The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways

Abstract

The nuclear receptor (NR) coactivator TIF2 possesses a single NR interaction domain (NID) and two autonomous activation domains, AD1 and AD2. The TIF2 NID is composed of three NR-interacting modules each containing the NR box motif LxxLL. Mutation of boxes I, II and III abrogates TIF2-NR interaction and stimulation, in transfected cells, of the ligand-induced activation function-2 (AF-2) present in the ligand-binding domains (LBDs) of several NRs. The presence of an intact NR interaction module II in the NID is sufficient for both efficient interaction with NR holo-LBDs and stimulation of AF-2 activity. Modules I and III are poorly efficient on their own, but synergistically can promote interaction with NR holo-LBDs and AF-2 stimulation. TIF2 AD1 activity appears to be mediated through CBP, as AD1 could not be separated mutationally from the CBP interaction domain. In contrast, TIF2 AD2 activity apparently does not involve interaction with CBP. TIF2 exhibited the characteristics expected for a bona fide NR coactivator, in both mammalian and yeast cells. Moreover, in mammalian cells, a peptide encompassing the TIF2 NID inhibited the ligand-induced AF-2 activity of several NRs, indicating that NR AF-2 activity is either mediated by endogenous TIF2 or by coactivators recognizing a similar surface on NR holo-LBDs.

Full text

The EMBO Journal Vol.17 No.2 pp.507–519, 1998
The coactivator TIF2 contains three nuclear
receptor-binding motifs and mediates
transactivation through CBP binding-dependent
and -independent pathways
Johannes J.Voegel, Matthias J.S.Heine,
Marc Tini, Vale
´
rie Vivat, Pierre Chambon
and Hinrich Gronemeyer
1
Institut de Ge
´
ne
´
tique et de Biologie Mole
´
culaire et Cellulaire,
(IGBMC)/CNRS/INSERM/ULP/Colle
`
ge de France, BP 163, 67404
Illkirch Cedex, C.U. de Strasbourg, France
1
Corresponding author
e-mail: hg@titus.u-strasbg.fr
J.J.Voegel and M.J.S.Heine contributed equally to this work
The nuclear receptor (NR) coactivator TIF2 possesses
a single NR interaction domain (NID) and two auto-
nomous activation domains, AD1 and AD2. The TIF2
NID is composed of three NR-interacting modules each
containing the NR box motif LxxLL. Mutation of
boxes I, II and III abrogates TIF2–NR interaction and
stimulation, in transfected cells, of the ligand-induced
activation function-2 (AF-2) present in the ligand-
binding domains (LBDs) of several NRs. The presence
of an intact NR interaction module II in the NID is
sufficient for both efficient interaction with NR holo-
LBDs and stimulation of AF-2 activity. Modules I and
III are poorly efficient on their own, but synergistically
can promote interaction with NR holo-LBDs and AF-2
stimulation. TIF2 AD1 activity appears to be mediated
through CBP, as AD1 could not be separated muta-
tionally from the CBP interaction domain. In contrast,
TIF2 AD2 activity apparently does not involve inter-
action with CBP. TIF2 exhibited the characteristics
expected for a bona fide NR coactivator, in both
mammalian and yeast cells. Moreover, in mammalian
cells, a peptide encompassing the TIF2 NID inhibited
the ligand-induced AF-2 activity of several NRs, indic-
ating that NR AF-2 activity is either mediated by
endogenous TIF2 or by coactivators recognizing a
similar surface on NR holo-LBDs.
Keywords: activation function/GRIP1/ligand-dependent
activation/NR box/transcription intermediary factors
Introduction
Nuclear receptors (NRs) represent a family of ligand-
inducible transcription factors which trigger complex
events during development, differentiation and homeo-
stasis. They control gene expression upon binding of small
hydrophobic ligands, such as steroid and thyroid hormones,
vitamin D and retinoids. All NRs display a modular
structure, with five to six distinct regions, termed A–F.
The N-terminal A/B region contains the activation function
AF-1, which can activate transcription constitutively.
Region C encompasses the DNA-binding domain (DBD),
which recognizes cognate cis-acting elements. Region E
© Oxford University Press
507
contains the ligand-binding domain (LBD), a dimerization
surface and the ligand-dependent transcriptional activation
function AF-2 (reviewed in Gronemeyer and Laudet,
1995; Kastner et al., 1995; Mangelsdorf and Evans 1995;
Mangelsdorf et al., 1995; Beato et al., 1995; Chambon,
1996).
In transiently transfected cells, both AF-1 and AF-2 of
several NRs activate transcription in a promoter- and cell-
dependent manner (Tora et al., 1989; Berry et al., 1990;
Nagpal et al., 1992, 1993). These findings, together
with the transcriptional interference/squelching observed
between the AFs of steroid receptors (Bocquel et al.,
1989; Meyer et al., 1989; Tasset et al., 1990), led to the
concept of transcriptional mediators/intermediary factors
(TIFs), which mediate AF activity to the transcriptional
machinery and chromatin template. Several putative coac-
tivator TIFs for NR AF-2s have been characterized (for
recent reviews, see Chambon, 1996; Horwitz et al., 1996;
Glass et al., 1997). In particular, Le Douarin et al. (1996)
have demonstrated that a 10 amino acid fragment of
TIF1α is necessary and sufficient to mediate interaction
with retinoid X receptor (RXR) in a ligand- and AF-2
integrity-dependent manner. Notably, within this TIF1α
fragment, these authors identified a LxxLLL motif, termed
the NR box, whose integrity is required for interaction
with NRs, and pointed out that this motif is conserved in
several other putative coactivators (Le Douarin et al.,
1996). TIF1α and several other putative coactivators do
not, or only very poorly, stimulate transactivation by NRs
in transiently transfected mammalian cells. In contrast,
the TIF2/SRC-1 family (On
˜
ate et al., 1995; Voegel et al.,
1996), the CBP/p300 family (Chakravarti et al., 1996;
Hanstein et al., 1996; Kamei et al., 1996; Smith et al.,
1996; for reviews see Eckner, 1996; Janknecht and Hunter,
1996; Shikama et al., 1997) and the androgen receptor
coactivator ARA70 (Yeh and Chang, 1996) have been
shown unequivocally to enhance AF-2 activity.
In addition to binding NRs, CBP/p300 can also interact
directly with SRC-1 (Kamei et al., 1996; Yao et al., 1996),
and both CBP and p300 have been shown to exert
histone acetyltransferase (HAT) activity (Bannister and
Kouzarides, 1996; Ogryzko et al., 1996). Moreover, CBP/
p300 can recruit p/CAF, which is itself a nuclear HAT
(Yang et al., 1996). However, apart from interacting with
coactivators in a ligand-dependent manner, NRs have also
been shown to interact, often in a ligand-independent
fashion, directly or indirectly with components of the
transcriptional machinery, such as TFIIB, TBP, TAFs or
TFIIH(Baniahmadetal.,1993;Jacqetal.,1994; Schulman
et al., 1995; May et al., 1996; Mengus et al., 1997;
Rochette-Egly et al., 1997).
We have reported previously that the 160 kDa human
nuclear protein TIF2 exhibits all of the properties expected
for a bona fide coactivator/TIF/mediator of NR AF-2; it

