A Transcriptional Silencing Domain in DAX-1 Whose Mutation Causes Adrenal Hypoplasia Congenita
Enzo Lalli,
Barbara Bardoni,
Emmanuel Zazopoulos,
Jean-Marie Wurtz,
Tim M. Strom,
Dino Moras and
Paolo Sassone-Corsi
Institut de Génétique et de Biologie Moléculaire
et Cellulaire (E.L., B.B., E.Z., J-M.W., D.M., P. S-C.) 67404
Illkirch-Strasbourg, France
Biologia Generale e Genetica
Medica (B.B.) Università di Pavia 27100 Pavia, Italy
Abteilung für Pädiatrische Genetik (T.M.S.)
Kinderpoliklinik der Ludwig Maximilians Universität 80336,
München, Germany
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ABSTRACT
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The DAX-1 gene encodes an unusual member of the
nuclear hormone receptor superfamily. Mutations in the human DAX-1 gene
cause X-linked adrenal hypoplasia congenita associated with
hypogonadotropic hypogonadism. We have shown that DAX-1 binds to
hairpin secondary structures and blocks steroidogenesis in adrenal
cells via transcriptional repression of the steroidogenic acute
regulatory protein (StAR) promoter. Here we have investigated the
molecular mechanism of DAX-1-mediated repression. We show that the
DAX-1 C terminus contains a potent transcriptional silencing activity,
which can be transferred to a heterologous DNA-binding domain. Deletion
analysis and modeling of DAX-1 structure identify two cooperating
domains required for the silencing function, one located within helix
H3 and the other within H12. The silencing function is cell- and
promoter-specific. Strikingly, two point mutations (R267P and
V269)
found in adrenal hypoplasia patients impair silencing. These findings
suggest that transcriptional silencing by DAX-1 plays a critical
role in the pathogenesis of adrenal hypoplasia congenita.
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INTRODUCTION
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The human DAX-1 gene was cloned from the DSS region localized in
Xp21, whose double dosage causes male-to-female sex reversal in
individuals with a 46,XY karyotype (1). Mutations in the DAX-1 gene
have been shown to be responsible for both adrenal hypoplasia congenita
(AHC) and hypogonadotropic hypogonadism (HHG) (2, 3, 4, 5). The permanent
zone of the adrenal cortex is absent in AHC patients, while abnormally
large fetal adrenal cells persist, resulting in structural
disorganization of the adrenal gland and low serum levels of
glucocorticoids, mineralocorticoids, and androgens. Individuals with
AHC fail to respond to ACTH treatment and require steroid hormone
replacement therapy for survival. HHG is a deficiency of production of
pituitary gonadotropins, FSH and LH, and is manifested at the time of
puberty. Males with HHG fail to undergo puberty unless treated with
testosterone (2, 3, 4, 5).
The DAX-1 protein has an unusual structure. The N-terminal portion can
be divided into three repeats of a 65- to 67-amino acid (aa) motif and
a fourth incomplete repeat. Extensive search has revealed no sequence
similarity between this domain and other known protein sequences. The
DAX-1 C terminus shares significant homology to the ligand-binding
domain (LBD) of some members of the nuclear hormone receptor
superfamily (2).
Recently we have shown that DAX-1 binds to DNA hairpin structures and
blocks steroidogenesis in adrenal cells by inhibiting the expression of
the steroidogenic acute regulatory protein (StAR) (6). Here we show
that the DAX-1 C-terminal domain is endowed with transcriptional
silencing activity. This property is restricted to a subset of the
members of the nuclear hormone receptor superfamily: the thyroid
hormone receptor (TR) and the related oncogene product v-erbA,
retinoic acid receptor (RAR), and the chicken ovalbumin upstream
promoter transcription factor (COUP-TF) (7, 8). Silencing domains in
nuclear receptors are located in the C terminus of the protein
(corresponding to the LBD). They function in the absence of ligand and
have a modular nature, since they can be transferred to a heterologous
DNA-binding domain. It has recently been shown that nuclear receptor
ligand-independent silencing activity is mediated by the recruitment of
corepressor factors termed TRACs (TR- and RAR-associated corepressors)
(9, 10, 11).
