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* Institut für Physiologische Chemie I, Heinrich-Heine-Universität, D-40001 Düsseldorf, Germany;
Genetisches Institut der Justus-Liebig-Universität, D-35392 Giessen, Germany; and
Georg-Speyer-Haus, D-60596 Frankfurt, Germany
2Correspondence: Institut für Physiologische Chemie I. Heinrich-Heine-Universität Düsseldorf, Postfach 10 10 07, D-40001 Düsseldorf, Germany. E-mail: carlberg{at}uni-duesseldorf.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,25(OH)2D3) signaling via combined contact
with coactivator proteins and specific DNA binding sites (VDREs), which
ultimately results in activation of transcription. In contrast, the
mechanisms of transcriptional repression via the VDR are less well
understood. This study documents VDR-dependent transcriptional
repression largely via histone deacetylase (HDAC) activity. Direct,
ligand-sensitive protein-protein interaction of the VDR with the
nuclear receptor corepressor (NCoR) and a novel corepressor, called
Alien, was demonstrated to be comparable but independent of the VDR
AF-2 trans-activation domain. Functional assays
indicated that Alien, but not NCoR, displays selectivity for different
VDRE structures for transferring these repressive effects into gene
regulatory activities. Moreover, superrepression via Alien was found to
be affected only in part by HDAC inhibitors such as trichostatin A.
Finally, for a dissociation of VDR-Alien complexes in
vitro and in vivo, higher ligand concentrations
were needed than for a dissociation of VDR-NCoR complexes. This
suggests that Alien and NCoR are using different interfaces for
interaction with the VDR and different pathways for mediating
superrepression, which in turn characterizes Alien as a representative
of a new class of corepressors. Taken together, association of the VDR
with corepressor proteins provides a further level of transcriptional
regulation, which is emerging as a complex network of protein-protein interaction-mediated control.—Polly, P., Herdick,
M., Moehren, U., Baniahmad, A., Heinzel, T., Carlberg, C. VDR-Alien: a
novel, DNA-selective vitamin D3 receptor-corepressor
partnership.
Key Words: gene regulation • repression • NCoR
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,25-dihydroxyvitamin D3
(1,25(OH)2D3), are
principally mediated through the vitamin D receptor (VDR)
(1)
, which is a member of the nuclear receptor
transcription factor superfamily (2)
. VDR binds as a dimer
to specific sequences in promoter regions of
1,25(OH)2D3 target
genes, commonly referred to as
1,25(OH)2D3 response
elements (VDREs) (3)
. The heterodimer partner for the VDR
is the retinoid X receptor (RXR) (4)
, which is the nuclear
receptor for 9-cis retinoic acid (5)
. Simple
VDREs consist of two hexameric nuclear receptor binding sites, and
VDR-RXR heterodimers can preferentially bind to directly repeated core
binding site arrangements with three spacing nucleotides (DR3-type
VDREs) or to inverted palindromic structures with nine intervening
nucleotides (IP9-type VDREs) (4)
. Moreover, complex VDREs
are known that are formed by more than two hexameric core binding sites
and may contain additional binding sites for members of other
transcription factor families (6)
.
The nuclear receptors for 3,5,3'-triiodothyronine
(T3), T3R, and
all-trans retinoic acid (RAR) display, in most cases,
conserved functions that are characteristic for the nuclear receptor
superfamily (3)
. A high degree of homology is present
between family members in their DNA binding domain (DBD) of 66–70
amino acids (aa), whereas a lesser degree of primary structure homology
is apparent in the carboxy-terminal ligand binding domain (LBD) of
250–300 aa. The DBD and the LBD are connected by a flexible hinge
region of 30–50 aa (7)
. Crystal structure analysis of six
presently characterized nuclear receptor LBDs has demonstrated a
conserved spatial structure formed by 11–12 -helices (8
, 9)
. The LBD has diverse functions, as it is not only involved in
ligand binding, but also in interaction with other nuclear receptors
for the formation of homo- and heterodimeric complexes and in contact
with nuclear mediator proteins, such as coactivators and corepressors,
for modulation of transcriptional activities. Contact points for
coactivators have been mapped in the activation function 2 (AF-2)
domain of helix 12 and in helix 3, whereas interaction regions for
corepressors have been suggested to be within helix 1 (CoR-box)
(10
, 11)
and helices 10 and 11 (12)
.
