Download A 55-Kilodalton Accessory Factor Facilitates Vitamin D Receptor

A 55-Kilodalton Accessory Factor
Facilitates Vitamin D Receptor DNA
Binding
Teruki Sone, Keiichi Ozono, and J. Wesley Pike*
Departments of Pediatrics (T.S., K.O., J.W.P.) and Cell Biology
(J.W.P.)
Baylor College of Medicine
Houston, Texas 77030
The interaction of the vitamin D receptor with a
vitamin D-responsive element (VDRE) derived from
the human osteocalcin promoter in vitro has been
shown to require a nuclear accessory factor (NAF)
derived from monkey kidney cells. In this report we
show that this factor is widely distributed in cells
and tissues, including those that do not express the
vitamin D receptor (VDR). NAF is required for VDR
binding to a variety of known VDREs. VDR and NAF
independently bind the VDRE weakly, as assessed
by elution profiles generated during VDRE affinity
chromatography. Together, however, both proteins
coelute from this column with a profile that indicates
a tighter strength of interaction. Analogous chromatography of the VDR derived from ROS 17/2.8
cells treated with 1,25-dihydroxyvitamin D3 in culture
also reveals a dual profile of weak and strong binding, suggesting that in vivo modifications are unlikely
to alter receptor DNA binding. NAF is a protein of 55
kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cross-linking
experiments suggest that the VDR and NAF together
form a heterodimer on a single VDRE with a mol wt
of 103 kDa. These data demonstrate that NAF is
required for VDR binding to specific DNA in vitro and
suggest the possibility that NAF may be required for
the transactivation capability of the VDR in vivo.
(Molecular Endocrinology 5: 1578-1586,1991)
INTRODUCTION
Transcriptional regulation of gene expression is mediated by a family of ligand-activated nuclear receptors
that include those for the steroid, thyroid, and retinoic
acid hormones (1-3). These receptors are DNA-binding
proteins that interact in a sequence-specific manner
with c/s-acting elements located in or near hormonesensitive promoters (3). The DNA sequence motifs that
mediate receptor response are generally comprised of
0888-8809/91/1578-1586$03.00/0
Molecular Endocrinology
Copyright © 1991 by The Endocrine Society
1578
direct or inverted repeats, such as those found in the
tyrosine aminotransferase (4), vitellogenin (5), laminin
B1 (6), GH (7, 8), and osteocalcin (9-12) genes. These
elements often mediate the action of a single hormone,
although within certain genes they may mediate the
action of several hormones whose receptors exhibit
related sequence specificity. Thus, for example, the
progesterone and glucocorticoid receptors recognize
identical responsive elements (13, 14), and thyroid,
retinoic acid and vitamin D receptors (VDR) also recognize very similar elements (6-12, 15-17). The hormonal specificity for transactivation demonstrated by
certain genes clearly suggests that determinants other
than the nucleotide sequence of the receptor-binding
site may be required. These determinants may include
half-site spacing, chromatin structure, receptor expression, and, perhaps most importantly, expression of
additional protein factors that participate in the activation process.
The DNA-binding domain of the steroid receptors is
comprised of two potential loop structures, each folded
about a single zinc atom (18). The three-dimensional
solution structure of this domain in the estrogen (19)
and glucocorticoid (20) receptors suggests that the
carboxyl side of each loop contains an a-helix. The first
helix is postulated to lie in the major groove and confer
specificity, whereas the second stabilizes the complex
(21). Certain of the receptors appear to bind to responsive elements as cooperative dimers (22-24). The highest affinity DNA binding interaction of the receptor,
however, requires the DNA-binding domain as well as
a separate and distinct domain located within the carboxy-terminal region (25-27). While the structure of this
domain remains largely uncharacterized, it is clear that
this domain participates not only in homodimer formation (26), but may also mediate formation of dimers
comprised of heterologous receptors (25, 27) or dimers
comprised of a specific receptor and unknown nuclear
proteins (28-34). Examples include retinoic acid-thyroid
receptor heterodimers (25, 30), heterodimers composed of the thyroid receptor and a protein designated
TRAP (32-34), and heterodimers between the retinoic
acid receptor and several different proteins that appear
to be expressed in a tissue-specific manner (31). In
VDR-NAF
each situation these proteins facilitate DNA binding of
their respective receptor partners. While the formation
of heterodimers may well increase the diversity and
complexity with which cells can respond to hormone,
the physiological relevance of these protein factors
remains unknown.