Here we present a structural model of the
DAX-1-silencing domain, based on the homology with the ligand-binding
domain of apo-RXR
and holo-RAR
(12, 13). The DAX-1-silencing
domain is bipartite in its nature, since the integrity of the
-helical modules H12 and H3 is required for its function. We show
that two different single amino acid mutations, responsible for the AHC
phenotype, abolish DAX-1-silencing activity. Our findings indicate that
a direct relationship exists between the loss of DAX-1 transcriptional
repression and AHC. Importantly, all DAX-1 mutations found in AHC
patients have the common feature to alter its C terminus.
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RESULTS
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Structure Modeling of the DAX-1 C Terminus
The three-dimensional structure of the LBD of
apo-RXR
, which has about 35% similarity with DAX-1 C terminus,
has recently been solved (12). Based on apo-RXR
and holo-RAR
(13)
structures, which have defined the existence of a common fold for the
LBD of nuclear receptors (14, 15), and on the alignments of the
sequences of human DAX-1 (2) and mouse Dax-1 (16), we have
been able to identify in DAX-1 C terminus the domains corresponding to
-helices 112, which represent the hallmark of the nuclear receptor
LBD structure (H112; Fig. 1
).
Surprisingly, we have found that helix H1 encompasses the region
previously defined as the last and incomplete repeat. In the light of
this finding, we have reconsidered our view of the DAX-1 structure.
Indeed, the three highly conserved repeats in the N terminus are
followed by a short stretch of five residues linking the third repeat
to H1. The structure of the human DAX-1 C terminus, based on the model
defined for apo-RXR
and holo-RAR
, is presented in Fig. 2
. Structural analysis of various LBDs
has shown that H1 is an integral part of the hormone-binding domain
(15), contacting H3, H5, and H8. The residues involved in H1 contacts
follow two alternative patterns: [(I,L)lx(A,I)Exx] in RXRs and
steroids receptors and [hxcAHxxT] in the RAR/TR subgroup (c, charged;
l, long side-chain; h, hydrophobic; x, any amino-acid residue).
In both cases, residues contained in the pattern anchor H1 to the LBD
core (15). The first hydrophobic residue (V193 in hRAR
; I235 in
hRXR
) is buried and forms key contacts together with the adjacent
histidine (H197 in hRAR
) or glutamate (E239 in hRXR
). This last
glutamate forms a buried salt bridge with R371 in H8 of hRXR
. In
hDAX-1, the residue corresponding to hRXR
R371 (S332 in hRAR
) is
a lysine (K382). This suggests the presence of a RXR-like pattern for
DAX-1 in H1. The pattern identified in DAX-1 is [(V,T)Sx(N,D)Qxx],
and the deeply buried glutamate forming the salt bridge with the lysine
in H8 is predicted to be located in H5 (E298). This glutamate residue
corresponds to a serine in hRXR
and to an arginine in hRAR
, which
both point to the H197 and E239 in H1 of hRXR
and hRAR
,
respectively. The residue preceding this histidine and glutamate in the
H1 pattern is an alanine, which is in close contact with an arginine at
the end of H3 (R246 as in hRAR
). This arginine is highly conserved
among many members of the nuclear receptor superfamily. In DAX-1, this
residue is replaced by a tyrosine (Y271 in hDAX-1), and either an
asparagine or an aspartate (N221 in hDAX-1) are substituted for the
alanine in the H1 pattern.
A remarkable characteristic of the DAX-1 LBD is the presence of an
unusually long insertion (26 amino acids) between H6 and H7 (Figs. 1
and 2
), which lies in the proximity of the predicted ligand-binding
pocket. This insertion represents an outstanding feature of DAX-1,
being absent in hRAR
, in hRXR
, and in most other members of the
nuclear receptor superfamily. The conservation of this insertion in
both human and mouse sequences suggests that it can play a relevant
role for DAX-1 function. Since structural predictions of loops whose
length is greater than about 10 residues are most likely to be
inaccurate, we have chosen to replace it in Fig. 2
by a residue stretch
similar in length to the hRAR
loop 67.
Another remarkable feature of DAX-1 is a conserved amphipathic
-helix motif in H12, whose integrity has been shown to be essential
for the function of the ligand-dependent AF-2 activation domain of
other nuclear receptors (17, 18).
