximate 56-fold
increase in receptor-mediated transcription. In sharp contrast, the
1423 and
1821 deletants that displayed little interaction
with TFIIB (Fig. 4
, A and B) are moderately (
1821) or severely
(
1423) impaired in their ability to activate transcription
compared with the WT control. Interestingly, the
513 and, to a
lesser extent, the
1517 mutant, both of which displayed an
enhanced association with TFIIB (Fig. 4
, A, B, and D), showed a
corresponding modest increase in transactivation in response to
1,25-(OH)2D3. In another
set of similar experiments (Fig. 5B
), also employing transfected COS-7
cells, R18A, R22A, and R22K point mutant hVDRs were evaluated. WT hVDR
mediates a 23-fold increase in transcription of the GH reporter gene in
the presence of ligand, while the R18A or R22A mutants exhibit only a
17-fold or 3-fold enhancement, respectively. A conservative replacement
(arginine to lysine) at residue 22 preserves the activity at WT levels,
suggesting that a basic charge is required at this position in hVDR for
full transcriptional activity. Because interaction of R18A and R22A
with TFIIB is attenuated (Fig. 4C
), we overexpressed TFIIB together
with these mutant hVDRs in a rescue experiment. The additional TFIIB
was able to boost the level of
1,25-(OH)2D3-elicited
transcription of WT receptor from 23- to 34-fold (Fig. 5B
). Moreover,
the activity of the R18A mutant, whose transactivation capacity is only
mildly affected (17-fold vs. 23-fold for WT hVDR), can be
almost completely restored by overexpression of TFIIB. However, the
severely affected R22A mutant, with a 3-fold response to ligand, is
boosted only slightly (up to 4-fold ligand stimulation) by TFIIB
overexpression. A similar analysis in HeLa cells (Fig. 5C
) and in a rat
osteoblast-like osteosarcoma cell line, ROS 2/3 (data not shown), also
employing the artificial VDRE, [CT4]4-TKGH
reporter, reveals a comparable pattern of transactivation by the mutant
hVDRs in that R22A-mediated transcriptional stimulation is diminished
compared with R18A and is less successfully rescued by excess TFIIB.
ROS 2/3 cells were further analyzed with a reporter construct
containing 1100 bp of upstream promoter sequence from the rat
osteocalcin natural promoter, which contains a single VDRE, linked to
the GH reporter gene (Fig. 5D
). Responsiveness to
1,25-(OH)2D3 of this
natural promoter construct was blunted compared to that occurring with
multiple copies of the VDRE (Fig. 5
, AC), but the relative pattern of
transcriptional activity displayed by the WT and mutant hVDRs was
similar. Most importantly, excess TFIIB nearly restored the
1,25-(OH)2D3
transcriptional responsiveness of R18A and R22A hVDRs (Fig. 5D
) without
potentiating activation by the WT receptor in this setting of a natural
promoter in a bone-derived cell type. These data support the contention
that Arg-18 and Arg-22, two hVDR residues situated just N-terminal of
the first DNA-binding zinc finger, play an important role in
1,25-(OH)2D3-elicited
transactivation of VDRE-regulated genes (Fig. 5
) via a mechanism that
includes recruitment and contact with TFIIB (Fig. 4
).

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Figure 5. Transcriptional Activity of N-Terminal hVDR Mutants
Is Related to the Magnitude of Receptor-TFIIB Interaction
A, COS-7 cells were cotransfected by calcium phosphate-DNA
coprecipitation with expression vectors for either the WT or internally
deleted hVDRs and a reporter plasmid containing four osteocalcin VDREs
linked to the human GH gene ([CT4]4-TKGH). Cells were
treated for 24 h posttransfection with 10-8
M 1,25-(OH)2D3 or ethanol vehicle.
The level of GH secreted into the culture medium, which serves as an
index of transcriptional activity, was assessed by RIA of each plate.
B, Expression vectors for either the WT or indicated point mutant hVDRs
and [CT4]4-TKGH were cotransfected into COS-7 cells as in
panel A followed by treatment with 10-8 M
1,25-(OH)2D3 for 24 h. An additional group
of cells was further cotransfected with an expression plasmid for TFIIB
in "rescue" experiments. The transcriptional activity of WT, R18A,
and R22A was similarly monitored in HeLa cells (panel C) and in a rat
osteoblast-like osteosarcoma cell line, ROS 2/3 (panel D), except
employing a reporter vector that contains approximately 1100 bp of
upstream sequence from the rat osteocalcin promoter in ROS 2/3. Each
treatment group consists of triplicate samples, and each panel is
representative of at least three independent experiments. Error
bars represent the SEM.
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Neither Expression nor DNA and Hormone Binding Are Affected in hVDR
Mutants Possessing Compromised Transcriptional Activity
One possible explanation for the reduced transcriptional
activation observed with some of the mutant hVDRs is that the
introduction of internal deletions or even point alterations within the
VDR molecule could lead to changes in protein stability. We therefore
examined the level of VDR protein expression (Fig. 6
) in transfected cells employed for the
transcriptional assays (Fig. 5
). Since assessment of transcriptional
activity involves assay of the culture medium, the same cells can be
lysed and analyzed by Western blotting with an anti-VDR monoclonal
antibody (9A7
). These immunoblots revealed that the internal
deletants were all expressed at levels comparable to the WT receptor
(Fig. 6A
). In fact, a mutant that displayed enhanced transcriptional
activity (
513) is slightly less expressed than the WT hVDR (Fig. 6A
, compare lanes 1 and 2), while a transcriptionally inactive mutant
(
1423) is somewhat enhanced in its expression (lane 3). Similarly,
the functionally defective point mutant VDRs (R18A and R22A) are well
expressed (Fig. 6B
, compare lanes 13), as are the R22K and E2A
mutants (data not shown). Since rescue experiments (Fig. 5
) involved
the overexpression of TFIIB, we assessed the expression of endogenous
and transfected TFIIB utilizing a polyclonal TFIIB antibody (Fig. 6C
).
