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Department of Biochemistry College of Medicine University of Arizona Tucson, Arizona 85724
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. These mutants included truncations of
the hormone-binding (
161–427, 134–427, 115–427, 84–427)
or DNA-binding (1–88) domains, as well as smaller internal
deletions in the N-terminal region of VDR (5–13, 14–23,
15–17, and 18–21). 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 1–427) 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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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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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 (1–88) 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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5–13 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 14–23 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 15–17 displaying enhanced (Fig. 4B, lane 8)
and 18–21 (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 18–23 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 5–8). 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 5–13 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 5–13 is
normalized to differences in expression of this mutant (input panel,
right), 5–13 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
14–23 and 18–21 deletants that displayed little interaction
with TFIIB (Fig. 4, A and B) are moderately (18–21) or severely
(14–23) impaired in their ability to activate transcription
compared with the WT control. Interestingly, the 5–13 and, to a
lesser extent, the 15–17 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, A–C), 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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) 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 (5–13) is slightly less expressed than the WT hVDR (Fig. 6A, compare lanes 1 and 2), while a transcriptionally inactive mutant
(14–23) 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 1–3), 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 4–6 vs. 1–3). Importantly, overexpression of
TFIIB does not affect the expression of WT or mutant hVDRs (Fig. 6B, lanes 4–6).
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, 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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, 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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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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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 257–355 (30). More recently, utilizing an alternative strategy,
the hormone-binding domain (hVDR residues 93–427) 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 1–92) 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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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 (1–21) 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 23–27 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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5–13, 4–23, 15–17, and 18–21.
Truncations produced were 161–427 (which contains amino acids
4–160), 134–427 (amino acids 4–133), 115–427 (amino acids
4–114), 84–427 (amino acids 4–83), and 1–88 (amino acids
89–427). Finally, one insertional mutation was created using M4 hVDR
(amino acids 4–427) 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
manufacturer’s 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 Ham’s 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 5–15% 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 (5–20%) 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 manufacturer’s
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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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R. Metivier, G. Penot, G. Flouriot, and F. Pakdel Synergism Between ER{alpha} Transactivation Function 1 (AF-1) and AF-2 Mediated by Steroid Receptor Coactivator Protein-1: Requirement for the AF-1 {alpha}-Helical Core and for a Direct Interaction Between the N- and C-Terminal Domains Mol. Endocrinol., November 1, 2001; 15(11): 1953 - 1970. [Abstract] [Full Text] [PDF]
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K. L. Sunn, T.-A. Cock, L. A. Crofts, J. A. Eisman, and E. M. Gardiner Novel N-Terminal Variant of Human VDR Mol. Endocrinol., September 1, 2001; 15(9): 1599 - 1609. [Abstract] [Full Text] [PDF]
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 A. L. Boskey Musculoskeletal Disorders and Orthopedic Conditions JAMA, February 7, 2001; 285(5): 619 - 623. [Abstract] [Full Text] [PDF]
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S. A. Gardezi, C. Nguyen, P. J. Malloy, G. H. Posner, D. Feldman, and S. Peleg A Rationale for Treatment of Hereditary Vitamin D-resistant Rickets with Analogs of 1alpha ,25-Dihydroxyvitamin D3 J. Biol. Chem., July 27, 2001; 276(31): 29148 - 29156. [Abstract] [Full Text] [PDF]
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