Intriguingly, all mutations in AHC patients have the common
feature to alter DAX-1 C terminus. Most of these mutations are
deletions, nonsense and frameshift mutations in the coding sequence
(2, 3, 4, 5). Two AHC patients present single amino acid changes (3). Both
mutations reside in the N-terminal portion of the putative LBD; in one
case arginine 267 is replaced by proline (3), while in the other
case a 3-bp deletion suppresses valine 269, leaving the remainder of
the sequence in frame (3). Structure prediction localizes the sites of
these mutations in H3, inside (V269) or immediately adjacent (R267) to
the conserved hydrophobic residues belonging to the core of the nuclear
receptor structure (Fig. 2
).
The C Terminus of DAX-1 Possesses Transcriptional Silencing
Activity
We have recently shown that DAX-1 represses StAR promoter
activity in Y-1 mouse adrenal cells (6). Repression is dependent on the
presence of a hairpin secondary structure, which functions as the
DAX-1-binding site, in the StAR promoter (located between positions
-61 and -27 in the human StAR promoter), and results in a complete
block of steroid production in Y-1 cells stably transfected with human
DAX-1 (6). To assess the impact that the R267P and
V269 mutations
have on DAX-1 transcriptional properties, we have studied the effect of
DAX-1 proteins harboring these mutations on StAR promoter activity in
Y-1 cells. While wild type DAX-1 efficiently represses both basal and
forskolin-stimulated StAR promoter expression, the presence of either
mutation in DAX-1 results in impairment of the repression effect (Fig. 3a
). This cannot be accounted for by
differences in DNA binding, since both mutant proteins bind with
comparable affinity as wild type DAX-1 to the StAR promoter hairpin
structure (Fig. 3b
). The mutated proteins are expressed at levels
comparable to the wild type DAX-1 in transfected cells (Fig. 3c
).

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Figure 3. Point Mutations in DAX-1 Which Abrogate Repression
of StAR Promoter
a, Top panel: Schematic representation of the DAX-1
protein. The three N-terminal repeats are indicated with
arrows. The C terminus is indicated in
black. The position of two point mutations found in two
AHC patients is indicated. Lower panel: Repression of
StAR promoter activity by DAX-1, R267P DAX-1, and V269 DAX-1. Y-1
cells were transfected with a plasmid carrying the luciferase gene
under the control of a 1.3-kb StAR promoter fragment (pGL1.3 kb StAR; 1
µg). StAR promoter activity is shown either in basal conditions or
after stimulation with 10 µg/ml forskolin for 16 h.
Histograms show cells cotransfected with the empty
expression vector pSG5 (1 µg), pSG.DAX-1 (1 µg), pSG.R267P DAX-1 (1
µg), and pSG. V269 DAX-1 (1 µg). StAR promoter activity
in the presence of cotransfected pSG5 is set as equal to 1. Values were
normalized using a cotransfected SV40-ß-galactosidase expression
plasmid and represent the average (±SEM) of three
experiments, each one performed in duplicate. b, Electrophoretic
mobility shift assay to test binding of GST.DAX-1, GST.R267P DAX-1, and
GST. V269 DAX-1 fusion proteins to an oligonucleotide encompassing
StAR promoter hairpin -67/-21 (6). Lane 1, No protein. Lane 2, GST
(0.9 µg). Lanes 35, GST.DAX-1 (0.3, 0.6, and 0.9 µg,
respectively). Lanes 68, GST.R267P DAX-1 (0.3, 0.6, and 0.9 µg,
respectively). Lanes 911, GST. V269 DAX-1 (0.3, 0.6, and 0.9 µg,
respectively). Protein-DNA complexes were analyzed in a 8% acrylamide
gel in 0.25 x Tris-borate-EDTA. c, Western blot analysis showing
equivalent expression of DAX-1 and of the mutant R267P and V269
proteins. Cells were transfected with 10 µg of the expression vectors
pSG5 (lane 1), pSG.DAX-1 (lane 2), pSG.R267P DAX-1 (lane 3), and
pSG. V269 DAX-1 (lane 4), respectively. The anti-DAX-1 2F4 monoclonal
antibody (6) was used for DAX-1 protein detection in total cell
lysates.
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Ligand-independent transcriptional repression within
the nuclear hormone receptor superfamily is restricted to TR (and its
variants), RAR and COUP-TF (7, 8). This activity resides in the LBD and
can be transferred to a heterologous DNA-binding domain. This prompted
us to investigate whether DAX-1 C terminus is provided with a
transferable silencing domain and how the R267P and
V269 mutations
may affect this property.