J.J.Voegel et al.
interacts directly with the LBDs of several NRs in an
agonist- and AF-2-integrity-dependent manner in vitro and
in vivo, harbours an autonomous activation function,
relieves NR autosquelching, and enhances the activity of
steroid NR AF-2s when overexpressed in transiently
transfected mammalian cells (Voegel et al., 1996). How-
ever, even though TIF2 interacts in an agonist- and AF-2
integrity-dependent manner with the retinoic acid and
retinoidX receptors (RAR and RXR), no stimulation of
RAR/RXR-induced transcriptional activation could be
observed in mammalian cells overexpressing TIF2 under
the experimental conditions used (Voegel et al., 1996).
Hong et al. (1996) originally described a partial cDNA
of the mouse homologue of TIF2, named GRIP1, and
recently reported the isolation of a full-length GRIP1
cDNA (Hong et al., 1997). Their results have confirmed
our previous observations and, furthermore, using the
yeast Saccharomyces cerevisiae as model system, they
have shown that transcriptional activation by the thyroid
hormone receptor (TR), RAR and RXR could also be
stimulated by GRIP1 co-expression, which suggests that
TIF2/GRIP1 could be a general coactivator for NRs
(Walfish et al., 1997).
Here we show that TIF2 contains an NR interaction
domain (NID) and two autonomous activation functions
(AD1 and AD2). Moreover, we performed a detailed
mapping of both the NID and the AD1, resulting in the
identification of (i) three redundant LxxLL motifs in the
NID and (ii) a CBP interaction domain (CID), which is
identical with AD1. The present results are discussed in
view of the integration of TIF2 function in the sequence
of events leading to activation of target gene transcription
by NRs.
Results
The TIF2 nuclear receptor interaction domain
comprises three binding modules, each containing
the NR box motif LxxLL
We have demonstrated previously that a fragment of TIF2
which encompasses residues 624–1287 (TIF2.1; Voegel
et al., 1996) interacts in an agonist- and AF-2-integrity-
dependent manner with several NRs in vitro and in vivo,
and stimulates the transcriptional activity of several NR
AF-2s, most likely via the activation function which was
identified in this fragment. To delineate further the TIF2
NID, we studied the interaction between a series of TIF2
deletion mutants and the oestrogen receptor (ER) or RARα
LBDs, using GST fusion protein-based in vitro assays. In
both cases, a NID was mapped to the central region of
TIF2 (amino acids 624–869 in mutant TIF2.5; see Figure
1A and B). The agonist-dependent TIF2–NR interaction
was also observed on DNA-bound NRs (C.Zechel, unpub-
lished observation). No additional NID could be identified
in the N- or C-termini of TIF2 (Figure 1A and B;
mutants TIF2.0, TIF2.2 and TIF2.7). In contrast, SRC-1,
a paralogue of TIF2, apparently harbours two distinct non-
contiguous NIDs located in the central and C-terminal
regions (On
˜
ate et al., 1995; Yao et al., 1996; Zhu et al.,
1996).
To delineate further the TIF2 NID, TIF2.5 was C-
terminally truncated to Pro775, yielding TIF2.34 which
also interacted with ER and RARα LBDs in a ligand-
508
dependent manner (Figure 2A and B). Upon further
truncation to Ser697, the resulting mutant TIF2.35 still
interacted with both ER and RARαLBDs but,surprisingly,
a ligand-dependent interaction was also found with
TIF2.36 (Figure 2A and B), thus indicating that the
TIF2 NID is composed of at least two autonomous NR-
interacting modules.
An alignment of the TIF2 NID amino acid sequence
present in TIF2.34 with the corresponding region of SRC-
1 (On
˜
ate et al., 1995) revealed three highly conserved
regions (Figure 2A). Interestingly, all three contain the
motif LxxLL (Figure 2C), originally identified in the so-
called NR box of TIF1α as the LxxLLL motif, which is
also present in RIP140 (Cavaille
`
s et al., 1995) and TRIP3
(Lee et al., 1995) (see Le Douarin et al., 1996 and Figure
2C). Importantly, 10 amino acid peptides comprising the
TIF1α or RIP140 NR boxes were sufficient for functional
interaction with RXR in a ligand- and AF-2 AD-integrity-
dependent manner, and mutation of the leucines at position
4 and 5 (LL→AA) of the TIF1α LxxLLL motif abrogated
TIF1α–RXR interaction (Le Douarin et al., 1996). The
functionalityof the RIP140 NR box was confirmed recently
(Heery et al., 1997).
To investigate the functional significance of the three
TIF2 NID motifs, the LL→AA mutation was introduced
in the context of both the full-length TIF2 and TIF2.1.
Mutation of all three motifs (TIF2m123, TIF2.1m123; see
Figure 2A; numbers following ‘m’ refer to the mutated
motifs) abrogated both the ligand-induced binding of
TIF2 or TIF2.1 to ER, RARα and RXRα (Figure 2D,
quantitation in Figure 2E, white bars) and the TIF2- or
TIF2.1-dependent stimulation of ligand-induced trans-
activation by ER and RXRα AF-2s (Figure 2E, black
bars). TIF2.1 constructs in which two NR box motifs were
mutated still exhibited both ER binding and stimulation
of AF-2 activity, in particular when the NR box motif II
was intact, suggesting that the three NR box-containing
modules are, at least inpart, functionallyredundant (Figure
2D and E; TIF2.1m12, m13 and m23). This redundancy
was obvious when only one NR box motif was mutated;
in contrast to TIF1α, which contains only one NR box
(Le Douarin et al., 1996), mutation of a single TIF2 NR
box did not abrogate ER binding and stimulation of ER
AF-2 activity. All three mutants (TIF2.1m1–m3) bound
to ER and stimulated oestradiol-dependent transactivation
by ER with efficiencies similar to TIF2.1 itself (Figure
2D and E). In the case of RARα and RXRα, the mutations
had, in general, a more deleterious effect on receptor LBD
binding and stimulation of AF-2 activity than in the case
of ER (Figure 2D and E). This may possibly reflect a
weaker interaction between TIF2 and either RARα or
RXRα than with ER. However, in spite of exhibiting in
general a lower activity, the patterns of NR binding and
stimulation of AF-2 activity of the NR box mutants were
similar for ER, RARα and RXRα, as mutation of motif
II was always more detrimental in double mutants than
mutation of motifs I and III (see Figure 2E). Importantly,
for both ER and RXRα, we observed a good qualitative
correlation between the effect of any of the various
mutations on TIF2.1–receptor binding in vitro and TIF2.1-
mediated stimulation of AF-2 activity (Figure 2E; note
the significant reduction in both parameters for ER/
TIF2.1m12, ER/TIF2.1m23 and RXR/TIF2.1m2 despite