The VDR is a member of the steroid receptor family
and mediates the genomic action of 1,25-dihydroxyvitarnin D3 [1,25-(OH)2D3] (1-3,35). Vitamin D-responsive
elements (VDRE) have been identified in the human (9)
and rat (10-12) osteocalcin (OC) genes and mouse
osteopontin (15) gene. Activation of the human OC
promoter requires the presence of a functional receptor
(36). The VDR has been shown to bind to each of these
response elements in vitro (10,12,15,37,38), although
the details of this binding are unknown. Recently, we
demonstrated that the interaction of the VDR with the
human OC VDRE requires the presence of a mammalian cell protein factor that we termed nuclear accessory
factor (NAF) (39). In this report we further characterize
the distribution and properties of this protein factor that
facilitates strong binding of the VDR to OC VDRE DNA
in vitro.
1579
VDR Requires a Mammalian
Cell Nuclear Factor
for VDRE Binding
VDR-
RESULTS
Cellular Distribution of NAF
We used a bandshift enhancement assay to identify the
presence of NAF activity in cellular extacts. As seen in
Fig. 1, while cellular extracts containing this activity do
not bind to a VDRE probe in the absence of VDR, the
addition of this protein, either from crude yeast cytosols
or as purified VDR, leads to the clear demonstration of
a protein-DNA complex. As with NAF alone, yeast VDR
independently does not generate a complex. These
experiments indicate that while both proteins together
generate a DNA complex of sufficient affinity for identification during bandshift analysis, neither protein independently forms equivalent complexes. We used this
assay to determine the presence of NAF or NAF-related
activity in the cultured cells and mouse tissues indicated
in Table 1. Clearly, this protein or one functionally
related to NAF is widely distributed. Although VDR-NAF
complexes comigrate independent of cell source, we
cannot conclude that NAF is identical, as small differences in mol wt are unlikely to be detected in the
bandshift assay. The fact that NAF is expressed in
mouse liver and in cells lines such as CV-1, neither of
which contains the VDR, suggests that the protein is
not coexpressed with the VDR.
Requirement for NAF on VDREs
VDR derived from tissue nuclear extracts has been
demonstrated to bind VDREs in the rat OC and mouse
osteopontin genes (10,15). We evaluated the requirement for NAF in VDR binding to these labeled elements
by incubating the VDR with or without extracts contain-
12
3 4
Fig. 1. Bandshift Enhancement Assay for VDR and NAF
Cellular extracts were incubated with VDR DNA probe and
then electrophoresed as described in Materials and Methods.
Lane 1, pAVhVDR-transfected COS-1 cell nuclear extract (1
ng protein). Lane 2, COS-1 cell nuclear extract (1 ng protein).
Lane 3, VDR-expressing yeast cytosol (0.1 ng protein). Lane
4, VDR-expressing yeast cytosol (0.1 ^g protein) and COS-1
cell nuclear extract (1 ^g protein).
Table 1. Cellular and Tissue Distribution of NAF or NAFRelated Factor
Mouse tissues
Liver, kidney
Cultured cells
Kidney fibroblasts (CV-1, COS-1)
Hepatoma (HepG2)
Cervical carcinoma (HeLa)
Breast cancer (T47Dco)
Osteosarcoma (ROS 17/2.8)
Calvaria(MC3T3-E1)
Fibroblasts (human primary)
Lymphoblasts (human, Epstein-Barr virus-transformed)
Spodoptera fugiperda (insect, Sf9)
ing NAF and resolving the protein-DNA complexes by
bandshift assay. As observed in Table 2, NAF was
required for binding of the VDR to all of these elements,
suggesting a generalized requirement for this protein.
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MOL ENDO-1991
1580
Table 2. Requirement of NAF for VDR Interaction with
VDREs
Sequence
H
NAF
Required
GGGGCA-3'
AGGACA-3'
GGTTCA-3'
AGTTCA-3'
+
+
+
+
Element
H
Human OC
Rat OC
Mouse OP
Mouse |8RAR
5'-GGGTGA ACG
5'-GGGTGA ATG
5'-GGTTCA
CGA
5'-GGTTCA CCGAA
Sequence is indicated where the half-sites are designated H
and the spacing nucleotides are designated S.