A Bipartite Silencing Domain in DAX-1
To identify the domains essential for transcriptional silencing in
DAX-1, a series of deletions of its C terminus (aa 207470) was
produced and fused in-frame to the yeast GAL4 DNA-binding domain (aa
1147). In addition, GAL4/DAX-1 fusion constructs containing either
the R267P or the
V269 mutation were generated, as well as constructs
where R267 and V269 were replaced by an alanine residue (Fig. 4
). The effect of these mutated DAX-1
proteins on transcription driven by two different basal promoters
[herpes simplex virus (HSV) thymidine kinase (tk) and rabbit
ß-globin promoters] was measured in two cell lines, mouse
L tk- fibroblasts and Y-1 adrenal tumor cells.
All GAL4/DAX-1 fusion proteins are expressed at similar levels in
transfected cells and bind to the cognate 17 mer sequence with
comparable affinities (data not shown).

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Figure 4. GAL4/DAX-1 Fusion Constructs Used in Transient
Transfection Assays
The yeast GAL4 DNA-binding domain (aa 1147) was fused in-frame with
DAX-1 sequences, as indicated in the figure.
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The fusion construct G4D 207470, which comprises H1-H12 of the DAX-1
C-terminus, is a potent silencer (generating about 15-fold repression)
of the ß-globin promoter, both in L tk- and Y-1 cells
(Fig. 5
). Conversely, the tk promoter is
silenced less efficiently (
10-fold) by G4D 207470 in L
tk- cells and poorly (
3-fold) in Y-1 cells (Fig. 5
).

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Figure 5. Transcriptional Repression Activity of the Various
GAL4/DAX-1 Fusion Constructs Shown in Fig. 4
Construct activity was tested on two different basal promoters (HSV tk
and rabbit ß-globin), in L tk- and Y-1 cells. Fold
repression is calculated compared with promoter activity in the
presence of cotransfected empty expression vector pSG5 (1 µg). In
each case, 1 µg reporter plasmid and 1 µg GAL4/DAX-1 fusion plasmid
were used, as indicated. Values were normalized using a cotransfected
SV40-ß-galactosidase expression plasmid. The mean value
(±SEM) of three different experiments is reported.
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Removal of the most C-terminal 19 amino acids from G4D 207470,
encompassing the last portion of the predicted H11, H12, and the short
loop between them, results in complete abrogation of silencing of both
promoters in both cell types. Further deletion of sequences
encompassing H10 to H7 has no effect on this loss of function (Fig. 5
).
When N-terminal deletions of G4D 207470 were examined, we observed
that deletion of aa 207244, corresponding to the predicted H1 and to
part of the loop between H1 and H3, significantly reduces silencing of
the ß-globin promoter in Y-1, but not in L tk- cells.
Considering the poor repression activity of G4D 207470 on the tk
promoter in Y-1 cells, it is difficult to evaluate the effect of G4D
245470 in this context. Conversely, no loss of silencing activity of
the tk promoter by G4D 245470 was detected in L tk-
cells. G4D 272470 and mutants with deletions spanning further in the
N terminus display loss of silencing (Fig. 5
).
Introduction of the R267P and
V269 mutations into G4D 245470
results in complete loss of silencing of both the tk and the ß-globin
promoters in L tk- cells and of the ß-globin promoter in
Y-1 cells. It was not possible to assess the effect that these
mutations have on silencing of the tk promoter in Y-1 cells since, as
already mentioned, G4D 245470 has negligible effect on the activity
of this promoter in this cell type. We have also generated additional
mutations at positions 267 and 269. While alanine substitution of R267
has no effect on silencing activity, mutation of V269 into alanine
results in complete loss of silencing (Fig. 5
).
Recruitment of Corepressors
The ligand-independent silencing effect of TR and RAR has been
shown to be mediated by the interaction of their LBDs with corepressor
molecules termed TRACs (9, 10, 11). Cotransfection of expression vectors
encoding either TR and RAR causes the recovery of basal promoter
expression silenced by unliganded GAL4/TR and GAL4/RAR (19). This
phenomenon is believed to be produced by titration of cellular
corepressors.
When full-length DAX-1 is cotransfected together with G4D 245470,
attenuation of tk promoter silencing is observed (Fig. 6
, a and b). This result implies that the
mechanism of transcriptional repression by DAX-1 involves interaction
with a corepressor molecule or a component of the basal transcriptional
machinery. Significantly, R267P and
V269 DAX-1 mutants are unable to
relieve silencing by G4D 245470 (Fig. 6b
), suggesting that the
impaired repression activity of these mutants can be accounted for by a
less efficient interaction with corepressor molecules than the DAX-1
wild type protein.