TIF2 functional domains
Fig. 1. Mapping of TIF2 domains. (A) Schematic representation of functional domains identified in TIF2 [NID, nuclear receptor interaction domain; CID,
CBP interaction domain; AD1 and AD2, two autonomous activation functions; bHLH, sequence similarity with basic helix–loop–helix motifs; PAS A, B,
sequence similarity with the Per Arndt-Sim (PAS) motifs; Q rich, a glutamine-rich sequence]. The various TIF2 constructs are denoted; expressed
residues are given in parentheses. Bold lines indicate expressed sequences. Constructs that score positive or negative for NR interaction, transactivation or
CBP binding are identified on the right by ‘1’ and ‘–’ signs respectively; nd, not determined. (B) Mapping of the NID of TIF2. GST pull-down
experiments were performed with
35
S-labelled in vitro translated TIF2 polypeptides and bacterially produced GST, GST–hERα(DEF) and GST–
hRARα(DEF) in the presence or absence of 1 µM of the cognate ligand (E2, oestradiol for ER; RA, all-trans-retinoic acid for RAR). (C) Analysis of the
transcriptional activity of GAL–TIF2 fusion proteins. Cos-1 and HeLa cells were transfected with 3 µg of plasmids expressing different regions of TIF2
fused to the DNA-binding domain of the yeast transcription factor GAL4 together with 1 µg of the (17m)
5
-G-CAT reporter plasmid. Fold inductions
above the GAL4 DBD value are indicated. The mean and standard deviation of at least three experiments are shown. A representative Western blot,
illustrating the expression levels of the GAL4–TIF2 fusion proteins, expressed from 10 µg of the corresponding expression vectors, is shown on the left.
The blot was revealed with mouse monoclonal antibodies 2GV3 and 3GV2 specific for the GAL4 DBD domain (White et al., 1992). (D) Mapping of the
CID of TIF2. GST pull-down experiments were performed with
35
S-labelled in vitro translated TIF2 polypeptides and bacterially produced GST and
GST–CBP* (expressing CBP residues 1872–2165). (E) Two-hybrid analysis of the CBP–TIF2 interaction in mammalian cells in vivo. HeLa cells were
transfected with 0.2 µg of the GAL4 or GAL4–CBP* expression vectors together with 0.2 µg of the VP16 or VP16–TIF2 expression vectors in the
presence of 1 µg of (17m)
5
-TATA-CAT reporter plasmid. Fold induction relative to the activity displayed by GAL–CBP* in the absence of a VP16 fusion
protein is indicated. The mean of three experiments is shown; in each case, values varied by ,20%.
509

J.J.Voegel et al.
Fig. 2. Mapping of the TIF2 NID. (A) Alignment of the TIF2 NID with the corresponding regions of SRC-1 and p/CIP, and description of NID
mutations. The three conserved regions are displayed with the corresponding amino acid numbers of hTIF2 (GenEMBL accession No. X97674),
full-length hSRC-1 (F-SRC-1; Takeshita et al., 1996; accession No. U59302) or p/CIP (Torchia et al., 1997; GenEMBL accession No. AF000581);
the leucines pertaining to the three NR box motifs (I, II and III) are boxed. The various deletion and leucine→alanine point mutation constructs are
denoted. (B and D) Interaction of TIF2 NID mutants with NRs in vitro. GST affinity chromatography experiments were carried out with
35
S-labelled
in vitro translated GAL4 DBD fusions of TIF2 deletion mutants (B), TIF2 or TIF2.1 point mutants (D) and bacterially expressed GST and GST
fusions of the ER(DEF) (6 1 µME
2
), RAR(DEF) (6 1 µM all-trans RA) and RXR(DE) (6 1 µM9-cis RA). For quantification of point mutant
interactions, see below. (C) Alignment of the NR boxes identified in several cofactors. The conserved leucines (cf. LeDouarin et al., 1996) are
boxed. For the TRIP3 sequence, see DDBJ/EMBL/GenBank accession No. L40410 and Lee et al., 1995. (E) Effect of TIF2 NID point mutations in
TIF2 and TIF2.1 on the stimulation of NR AF-2 activity. Cos-1 cells were co-transfected with 1 µg of the (17m)
5
-TATA-CAT reporter, 0.2 µgof
Gal–hERα(DEF) or Gal–mRXRα(DE), and 2.5 µg of the TIF2.1 wild-type or mutated fragments or 0.25 µg of the wild-type or mutant TIF2, as
indicated. The reporter gene activation relative to the TIF2.1 (top panels) or TIF2 (bottom panels) wild-type activity and in the presence (‘1’) of
1 µM estradiol or 9-cis-RA, respectively, is indicated for each mutant (black bars); for comparison, in vitro binding of the respective mutants relative
to TIF2.1 wild-type binding in the presence of ligand is indicated by the white bars. Each bar represents the mean value obtained from at least three
interaction or at least four transactivation experiments, respectively; standard deviations are indicated. The expression levels of TIF2 mutants in the
cells were verified by Western blot (not shown) with mouse monoclonal antibody 3Ti3F1, which is directed against an epitope outside the mutated
area.
510

TIF2 functional domains
some quantitative differences; in all other cases there is
also a good quantitative correlation between NR binding
and stimulatory activities of TIF2.1 mutants within the
variations of the assays), supporting a mechanism whereby
the stimulation of AF-2 activity by TIF2 involves TIF2–
NR interaction through the NR holo-LBD–TIF2 NR box
interface(s).
TIF2 contains two autonomous transcription
activation functions
Transient transfection assays witha GAL4 reporter plasmid
and chimeras containing various TIF2 fragments linked
to the GAL4 DBD demonstrated the presence of two
autonomous transcription activation domains in the C-
terminal 460 amino acids of TIF2, termed AD1 and AD2
(delineated by mutants TIF2.8, TIF2.12 and TIF2.2 in
Figure 1A and C). The N-terminal AD1 (amino acids
1010–1131), which is present in TIF2.1, showed a stronger
activity than the C-terminal AD2 (amino acids 1288–
1464) (compare TIF2.8 and TIF2.12 with TIF2.2 in Figure
1A and C). The weaker activity of AD2 (relative to AD1)
could be due to a lower expression level of the GAL–
TIF2.2 fusion protein (compare with GAL–TIF2.8 and
GAL–TIF2.12 in Figure 1C). Both TIF2 activation func-
tions were active in Cos-1 and HeLa cells (Figure 1C).
However, the minimal AD1 (TIF2.8) and AD2 (TIF2.2)
constructs exhibited some cell-specific activities, as GAL–
TIF2.8 was more active in HeLa than in Cos cells, whereas
the opposite was observed for GAL–TIF2.2 (Figure 1C).
Interestingly, the glutamine-rich region of TIF2 could
neither activate transcription on its own when fused to
the GAL4 DBD (Figure 1A and C; mutant TIF2.6) nor
was it required for transcriptional activation by AD1 or
AD2. No activation function could be detected in the N-
terminal part of TIF2 (see Figure 1A and C; mutant
TIF2.0).
We conclude from these data that the NID and the two
transcription activation functions of TIF2 correspond to
distinct modular domains, since TIF2.5 can bind to NRs,
but cannot activate transcription, whereas TIF2.2 and
TIF2.8 cannot bind NRs but are able to activate transcrip-
tion (Figure 1A).
In TIF2 the activation domain-1 is indistinguishable
from the CBP interaction domain
Recently, CBP and p300, originally identified as coactiv-
ators of the transcription factor CREB, were shown to act
as general integrators of multiple signalling pathways,
including activation via agonist-bound RARα and TR (for
reviews and references, see Eckner, 1996; Janknecht and
Hunter, 1996; Glass et al., 1997; Shikama et al., 1997).
Furthermore, it was reported that SRC-1, which belongs
to the same gene family as TIF2, interacts with CBP and
p300 (Hanstein et al., 1996; Kamei et al., 1996; Yao et al.,
1996). Using GST fusion protein-based interaction and
animal cell-basedtwo-hybrid assays, we therefore analysed
whether TIF2 could also interact with CBP. In the two-
hybrid system, only the central TIF2.1 fragment, but not
the N-terminal TIF2.0 or the C-terminal TIF2.2 fragments
(Figure 1A), scored positive for interaction with GAL–
CBP* (containing amino acids 1872–2165 of CBP, which
encompass the SRC-1-interacting domain of CBP; Figure
1E). A GST–CBP* fusion protein was expressed in
511
Escherichia coli and used for pull-down assays with
in vitro-translatedTIF2 polypeptides(Figure 1D).TIF2 did
interact with CBP and, interestingly, the CID apparently
overlapped the AD1 activation domain of TIF2 (compare
Figure 1A, C and D; mutants TIF2.8 and TIF2.12). The
interaction of TIF2 with CBP was direct, as a purified
E.coli-expressed TIF2.1 protein also interacted with GST–
CBP* (data not shown). Only this region of TIF2 interacted
significantly with GST–CBP*, thus suggesting that the
TIF2 AD1 activity may originate from the recruitment of
CBP. Deletion mutants encompassing further N-terminal
regions (Figure 1A and D; mutants TIF2.10 and TIF2.4)
or the C-terminal AD2 activation domain (Figure 1A
and D; mutant TIF2.2) did not bind to GST–CBP*.
Furthermore, TIF2.2 also did not interact with full-length
CBP (data not shown), suggesting that the activity of TIF2
AD2 is mediated by (a) factor(s) distinct from CBP.
To investigate whether the CID of TIF2 could be
separated from the AD1 activation domain, the ability of
a series of GAL–TIF2 truncation mutants to activate a
GAL4 reporter was compared with their ability to interact
with the GST–CBP* protein in vitro (Figure 3). TIF2.13
(which encompasses Pro1011–Ser1122) exhibited potent
transcriptional activity, comparable with that of larger
TIF2 fragments (compare Figures 1 and 3). Removal of
26 C-terminal (TIF2.15) or 20 N-terminal (TIF2.18) amino
acid residues reduced transcriptional activity only weakly
(Figure 3A and B). Note that TIF2.13 also interacted with
CBP in vivo, as shown by two-hybrid assay in transfected
mammalian cells (Figure 4C).
While the internal deletion of residues Asp1061–
Ala1070 (TIF2.19) had only a minor effect on the ability
of TIF2.13 to transactivate, deletion of the Glu1071–
Leu1080 segment (mutant TIF2.20) significantly reduced
TIF2 AD1 transcriptional activity. These residues belong
to a sequence predicted to fold into an α-helical structure
which is highly conserved between TIF2 and SRC-1 (H1,
grey bar in Figure 3A). The involvement of this region in
transactivation was confirmed by the analysis of mutants
TIF2.21–TIF2.31 (Figures 3A and B). All constructs
containing the TIF2 wild-type sequence from Glu1071 to
Ile1096 stimulated transcription, whereas even a deletion
of only some of these residues significantly reduced, but
did not abrogate, transcriptional activation. On its own,
this α-helical H1 peptide transactivated very poorly, and
had to be incorporated into additional upstream and/
or downstream TIF2 sequences to generate significant
transcriptional activity (Figure 3A and B; compare mutants
TIF2.13, TIF2.21 and TIF2.31, and data not shown). In
particular, an adjacent predicted α-helical region (H2, grey
bar in Figure 3A) contributes significantly to TIF2 AD1
activity, since deletion of this region severely reduced
AD1-dependent transactivation (compare TIF2.18 and
TIF2.21 in Figure 3A and B). Moreover, the H2 region
can apparently function even when H1 is partially deleted
(mutant TIF2.20), suggesting that the AD1 surface may
be composed of partially redundant structural elements.
Importantly, in all cases, AD1 activity coincided with
CBP interaction, since transcriptionally inactive constructs
did not interact with CBP (TIF2.24, TIF2.27 and TIF2.29
in Figure 3A–C), while transcriptionally active mutants
also bound CBP.
To investigate whether the leucine motif (LLxxLxxxL)