Overexpression of TFIIB leads to a dramatic enhancement in the level of
detected protein above endogenous amounts in transfected cells (Fig. 6C
, lanes 46 vs. 13). Importantly, overexpression of
TFIIB does not affect the expression of WT or mutant hVDRs (Fig. 6B
, lanes 46).
The residues implicated in TFIIB contact and in mediating a
transcriptional response to
1,25-(OH)2D3 are located in
the N-terminal domain of hVDR, in close proximity to the DNA binding
zinc fingers of the receptor. We therefore performed a
hormone-dependent electrophoretic mobility shift assay with extracts
from COS-7 cells expressing limiting amounts of VDR proteins that
approximate levels found in
1,25-(OH)2D3 target tissues
(3, 45). Under these near-physiological conditions, both the
heterodimeric DNA binding and the hormone association of the WT and
mutant receptors can be assessed. As shown in Fig. 7A
, both the WT and hVDR mutants formed a
VDR-containing shifted complex (lanes 3, 6, and 8) that was enhanced by
the addition of the
1,25-(OH)2D3 ligand (lanes
4, 7, and 9). Significantly, the level of augmentation by
1,25-(OH)2D3 (
4-fold)
based on densitometric scanning of the autoradiographs in Fig. 7A
was
similar for the WT receptor and each mutant hVDR (data not shown).
Also, the level of expression of each receptor was essentially
equivalent and unmodified by ligand as monitored by immunoblotting
(data not shown). The nonspecific (NS) nature of the two lower
protein-DNA complexes was deduced by their appearance in the lanes that
used extracts from non-VDR transfected cells (lanes 1 and 2), as well
as by their lack of elimination by the VDR antibody (lane 5). Similar
data were obtained when employing the R18A or R22A hVDR (Fig. 7B
).
Taken together, these results strongly suggest that deletion of hVDR
residues between positions 15 and 21 or modification of amino acids 18
or 22 to alanine does not decrease heterodimeric DNA binding by the VDR
protein. Therefore, altered VDRE association cannot account for the
attenuation of transactivation by these mutants. Additionally, the
nearly equivalent enhancement in DNA binding elicited by the
1,25-(OH)2D3 hormone
suggests that ligand binding properties of the mutant hVDRs remain
unchanged.

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Figure 7. DNA and Hormone Binding Are Not Affected in hVDR
Mutants Possessing Compromised Transcriptional Activity
A, A gel mobility shift assay was performed using extracts (5
µg total protein) from COS-7 cells transfected with WT or internally
deleted hVDR expression plasmids (0.1 µg) along with a labeled
oligonucleotide containing the VDRE from the rat osteocalcin gene as
described in Materials and Methods. Extracts were
incubated with 10-7 M
1,25-(OH)2D3 (lanes 2, 4, 5, 7, and 9) or
ethanol vehicle (lanes 1, 3, 6, and 8) for 0.5 h at 22 C before
incubation with the VDRE oligonucleotide. Lane 5 also contained an
anti-VDR monoclonal antibody (9A7 ) that is directed against an
epitope in the DBD and disrupts the interaction of VDR with the VDRE
(12 ). Lanes 1 and 2 contain extracts from COS-7 cells transfected with
the expression vector lacking the hVDR insert. Arrows
indicate the migration of the specific VDR-VDRE complex (as a
heterodimer with RXR), nonspecific complexes (NS), or free probe. B,
The gel mobility shift assay was performed as in panel A but employing
point mutant hVDRs.
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The Polymorphic N Terminus of hVDR Affects Both the Interaction
with TFIIB and the Level of Transactivation
The N-terminal hVDR FokI polymorphism results in the
formation of either a full-length 427-amino acid hVDR (denoted either
"f" to indicate the presence of the FokI restriction
site or designated "M1" for translation from the first methionine
in the primary sequence), or a shorter 424-amino acid protein (termed
either "F" for the absence of the FokI site or named
"M4" to indicate translational initiation from the methionine at
the fourth position in the primary sequence). These isoforms have
previously been associated with differences in BMD in diverse
populations (36, 37, 38, 39, 40, 41). Having identified specific residues located near
the N terminus of hVDR as important for TFIIB interaction and
transactivation, we next investigated the potential impact, if any, of
this polymorphic N terminus on the TFIIB contact domain nearby in the
primary sequence. First, extracts from human fibroblasts were evaluated
by Western blotting to determine the extent of expression of the two
endogenous hVDRs in these cells. As shown in Fig. 8A
, both polymorphic forms of the
receptor are well expressed (lanes 1 and 2), and in the case of the
heterozygote, both proteins are apparently synthesized (lane 3). The
electrophoretic mobility of the M1 and M4 hVDRs can be distinguished in
these denaturing gels, with the shorter M4 receptor displaying a
slightly faster migration (compare lane 2 to 1). We then constructed
expression vectors containing the coding sequence for both the M1 and
M4 hVDRs. Western blot analysis of extracts from COS-7 cells
transfected with either of these plasmids demonstrates the
expression of the appropriate hVDR isoform (Fig. 8B
), with little
difference in the expression levels of either species. As in the case
of the endogenous hVDR, the overexpressed polymorphic receptors display
distinct electrophoretic mobilities (compare lanes 1 and 2 or 4 and 5).
Similar to the experiments shown in Fig. 7
, we evaluated the
heterodimeric DNA binding activity and hormone binding capacity of the
M1 and M4 hVDRs in the hormone-dependent gel mobility shift assay and
found no detectable differences in either DNA or
1,25-(OH)2D3 association
between these two receptor isoforms (data not shown).