Many coactivators and corepressors, generally grouped as families, have
been identified, characterized, and studied in the context of
transcriptional activation or repression (13)
. The nuclear
corepressor (NCoR) (11)
and silencing mediator for
retinoid and thyroid hormone receptors (SMRT) (10)
were
the ‘founding’ members of the corepressor family, which has grown to
include variants of these like RIP13a, RIP13
1, and SMRTe (14
, 15)
, others such as SUNCoR (16)
, and the most
recent member, Alien (17)
. Interaction of corepressors
with nuclear receptors has been described, especially for
T3R and RAR, but also for orphan members of the
nuclear receptor superfamily such as RevErb (18)
.
Several mechanisms are postulated to be operating to mediate
repression. Identification of several histone deacetylases (HDACs) has
provided a molecular link between histone deacetylation, corepressor
activity and transcriptional regulation (13
, 19)
. In
addition, corepressor interaction with components of the basal
transcriptional machinery, which represent alternative pathways to
HDAC-nuclear receptor interaction, has also been suggested (20
, 21)
.
Several reports indicate a repressive function involving the VDR that
includes direct repression mediated by VDR contacts with DNA sequences
within target genes (22
23
24)
, competitive VDR-squelching
activities resulting in heterodimer formation with other transcription
factors (25
, 26)
, VDR-mediated transrepression of thyroid
and retinoid signaling, and hence another form of receptor squelching
(27
28
29)
, and the recent report describing inhibition of
Smad3-mediated potentiation of VDR function by Smad7 (30)
.
It has previously been demonstrated that the VDR can interact with
SMRT, NcoR, and the NCoR variant RIP131 (31
, 32)
, but
compared with other nuclear receptors, VDR-corepressor interaction is
less well understood.
In this report, VDR-mediated repression and corepressor-mediated superrepression were studied in solution and on VDREs. VDR was confirmed to interact effectively with NCoR and was demonstrated for the first time to interact with the novel corepressor Alien. VDR-Alien interaction was found to be comparable to T3R-Alien interaction. VDR-Alien complexes showed DNA selectivity by mediating repression only through DR3-type VDREs and not through IP9-type VDREs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 34)
, four copies of the simple
DR3-type VDRE from the rat atrial natriuretic factor (ANF) gene
promoter (35)
, and four copies of the simple IP9-type VDRE
from the mouse c-fos promoter (36)
were fused
with the thymidine kinase (tk) minimal promoter driving the
luciferase reporter gene; the core sequences of the VDREs are given in
Fig. 1
and Fig. 3
.
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GAL4 fusion constructs
The DBD of the yeast transcription factor GAL4 (aa 1–147) was
fused with the cDNA of the LBD of human VDR (aa 109–427), with the
cDNA of the LBD of human T3Rß1 (aa 165–461)
(11)
, and with the cDNA of the first repression domain of
NCoR (aa 1–393) (11)
. For the mammalian one-hybrid
assays, the luciferase reporter gene was driven by three copies of the
GAL4 binding site fused to the tk promoter
(11)
.
Yeast two-hybrid expression constructs
The fusion protein construct
pLexA-VDR80–427 was created by subcloning a
region of the cDNA of human VDR spanning from aa 80–427 (hinge region
and LBD) into the pGilda vector (Clontech, Heidelberg, Germany)
containing the LexA-DBD. pJG4–5-h-Alien (17)
and
pJG4–5-NCoR2240–2453 (11)
were
constructed by fusing the full-length cDNA of Alien (305 aa) or a
region of the cDNA coding for the nuclear receptor interaction domain
of NCoR (spanning from aa 2240–2453), respectively, to the activation
domain of pJG4–5. The ß-galactosidase reporter gene vector used was
pSH18–34 (17)
.