Coexpression of RAR
does not relieve silencing by G4D 245470 (Fig. 6
, a and b). This result suggests that transcriptional repression
caused by the DAX-1 C terminus may involve interaction with different
molecules than the TRAC corepressors that have been described to
interact with TR and RAR in the absence of ligand.
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DISCUSSION
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The unusual structure of the DAX-1 protein is suggestive of
complex regulatory functions. We have recently shown that DAX-1 binds
to hairpin DNA structures and suppresses steroidogenesis in adrenal
cells via repression of StAR expression (6). Here we identify
transcriptional silencing as another important regulatory property of
DAX-1. Based on the structural model of DAX-1 C terminus, we show that
two domains are necessary for this activity: a N-terminal domain, which
minimally requires H3, and a C-terminal domain, including the final
portion of H11, H12 and the intermediary short loop. Fusion of H1
sequences to the N-terminal silencing domain has a cell- and
promoter-specific effect in reinforcing transcriptional repression.
Single amino acid mutations R267P and
V269, located in the predicted
H3, abolish DAX-1-silencing function, either in the context of the
natural protein (Fig. 3
) or when the C terminus is fused to a
heterologous DNA-binding domain (Fig. 4
). Remarkably, substitution of
R267 with alanine has no effect, while alanine replacement of V269
causes loss of silencing (Fig. 4
). While deletion or mutation of V269
is likely to affect the stability of the conserved hydrophobic core
(12, 13, 14, 15), R267 is predicted to be exposed on H3 surface (Fig. 2
). Here
we show that residues of different nature (charged vs.
hydrophobic) can occupy this position without producing loss of
transcriptional silencing, indicating that the effect of the R267P
mutation is probably due to distortion in the spatial arrangement of
the final portion of H3.
Unliganded RAR and TR, as well as the orphan receptor COUP-TF, act as
transcriptional repressors (7, 8); the domain responsible for their
silencing function resides in the C terminus, corresponding to the LBD
(7, 8). v-erbA and kindred S TR are variants of TR harboring mutations
in their C terminus that impair hormone binding but that do not affect
silencing activity (7, 20). Constitutive silencing by v-erbA and
kindred S TR is believed to be important in the pathogenesis of
erythroid transformation and generalized thyroid hormone resistance,
respectively (21, 20). Remarkably, all mutations in DAX-1 causing
AHC/HHG reported to date have, as a common feature, the production of
C-terminally modified proteins (2, 3, 4, 5). Based on our deletion analysis,
the result is invariably the impairment of transcriptional silencing by
DAX-1. This represents a novel example of loss of transcriptional
repression by a member of the nuclear receptor superfamily associated
with a pathological situation.
Our analysis shows that DAX-1 contains a transferable silencing domain
that is able to repress the activity of various promoters, when
appropriately tethered in their vicinity. This finding may be relevant
to the understanding of the pathogenetic mechanism of AHC/HHG. Indeed,
it is likely that DAX-1 modulates the expression of a set of genes
involved in adrenal gland development. Some of these genes may be
distinct from those whose expression is characteristic of the
differentiated steroidogenic function of the adrenal cortex
(i.e. StAR). Our data show that promoters containing a
diverse array of elements supporting basal transcription can be a
target for regulation by DAX-1. Several studies in
Drosophila demonstrated the importance of transcriptional
repressors in regulating developmental cascades. For example,
even-skipped is a homeodomain protein genetically defined as a
repressor of segmentation-controlling genes (22), and the
ecdysone-induced orphan receptor E75B regulates metamorphosis by
repressing the function of another orphan receptor, DHR3 (23). DAX-1
provides one of the rare known examples of mammalian transcriptional
repressors whose loss of function is associated with a congenital
disease. Another case is represented by the WT1 tumor suppressor gene.
A point mutation in a WT1 allele still present in a patient affected by
WAGR (Wilms tumor, aniridia, genitourinary malformations and mental
retardation) syndrome has been described (24), which converts glycine
201 into aspartic acid. The consequence is the transformation of the
product encoded by the mutated WT1 allele from a transcriptional
repressor into an activator (24).