NAF was probably present in the protein-DNA complexes previously reported (10, 15). Interestingly, VDR
also binds to the mouse retinoic acid receptor /3 gene
retinoic acid response element (RARE) in the presence
of NAF despite the fact that this gene is not activated
by vitamin D (16). We do not find this in vitro interaction
of the VDR surprising in view of the distinct homology
observed between this RARE and that of the VDREs,
particularly with the element found within the osteopontin gene (15). In the latter case, the nucleotide spacing
between the two GGTTCA half-sites [3 basepairs (bp)
in the VDREs and 5 bp in the RARE] represents the
only fundamental different between the two elements.
Interaction of VDR and NAF with VDRE Affinity
Resins
We examined the binding properties of the VDR and
NAF further through VDRE affinity chromatography. In
contrast to the results obtained by bandshift analysis,
the yeast VDR alone was capable of weakly binding
concatemerized human osteocalcin VDREs, binding under low salt conditions, and eluting during a linear
gradient at 0.13 M KCI (Fig. 2A). NAF was equally
capable of a similar weak interaction on the VDRE when
extracts were chromatographed and aliquots of each
fraction were subjected to bandshift analysis in the
presence of added VDR (Fig. 2B). In contrast, chromatography of a combination of both yeast VDR and
nuclear extracts containing NAF leads to the elution of
two peaks of VDR activity, one at 0.13 M and a stronger
interaction at 0.26 M (Fig. 2C). Bandshift assay of
individual fractions for NAF activity clearly revealed that
NAF activity was associated only with the high affinity
peak (Fig. 2D). These results suggest that both proteins
independently bind VDRE DNA. Together, however,
they coelute from the VDRE affinity column, and their
desorption requires higher ionic strength.
receptor elution. 1,25-(OH)2D3 is capable of regulating
the phosphorylation state of the VDR when added to
intact cultured cells (40, 41), and this modification as
well as the capacity of the hormone to up-regulate the
receptor (42) might feasibly affect DNA binding. As
observed in Fig. 3, both weak and strong interactions
were evident upon chromatographic analysis, and their
elution profiles were not significantly different from
those identified when the VDR was labeled in vitro. The
relative distribution of both peaks was somewhat similar, and bandshift analysis indicated that the high affinity
complex comigrates with the known VDR-NAF complex
(data not shown). The results of this experiment suggest that formation of the VDR-hormone complex in
culture does not substantially alter its capacity to interact with the VDRE independently, and that the VDRNAF complex does not demonstrate an interaction different from that observed after in vitro VDR labeling.
These data also suggest that NAF activity is insufficent
in these experiments to saturate endogenous levels of
the VDR. Thus, the protein probably exists in concentrations that correspond to those of normal VDR.
Physical Properties of NAF
Additional chromatographic analyses revealed that NAF
bound a variety of ion exchange and affinity resins,
including diethylaminoethyl Sephadex, phosphocellulose, calf thymus DNA-cellulose, and heparin-agarose
(data not shown). Moreover, the protein was eluted
from calf thymus DNA cellulose with p-chloromercuribenzene sulfonate, a property characteristic of the VDR
(43) as well as several of the other steroid receptors
(44, 45). We partially purified NAF from HeLa cell cytosols and then determined its molecular mass after
resolution by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE; see Materials and Methods). As observed in Fig. 4A, bandshift analysis of
protein fractions resolved by SDS-PAGE revealed a
precise molecular mass of 55 kDa. This size estimate
differed from that of the 48-kDa VDR simultaneously
determined by Western blot analysis of a parallel electrophoretic lane (Fig. 4B), suggesting that NAF is not
simply a modified form of the VDR. Importantly, the
capacity of renatured NAF to facilitate DNA binding of
exogenously added VDR in the absence of added ATP
also suggests that NAF is not a protein kinase. Finally,
the observation that the VDR-NAF-DNA complex derived from partially purified NAF comigrated during
bandshift analysis with unpurified material (Fig. 4A, lane
c) indicates that NAF was not proteolyzed during the
enrichment procedure.
Formation of 1,25-(OH)2D3-Receptor Complex in
Culture
VDR-NAF Cross-Linking
We treated confluent ROS 17/2.8 cells with radiolabeled 1,25-(OH)2D3 and chromatographed the resulting
1,25-(OH)2D3-receptor complex on the VDRE affinity
column to determined whether the formation of the
1,25-(OH)2D3-receptor complex in intact cells might alter
VDR was translated in vitro in the presence of [35S]
methionine and then incubated with VDRE DNA endfilled with biotinylated 11-dUTP. Precipitation of the
VDRE with immobilized streptavidin followed by SDSPAGE revealed the presence of a VDR protein predom-
VDR-NAF
1581
0.13
0.13
0.26
10
20
30
40
50
60
Relative Fraction Number
D.