In vitro translation/mammalian constructs
The cDNAs for human VDRwt
(37)
, human VDR413Stop
(38)
, human RXR (5)
, and chicken
T3R (39)
were subcloned into the
SV40 promoter-driven pSG5 expression vector (Stratagene, Heidelberg,
Germany). The cDNA for human Alien (17)
was subcloned into
the pT7ßSal vector and the RSV promoter-driven pABgal expression
vector. The cDNA of mouse NCoR (11)
was subcloned into the
CMV promoter-driven CMX expression vector and the cDNA of the
carboxy-terminal region of mouse NCoR (aa 1629–2453) was subcloned
into the vector pBIIKS (Stratagene) (11)
.
GST fusion protein constructs
The cDNA of human Alien (17)
and two cDNA fragments
of mouse NCoR (spanning from aa 1954–2215 and aa 2218–2453,
respectively) (11)
were subcloned into the GST fusion
vector pGEX (Amersham-Pharmacia, Freiburg, Germany).
Transfection and luciferase assays
Cos-7 SV40-transformed African green monkey kidney cells were
seeded into 6-well plates (105 cells/ml) and
grown overnight in phenol red-free DMEM supplemented with 10%
charcoal-treated fetal bovine serum (FBS). Liposomes were formed by
incubating 1 µg of the reporter plasmid, 1 µg of each pSG5-based
receptor expression vectors for VDR and RXR, 1 µg of the expression
vectors for Alien or NCoR and 1 µg of the reference plasmid pCH110
(Amersham-Pharmacia) with 15 µg
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate
(DOTAP, Roche Diagnostics, Mannheim, Germany) for 15 min at room
temperature in a total volume of 100 µl. After dilution with 900 µl
phenol red-free DMEM, the liposomes were added to the cells. Phenol
red-free DMEM supplemented with 30% charcoal-treated FBS (500 µl)
was added 4 h after transfection. At this time,
1,25(OH)2D3 (100 nM or
graded concentrations), ethanol (0.1%), or the HDAC inhibitor
trichostatin A (TSA; 20 nM) were also added. For the mammalian
one-hybrid assay, HeLa human cervix carcinoma cells were cultured,
seeded, and transfected under the same conditions as Cos-7 cells, but
expression vectors for the respective GAL4 fusion proteins and a GAL4
binding site-driven luciferase reporter gene construct were used in
transfections. For both types of assays, the cells were lysed 16 h
after onset of stimulation using the reporter gene lysis buffer (Roche
Diagnostics) and the constant light signal luciferase reporter gene
assay was performed as recommended by the supplier (Roche Diagnostics).
The luciferase activities were normalized with respect to
ß-galactosidase activity and induction or repression factors were
calculated as the ratio of luciferase activity of ligand-stimulated
cells to that of solvent controls.
Yeast two-hybrid assays
Yeast two-hybrid assays were performed as described previously
(12
, 40)
. The EGY48 yeast strain was transformed with the
pLexA-VDR80–427 as the bait, pJG4–5-h-Alien and
pJG4–5-NCoR as the activator vectors and pSH18–34 as the reporter
vector. Solvent or
1,25(OH)2D3 (at a final
concentration of 500 nM) was added to the assay to test ligand-mediated
dissociation effects. ß-Galactosidase reporter gene activity was
determined 16 h after onset of stimulation. The intensity of
solvent-treated or ligand-treated VDR-corepressor interaction was
expressed as Miller units.
In vitro protein translation
In vitro translated VDR, VDR413Stop,
and T3R proteins were generated by transcribing their
linearized pSG5-based cDNA expression vectors with T7 RNA
polymerase and translating these RNAs in vitro using
rabbit reticulocyte lysate as recommended by the supplier (Promega,
Mannheim, Germany). In vitro translated Alien and NCoR
proteins were generated from the respective expression plasmids using
the TNT rabbit reticulocyte system as recommended by the supplier
(Promega). [35S]-labeled in vitro
translated proteins were routinely checked for equal loading by
electrophoresis through a 10% sodium dodecyl sulfate
(SDS)-polyacrylamide gel, subsequent gel drying, exposure to a Fuji
MP2040S PhosphorImaging screen, and quantification with the use of a
Fuji FLA2000 reader (Tokyo, Japan) using Image Gauge software (Raytest,
Sprockhövel, Germany).