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Figure 8. Both N-Terminal Polymorphic Variants of hVDR Are
Expressed in Human Fibroblasts and in Transfected Cells
A, Whole-cell extracts (40 µg protein equivalents) of human
fibroblasts were analyzed by immunoblotting employing the anti-VDR
monoclonal antibody, 9A7 . The genotype (see Materials and
Methods for details) of each fibroblast line is indicated along
with the corresponding protein form. The arrows reveal
the positions of the faster migrating M4 hVDR (424 amino acids) and the
slower migrating M1 receptor variant (427 amino acids). B, Whole-cell
extracts (40 µg protein equivalents) of COS-7 cells transfected with
the M1 (lanes 2 and 5), M4 (lanes 1 and 4), or both M1 and M4 (lanes 3
and 6) hVDR expression plasmids were analyzed as in panel A. Two
different amounts of expression plasmid (1.0 and 3.0 µg) were used in
the transfections.
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Since both polymorphic forms of hVDR are well expressed, either
endogenously in cultured human fibroblasts, or in transfected cells, we
tested the transcriptional activity in response to varying doses of
1,25-(OH)2D3 of these two
receptors in different transfected cell lines. Figure 9A
illustrates that in transfected COS-7
cells, 1,25-(OH)2D3
(10-9 and 10-8
M) treatment results in an approximate 27- and 78-fold
increase, respectively, in receptor-mediated transcription with the M4
receptor. In contrast, the M1 protein only displayed an 8.5- and
38-fold increase in activity at these two ligand concentrations.
Interestingly, overexpression of TFIIB did not lead to a statistically
significant potentiation of transcription with the more active M4, but
did increase the activity of M1 (up to 13.3- and 66-fold). Similar
results were obtained with the [CT4]4-TKGH
reporter in HeLa cells (Fig. 9B
) and in murine P19 embryonal carcinoma
(EC) cells or a rat osteoblast-like osteosarcoma cell line, ROS 2/3
(data not shown). ROS 2/3 cells were further analyzed with a reporter
construct containing 1100 bp of the rat osteocalcin natural promoter
linked to the GH reporter gene (Fig. 9C
). In this experiment,
1,25-(OH)2D3
(10-9 and 10-8
M) treatment results in an approximate 3.0- and 3.5-fold
increase, respectively, in receptor-mediated transcription with the M4
receptor, but only a 1.8- and 2.6-fold effect was noted with M1. Again,
the overexpression of TFIIB does not enhance transactivation by M4
(2.4- and 3.6-fold hormone effect) but does lead to a small but
significant elevation in M1 activity (3.0- and 3.4-fold stimulation).
Examination of the sequence differences between the M1 and M4 hVDR
reveals that the three amino acid N-terminal extension of M1 (MEA-)
contains a glutamate at position 2. Replacement of this residue with an
uncharged alanine residue results in the complete restoration of
1,25-(OH)2D3-elicited
transcription (at both 10-9 and
10-8 M hormone) in transfected ROS
2/3 cells (Fig. 9D
).

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Figure 9. Transcriptional Activity of the f/M1 Polymorphic
hVDR Is Reduced as Is Association of this Variant Receptor with hTFIIB
Expression vectors for either the M1 (427 amino acids) or M4 (424
amino acids) polymorphic hVDR and [CT4]4-TKGH were
cotransfected into COS-7 (panel A) or HeLa (panel B) cells as in Fig. 5
followed by treatment with the indicated concentration of
1,25-(OH)2D3 for 24 h. A parallel group of
cells was further cotransfected with an expression plasmid for human
TFIIB in "rescue" experiments (+TFIIB). The transcriptional
activity of M1 and M4 hVDRs was also monitored in a rat osteoblast-like
osteosarcoma cell line, ROS 2/3 (panel C), employing a reporter vector
that contains approximately 1100 bp of upstream sequence from the rat
osteocalcin promoter linked to the GH reporter gene. D, Expression
vectors for M1 and M4 polymorphic hVDR, or a mutant M1 (containing an
alanine at position 2 instead of a glutamic acid; E2A) along with a
natural osteocalcin promoter-reporter construct were cotransfected into
ROS 2/3 cells as in panel C, followed by treatment with the indicated
concentration 1,25-(OH)2D3 for 24 h. Each
treatment group consists of triplicate samples, and each panel is
representative of at least three independent experiments. Error
bars represent the SEM. E, M1 (lanes 2 and 4) and
M4 (lanes 1 and 3) hVDR proteins were generated in the IVTT system and
incubated with 20 µl of GST-hTFIIB-S for 1 h at 4 C. The beads
were then washed and analyzed as described in the legend to Fig. 2 . The
amount of extract analyzed as input was 5% of the amount used in the
pull-down reactions.
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Finally, we employed the in vitro coprecipitation assay to
determine directly if the two hVDR isoforms display a differential
level of interaction with TFIIB. Figure 9E
depicts the association
between M4 (lanes 1 and 3) and M1 (lanes 2 and 4) hVDR and a GST-TFIIB
fusion protein bound to Sepharose beads. The M4 hVDR displays a 2-fold
greater interaction with TFIIB compared with the M1 protein, based on
quantitative densitometric scanning of the images shown in Fig. 9E
and
taking into account the minor difference in hVDR protein expression as
depicted in the input panel. These results imply that the observed
differences in transactivation capacity between M1 and M4 hVDR are
likely the result of a polymorphic N terminus that selectively
modulates the interaction of hVDR with TFIIB.
 |
DISCUSSION
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The VDR is a ligand-dependent transcription factor that
regulates a number of genes, many of which are involved in calcium and
phosphate mineral homeostasis and bone remodeling. Like many other
members of the nuclear receptor superfamily, the VDR binds to DNA as a
heterodimer with RXR and controls the transcription of its target genes
by interacting with other coregulatory proteins. One of these proteins,
basal transcription factor IIB, has previously been shown to interact
both physically and functionally with VDR (30, 31). In the present
study, we have localized within the N-terminal segment of VDR a
critical site of interaction with TFIIB and demonstrated that key,
evolutionarily conserved residues in this region are required for both
TFIIB association and
1,25-(OH)2D3-dependent
transactivation. We also explored the relevance of these findings to a
naturally occurring polymorphism at the N terminus of hVDR that results
in two structurally distinct isoforms of the receptor. Not only do
these two forms (f/M1 and F/M4) display a differential pattern of
response to 1,25-(OH)2D3 in
eliciting VDRE-mediated transactivation in a number of cell lines
including osteoblasts, but they also exhibit a positive correlation
between their transcriptional activity and their ability to interact
with TFIIB. Because these hVDR isoforms have previously been associated
with differences in BMD in certain populations, one factor contributing
to a genetic predisposition to osteoporosis therefore may involve the
varying potency in interaction between polymorphic hVDRs and the basal
transcriptional machinery.