J.J.Voegel et al.
Fig. 3. Mapping of the TIF2 AD1 and interaction of the AD1 domain
with CBP. (A) Alignment of the TIF2 AD1 with the corresponding
regions of SRC-1 and p/CIP. Description of TIF2 AD1 deletion
mutants and their properties. The regions of TIF2 and hSRC-1
predicted to fold into α-helices (H1 and H2) are indicated (PHD and
SOPM program; Geourjon and Deleage, 1994; Rost and Sander, 1994).
To facilitate correlation, the transcriptional activities of GAL–TIF2
constructs on a GAL4 reporter and the abilities of the various TIF2
mutants to interact with CBP in GST pull-down experiments are given
on the right in a semi-quantitative fashion (see also B and C);
transcriptional activity is categorized arbitrarily as strong (‘11’,
.350-fold), reduced (‘1’, .100-fold), weak (‘w’, 10–35-fold), none
(‘–’); due to the limited possibility to quantitate GST pull-down data,
CBP interaction is displayed as either strong (‘1’), weak (‘w’) or
none (‘–’); nd, not determined. (B) Transcriptional activation of TIF2
AD1 mutants. Cos-1 and HeLa cells were co-transfected with 3 µgof
plasmids µexpressing different mutants of the TIF2 AD1 fused to the
DNA-binding domain of the yeast transcription factor GAL4 together
with 1 µg of the (17m)
5
-G-CAT reporter plasmid. Fold inductions
above the activation seen with the GAL4 DBD alone are indicated.
The values represent the mean of at least three experiments. Note that
all GAL4–TIF2 fusion proteins were expressed at similar levels, as
revealed by Western blot with antibodies directed against the GAL4
DBD (data not shown). (C) Interaction of TIF2 AD1 mutants with
CBP in vitro. GST pull-down experiments were performed with
35
S-labelled in vitro translated TIF2, the CID mutant TIF2(LLL) or the
indicated GAL–TIF2 fusion proteins and bacterially produced GST or
GST–CBP*. Note, that the GAL4 DBD on its own does not interact
with the GST–CBP* affinity matrix.
in the H1 region is required for both AD1 transcriptional
activity and interaction with CBP, we introduced point
mutations into full-length TIF2 and TIF2.13, converting
the three conserved hydrophobic leucines to alanines
[TIF2.13(LLL) in Figure 3A and TIF2(LLL)]. Interes-
tingly, this mutation dramatically reduced AD1 activity
[compare GAL–TIF2.13 and GAL–TIF2.13(LLL) in
Figure 3B], while a mutation of the adjacent Asp–Gln
sequence, which is also conserved [Figure 3A;
TIF2.13(DQ)], had very little, if any, effect (Figure 3B).
Again, AD1 activity (Figure 4A) and interaction with
CBP in vivo (Figure 4C), as well as in vitro (Figure
4D), were correlated, since GAL–TIF2.13(DQ), which
transactivated as efficiently as wild-type GAL–TIF2.13,
interacted strongly with CBP, whereas the transcriptionally
inactive GAL–TIF2.13(LLL) interacted very weakly with
512
CBP. Notably, TIF2(LLL) was also unable to interact
efficiently with CBP (Figure 3C), supporting a critical
role for these leucine residues in shaping the CID also in
the context of the full-length TIF2 protein. Finally, co-
expressed TIF2(LLL) partially impaired the stimulatory
effect of TIF2 on the ligand-induced ER AF-2 activity of
GAL–ER (Figure 4B), suggesting that the integrity of these
leucines is required for at least part of the transcription
stimulatory effect exerted by TIF2. That TIF2(LLL) still
exhibited some stimulatory activity on ER AF-2 is not
surprising in view of the existence of the second activation
domain AD2 (see above). In addition, we presently also
do not exclude that although mutating the three leucines
abrogates TIF2–CBP interaction in vitro, in vivo this
mutation may not be sufficient to inhibit fully the formation
of a complex comprising, for example, holo-NR–CBP–