0.13
20
30
40
50
Fraction Number
60
0.26
10
20
30
40
50
60
Relative Fraction Number
0.5
0.4
0.3
0.2
0.1
0
Fig,. 2. VDRE Affinity Chromatography of the VDR and NAF
Cellular extracts from COS-1 cells or yeast were incubated for 3 h at 4 C with or without 1,25-(OH)2-[3H]D3 (4 nM) and then
chromatographed on a VDRE-Sepharose column (5 ml) containing concatemerized VDRE oligonucleotide (25 Mg/ml). Samples were
applied in TD buffer containing 0.05 M KCI, washed, and then eluted with TD buffer containing a linear gradient of KCI. Aliquots
(0.5 ml) were quantitated for tritium by liquid scintillation spectrophotometry or asssessed for NAF activity by bandshift assay in
the presence of added yeast VDR. A, Chromatography of VDR-containing yeast cytosol (140 ng cytosol protein; 1 pmol VDR)
prelabeled for 3 h at 4 C with 1,25-(OH)2D3. B, Chromatography of COS-1 cell extracts (3 mg cytosol protein) and assessment of
relative NAF activity. The NAF-VDR-VDRE complex was excised and quantitated. C, Chromatography of prelabeled VDR-containing
yeast cytosol sample, as in A, premixed with nuclear extract (140 ^g protein) derived from nontransfected COS-1 cells, as in B. D,
Assessment of NAF activity in aliquots of the chromatography in C. VDR was added in excess to each aliquot.
0.14
0.26
20
30
40
Fraction Number
60
Fig. 3. Formation of 1,25-(OH)2D3-VDR Complexes in Culture
Does not Alter the VDR-NAF Profile
ROS 17/2.8 cells were incubated with 1,25-(OH)2D3 in culture, as described in Materials and Methods. Cytosol was
prepared, and 0.86 mg protein containing 0.94 pmol labeled
VDR was chromatographed on a VDRE affinity column, as
described in Fig. 2.
inantly when HeLa cell extracts were included in the
incubation (Fig. 5). Incubation with unprogrammed lysates did not result in precipitable 35S-labeled protein.
Most importantly, when these precipitates were sub-
jected to the cross-linking reagent bismaleimidohexane
(BMH), a VDR-related cross-linked band at 103 kDa
was evident (Fig. 5). This size is significantly greater
than that of a VDR homodimer at 96 kDa. Larger crosslinked species are also evident (J 20-135 kDa), which
may reflect either nonspecific interactions or additional
heterogeneous protein-protein interactions with the
VDR/NAF complex. This 103-kDa cross-linked complex
was similarly observed when VDR was incubated with
extracts of COS-1, ROS 17/2.8, and mouse fibroblasts
(data not shown). These observations are, therefore,
consistent with a molecular mass for NAF of approximately 55 kDa. More importantly, they suggest that the
interaction of the VDR on the OC VDRE probably
occurs as a heterodimer with NAF.
DISCUSSION
We have previously reported the existence of a trypsinand heat-sensitive component in mammalian cells which
facilitates the binding of the VDR to OC VDRE DNA
(39). In this report we describe several additional prop-
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MOL ENDO-1991
1582
A.
VDR/NAF
VDR
<Fxn7)
-VDR
46
1 2
3 4 5 6 7 8 9
Fraction
10 11 12
C
Fig. 4. Estimate of Molecular Mass of NAF after SDS-PAGE
A, HeLa cell-derived samples enriched for NAF were electrophoresed, transferred to Immobilon P, and evaluated for
NAF migration, as described in Materials and Methods. NAF
migrated in fraction 4 corresponding to a molecular mass of
54-56 kDa relative to standard proteins. As NAF does not
bandshift independently of the VDR, the protein/DNA band
designated NAF contains exogenously added VDR for assessment. Lane C represents migration of crude unenriched HeLa
cell extracts containing NAF combined with added yeast VDR.