GST pull-down assays
Bacterial overexpression of GST-Alien,
GST-NCoR1954–2215, and
GST-NCoR2218–2453 was facilitated in the
Escherichia coli BL21(DE3)pLysS strain (Stratagene).
GST-Alien fusion protein expression was performed with
isopropyl-ß-D-thio-galactopyranoside (IPTG, 1.25 mM) for 3 h at
30°C and GST-NCoR1954–2215 and
GST-NCoR2218–2453 expression with IPTG (1.25 mM)
for 3 h at 37°C. The fusion proteins were checked for equal
loading by Coomassie brilliant blue staining. GST pull-down assays were
performed by coincubation of GST-Alien,
GST-NCoR1954–2215, or
GST-NCoR2218–2453 fusion proteins with in
vitro translated [35S]-labeled
VDRwt, [35S]-labeled
VDR413Stop, or
[35S]-labeled T3R.
Nuclear receptors were preincubated for 20 min at room temperature with
solvent (ethanol or DMSO) or the respective ligands (10 µM or graded
concentrations) prior to addition of a 50% GST-Alien-,
GST-NCoR1954–2215-, or
GST-NCoR2218–2453-Sepharose bead slurry in PPI
buffer (20 mM HEPES, pH 7.9, 200 mM KCl, 1 mM EDTA, 4 mM
MgCl2, 1 mM dithiothreitol, 0.1% Nonidet P-40
and 10% glycerol). All GST fusion-Sepharose slurries were preblocked
in PPI buffer containing bovine serum albumin (1 µg/µl) prior to
use in pull-down assays. VDR or T3R proteins that
were not bound to GST fusion proteins were washed away with PPI buffer.
In vitro translated VDR or T3R used
(10% of input) and bound to GST-Alien,
GST-NCoR1954–2215, or
GST-NCoR2218–2453 was detected by
electrophoresis through a 10% SDS-polyacrylamide gel, subsequent gel
drying, exposure to a Fuji MP2040S PhosphorImaging screen, and
quantified with the use of a Fuji FLA2000 reader using Image Gauge
software.
Immunoprecipitation assay
Immunoprecipitation assays were performed with in
vitro translated, [35S]-labeled Alien or
[35S]-labeled NCoR with an anti-VDR polyclonal
antibody (directed against the carboxy-terminal region of the VDR;
Santa Cruz, Heidelberg, Germany). In vitro translated
[35S]-labeled Alien and
[35S]-labeled NCoR (input 100%), in
vitro translated VDR, and either ethanol solvent or
1,25(OH)2D3 (10 µM)
were incubated with anti-VDR antibody for 20 min at room temperature,
followed by an overnight incubation at 4°C in IP buffer (20 mM HEPES,
pH 7.9, 200 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40,
and 10% glycerol) containing a mixture of protease inhibitors (Roche
Diagnostics). After this incubation, 50 µl of Protein Sepharose A
beads (Amersham-Pharmacia) was added to the reaction mixture and
further incubated at 4°C with constant rotation. Alien or NCoR
proteins that were not bound to VDR-coupled anti-VDR were vigorously
washed away with a series of NET-N buffer (150 mM NaCl, 20 mM Tris-HCl,
pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet P-40, 10%
glycerol, and protease inhibitors), IP buffer and NET buffer (150 mM
NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 10%
glycerol) washes.