TIF2 functional domains
Fig. 4. Identification of TIF2 AD1 mutants which are impaired in both transcriptional activation and interaction with CBP. (A) Transcriptional
activation by TIF2.13 and TIF2.13 mutants. Cos-1 and HeLa cells were co-transfected with 3 µg of plasmids expressing the TIF2.13 region and the
indicated TIF2.13 mutants fused to the DNA-binding domain of the yeast transcription factor GAL4 together with 1 µg of the (17m)
5
-G-CAT
reporter plasmid. Fold inductions above the GAL4 DBD 1-fold value are indicated. The mean and standard deviation obtained from at least four
experiments are shown. The expression levels of the GAL4–TIF2.13 fusion proteins were confirmed by Western blotting (data not shown). (B) The
CID mutant TIF2(LLL) is partially deficient in stimulating ligand-induced ER AF-2 activity and its co-expression decreases the TIF2-dependent
stimulation. Cos-1 cells were co-transfected with 1 µg of (17m)
5
-TATA-CAT reporter, 0.2 µg of GAL-ERα(DEF) (‘GAL-ER’) and the indicated
amounts of TIF2 constructs in the presence of 1 µM oestradiol. Mean and standard deviation of five experiments are represented as fold induction of
the oestradiol-induced GAL–ER activity. Note that TIF2(LLL) contains an intact AD2 function. (C) Interaction of TIF2.13 wild-type and TIF2.13
mutants with CBP in mammalian cells revealed by two-hybrid analysis. HeLa cells were transfected with 0.2 µg of GAL4 or GAL–CBP* expression
vectors together with 0.2 µg of VP16 or VP16–TIF2.13 expression vectors in the presence of 1 µg of (17m)
5
-TATA-CAT reporter plasmid. Data are
represented as fold induction of the activity seen with GAL–CBP* alone. The mean and standard deviation obtained from 10 experiments are shown.
The expression levels were confirmed by Western blotting with antibodies directed against GAL4 DBD and VP16 AAD (data not shown). (D)
Interaction of TIF2.13 wild-type and TIF2.13 mutants with CBP in vitro. GST pull-down experiments were performed with
35
S-labelled in vitro
translated VP16–TIF2.13 polypeptides and bacterially produced GST and GST–CBP*. Note that the VP16 activation domain on its own does not
interact with GST–CBP*.
TIF2, which may be stabilized by further protein–protein
interactions. Together, the above results indicate that (i)
CBP mediates the AD1 activity of TIF2, (ii) the H1 motif
is critically involved in, but not sufficient for, efficient
CBP binding and AD1 activity and (iii) the integrity of a
leucine-rich motif within H1 is required for an efficient
CID/AD1 function.
TIF2 expression in yeast strongly enhances the
AF-2 activity of ER, RARα or RXRα, but has no
effect on ER AF-1 activity
The observation that animal transcriptional activators,
such as the human ER (Metzger et al., 1988), are also
active in the yeast S.cerevisiae demonstrated that the
basic principles of transcriptional enhancement have been
conserved from yeast to man. We therefore investigated
whether TIF2 could enhance transcriptional activation by
various NR constructs expressed in S.cerevisiae. Both
NRs and TIF2.1 were expressed from multicopy plasmids
in the yeast strain PL3(α), which contains a URA3
513
reporter gene under the control of three oestrogen response
elements [(ERE)
3
-URA3; Pierrat et al., 1992].
As expected from previous studies (Metzger et al.,
1988, 1992; Pierrat et al., 1992, 1994), the full-length ER
(HEG0) induced orotidine-59-monophosphate decarb-
oxylase (OMPdecase) activity in a ligand-dependent man-
ner (Figure 5, lanes 1 and 3). Interestingly, the
transcriptional activity of ER was enhanced further by co-
expression of the TIF2.1 fragment (Figure 5, compare
lanes 3 and 4). In the absence of hormone, TIF2.1 had no
significant effect on ER-induced transcriptional activation
(Figure 5, compare lanes 1 and 2). Essentially the same
results were observed for HEG19 which is devoid of the
N-terminal region A/B, indicating that TIF2 exerts its
effect on the ligand-dependent ER AF-2 (Figure 5, lanes
5–8). In contrast, neither the AF-1 activity of HE15 (which
encompasses the ER regions A, B and C; Kumar and
Chambon, 1988) nor the AF-2a activity of the HE179-
338 construct (Pierrat et al., 1994) were stimulated by co-
expressing TIF2.1 (Figure 5, lanes 9–12). This is in

J.J.Voegel et al.
Fig. 5. The TIF2.1 coactivator fragment efficiently stimulates the ligand-dependent AF-2s of ER, RAR and RXR in yeast. No stimulatory effect of
TIF2.1 on the isolated AF-1 of ER (HE15) is observable. Plasmids expressing different regions of hERα (white), hRARα (grey) and mRXRα
(black) fused to the ER DBD [hERα(C)] were introduced into the yeast reporter strain PL3(α) together with TIF2.1 as indicated. Transformants were
grown in the presence or absence of 1 µM of the cognate ligand (oestradiol for ER, all-trans-RA for RAR, 9-cis-RA acid for RXR). OMPdecase
activities determined on each cell-free extract are expressed in nmol/min/mg protein; the mean and standard deviation of at least four experiments
are shown. The ER(C)–RAR(DEF) and ER(C)–RXR(DE) expression vectors contain N-terminally the ER(F) epitope tag for Western blot detection;
this peptide is known not to exert any transcriptional activity.
agreement with the results obtained in mammalian cells
and with the observation that an intact LBD is required
for TIF2.1 to interact with the ER (Voegel et al., 1996).
TIF2.1 also stimulated the AF-2 activity of the liganded
RXRα(DE) region in yeast (Figure 5, compare lanes 19 and
20; Heery et al., 1993). This enhancement was ligand-
dependent; no activation via the RXRα(DE) region was
observed when the ER(C)–RXRα(DE) chimera was co-
expressed with TIF2.1 in the absence of ligand (Figure 5,
comparelanes18 and20).Again theseobservationsparallel
those made in HeLa and Cos-1 cells (see Figure 6D).
Surprisingly,evenintheabsenceofligand,andincontrast
to the observations made with ER and RXRα, TIF2.1 very
efficiently enhanced transactivation by the RARα AF-2
(Figure 5, lanes 13 and 14). The addition of retinoic acid
further increased this transcriptional activation (Figure 5,
lanes 14 and 16). Note that, as previously reported (Heery
et al., 1993), both RARα and RXRα
AF-2 on their own poorly activated transcription from the
URA3 reporter.
The isolated TIF2 NID inhibits the AF-2 activity of
several NRs in transfected cells
As previously shown, overexpression of the TIF2.1 frag-
ment, which contains both the NID and AD1 functions,
stimulates ER AF-2 activity in Cos-1 cells (Figure 6A,
lanes 2 and 3; Voegel et al., 1996). This stimulation was
due to a direct interaction between the ER LBD and the
NID of TIF2, as is apparent from the observation that
overexpression of the TIF2.5 mutant (which contains the
isolated NID, but lacks AD1; see Figure 1A) prevented
the stimulatory effect of TIF2.1 (Figure 6A, compare lanes
3 and 4). Note that in the presence of TIF2.1, TIF2.5
514
overexpression decreased the transactivation by the ER
AF-2 even below the level observed in the absence of
TIF2.1 (Figure 6A, compare lane 4 with lane 2). This
level of activity presumably originates from endogenous
Cos-1 coactivators, thus suggesting that these mediators
either correspond to endogenous TIF2s or interact with
the ER holo-LBD through surfaces which are identical to,
or in the direct vicinity of, the TIF2 NID interaction
surface. Indeed, the ligand-induced ER AF-2 activity could
be rescued from TIF2.5 inhibition and further enhanced
(Figure 6B, lane 3) by co-expressing TIF2 (lane 5) or
TIF2.1 (lane 4). Moreover, SRC-1 could also relieve the
inhibition by TIF2.5 (data not shown), suggesting that
TIF2 and SRC-1 may interact with a common, or adjacent,
surface(s) on the ER LBD.
We previously reported an agonist-dependent interaction
of TIF2 with RAR and RXR LBDs, which was dependent
on the integrity of the NR AF-2 AD core, but failed to
observe a stimulatory effect of TIF2 on the transcription
activation of a (17m)
5
-globin-promoter-CAT reporter by
GAL–RAR LBD or GAL–RXR LBD fusion proteins
(Voegel et al., 1996). Since this failure was likely to be
due to the presence of sufficient amounts of endogenous
mediators for achieving maximal transactivation from this
reporter gene, we modified the transfection conditions and
used a reporter construct bearing a minimal promoter. A
clear TIF2 and TIF2.1 stimulatory activity for RXRα AF2
was observed in HeLa and Cos-1 cells when using the
(17m)
5
-TATA-CAT reporter (Figure 6D, compare lanes 3–
6 and 11–14). This stimulatory effect was less marked
with RARα AF-2 and could be observed reproducibly
only with the TIF2.1 fragment in Cos-1 cells (Figure 6E,
compare lane 10 with lanes 13 and 14; note that TIF2.1