Both unenriched and enriched NAF samples comigrate. B,
Western blot detection of the VDR. An unfractionated electrophoretic lane identical with regard to protein sample in A was
subjected to Western blot analysis using the anti-VDR monoclonal antibody 9A7. VDR migrated in fraction 7 (designated
VDR/Fxn 7) at 47-48 kDa relative to fractions in A.
erties of this nuclear protein(s). Its wide cellular and
tissue distribution is consistent with the almost ubiquitous expression of the VDR, although its expression in
certain cell types and in the liver indicates that it is not
coexpressed with the VDR and, therefore, may retain
an independent function. As such, its ubiquitous distribution may be analogous to certain proteins, such as
E12, which participates in heterodimer formation and
the function of MyoD (46). The concentration of NAF in
cells has not been measured. Nevertheless, its activity
is sufficient in cellular extracts to promote tight binding
of approximately 40-50% of endogenous receptor in
ROS cells after their labeling in situ with 1,25-(OH)2D3.
Thus, NAF may exist in concentrations sufficient to
influence VDR binding to specific DNA in vivo. The
protein in Hela cells appears to be approximately 55
kDa, as judged by SDS-PAGE. Furthermore, the capacity of the protein to promote VDR binding to VDRE in a
bandshift assay after SDS-PAGE and denaturation/
renaturation suggests that the protein is indeed a factor
that forms an association with the VDR rather than
modifying the protein.
Our studies demonstrate that while both VDR and
NAF bind weakly to VDRE DNA, together they bind to
the VDRE with a strength of interaction that exceeds
that of the individual proteins. Cross-linking experiments demonstrate that the two proteins can be coupled to form a heterodimer while bound to the VDRE.
The lack of ability of either VDR or NAF alone to bind
with similar high affinity suggests that neither is capable
24-
1 2
3 4
Lane
Fig. 5. Cross-Linking of VDR-NAF Heterodimers
[35S]Methionine-labeled VDR was prepared through in vitro
translation and then mixed with biotinylated VDRE oligonucleotide in the absence or presence of NAF. Samples were
incubated for 30 min at room temperature in bandshift buffer
containing 0.1 M KCI and 100 ng poly(dl-dC) and protein-DNA
complexes precipitated with streptavidin-agarose. The precipitate was washed with 20 mM Hepes-NaOH (pH 7.9), 50 mM
NaCI, and 20% glycerol. A sample containing NAF was treated
with 1 mM BMH for 10 min at room temperature, subjected to
SDS-PAGE, and fluorographed at - 7 0 C. Unprogrammed
lysate failed to produce precipitable 35S. VDR precipitated in
the presence (lane 1) and absence (lane 2) of NAF. NAF derived
from HeLa cell extracts in the presence (lane 3) and absence
(lane 4) of BMH is shown.
independently of an analogous strong homodimer interaction. Hypothetically, however, both proteins may interact independently on each half-site. The inability to
form strong homodimers, nevertheless, limits their affinity and perhaps residence time. In contrast, appropriate
alignment of a molecule of VDR and NAF on each halfsite of the two direct repeats leads to heterodimer
formation and, in turn, high affinity association. Alternatively, dimers may form independently of DNA and
then interact with high affinity on the VDRE. Importantly,
the labeling of endogenous VDR in ROS cells with 1,25(OH)2D3 also leads to a protein that interacts with NAF.
This receptor is likely to retain all of the appropriate
modifications that might occur in normal cells. Moreover, this experiment discounts the possibility that inappropriate or absent modification of VDR in yeast (or
after in vitro translation) prevents VDR homodimer formation, but not NAF heterodimer formation. We conclude from these data that the VDR and NAF preferentially form heterodimers in vitro and speculate that
these dimers may be fundamental to the regulation of
gene expression by 1,25-(OH)2D3 in vivo. The capacity
of the VDR to form heterodimers might well extend and
VDR-NAF
diversify the action of this transcription factor on cellular
processes that are regulated by the vitamin D hormone.
Protein factors that form heteromers with thyroid
hormone receptors (TR) have been identified recently
(28-34). In the case of the TR, this activity has been
found in a variety of cells, including GH3, 235 cells, F9,
and JEG3 cells (29). Consistent with our findings for
NAF, several of these factors are expressed independently of TR, suggesting that these factors may retain
independent functions. One such protein of 63 kDa has
been designated TR auxiliary protein (TRAP) (29, 3234). As with VDR and NAF, both TRAP and TR bind
directly to the GH element independently, although both
together interact in a much tighter fashion (32, 33).