Initial Mapping of a TFIIB Contact Site in VDR
Previous studies have identified TFIIB as a VDR-interacting
protein, employing both pull-down assays (30, 31, 32, 33, 34) and the yeast
two-hybrid system (31, 34). Yeast two-hybrid data from our laboratory
confirm this interaction (C. Encinas and P. W. Jurutka,
unpublished data). Furthermore, mapping studies have been performed
using truncation and deletion methodology with both the GST pull-down
(30) and yeast assays (31, 34). The results of these initial mapping
experiments pointed to the hormone-binding domain in hVDR as possessing
one site of interaction for TFIIB, likely encompassing primarily amino
acids 257355 (30). More recently, utilizing an alternative strategy,
the hormone-binding domain (hVDR residues 93427) was employed to
generate a random point mutant library that was used to screen for
TFIIB interaction-deficient mutants in the yeast two-hybrid system. The
results implicated 11 individual residues between amino acids 228 and
345 in VDR, the majority of which are hydrophobic, as being critical
for TFIIB association (46).
Multiple TFIIB Interaction Sites and Functional
Significance
Several factors argue, however, that the C-terminal domain of hVDR
may not be the only site of TFIIB interaction. The limitation of using
gross deletions and truncations (30, 31) is that, once any critical
interacting region has been removed, other, perhaps equally important,
domains may go undetected. Also, in the above cited point mutant screen
with the hVDR hormone-binding region (46), the entire N-terminal region
of hVDR (residues 192) was not present during the analysis. These
caveats, plus the current results using small N-terminal deletions and
point mutations, lead us to propose that, in addition to hydrophobic
amino acids within the hormone-binding region of hVDR, charged residues
in the N-terminal domain of the molecule are required for optimal TFIIB
association and functionally competent VDR-mediated transactivation
(see also Fig. 10
).

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Figure 10. Model of Interaction between hVDR and TFIIB and
Its Consequences for Transactivation by the hVDR-RXR Complex on the
VDRE
M1 and M4 hVDR refer to genetic variants of the hVDR gene, also
distinguished by the presence (f) or absence (F) of a
FokI site in the genomic DNA. A, The M4 variant of hVDR,
lacking a Glu residue at position 2, can interact optimally with hTFIIB
via two conserved basic residues (Arg-18 and Arg-22) in the Site II
(denoted "II") N-terminal interaction domain. This interaction with
hTFIIB is augmented by a previously described Site I interacting
region, denoted "I", located within the C-terminal ligand-binding
domain of hVDR (30 34 ). TFIIB is thereby efficiently delivered to the
transcriptional initiation complex, leading to robust transcriptional
activation. Also part of the activation process is the dissociation of
a corepressor ("CoR"), as well as the binding of a coactivator
(CoA), like SRC-1, which aids in derepression of the gene by
acetylating histones and reorganizing chromatin in the promoter region
(as suggested by the "dissociation" of histones; see text for
references). B, The M1 variant of hVDR contains three additional amino
acids at the extreme N terminus, including a Glu residue (Glu-2), which
is proposed to weaken the interaction of Arg-18 and Arg-22 with hTFIIB.
Subsequent steps in transactivation by the hVDR-RXR complex on the VDRE
can still occur but are attenuated by the weaker nature of the f/M1
hVDR contact with hTFIIB in the Site II region.
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The functional significance of TFIIB interaction with VDR is supported
by transactivation studies that demonstrate a cooperative effect of
these two molecules. In experiments employing P19 EC cells,
1,25-(OH)2D3 treatment
results in only a minor stimulation of reporter gene expression when
either hVDR or TFIIB alone is cotransfected into these cells.
Concomitant overexpression of VDR and TFIIB causes a dramatic
(>30-fold) enhancement in reporter gene transcription (30). Another
report (34) revealed that expression of a TFIIB mutant that was
nonfunctional but still retained the ability to interact with hVDR
resulted in a selective dominant negative effect on
1,25-(OH)2D3-elicited
transcription in an osteoblast-like cell line. This inhibition could be
reversed by simultaneous expression of WT TFIIB. Neither an inhibition
nor a stimulation was observed with glucocorticoid-induced
transcription of a glucocorticoid responsive element-reporter
construct, indicating that excess TFIIB did not exert nonspecific
effects on transcription. Thus, one requirement for
1,25-(OH)2D3-mediated
transcription appears to be specific physical interaction of hVDR with
TFIIB, which we propose involves both C- and N-terminal domains in the
receptor.
Previous investigations of the putative functional role for the N
terminus of hVDR have suggested that, unlike some of the other nuclear
receptors that possess a larger N-terminal domain, perhaps this region
of the hVDR may be functionally dispensable. Removal of N-terminal
residues up to and including amino acid 21 in hVDR did not have a
significant impact on transcriptional activity in transfected COS-1
cells using a human osteocalcin gene promoter-reporter construct (47).
However, this hVDR construct still possesses Arg-22, a residue that is
vital for transcriptional activity in all of the cell lines we tested.
One might therefore deduce from our results that exposure of Arg-22 at
the extreme N terminus of the (
121) truncation mutant (47) might
accentuate its role, even obviating the need for Arg-18, the other
basic residue we found to play a significant role in hVDR-TFIIB
binding.