[35S]-Corepressor-VDR-anti-VDR-antibody
interaction was detected by electrophoresis through a 10%
SDS-polyacrylamide gel, subsequent gel drying, exposure to a Fuji
MP2040S PhosphorImaging screen, and quantification with the use of a
Fuji FLA2000 reader and Image Gauge software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, together with the expression vectors for VDR and RXR
into Cos-7 cells, were performed to assess the ability of VDR to
mediate repression (Fig. 1A
). The complex VDRE was chosen
because an internal AP-1 site provides this response element with a
relatively high basal activity. In the absence of
1,25(OH)2D3, increasing
concentrations of the VDR expression vector augmented the intrinsic
repression activity of the VDR, whereas overexpression of RXR was
without significant effect. Moreover, increasing amounts of
overexpressed VDR resulted in an increased repressing effect. This
suggests that VDR-triggered repression is not only mediated by VDR-RXR
heterodimers, but also by other VDR-containing protein complexes, such
as VDR homodimers and VDR-RAR heterodimers. In fact, the formation of
both types of dimeric complexes has previously been shown on the
complex VDRE chosen here (34
, 37)
. In the presence of the
HDAC inhibitor TSA, VDR-mediated repression was found to be completely
abolished. To test whether this repression was in fact mediated by the
VDR, fusion proteins, each containing the DBD of GAL4 and either the
LBD of VDR, the LBD of T3R or the first
repression domain of NCoR, respectively, were overexpressed in HeLa
cells. GAL4 binding site-driven luciferase reporter gene activity was
then determined. In this mammalian one-hybrid assay, the VDR-LBD showed
28.6-fold repression, the T3R-LBD mediated
9.7-fold repression, and the repression domain I of NCoR displayed
45.1-fold repression.
VDR-corepressor interactions were assessed, in vitro,
with the use of bacterially overexpressed GST fusion proteins. GST
pull-down assays demonstrated that VDR can interact with the
corepressor Alien in a ligand-dependent manner (Fig. 2A
). The intensity of the VDR-Alien interaction appeared to be similar to
the T3R-Alien interaction. A ligand-mediated
dissociation of the VDR-Alien complex was seen on treatment with
1,25(OH)2D3 (10 µM),
where VDR-Alien interaction appeared to be reduced by 50–60% in the
presence of 1,25(OH)2D3,
which was slightly weaker than T3-mediated
dissociation of T3R-Alien interaction. As a
control, VDR and T3R interaction with the two
established nuclear receptor interaction domains of NCoR, i.e.,
NCoR1954–2215 and
NCoR2218–2453, was also tested in GST pull-down
assays (Fig. 2B
). VDR showed comparable, ligand-dependent
interaction with both NCoR interaction domains. A similar range of
ligand-dependent interaction with NCoR1954–2215
was also found with T3R, but with
NCoR2218–2453, T3R displayed an interaction
that was three- to fourfold more effective. The role of the VDR AF-2
domain for an interaction with corepressors was investigated using
VDR413Stop, an AF-2 deletion mutant of the VDR
(Fig. 2C
), and a range of VDR AF-2 point mutants spanning
the entire VDR AF-2 domain from aa 417 to 423 (data not shown) in
ligand-dependent GST pull-down assays. When assessing
VDR413Stop, the effect of ligand-dependent
dissociation of Alien or NCoR was still present. Furthermore, when
checking the VDR AF-2 point mutated series, it was shown that not one
mutant demonstrated a loss of ligand-dependent dissociation from either
Alien or NCoR (data not shown). Taken together, this suggested that
VDR-Alien and VDR-NCoR interaction was independent of the AF-2 domain.
As an additional confirmation of VDR-Alien and VDR-NCoR interaction, an
antibody directed against the carboxy-terminal region of the VDR, was
used in immunoprecipitation assays with in vitro translated
[35S]-labeled Alien or
[35S]-labeled-NCoR1629–2453
(Fig. 2D
). In this assay, VDR-corepressor interaction
also appeared to be ligand sensitive as the addition of ligand resulted
in a dissociation of 50% of Alien and 26% of NCoR from the
VDR-antibody complex. Moreover, an antibody directed against the
amino-terminal region of the VDR was also tested and confirmed
VDR-Alien and VDR-NCoR interaction (data not shown). Finally, yeast
two-hybrid assays were performed in order to assess VDR-corepressor
association in vivo (Fig. 2E
). Yeast cells were
transformed with expression vectors for
LexADBD-VDR80–427 fusion
protein as the bait and activation domain fusion proteins containing
Alien or NCoR2240–2453 as activators.