TIF2 functional domains
Fig. 6. Co-expression of the isolated NID of TIF2 inhibits the ligand-induced activity of the transcription activation function AF-2 in the ER, RXR
and RAR LBDs, while TIF2 and TIF2.1 stimulate NR AF-2 activity. (A) Expression of the NID-containing TIF2.5 lacking the AD1 and AD2
activation functions reverses the stimulatory effect of the potent coactivator fragment TIF2.1. Cos-1 cells were co-transfected with 1 µgofthe
(17m)
5
-TATA-CAT reporter and 0.2 µg of GAL–ERα(DEF) expression vector in the presence or absence of 1 µM oestradiol. Where indicated,
0.1 µg of TIF2.1 and 2.5 µg of TIF2.5 expression vectors were co-transfected in addition. (B) Co-expressed TIF2 or TIF2.1 rescue the ligand-
induced ER AF-2 activity from TIF2.5-mediated repression. Cos-1 cells were co-transfected with 1 µg of (17m)
5
-TATA-CAT reporter, 0.2 µgof
GAL–ERα(DEF) and the indicated amounts of the TIF2 constructs. (C–E) Full-length TIF2 and the coactivator fragment TIF2.1 enhance, whereas
TIF2.5/NID blocks, the ligand-induced AF-2 activity of the ER, RXR and RAR LBDs. Cos-1 and HeLa cells were co-transfected with 1 µgofthe
(17m)
5
-TATA-CAT reporter and 0.2 µg of the expression vector encoding the respective GAL DBD fusion of hERα(DEF), mRXRα(DE) or
mRARα(DEF) in the presence or absence of 1 µM ligand (E2, oestradiol; 9C-RA, 9-cis-RA; T-RA, all-trans-RA), together with 0.25 or 2.5 µgof
TIF2, TIF2.1 and TIF2.5 expression vectors. (A–D) The mean value of induction obtained from the quantitation of at least three experiments
(relative to the respective receptor LBD activity in the absence of recombinant TIF2) is indicated below each panel. Similar expression levels for
TIF2.1 and TIF2.5 were verified routinely by Western blotting with mouse monoclonal antibody 3Ti3C11 directed against a region of TIF2.5 (not
shown).
and TIF2.5 are expressed at a .10-fold higher level than
TIF2; data not shown).
Assuming that TIF2 or coactivators recognizing the
TIF2-interacting surface on NR LBDs mediate the AF-2
function of NRs, the NID-containing TIF2.5 should exert
its inhibitory activity not only on ER, but also on other
NRs, independently of the cellular context. We therefore
515
analysed the effect of TIF2.5 on the AF-2 activity of ER,
RXRα and RARα in HeLa and in Cos-1 cells (Figure
6C–E). In all cases, TIF2.5 expression led to a dose-
dependent inhibition of the NR AF-2 activity, indicating
that the endogenous mediators were competed out by the
isolated overexpressed TIF2 NID, and strongly suggesting
that TIF2 or transcriptional intermediary factors recogniz-