The variable degree of silencing by the DAX-1 C terminus, depending on
the promoter and cell type, represents functional evidence that
additional factors are needed to mediate repression by DAX-1. These
might belong to the family of corepressor factors (TRACs) that are able
to form a complex with unliganded TR/RXR and RAR/RXR heterodimers
(9, 10, 11). Corepressors are released when specific ligands bind to the
receptors, allowing recruitment of coactivators (11). Multiple modes of
interaction of unliganded nuclear receptors with TRAC corepressors
exist (9, 10, 25). DAX-1, however, lacks sequence similarity with
motifs that have been shown to be required for interaction of TR/RAR
(9, 10) and RevErb (25) with corepressors. In addition, repression by
G4D 245470 can be relieved by cotransfected DAX-1, but not RAR
(Fig. 6
), suggesting that distinct factors are required to mediate
silencing by DAX-1. The abundance of these mediating factors might
possibly vary according to the cell type. The particular promoter
structure might also influence the efficiency of their recruitment,
depending on the specific set of transcription factors bound to the
promoter. This could explain the difference in silencing efficacy of
the tk promoter by G4D 207470 in L tk- as compared with
Y-1 cells. In addition, our data suggest that H1 sequences, which are
absent in G4D 245470, can significantly increase the availability of
mediating factors to the DAX-1-silencing domain when they are either
present in limiting amounts or inefficiently recruited to the
promoter.
Recent results indicate a direct and specific in vitro
interaction between the transactivator SF-1 and DAX-1 (26). While
analogous in vitro results have been obtained in our
laboratory, we have not been able, by using several experimental
approaches, to demonstrate an in vivo interaction between
SF-1 and DAX-1 (Ref. 6 and our unpublished results). On the other hand,
we have shown that DAX-1-mediated repression of both the
dax-1 and the StAR promoters is dependent on
specific binding of DAX-1 to DNA hairpin structures (6). It is
conceivable that SF-1 and DAX-1 association may be possible in
vitro under some experimental conditions that do not exist in
physiological situations. It is also possible that tissue-specific
bridging factors may exist that could facilitate SF-1 and DAX-1
association.
In conclusion, one mechanism by which DAX-1 exerts transcriptional
repression is the recruitment of a powerful silencing domain to target
promoters via binding to hairpin DNA structures (6). Due to the
presence and conservation in the DAX-1 C terminus of a potential AF-2
domain, it is still possible that a ligand may induce a switch in DAX-1
function from a repressor to an activator.
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MATERIALS AND METHODS
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Sequence Alignment and Model Building
These were performed as described (15), using the ClustalW 1.5
(27) and Modeller (28) packages. The model presented relies on the
comparison of the nonliganded human RXR
(hRXR
) and liganded human
RAR
(hRAR
) crystal structures, combined with sequence analysis.
In this respect DAX-1 exhibits about 20% and 16% sequence identity
and 35% and 29% sequence similarity with hRXR
and hRAR
,
respectively. In DAX-1 the conserved regions (from helix H3 to H5 and
from helices H7 to H11) are clearly identified, and, in addition, DAX-1
also has a conserved C-terminal amphipathic
-helical domain (helix
H12). We could clearly identify the conserved regions among nuclear
receptors, which constitute the anchoring points on which the model
relies. The conserved regions identified suggest that the fold is
conserved and that a good starting model can be obtained, except
for the loop 67 region. The loop 67 in DAX-1 is a rather long loop
(30 amino acids) that cannot be modeled reliably. The importance of the
loop 67 for DAX-1 structure and function requires further
investigation. In the absence of any three-dimensional (3D)
experimental data, we preferred not to include the loop in the model,
as it brings no further information concerning the mutants discussed.
Structural modeling was performed according to the sequence alignment
shown in Fig. 1
and taking the liganded hRAR
crystal structure (13)
as a landmark. To obtain the final model, we first minimized the
structure obtained from Modeller with the CHARMM package (MSI Inc., San
Diego, CA). The minimization is conducted in two steps, each consisting
of 1000 steps of the Powell algorithm. The C
atoms were first
restrained by a harmonic potential of 30 kcal/Å2 and then
released to give the final structure. The united atom force field was
used. The final structure was then analyzed with PROCHECK (29), which
shows that more than 90% of the residues in the Ramachandran plot are
in the most favored regions and that main-chain and side-chain
parameter statistics are inside the range of or better than the
statistics derived from crystal structures solved at a resolution of
2Å (data not shown). The quality/validity of the 3D model can also be
assessed by how well the DAX-1 sequence fits the native fold of the
hRAR
crystal structure. The program PROSAII (version 3.0) (30) gives
a Z-score for Cß potentials of -4.2, which is in the range observed
for crystal structures of the same size (range from -4 to -9; hRAR
Z-score equal to -8.4 and hRXR
Z-score equal to -6.9).