ß-galactosidase reporter gene assays demonstrated VDR-Alien and
VDR-NCoR interaction and an ligand-dependent dissociation of these
complexes.
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Ligand-driven, DNA-independent VDR-corepressor dissociation was
assessed in GST pull-down assays in the presence of graded
concentrations of
1,25(OH)2D3 (Fig. 3A
). On close examination of the dose-dependent dissociation
of VDR-corepressor interaction, VDR-NCoR interaction appeared to be
slightly more ligand-sensitive, i.e., more susceptible to
1,25(OH)2D3-mediated
dissociation with a half-maximal inhibition
(IC50) value of 330 nM, than VDR-Alien
interaction with an IC50 value of 400 nM.
However, these differences in the IC50 values
were not found to be statistically significant. Incomplete
VDR-corepressor dissociation was observed. To assess the functional
consequences of DNA-dependent effects of corepressor-VDR interaction,
luciferase reporter gene assays were performed from Cos-7 cells that
were transfected with expression vectors for VDR, RXR, Alien, or
NCoR1–2453 together with luciferase reporter
gene constructs driven by the DR3-type VDRE of the rat ANF gene
promoter (35)
(Fig. 3B
) or IP9-type VDRE of the
mouse c-fos gene promoter (36)
(Fig. 3C
). Cells were then stimulated for 16 h with graded
1,25(OH)2D3
concentrations. ß-Galactosidase normalized luciferase reporter gene
activities were expressed as fold induction and provided typical
dose-response curves. The overexpression of NCoR and Alien appeared to
be similar as judged by Western blotting (data not shown). On DR3-type
VDREs, an overexpression of Alien as well as of NCoR reduced reporter
gene activity over the whole concentration range from 0.01–100 nM, but
did not significantly affect the half-maximal activation
(EC50) value of 4 nM (Fig. 3B
). On
IP9-type VDREs, an overexpression of NCoR resulted in similar effects,
but shifted the EC50 value from 1.4 nM to 3.2 nM
(Fig. 3C
). In all three dose-response curves, the
overexpression of corepressors resulted in a repressive effect, even at
saturating ligand concentrations. This indicates that there is also an
incomplete dissociation of VDR-corepressor interaction in
vivo. An overexpression of Alien on IP9-type VDREs did not provide
a significant repressive effect on gene activation or a shift in ligand
sensitivity.
Luciferase reporter gene assays performed with overexpressed Alien and
NCoR on DR3- and IP9-type VDREs in direct comparison (Fig. 4
) could confirm the selectivity of Alien to mediate ‘superrepression’
of VDR-RXR heterodimer activity only via DR3-type VDREs. Moreover, in
this experimental series, the effect of TSA was demonstrated to
partially relieve the NCoR-mediated superrepression. In contrast, the
incomplete reduction of the repressive effect of VDR-Alien on DR3-type
VDREs suggested that Alien is relatively TSA insensitive and is using,
at least in part, alternate molecular pathways.