Cross-linking studies support the formation of a TRAPTR heterodimer (34). The TR, however, can also form
independent homodimers on TREs (30), a finding apparently different from that for the VDR. TR interaction
with TRAP can be influenced by mutating specific regions within the complex TRE motif (33). These studies
indicate that certain of the TRE domains interact preferentially with TRAP, and others with the TR. Thus, the
relative orientation of each protein on repeated, but
dissimilar, elements may be important for binding interaction. This may be true for VDR and NAF on the
osteocalcin gene (12, 37), where the two half-sites are
nonidentical. It is unlikely to be true, however, for the
response element in the osteopontin gene, where the
two half-sites are the same (15). In the latter case,
unless flanking sequence contributes to preference, it
seems probable that orientation may be determined by
the proper alignment of the dimerization domains of
VDR and NAF.
TR deletion analysis suggests that dimer formation
is mediated by a region within the carboxy-terminal half
of the TR as well as through a region immediately
down-stream of the two zinc-binding fingers (32). In the
estrogen (ER) and glucocorticoid (GR) receptors, the
weak zinc finger dimerization motif is sufficient to mediate homodimer formation of the DNA-binding region
(22-24). However, small deletions in the distant Cterminal region also block DNA binding of the ER,
suggesting the presence of a separate dimerization
domain (26). The organization of this independent dimerization motif in TR and ER has been suggested (25,
30). It remains to be determined whether NAF interaction with VDR occurs through an analogous domain,
although these regions in the VDR show significant
homology to those in the TR (47).
Similar observations have been made for the retinoic
acid receptor (RAR). Addition of nuclear extracts from
a variety of cell types has revealed enhanced RAR
binding (31). Cross-linking studies indicate that these
binding factors are represented by several different
sized proteins that are expressed in an apparently cellspecific manner (31). As with the TR, dimerization appears principally a function of the carboxy-terminal portion of the RAR (30). Is NAF identical to the factors
being described for either the TR or RAR? On the basis
of a molecular mass of 55 kDa, it seems unlikely that
1583
NAF and TRAP are identical. In contrast, NAF is similar,
if not identical, in size to at least one of the factors in
HeLa cells observed to form dimers with RAR (31). It is
likely that resolution of the identity of NAF will come
through the molecular cloning of this factor.
We have described a generally ubiquitous protein
activity that forms a heterodimer with the VDR on
natural VDREs. This protein appears to serve the same
cooperative binding function with the VDR as do factors
that form complexes with the TR and RAR. If this
protein is essential for transcription activation by 1,25(OH)2D3, which remains to be demonstrated, it adds an
additional complexity to the mechanism by which the
vitamin D hormone functions to control gene expression.
MATERIALS AND METHODS
Plasmid Constructions
Recombinant human (h) VDR was expressed in mammalian
cells using the pAVhVDR expresssion vector, as previously
reported (36). The yeast hVDR expression vector YEpV1 was
used to produce the full-length hVDR in Saccharomyces cerevisiae (48).
Cell Lines
The cell lines investigated were cultured by standard techniques in the appropriate medium containing serum supplements as well as penicillin G (100 U/ml) and streptomycin (100
Mg/ml). Cell lines included COS-1, CV-1, ROS 17/2.8, HeLa,
HepG2, T47DCo, MC3T3-E1, primary human fibroblasts, Epstein-Barr-virus transformed human lymphoblasts, and SF9
insect cells. Suspension HeLa cells were grown in Spinner
cultures in S-minimal Eagle's Joklik Medium supplemented
with the above antibiotics and 5% horse serum. For mammalian cell VDR expression, COS-1 fibroblasts were plated in
100-mm dishes and transfected 24 h later with 20 ^g
pAVhVDR using diethylaminoethyl dextran methods (49).
The Saccharomyces cerevisiae cell strain BJ3505 (MATa
Pep4:HIS3 Prb1-A1.6R His3,Lys2-208,Trp1-A101 lira3"52) was
used as a recipient host for the hVDR cDNA derivatives of
YEp46 and transformed as previously described (48). YEpV1 transformed yeast were cultured in flasks and induced with
0.1 mM CuSO4 in the presence or absence of 1,25-(OH)2D3,
as previously described (38).
Cellular Extractions
Cultured mammalian cells were harvested from plates with
trypsin or by centrifugation and washed twice with PBS. The
cells were resuspended on ice in TD buffer [10 mM Tris-HCI
(pH 7.6) and 5 mM dithiothreitol], allowed to swell, and then
lysed with 20 strokes of a Dounce homogenizer (Kontes Co.,
Vineland, NJ). The samples were brought to a final concentration of 0.3 M KCI using a 3-M KCI stock prepared in 10 mM
Tris-HCI, pH 7.6, and then subjected to ultracentrifugation.