Plasmids
Construction of the pSG5-based human DAX-1 expression vector
(pSG.DAX-1) has been described (2). The DAX-1-coding sequence (from
position 3 to 1197 with respect to the translation start site) was
PCR-amplified from genomic DNA of patients 2115 (
V269) and 2687
(R267P) (3). To insert each mutation into the wild type-coding
sequence, generating pSG.
V269 DAX-1 and pSG.R267P DAX-1, the
amplified DNA was excised with BspEI-PvuII and cloned into
BspEI-PvuII-digested pSG.DAX-1.
The vector pG4MpolyII (31) was used to generate fusion constructs
between the sequence encoding for the yeast GAL4 (aa 1147)
DNA-binding domain and various portions of the DAX-1 C terminus. DAX-1
sequences were PCR-amplified from plasmid pSG.DAX-1 using the
appropriate primers and cloned into the
KpnI-BamHI sites of pG4MpolyII. pG4D R267P and
pG4D
V269 were constructed by PCR amplification of the sequence
encoding for aa 245470 from pSG.R267P DAX-1 and pSG.
V269 DAX-1,
respectively, and subsequent insertion into the
KpnI-BamHI sites of pG4MpolyII. PCR mutagenesis
was used to introduce the mutations R267A and V269A into pG4D 207470.
Each plasmid was verified by sequencing.
DAX-1-, R267P DAX-1-, and
V269 DAX-1-coding sequences were PCR
amplified from pSG.DAX-1, pSG.R267P DAX-1, and pSG.
V269 DAX-1,
respectively, and cloned into pGEX 4T-3 (Pharmacia, Piscataway, NJ),
for glutathione-S-transferase (GST) fusion protein
expression. Each plasmid was verified by sequencing.
pGL1.3 kb StAR (32), 2x17mer-tk-chloramphenicol acetyltransferase (CAT)
(33) (which has two GAL4 sites cloned upstream the -105/+51 HSV tk
promoter), and 5x17mer-globin-luc (which has five GAL4 sites cloned
upstream from the -109/+10 rabbit ß-globin promoter) were used as
reporter plasmids in transient transfection assays.
DAX-1 Protein Expression in Escherichia coli and
Electrophoretic Mobility Shift Assay
These were performed according to the described methods (6, 25).
In the electrophoretic mobility shift assay, the labeled
oligonucleotide
5'-TTGCACAGTGAGTGATGGCGTTTTTAT-C-TCCTGATGATGATGCACAGCCTTCAGCGGGGGACAT-TTAAGACGCAGAA
-3', encompassing StAR promoter hairpin -67/-21 (6) was used as a
probe.
Protein Analysis
Western blotting was performed as described in Ref. 6, using the
anti-DAX-1 monoclonal antibody 2F4 raised in our laboratory for DAX-1
protein detection (6).
Transient Transfection Assays
Y-1 mouse adrenal cells were transfected by the calcium
phosphate method, as described previously (2, 6). L tk-
mouse fibroblast cells were transfected by the
diethylaminoethyl-dextran method (34). CAT and luciferase assays were
performed as described (6, 31).
 |
ACKNOWLEDGMENTS
|
---|
We thank D. M. Stocco, T. Meitinger, and G. Camerino for
discussions; J. F. Strauss III for the gift of the plasmid pGL1.3
kb StAR; and E. Heitz, S. Vicaire, M. Acker, F. Ruffenach, and C.
Werlé for technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. P. Sassone-Corsi, Institut de Genetique et de Biologie Molecular et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch Cedex, C.U. Strasbourg, France.
E. L. was supported by a Telethon Italy Fellowship. B. B. was
supported by an EMBO short term fellowship. This study was supported by
grants from CNRS, INSERM, CHUR, Rhône-Poulenc Rorer (Bioavenir),
and Association pour la Recherche sur le Cancer to P. S.-C.
Received for publication August 7, 1997.
Revision received September 22, 1997.
Accepted for publication September 25, 1997.
 |
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