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An additional difference between Alien and NCoR became obvious when
their superrepressive effects on DR3-type, VDRE-bound VDR-RXR
heterodimers in Cos-7 cell reporter gene assays were normalized. At a
concentration of 0.3 nM
1,25(OH)2D3, Alien still
displayed 70% repression whereas NCoR showed only 40% of its
repression potential (Fig. 5
). In contrast, at 10-fold higher ligand concentration (3 nM) for both
proteins, less than 15% of their superrepressive activity remained.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 42)
in the absence of ligand, class II nuclear
receptors are complexed with corepressor molecules that keep chromatin
condensed and therefore have to be released before transcriptional
activation can occur. This suggests that a primary nuclear
hormone-responding gene is in a repressed state until ligand actively
destabilizes the nuclear receptor-corepressor complex. This report
describes VDR as a nuclear receptor with intrinsic repression activity
that, as being sensitive to TSA treatment, appears to act through the
established Sin3-HDAC pathway (43
44
45)
. On natural VDREs,
full-length VDR has relatively weak silencing activity compared with
T3R and RAR (11
, 16)
, but in the
mammalian one-hybrid system the VDR-LBD demonstrated more potent
repressive activity than T3R. On the complex VDRE
of the human osteocalcin gene promoter, VDR is known to act as a
homodimer as well as a heterodimer (37
, 46)
; however,
overexpression of RXR was found to be without additional effect on
repression. This suggests that in addition to VDR-RXR heterodimers,
other VDR-containing nuclear complexes can also serve as mediators of
VDR-triggered repression. Moreover, NCoR was confirmed to directly
contact the VDR with similar binding affinity with both of its nuclear
receptor interaction domains. The VDR-NCoR complex dissociates with
increasing 1,25(OH)2D3
concentrations, suggesting that VDR-NCoR interaction is valid and can
be compared to established T3R- or RAR-NCoR
interactions (10
, 11
, 17)
.
The most significant finding of this report is the demonstration of a
protein-protein interaction of VDR with the novel corepressor Alien,
both in vivo and in vitro. Compared to NCoR (2453
aa and a molecular mass of 270 kDa), Alien is much smaller (305 aa and
a molecular mass of 34 kDa) and shows no obvious sequence homology to
known corepressor molecules (17)
. Alien is known to
interact with T3R and ecdysone receptor but not
with RAR (17)
. This study shows that in its functionality
as a corepressor of VDR, Alien appears to be at least as potent as
NCoR. Alien even appears to interact more effectively with VDR than
NCoR does with VDR, as higher
1,25(OH)2D3
concentrations are needed to dissociate the VDR-Alien complex than
VDR-NCoR complexes both in vitro and in vivo.
However, the most interesting characteristic of Alien is that it
mediates repression only from VDR-RXR complexes that are bound to
DR3-type VDREs and not from those bound to IP9-type VDREs. The
protein-DNA complexes that are formed on these two types of response
elements are clearly distinct. On DR3-type elements, the DBDs of VDR
and RXR bind to the same side of the DNA, they contact each other and
show a head-to-tail orientation; on IP9-type elements, the two DBDs
bind to opposite sides of the DNA at a distance that is too wide for a
direct contact of the core DBDs (1
, 7)
. Since the LBDs of
VDR and RXR in both types of protein-DNA complexes are assumed to
interact in a similar fashion, it is more likely that a differential
effect of Alien is due to its interaction with the hinge region rather
than with the LBD of VDR. The conformation of VDR-RXR heterodimers on
IP9-type VDREs suggests that the interaction interface of VDR with
Alien may not be accessible due to steric hindrance. For interaction
with nuclear receptors, NCoR requires the so-called CoR-box, a
conserved aa sequence within helix 1 of the LBD, within nuclear
receptors such as T3R and RAR (11)
.
This suggests that Alien and NCoR are using different interaction
interfaces within the VDR.
Both in vitro GST pull-down as well as in vivo
mammalian reporter gene assays suggested that, at saturating ligand
concentrations, there is incomplete dissociation between VDR and
corepressors. An explanation for this effect may be that there are two
forms of VDR-corepressor complexes, of which only one is ligand
sensitive and behaves as described in the present model of nuclear
hormone signaling (41
, 42)
. In contrast, in the
ligand-insensitive complex type, the VDR molecule is blocked by the
corepressor and is probably not able to participate in the activation
process via coactivators in a fashion similar to what has been shown
for RAR-RXR heterodimers bound to a DR1-type response element
(47)
. Therefore, it can be hypothesized that in this
corepressor-complexed form, VDR may take alternate functions such as
ligand-dependent down-regulation of gene activity of, for example, the
parathyroid hormone gene.
In summary, an alternate pathway for repression involving the VDR has been presented. The novel interaction between the VDR and Alien, which is representative of a new class of corepressor, presents a mechanism for repression that appears to be VDRE selective.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 27, 1999. Accepted for publication November 11, 1999.
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REFERENCES |
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