J.J.Voegel et al.
ing the same or overlapping surfaces mediate NR AF-2
activity in these transfected cells (Figure 6C–E, compare
lane 2 with lanes 7 and 8; lane 10 with lanes 15 and 16).
Discussion
TIF2 is a coactivator for the AF-2s of nuclear
receptor holo-LBDs in transfected animal and
yeast cells
We have reported previously that TIF2 fulfils all of the
criteria that are expected for a coactivator of the ER AF-2
functions (Voegel et al., 1996). We show here that TIF2
can also be a coactivator for non-steroid receptors, as the
AF-2 activity of both RARα and RXRα was activated by
TIF2 overexpression in both animal and yeast cells. This
enhancement of AF-2 activity, which was particularly
strong in yeast cells, has also been observed recently for
GRIP1, the mouse homologue of TIF2 (Hong et al.,
1997). These observations suggest that yeast cells contain
coactivators which only poorly mimick the action of
mammalian NR coactivators. As yeast cells apparently do
not contain a CBP homologue, it will be interesting to
investigate which yeast factor(s) mediates the activity of
TIF2. Note in this respect that ER(C)–TIF2.1 and LeXA
DBD–TIF2.1 are strong transactivators in yeast and that
the LLL mutation in TIF2.13 impairs AD1 activity in
both mammalian and yeast cells (our unpublished results),
suggesting a similar recognition of the TIF2 AD1 surface
by presently unknown yeast factor(s).
Interestingly, the expression of TIF2 in yeast led to a
marked stimulation of transactivation by the unliganded
ER(C)–RARα(DEF), which was not observed with ER or
RXRα unliganded LBDs. Structural studies have revealed
that binding of the ligand results in a conformational
change of the LBD, which generates the surface(s) for
coactivator binding (Renaud et al., 1995). Our present
result, therefore, suggests that a high level of coactivators
might, in the absence of ligand, drive the LBD of some
receptors into a holo-LBD-like conformation, thus giving
rise to ligand-independent transcriptional activity. By ana-
logy, one could speculate that high levels of co-repressors
could ‘lock’ NR LBDs in the apo-LBD conformation. It
would therefore be interesting to investigate whether high
levels of co-regulators might lead to constitutive activity
(even in the presence of antagonists) or, conversely, to
lack of inducibility of NRs in some pathological states.
The NID between NRs and TIF2 comprises three
partially redundant modules each containing the
NR box motif LxxLL
Our present structure–function analysis reveals that TIF2
contains a single NID. In contrast, the other TIF2 family
member, SRC-1, was reported to contain two NIDs (On
˜
ate
et al., 1995; Yao et al., 1996; Zhu et al., 1996). However,
only one of the two SRC-1 NIDs is most probably
homologous to the TIF2 NID characterized here (see
Figure 2A). The TIF2 NID is composed of three modules,
and we have shown that the C-terminal and the two N-
terminal modules can bind in a ligand-dependent manner
to the NRs tested in this study, suggesting that each module
can mediate NR binding independently. Interestingly, these
modules contain the NR box motif LxxLL originally
recognized as the motif LxxLLL (Figure 2C) within a 10
516
amino acid NR-binding peptide of TIF1α that was critical
for TIF1α–NR interaction and conserved in RIP140 and
TRIP3 (Le Douarin et al., 1996 and references therein;
see also Results). Moreover, the TIF1α and RIP140
modules were shown to interact functionally with NRs
(Le Douarin et al., 1996). Confirmingour original observa-
tions, the implication of NR box motifs in NR–coactivator
binding and their presence in a number of different
coactivators was pointed out in two subsequent reports
(Heery et al., 1997; Torchia et al., 1997). Note that all
three TIF2 NR box motifs described here are conserved
in the recently discovered TIF2 paralogue p/CIP (Torchia
et al., 1997).
In contrast to TIF1α, for which mutation of leucines to
alanine at positions 4 and 5 of its single NR box motif
(LL→AA) abrogates NR binding, mutation of all three
motifs is required in the TIF2 NID to abrogate NR binding,
indicating that each of these motifs can contribute to a
TIF2 surface that interacts with a cognate surface of NR
holo-LBDs. That the NR boxes of TIF2 exhibit functional
redundancy is supported by the observation that the
LL→AA mutation in any of the three TIF2 NID motifs
apparently did not (in the case of the ER) or only weakly
(in the case of RARα and RXRα) reduce the efficiency
of NR interaction. Moreover, in the TIF2.1 environment,
any single intact NR box motif on its own (i.e. when
the two other motifs were mutated) was sufficient for
interaction with the holo-ER LBD, although only motif II
on its own could bring about a nearly wild-type NR
binding efficiency. In contrast, for RARα and RXRα
interaction, mutants with single intact NR boxes were five
(box II) to 20 (box I or III) times less efficient than the
wild-type TIF2 containing the three NR boxes.
Crystallographic studies will be necessary to distinguish
between two possible models, in which the three NR box
motifs (i) contribute to the formation of a tripartite NID
surface that specifically recognizes a cognate holo-NR
LBD surface, or (ii) belong to independent surfaces which
each can interact, albeit with different efficiencies, with
the same holo-NR surface. The second model could allow
TIF2 to interact cooperatively with both partners of NR
homo- or heterodimers, thus rendering transactivation
by NRs sensitive to small variations in TIF2 levels.
Furthermore, for both ER and RXRα, the effects of NR
box mutations on NR binding and stimulation of AF-2
activity were correlated, thus supporting the conclusion
that the transcriptional effect of TIF2 involves the forma-
tion of an NR box–NR LBD interaction interface.
The motif LxxLL has been found in a number of
other NR coactivators (see above), thus suggesting some
similarity in the mode of NR–coactivator interactions.
However, this does not exclude NR-specific modulation
of these interactions,as theNR box-surrounding sequences
are highly variable.
TIF2 contains both a CBP-mediated and a CBP-
independent activation function
The two TIF2 activation functions (AD1 and AD2) appar-
ently operate through different transcriptional activation
cascades. While the TIF2 AD1 activation domain could
not be separated by mutational analysis from the TIF2
domain which interacts in vitro and in vivo with a region
of the CBP surface which also mediates SRC-1 binding

TIF2 functional domains
(Kamei et al., 1996), neither this region nor full-length
CBP interacted with the TIF2 AD2. We currently are
attempting to define the mechanism mediating TIF2 AD2
activity. That the two TIF2 activation functions may
operate through distinct pathways is also suggested by
the differential cell specificity of the minimal fragments
exhibiting AD1 (e.g. TIF2.8, TIF2.7, TIF2.9 and TIF2.12)
and AD2 activity (TIF2.2). While TIF2.2 is more active
in Cos-1 than in HeLa cells, all of the minimal fragments
containing AD1 are more active in HeLa cells. It is
tempting to speculate that the coactivator activity of TIF2
may be modulated cell-specifically by the differential
efficiency of its two ADs.
The core of AD1 (TIF2.31) on its own is a very poor
transactivator and binds CBP only weakly, thus requiring
additional surrounding sequences to generate a fully active
(i.e. efficient CBP-binding) surface. However, mutational
analysis of the AD1 corein thecontext ofa strongactivator
fragment (TIF2.13) reveals the critical importance of
transactivation and CBP binding in vivo and in vitro of
three leucine residues (Figure 4). These leucines belong
to a fully conserved LLxxLxxxL motif, embedded in a
highly conserved region of all three members of the TIF2
coactivator family (Figure 3A; Torchia et al., 1997), which
is distinct from the LxxLL NR box motif.
Role of TIF2 in nuclear transactivation by nuclear
receptor AF-2
The overall picture emerging from recent studies on the
mechanisms by which nuclear receptors modulate target
gene transcription involves three subsequent steps, (i) the
ligand-induced transconformation of the NR LBD, which
results in (ii) the dissociation of co-repressors and forma-
tion of NR–coactivator complexes, which themselves or
(iii) through interaction with additional downstream factors
(e.g. CBP, p300, p/CAF) modulate the acetylation status
of core histones and, thus, chromatin condensation/decon-
densation (see Introduction). Histone acetylation on its
own is, however, insufficient for transcription activation
(Wong et al., 1997), and a simultaneous or subsequent
fourth event comprises the direct and/or indirect recruit-
ment of elements of the transcription machinery (e.g.
TFIIB, TBP, TAFs, TFIIH; Baniahmad et al., 1993; Jacq
et al., 1994; Schulman et al., 1995; May et al., 1996;
Mengus et al., 1997; Rochette-Egly et al., 1997). Note
that such interactions do not need to be ligand-dependent,
if the primary function of the liganded LBD (AF-2) is to
regulate DNA accessibility through chromatin remodel-
ling. Indeed, most of the reported interactions between
NRs and general transcription factors occur in a ligand-
independent manner.
Within this sequence of events, TIF2 can apparently
fulfil at least two mediator functions: (i) as a ‘bridging
factor’ between the AF-2 function of NRs and CBP via
its AD1 activation domain and (ii) as a transcriptional
mediator through as yet unknown CBP binding-independ-
ent mechanisms via its AD2 function. We have not been
able to detect HAT activity of bacterially expressed TIF2
fragments under conditions where bacterially expressed
and purified yeast GCN5 was highly active (our unpub-
lished results).
Finally, it is important to stress that our present data
demonstrate that TIF2 interacts withNRs througha surface
517
(NID) that is critical for NR AF-2 activity, as the ligand-
induced transcription of several NR AF-2s was blocked
by the isolated NID (TIF2.5) and could be rescued by co-
expressing TIF2, TIF2.1 or SRC-1. This observation
clearly establishes that, at least in transfected cells, TIF2,
SRC-1or other coactivators which interact with anoverlap-
ping, if not identical, holo-LBD surface, are essential in
mediating the NR AF-2 activation function. This is in
keeping with the presence of three NR box motifs in the
TIF2 NID, and of at least one conserved LxxLL NR box
motif in all bona fide coactivators described to date. It
will be challenging to develop compounds that block the
NR box–NR interaction as an alternative to NR antagonists
for the use in endocrine therapies; in view of the divergent
environment, different number and functional redundancy
of coactivator NR boxes, it may be possible to identify
compounds that interfere in a receptor-selective manner
with coactivator binding. Similarly, blocking the inter-
action between coactivators and CBP (e.g. by expressing
the TIF2 CID, cognate synthetic peptides or functionally
equivalent compounds) may allow inhibition of CBP-
mediated, as well as the revealing of CBP-independent,
pathways in NR signalling.
Materials and methods
Plasmids
All recombinant DNA work was performed according to standard
procedures (Ausubel et al., 1993). GST fusions and GAL4 DBD fusions
of NRs have been expressed from the following previously described
plasmids: GAL (GAL4 DBD, pG4MpolyII, amino acids 1–147; Tora
et al., 1989), GAL–ER(DEF) [GAL4DBD-hERα(DEF), GAL–ER(147/
282), amino acids 282–595; Webster et al., 1989], GAL–RXR(DE)
[GAL4DBD-mRXRα(DE), amino acids 206–466; Allenby et al., 1993],
GAL–RAR(DEF) [GAL4DBD-mRARα(DEF)-hERα(F), amino acids
154–462 of RAR plus 553–595 of ER; Allenby et al., 1993], GST
(pGEX2T; Pharmacia), GST–ER [pGEX2T-hERα(DEF), also called
pGEX2T-HE14G, amino acids 282–595], GST–RXR [pGEX2T-
mRXRα(DE), amino acids 205–467] and GST–RAR (pGEX2T-
hRARα(DEF), amino acids 153–462] (all LeDouarin et al., 1995a). The
reporter plasmids (17m)
5
-TATA-CAT (May et al., 1996) and (17m)
5
-G-
CAT [(17m)
5
-β-globin-CAT; Durand et al., 1994] each contain five
copies of the GAL4 response element in front of a simple TATA motif
or of the β-globin promoter, respectively, upstream from the CAT
reporter gene.
In yeast, the hERα constructs were expressed from the following
YEp90-based plasmids: HEG0 (hERα, YEp90-HEG0, amino acids
1–595), HE15 (YEp90-HE15, amino acids 1–282), HEG19 (YEp90-
HEG19, amino acids 179–595) (all Pierrat et al., 1992) and HE179-338
(YEp90-HE179-338; Pierrat et al., 1994). From the yeast multicopy
plasmid pBL1 (LeDouarin et al., 1995b), which codes for ER(F)-epitope-
tagged ER(C) fusions, the following plasmids were expressed: ER(C)–
RAR(DEF) [pBL1-hRARα(DEF), amino acids 154–462] and ER(C)–
RXR(DE) [pBL1-mRXRα(DE), amino acids 205–467] (both vom Baur
et al., 1996).
TIF2 was expressed in yeast from the multicopy plasmid pAS3 (gift
from B.LeDouarin), which is a derivative of YEp90 containing the LEU2
marker. TIF2 constructs for transient transfection and in vitro translation
were obtained by PCR amplification of the indicated regions of TIF2
followed by subcloning in pSG5 (Green et al., 1988). The GAL4(1–
147) and VP16 chimeras were constructed by PCR amplification of the
indicated regions of TIF2 followed by subcloning in pG4MpolyII (Tora
et al., 1989) and NVP16 (Nagpal et al., 1993), respectively. For in vitro
binding assays, the indicated cDNAs were fused to GST in the pGEX-
2TK plasmid (Pharmacia). GAL–CBP* and GST–CBP* were obtained
by subcloning a PCR-amplified fragment coding for amino acids 1872–
2165 of mCBP in pG4MpolyII or pGEX-2TK, respectively. Details
concerning the plasmid constructions, all of which were verified by
sequencing, are available on request.