Other Nuclear Receptors Bind to TFIIB
The present proposal that hVDR has a second, N-terminal
interaction domain with TFIIB, and that this domain contains crucial
basic residues, has a precedent from the results concerning
interaction between TR and TFIIB. Chicken TR
contains 50 residues
N-terminal of the first zinc finger, including a cluster of basic amino
acids (KRKRK) at positions 2327 that were shown to be crucial for
both TFIIB interaction and transactivation by this receptor (48). Thus,
like hVDR, chicken TR
requires N-terminal basic residues for
contacting TFIIB and for transactivation. The fact that this cluster of
positively charged amino acids is well conserved among all known TR
homologs from vertebrate species [including fish (GenBank
accession no. BAA08201)] is indeed consistent with such an important
functional role for these residues. Significantly, positively charged
residues are conserved at positions corresponding to 18 and 22 in all
published VDR sequences, ranging from human to the recently cloned
Xenopus VDR (GenBank accession no. AAB58585),
strongly implying that these residues must subserve some crucial
function.
Moreover, human TRß also interacts specifically with TFIIB and,
similar to our proposal for VDR (Fig. 10
), deletion analysis revealed two
contact sites in the TRß molecule, one located in the N-terminal
region and the other positioned in the ligand-binding domain (49). Each
of these two distinct regions in TRß was demonstrated to interact
with different sites in TFIIB (49). It therefore appears that VDR and
TR, in addition to sharing other features, such as heterodimerization
with RXR on direct-repeat responsive elements as well as some sequence
homology (27%), also possess dual interaction interfaces with TFIIB.
Of course, it is possible that, in the tertiary structures of
full-length TR and VDR, these two interaction regions combine to form a
single docking scaffold.
A further example of a nuclear receptor that interacts with TFIIB via
an N-terminal domain is the homodimerizing ER (50). This study of ER,
as well as a similar evaluation of the orphan nuclear receptor
hepatocyte nuclear factor-4 (HNF-4) (51), concludes that association of
TFIIB with the respective receptor facilitates the assembly of
transcriptional preinitiation components, particularly the TATA-box
binding protein (TBP). Thus, a picture of nuclear receptor
transactivation is beginning to emerge in which TFIIB plays a crucial
role.
Significance of the TFIIB Interaction in Transactivation by VDR
There appear to be at least two distinct sets of protein-protein
interactions involved in transcriptional activation by hVDR. One is
represented by a group of coactivators that bind to a cleft comprised
of the Tyr-236 to Lys-246 (helix 3) region combined with the helix 12
AF-2 platform in VDR (52) and effect derepression of chromatin
nucleosome organization via HAT-catalyzed displacement of histones at
the active promoter site. In our current model, contact by these
coactivators with components of TBP-TAF and RNA polymerase II, with the
possible participation of a cointegrator analogous to CBP/p300
(53), facilitates transcriptional activation. A second crucial
interaction appears to be the recruitment of TFIIB to the promoter via
the VDR-RXR heterocomplex (see Figs. 2
and 10
). The delivery of TFIIB
would then stabilize the RNA polymerase II preinitiation complex and
allow for repeated rounds of transcription of the regulated target
gene.
Clinical Impact and Functional Significance of hVDR
Polymorphisms
The FokI site polymorphism in exon 2 of the hVDR gene
is distinct from all other reported VDR polymorphisms in that the two
biallelic variants actually differ in protein sequence (f/M1 being 3
amino acids longer). The shorter F/M4 receptor apparently arose after
the divergence of hominids from apes and has been dubbed a
"neomorph" (1), yet it presently constitutes approximately 65% of
VDR alleles in human subjects (36, 37, 38, 39, 40, 41, 54, 55). This predominance of
the relatively recent F/M4 allele suggests an evolutionary advantage in
humans. In support of this notion, the African-American population,
which has a significantly lower incidence of osteoporosis than
Caucasians, also has a greater prevalence (>80%) of the F/M4 variant
(38).
A direct association between the VDR FokI polymorphism and
BMD has been reported in several studies. In a group of
Mexican-American/Caucasian women, subjects with the ff genotype had a
12.8% lower BMD at the lumbar spine than FF subjects, with
heterozygotes possessing an intermediate BMD (36). This study also
showed an increased 2-yr rate of bone loss from the femoral neck in the
ff vs. FF women. Similarly, in a population of premenopausal
Japanese women, BMD in the lumbar spine was 12% less for the ff
genotype vs. FF (37). Recently, in a large cohort that
consisted of a group of 400 postmenopausal women of Italian descent,
the FF genotype was associated with enhanced lumbar BMD (41).
Interestingly, the effect of the FF genotype on enhanced lumbar BMD was
greatest in women within 5 yr of menopause, progressively declining
afterward, and the low BMD ff genotype was significantly
overrepresented in patients with osteoporotic vertebral fractures
(relative risk 2.58). In yet a fourth population of American
premenopausal women (both Caucasian and African-American), ff women had
femoral neck BMD that was 7.4% lower than that of FF women (38). The
association of BMD and the FokI polymorphism also has been
extended to include a population of healthy growing children. Subjects
that were FF homozygotes had a total body BMD that was 8.2% higher and
a mean calcium absorption that was 41.5% greater than ff individuals
(40). Thus, the FokI polymorphism may be related to several
VDR-dependent parameters of bone metabolism, including intestinal
calcium absorption and BMD. However, one study found no significant
relationship between the F/f genotype and BMD in a population of French
premenopausal women (55), although as the authors point out, the
association of BMD and this polymorphism may be masked by various
regional factors, such as a high calcium diet. Indeed, an influence of
dietary calcium on the impact of the FokI polymorphism on
BMD has been suggested by another group (39).
Comparative activities of the M1 vs. M4 hVDRs have been
investigated directly via cotransfection of expression plasmids for
these isoforms together with
1,25-(OH)2D3-responsive
reporter vectors. One such report (37) suggested a 1.7-fold greater
activity of F/M4 over f/M1 hVDR in transfected HeLa cells. We observe
(Fig. 9
) that the F/M4 hVDR isoform is 1.5- to 2.5-fold more
transcriptionally active than the f/M1 protein, with the most marked
difference resulting when the isoforms are assayed in osteoblast-like
cells employing a VDRE in the setting of its natural, bone-specific
promoter. However, in another study (56), the activities of hVDRs were
comparable, both in transfected COS-7 cells employing reporter gene
constructs and utilizing Northern blot analysis of vitamin D
24-hydroxylase mRNA induction by
1,25-(OH)2D3 in human
fibroblasts. The disparate results observed between Gross et
al. (56) and our present report or that of Arai et al.