Aliquots of cytosol were stored frozen at - 7 0 C. Untransfected
HeLa cells and COS-1 cells were used to obtain NAF. Nuclear
extracts were prepared as previously described (50). These
preparations contained undetectable endogenous VDR compared to pAVhVDR-transfected COS-1 cells.
Yeast cells were cultured as described above, collected by
centrifugation, and washed three times in TD buffer. Cells
were lysed by glass bead disruption in the same buffer con-
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MOL ENDO-1991
1584
taining 0.3 M KCI, and cytosols were prepared as described
previously (38).
Chromatographic Enrichment of NAF and Estimates of
MolWt
Intact Cell Incubation with 1,25-(OH)2D3
NAF was enriched from crude HeLa cell cytosol through an
initial precipitation with crystalline ammonium sulfate at 40%
saturation. After precipitation, the protein pellets were dissolved in TD buffer and then adsorbed to calf thymus DNA
cellulose. The resin was washed with Tris-HCI, pH 7.6, and
0.05 M KCI, then eluted using 1 mM pCMBS in the above
buffer, a procedure that dissociates NAF from immobilized
DNA. After pCMBS elution, fractions containing NAF were
precipitated with ammonium sulfate at 40% saturation. The
precipitated sample was dissolved and further chromatographed on a VDRE affinity column, as outlined above. The
NAF-containing sample was mixed with excess highly purified
yeast-derived VDR (38) to promote tight VDRE binding by
NAF. The VDR-NAF peak that eluted at 0.26 M KCI was
precipitated with 6% trichloroacetic acid and then subjected
to SDS-PAGE. After electrophoresis, the resolved proteins
were transferred to an Immobilon-P (Millipore Corp., Bedford,
MA) membrane at 4 C. The membrane was then sectioned
into 2-mm segments (a preliminary experiment revealed that
NAF migrated between the protein standards BSA and ovalbumin), and the adsorbed proteins from each fraction were
eluted for 2 h at room temperature with 50 mM Tris-HCI (pH
7.9), 1 % Triton X-100, 2% SDS, 5 mM dithiothreitol, and 0.2
mg/ml BSA. The eluted proteins were precipitated with 80%
aqueous acetone, dissolved in 6 M guanidine-HCI containing
50 mM Tris-HCI (pH 7.9), 0.15 M NaCI, 20% glycerol, 5 mM
dithiothreitol, 0.5 mM EDTA, and 50 HM ZnCI2, and then
dialyzed overnight in TD buffer containing 20% glycerol and
0.1 M KCI. Individual fractions were assessed for NAF activity
through bandshift analysis in the presence of added yeast
VDR. An identical protein sample was electrophoresed in
parallel, transferred to Immobilon-P, and then subjected to
Western blot analysis using the anti-VDR monoclonal antibody
9A7, as previously described (38).
ROS 17/2.8 cells were plated onto 100-mm dishes in Ham's
F-12 medium containing 10% fetal calf serum and allowed to
grow to confluency. Confluent dishes of cells were washed
several times with serum-free medium and then incubated with
the same medium containing 10% fetal calf serum and 20 nM
1,25-(OH)2-[3H]D3 (90 Ci/mmol) overnight at 37 C. Cells were
harvested, washed with PBS, and then lysed in TD buffer
containing 0.3 M KCI using Dounce homogenization. Hydroxylapatite was used to assess 1,25-(OH)2D3-binding activity
(38). Cytosolic extract was chromatographed on a 5-ml VDRE
affinity column, as outlined below.
Gel Retardation Assay
Bandshift analysis was used to assess the capacity of the
VDR to specifically bind a human osteocalcin VDRE. A synthetic duplex oligonucleotide of the sequences 5'TTGGTGACTCACCGGGTGAACGGGGGCATT-3' (9, 37) was
labeled with [«-32P]dATP ( 1 - 5 x 1 0 8 cpm/^g) using a filling-in
reaction of H/ndlll restriction ends. We also examined the
interaction of the VDR with the rat osteocalcin VDRE (5'CTGGGTGAATGAGGACATT-3') (10-12), the mouse osteopontin VDRE (5'-AGGTTCACGAGGTTCACGTCT-3') (15),
and the RARE from the mouse RAR /3 gene (5'AAGGGTTCACCGAAAGTTCATC-3') (16). DNA probes were
isolated on an 8% polyacrylamide gel and stored at - 7 0 C.