J.J.Voegel et al.
GST pull-down assays with [
35
S]methionine-labelled
proteins
DNA was transcribed and translated in vitro using the TNT T7-coupled
reticulocyte lysate system (Promega) following the instructions of the
manufacturer. The reticulocyte lysate containing the
35
S-labelled protein
(2 µl) was then incubated as described (vom Baur et al., 1996) with
GST, GST–CBP* or GST–hERα(DEF) and GST–hRARα(DEF), in the
presence or absence of 1 µM E2 and all-trans-retinoic acid (T-RA),
respectively. Bound proteins were recovered in SDS sample buffer and
revealed by fluorography (Amplify, Amersham) of SDS–polyacryl-
amide gels.
Transactivation assays
Yeast PL3(α) (Pierrat et al., 1992) transformants were grown exponenti-
ally in the presence or absence of ligand for about five generations in
selective medium containing uracil. Yeast extracts were prepared and
assayed for OMPdecase activity as described previously (Pierrat et al.,
1992). Transient transfections of HeLa and Cos-1 cells and CAT assays
were performed as described (Bocquel et al., 1989). Quantitative data
on CAT reporter expression were obtained either by phosphoimager
analysis (BAS2000,Fuji) of
14
C-labelled CATreaction products separated
by thin-layer chromatography, or using the CAT ELISA kit (Boehringer
Mannheim). In all cases, CAT activities were normalized to the β-
galactosidase concentrations resulting from co-transfection of 1 µgof
pCMVβGal (gift from T.Lerouge) as internal control.
Western blotting and antibodies
Expression levels of recombinant proteins in transfected cells were
determined by standard SDS–PAGE and subsequent semi-dry transfer
(MilliBlot, Millipore) to nitrocellulose membranes. Proteins were
revealed by chemiluminescent Western blot (SuperSignal, Pierce). The
following mouse monoclonal antibodies were used: 3Ti3F1 directed
against a TIF2 epitope within residues 940–1010, 3Ti3C11 against an
epitope between residues 624 and 869, 2GV3 and 3GV2 (White et al.,
1992) against the GAL4 DBD, and 2GV4B7 (gift from Y.Lutz) against
the VP16 activation domain.
Acknowledgements
We thank A.Pornon and Cathie Erb for help in transfection, OMPdecase
assays and GST pull-down experiments, and J.M.Garnier and his team
for construction of the TIF2 NR box mutants. We thank D.Bonnier,
I.Cheynel, G.Eisenmann and F.Granger for technical help; Y.Lutz for
antibody production; I.Davidson and his collaborators for the (17m)
5
-
TATA-CAT reporter; B.Le Douarin, E.vom Baur and R.Losson for yeast
strains and plasmids; T.Lerouge for the pCMVβGal plasmid; M.-J.Tsai
for SRC-1 and R.Goodman, P.Angel and C.K.Glass for CBP vectors;
and the DNA, peptidesynthesis and sequencing, cell culture, photography
and artwork services for help. J.J.V., M.J.S.H. and M.T. are recipients
of C.I.E.S., Boehringer-Ingelheim and H.F.S.P. fellowships, respectively.
This work was supported by funds from the Institut National de la Sante
´
et de la Recherche Me
´
dicale, the Centre National de la Recherche
Scientifique, the Centre Hospitalier Universitaire Re
´
gional, the Associ-
ation pour la Recherche sur le Cancer, the Fondation pour la Recherche
Me
´
dicale, the Ministe
`
re de la Recherche et de la Technologie, the
Colle
`
ge de France, the International Human Frontier Science and the
EC BIOMED (contract BMH4-96-0181 to H.G.) Programmes, and
Bristol-Myers-Squibb.
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Received June 26, 1997; revised October 28, 1997;
accepted October 29, 1997
Note added in proof
In parallel with the present work, Kalkhoven et al. [(1998) EMBO J.,
17, 232–243] identified a similar functional domain structure in the TIF2
orthologue SRC-1. The human homologue of mouse p/CIP, named AIB1/
RAC3/ACTR, has recently been identified by three groups independently
[Anzick et al. (1997), Science, 277, 965–968; Li et al. (1997), Proc.
Natl Acad. Sci. USA, 94, 8479–8484; Chen et al. (1997), Cell, 90,
569–580].