(37) may be because of differences between the reporter constructs used
in each study. While we employed both a synthetic rat osteocalcin VDRE
and a construct containing the natural promoter region from the
osteocalcin gene, Gross et al. (56) used a synthetic human
osteocalcin reporter and a construct that contained a large portion of
the vitamin D 24-hydroxylase promoter. Importantly, the negative study
of Gross et al. (56), and the earlier work by Arai et
al. (37) demonstrating a functional difference between M1 and M4
hVDR, were both conducted using transient transfection experiments with
a single cell line. In the present study, the functional variance
between M1 and M4 hVDR was derived from experiments in four different
cell lines, including an osteoblast-like osteosarcoma line (ROS 2/3).
In addition, we have provided a functional linkage between the variance
in M1/M4 hVDR activity and the differential interaction of these two
isoforms with TFIIB, an observation that is consistent with the
proximity of the hVDR polymorphic N terminus and the novel TFIIB
interaction domain localized within the N-terminal segment of hVDR
described in the present study. Finally, recent investigations in our
laboratory have found that the F/M4 VDR is more transcriptionally
active than the f/M1 receptor by analyzing endogenous VDR function in
17 different human fibroblast lines with varying genotypes at the
FokI locus but a constant genotype in terms of relevant VDR
polymorphisms in the 3'-UTR (G. K. Whitfield, L. S. Remus,
P. W. Jurutka, H. Zitzer, A. K. Oza, H. T. L. Dang, C. A.
Haussler, M. A. Galligan, M. L. Thatcher, and M. R. Haussler,
unpublished data). Therefore, we conclude that the F/M4 neomorph
represents a more transcriptionally potent VDR isoform.
Integrative Model for Transactivation by Polymorphic hVDRs
Figure 10
represents a schematic working model for transcriptional
control by 1,25-(OH)2D3
that incorporates the new findings presented in this manuscript. The
model highlights a novel role for conserved basic residues in the
N-terminal region of VDR for contacting TFIIB and provides an
explanation for the differential effect of the polymorphic N terminus
on both the interaction of the receptor with this basal transcription
factor and on the activity of VDR. Panel A (far left)
depicts the shorter 424 amino acid F/M4 hVDR in association with TFIIB
at two sites within the receptor (the three molecules of TFIIB do not
imply that this number of TFIIB molecules interact with one hVDR but
rather are meant to indicate the difference in the level of interaction
between TFIIB and M4 vs. M1 hVDR). Site I is localized in
the C-terminal hormone-binding region of hVDR (30, 34) while site II
represents the N-terminal domain identified in the present results,
specifically conserved basic residues Arg-18 and Arg-22 (denoted by
++). Also associated with VDR is a corepressor (CoR), likely SMRT
(signal mediator and repressor of transcription), which is
thought to contact the receptor in the C-terminal ligand-binding domain
(57). The VDR-TFIIB complex is loosely bound to DNA via the VDR DNA
binding zinc fingers, but the regulated gene is illustrated in the
repressed state because of chromatin nucleosome structure (shown
schematically as an association of DNA and histones).
Upon binding to the
1,25-(OH)2D3 ligand (panel
A, middle), the M4 VDR is postulated to undergo a
conformational change with the following consequences: 1) release of
TFIIB from site I (34), 2) release of the corepressor and concomitant
association of VDR with the RXR heteropartner (11, 45, 57) and a
coactivator/HAT protein (CoA), the latter interaction being facilitated
by the AF-2 of VDR (18, 19, 20, 58), and 3) derepression of the target gene
by coactivator/HAT-mediated chromatin nucleosome reorganization
(schematically depicted as a release of the histones). As a result of
this derepressive effect, the active RXR-VDR-TFIIB complex can now
associate with high-affinity VDRE binding sites located in the promoter
region of the regulated gene (panel A, right) and
"deliver" TFIIB to the preinitiation complex (PIC), resulting in
stabilization of the PIC followed by subsequent rounds of RNA
polymerase II-directed transcription of the downstream target gene. It
is conceivable that the TFIIB delivery process is also facilitated by a
VDRE (DNA)-induced change in the conformation of the N-terminal domain
of VDR, thereby releasing TFIIB as the rate-limiting factor in the
formation of the PIC.
Panel B depicts a similar mechanism of action for the longer 427-amino
acid M1 hVDR. However, this isoform of VDR does not interact with TFIIB
as well as M4 hVDR (schematically denoted as only two molecules of
TFIIB), presumably because of the presence of a negatively charged
glutamate (minus sign enclosed by circle) localized within
the three amino acid N-terminal extension of M1 hVDR. We speculate that
the molecular mechanism whereby the negatively charged Glu-2 residue
attenuates TFIIB binding could involve either an intermolecular
repulsion between f/M1 hVDR and presumed negative residues in TFIIB
that bridge to VDR Arg-22/18, or a nonproductive intramolecular
interaction of Glu-2 in f/M1 hVDR with the Arg-22/18 TFIIB site II,
thus precluding TFIIB contact. Regardless, the hypothesized net result
is that the M1 isoform delivers less TFIIB to the PIC, with a
subsequently reduced amount of transcriptional initiation and mRNA
synthesis from the target gene. Experimentally, the activity of M1 hVDR
can be raised to the level of the more potent M4 receptor either by 1)
overexpression of TFIIB to boost the endogenous levels of this protein
and thus overcome the lower affinity for hVDR and the resulting
diminished local concentrations that are delivered by M1 hVDR (Fig. 9
),
or 2) by "neutralization" of the glutamic acid residue at position
2 in M1 hVDR via mutagenesis to an alanine (Fig. 9D
).