Protein extracts were incubated with DNA probe in 20 n\ B
buffer [5 mM Tris-HCI (pH 7.9), 15 mM HEPES-NaOH (pH 7.9),
3.5 mM MgCI2, 5 mM EDTA, 10% glycerol, 0.1% Tween-20,
and 5 mM dithiothreitol] containing 100 mM KCI and 2 fig
poly(dl-dC) or as indicated in the figure legends. DNA-binding
species were resolved on a 5% polyacrylamide gel prepared
in Tris-glycine buffer at 30 mAmp constant current, as outlined
previously (38, 39). Gels were dried and then subjected to
autoradiography for 3-16 h at - 7 0 C with intensifying screens.
Appropriate regions of the gel were excised and 32P quantitated by liquid scintillation spectrophotometry.
General and VDRE DNA Affinity Chromatography
We prepared human osteocalcin VDRE affinity resins by methods described by Kadonaga and Tjian (51). Synthetic complementary oligonucleotides were annealed, subjected to 5'phosphorylation, and then concatemerized in reactions using
DNA ligase. Low specific activity [Y-32P]dATP was included in
the reaction to determine DNA uptake. The concatemerized
DNA was incubated with CNBr-activated Sepharose 4B overnight at room temperature with gentle mixing. The resins were
briefly incubated with ethanolamine and then washed and
equilibrated in TD buffer containing 0.05 M KCI. Quantitation
of an aliquot of the prepared resin indicated 25-50% uptake
of DNA. Mammalian cell or yeast cytosols were prelabeled
with 1,25-(OH)2-[3H]D3 for 3 h at 4 C to form 1,25-(OH)2D3VDR complexes. Samples were applied to the columns after
dilution with TD buffer to a final salt concentration of approximately 0.05 M KCI (determined by conductivity), washed, and
then eluted with a linear KCI gradient prepared in TD buffer.
Radiolabeled 1,25-(OH)2D3 was estimated by liquid scintillation
spectrophotometry from an aliquot of each fraction. Chromatography on DEAE-Sephadex, heparin-Sepharose, phosphocellulose, and calf thymus DNA cellulose was carried out as
outlined previously (52). Selective elution of NAF from calf
thymus DNA cellulose with 1 mM p-chloromercuribenezene
sulfonate (pCMBS) was carried out as previously described
(38).
Cross-linking the VDR and NAF
VDR mRNA-programmed reticulocyte lysates were prepared
as previously described (39). Precipitation and cross-linking
studies were carried out using the method of Glass and coworkers (27). VDR-containing lysates were incubated with
HeLa cell extracts and 0.5 pmol biotinylated VDRE in B buffer
containing 0.1 M KCI and 100 Mg/ml poly(dl-dC) for 30-min
incubation at room temperature. Streptavidin-agarose (20 fi\
of a 50% slurry in B buffer containing 0.1 M KCI) was added
for an additional 1 h at 4 C. Bound protein-DNA complexes
were extensively washed and then resuspended in 20 mM
HEPES-NaOH (pH 7.9), 50 mM NaCI, 20% glycerol, and 1 mM
bismaleimidohexane for 10 min at room temperature. The
reaction was stopped with /3-mercaptoethanol, and the protein
products were subsequently analyzed by SDS-PAGE. Gels
were treated with EN3HANCE (New England Nuclear, Boston, MA) and fluorographed at - 7 0 C.
Reagents
Modifying and restriction enzymes were obtained from Boehringer Mannheim (Indianappolis, IN). Synthetic oligonucleotides
were purchased from National Biosciences, Inc. (Hamel, MN).
[«-32P]dATP, [7-32P]dATP, and [125l]protein-A were obtained
from ICN-Flow (Costa Mesa, CA). [35S]Methionine and 1,25(OH)2-[26,27-3H]D3 (90 and 157 Ci/mmol) were obtained from
Amersham International (Arlington Heights, IL). Cyanogen bromide-activated Sepharose was purchased from Sigma Chemical Co. (St. Louis, MO).
VDR-NAF
1585
Acknowledgments
Fteceived June 18, 1991. Revision received July 26, 1991.
Accepted August 7,1991.
Address requests for reprints to: Dr. J. Wesley Pike, Ligand
Pharmaceuticals, Inc., 9393 Towne Center Drive, Suite 100,
San Diego, California 92121.
This work was supported by NIH Grant DK-38130 and the
Robert Welch Foundation.
* Established Investigator of the American Heart Association.
16.
17.
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