In summary, we have elucidated a novel domain in hVDR, located near the
N terminus and adjacent to the DNA-binding zinc finger motif, that is
required for
1,25-(OH)2D3-elicited
transcriptional activity. Within this region, two basic residues,
Arg-18 and Arg-22, were identified as critical for transactivation and
contact with the basal transcription factor IIB. A polymorphic variant
of hVDR that encodes a shorter, 424-amino acid protein (F/M4), which
has been associated with enhanced BMD in diverse populations, is more
transcriptionally active and is shown herein to associate more avidly
with TFIIB compared with the 427-amino acid f/M1 isoform. Given the
central role of
1,25-(OH)2D3 in calcium and
mineral homeostasis, the varying potency of interaction between
polymorphic hVDRs and components of the basal transcriptional machinery
is likely one of several factors contributing to a genetic
predisposition to osteoporosis.
 |
MATERIALS AND METHODS
|
---|
Construction of Mutant hVDR Plasmids
The hVDR expression vector, pSG5-hVDR (59), was employed in
synthesizing point mutants by in vitro site-directed
mutagenesis. Alteration of specific residues, deletions, as well as
truncations of hVDR, were created via the method outlined in the
Chameleon Double-Stranded, Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA) employing double-stranded hVDR
cDNA [which contains amino acids 4427, designated M4; see Baker
et al. (44)]. The mutations generated in M4 hVDR for this
study include the following point mutations: R18A, R22A, and R22K and
internal deletions
513,
423,
1517, and
1821.
Truncations produced were
161427 (which contains amino acids
4160),
134427 (amino acids 4133),
115427 (amino acids
4114),
84427 (amino acids 483), and
188 (amino acids
89427). Finally, one insertional mutation was created using M4 hVDR
(amino acids 4427) cDNA to include all 427 residues of the f hVDR
(designated M1 or full-length hVDR). M1 hVDR cDNA was then used to
construct E2A, a point mutant with alanine in place of glutamic acid at
position 2.
Transfection of Cultured Cells and Transcriptional Activation
Assay
COS-7 monkey kidney epithelial cells (800,000 cells per 60-mm
plate) were transfected with 0.1 µg of WT or mutant pSG5-hVDR
expression plasmid (59) and 10 µg of a reporter plasmid
([CT4]4-TKGH) containing four copies of the rat
osteocalcin VDRE (16) inserted upstream of the viral thymidine kinase
promoter-GH reporter gene (Nichols Institute Diagnostics,
San Juan Capistrano, CA) by the calcium phosphate-DNA coprecipitation
method as described previously (60). The pTZ18U plasmid was used as
carrier DNA, and each transfection contained a constant amount of total
DNA (20 µg). In TFIIB "rescue" experiments, an additional 0.2
µg of pSG5-human (h)TFIIB vector was cotransfected into the cells.
Sixteen hours later, the transfected cells were washed, then refed in
DMEM (Life Technologies, Gaithersburg, MD) supplemented
with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and
various concentrations of
1,25-(OH)2D3 in ethanol
vehicle. After 24 h of incubation at 37 C, the level of GH
secreted into the culture medium was assessed by RIA using a commercial
kit (Nichols Institute Diagnostics) according to the
manufacturers protocol. Treatment of HeLa cells was carried out in a
similar manner except that they were transfected with 1.5 µg of
pSG5-hVDR (or mutant) expression plasmid and, when indicated, 1.5 µg
of pSG5-hTFIIB. These cells were cultured in MEM supplemented with 10%
FBS and antibiotics. In some experiments a rat osteoblast-like
osteosarcoma cell line, ROS 2/3 (61), which contains only 100 VDR
molecules per cell (62, 63), was employed. The ROS 2/3 cells were
transfected with 1.0 µg of WT or mutant pSG5-hVDR in the absence or
presence of 1.0 µg pSG5-hTFIIB. The cells were later washed and then
refed in DMEM and Hams F-12 (DMEM/F12, 1:1) supplemented with 10%
FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and various
concentrations of
1,25-(OH)2D3 in ethanol
vehicle.
Preparation of Cellular Extracts and Immunoblotting
Transfected COS-7 cells (as described above) were lysed
directly in loading buffer (2% SDS, 5% ß-mercaptoethanol, 125
mM Tris-HCl, pH 6.8, and 20% glycerol), and 40 µg of
cellular protein were run on 515% gradient SDS/polyacrylamide gels.
After electrophoretic fractionation, proteins were electrotransferred
to Immobilon-P membranes (Millipore Corp., Bedford, MA)
using a Transblot apparatus (Bio-Rad Laboratories, Inc.
Richmond, CA) in 25 mM Tris-HCl, pH 7.4, 192 mM
glycine, 0.01% SDS, and 20% methanol. The membrane was then blocked
by incubation for 3 h with 3% blotto (3% dry milk, 10
mM Tris-HCl, pH 7.5, 150 mM NaCl).
Immunodetection of bound hVDR or hTFIIB proteins was then performed
using the monoclonal anti-VDR antibody, 9A7
(64) or an anti-TFIIB
polyclonal antibody (SI-1; Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA). After the first antibody treatment, the Immobilon-P
membrane was washed and treated at room temperature for 1 h with
goat antirat IgG conjugated to biotin followed by four 15-min washes. A
5-ml mixture of biotinylated alkaline phosphatase and neutravidin
(Pierce Chemical Co., Rockford, IL; in a ratio of 1:4) was
preincubated for 45 min at 22 C in 1% blotto. The mixture was diluted
to 30 ml with 1% blotto and added to the membrane for a 2-h incubation
with rocking at room temperature and then was washed four more times,
followed by a fifth wash with biotin blot buffer (0.1 M
Tris-HCl, pH 9.5, 0.1 M NaCl, 2 mM
MgCl2, 0.05% Triton X-100). Finally, the blot
was exposed to color reagent containing 50 µg/ml of
5-bromo-4-chloro-3-indolyl-phosphate and 100 µg/ml of 4-nitroblue
tetrazolium chloride. The color reaction was stopped by washing with
distilled water.
Preparation of Cellular Extracts and Gel Mobility Shift
Assays
The hVDR proteins used for gel mobility shift assays were
obtained from whole-cell extracts of COS-7 cells transfected with WT or
mutant pSG5-hVDR plasmids. The transfected cells were washed and then
refed in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100
µg/ml streptomycin but in the absence of
1,25-(OH)2D3. After 24
h of incubation at 37 C, the cells were washed twice with PBS (136
mM NaCl, 26 mM KCl, 8 mM
Na2HPO4, 1.5 mM
KH2PO4, pH 7.2), and
scraped into 200 µl of KETZD-0.3 buffer (10 mM Tris-HCl,
pH 7.6, 1 mM EDTA, 0.3 mM
ZnCl2, 0.3 M KCl, 10% glycerol, 1
mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, 15 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml
pepstatin A). After sonication, samples were centrifuged at 16,000
x g for 15 min at 4 C and the hVDR-containing supernatant
was used in electrophoretic mobility shift assays as described
previously (45, 65). Briefly, 5 µl of transfected COS-7 cell lysate
were incubated with 10-7 M
1,25-(OH)2D3 in DNA-binding
buffer [10 mM Tris-HCI, pH 7.6, 150
mM KCI, 1 mg/ml acetylated BSA, 50 µg/ml
poly(deoxyinosinic-deoxycytidylic acid)] for 30 min at 22 C followed
by the addition of 0.5 ng of 32P-labeled rat
osteocalcin VDRE
(5'-AGCTGCACTGGGTGAATGAGGACATTACA-3';
half-sites comprising an imperfect direct repeat are
underlined) and incubation for another 20 min.
Electrophoresis and autoradiography conditions were as described
elsewhere (66).
GST Coprecipitation and Coimmunoprecipitation Assays
Human transcription factor IIB (hTFIIB)-GST fusion protein
was expressed from pGEX-2T-hTFIIB (49), and GST alone was expressed
from pGEX-4T, both in Escherichia coli strain DH5
. The
overexpressed proteins were coupled to glutathione Sepharose (1.0 µg
protein/µl resin) according to the protocol of the manufacturer
(Pharmacia Biotech, Uppsala, Sweden) and stored as a 50%
slurry in KETZD-0.3 buffer (0.3 M KCl) containing 30%
glycerol at -20 C. For the GST pull-down assays, the desired cDNAs for
WT or mutant hVDRs or human RXR
were used to generate
[35S]methionine-labeled proteins, utilizing the
TNT Coupled Reticulocyte Lysate kit, an in vitro
transcription/translation system (Promega Corp., Madison,
WI). The GST or GST-TFIIB Sepharose beads (20 µl each) were incubated
in KETZD-0.15 buffer containing 0.1% Tween-20, 150
mM KCl, 1 mg/ml BSA, and the following protease
inhibitors: aprotinin, leupeptin, pefabloc SC, and pepstatin (wash
buffer) at 4 C for 1 h on a rocking platform. The desired
35S-labeled protein(s) was then incubated with
the beads in the absence or presence of
1,25-(OH)2D3
(10-6 M). Next, the
unbound proteins were washed from the beads four times with 1 ml each
of wash (KETZD-0.15) buffer. The bound proteins were extracted from the
beads into 40 µl loading buffer, boiled for 3 min and separated by
gradient (520%) SDS-PAGE, and visualized via autoradiography. The
amount of extract analyzed as input was 5% of the amount used in the
coprecipitation reactions. For coimmunoprecipitation assays, TFIIB and
WT or mutant hVDRs were overexpressed in COS-7 cells (as described
above), followed by preparation of cellular extracts in KETZD-0.3
buffer employing sonication. The lysates were incubated with 2 µg of
anti-TFIIB polyclonal antibody and 25 µl of Protein A/G-Plus Agarose
(Santa Cruz Biotechnology, Inc.) for 2 h at 4 C. The
immunoprecipitates were then washed extensively in wash buffer and
resuspended in 50 µl loading buffer, followed by immunodetection of
TFIIB-bound VDRs (as described above).
Genotyping of Human Fibroblasts
DNA was prepared from cultured human fibroblasts
(107 cells) using the QIAmp tissue kit
(QIAGEN, Valencia, CA) according to the manufacturers
instructions. The isolated genomic DNA (500 ng) was dissolved in a
total volume of 50 µl that also included 100 ng each of primer 2a and
2b (36), 5 µl of 10x buffer (Perkin-Elmer Corp.,
Norwalk, CT) with 1.5 mM MgCl2 and
2.5 mM each of dATP, dCTP, dTTP, and dGTP and 0.25 ml
Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN). PCR conditions were 10 cycles
at 94 C for 30 sec, 60 C for 60 sec (with -0.1 C/cycle) and 72 C for
60 sec. This was followed by 25 cycles at 94 C for 30 sec, 59 C for 60
sec, and 72 C for 60 sec. Approximately 200 ng of unpurified PCR
product were then incubated with 1 µl FokI enzyme
(New England Biolabs, Inc., Beverly, MA) and 1 µl 10x
buffer in a total volume of 10 µl for 1.5 h at 37 C. The
digestion mixture was then electrophoresed on a 4% NuSieve (3:1)
agarose gel in Tris-borate-EDTA buffer and analyzed as described
previously (36).
 |
ACKNOWLEDGMENTS
|
---|
We thank Milan Uskokovíc of Hoffmann-LaRoche Inc. for kindly providing us with 1,25-dihydroxyvitamin
D3 for our studies. We are grateful to Keiko
Ozato and Jorge Blanco for supplying the GST-TFIIB vector used in this
study. We also acknowledge Michelle Thatcher and Sanford Selznick for
their technical expertise.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Mark R. Haussler, Ph.D., Department of Biochemistry, College of Medicine, University of Arizona, Tucson, Arizona 85724.
This work was supported by NIH Grants (AR-15781 and DK-33351) to
M.R.H.
Received for publication September 18, 1999.
Revision received December 6, 1999.
Accepted for publication December 15, 1999.
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