(Received for publication, July 15, 1996, and in revised form, December 27, 1996)
From the University of Montpellier and INSERM, Hormones and Cancer (U148), 60 rue de Navacelles, 34090 Montpellier, France
Hormonal regulation of gene activity is mediated
by nuclear receptors acting as ligand-activated transcription factors.
Intermediary factors interacting with their activation functions are
required to mediate transcriptional stimulation. In search of such
receptor interacting proteins, we have screened a human cDNA
expression library and isolated a human protein that interacts in
vitro with transcriptionally active estrogen receptors (ER).
Sequence analysis reveals that this protein is the human homolog of
mouse TIF1 (transcription intermediary factor 1) shown to enhance
nuclear receptor ligand-dependent activation function 2 (AF2) in yeast. We have characterized the nuclear receptor binding site
on hTIF1 and shown that a region of 26 residues is sufficient for
hormone-dependent binding to the estrogen receptor. As
shown by point mutagenesis, the AF2 activation domain of ER is required
for the binding of hTIF1 but not sufficient, since a short region
encompassing the conserved amphipathic -helix corresponding to this
domain fails to precipitate hTIF1. We also demonstrate that hTIF1
association with DNA-bound ER requires the presence of estradiol.
Finally, we show that the interaction of hTIF1 with receptors is
selective since strong in vitro
hormone-dependent binding is only observed with some members of the nuclear receptor superfamily.
The estrogen receptor (ER)1 belongs to a superfamily of nuclear receptors that function as ligand-dependent transcription factors (1, 2). Transcription is mediated by means of two activation regions, AF1 located in the NH2-terminal domain and AF2 located in the hormone binding domain, whose activities vary depending on the responsive promoter and cell type (3-5). These two activation regions appear to function independently on certain promoters, but, in some cases, both are required for full transcriptional activity (4). Recently, a ligand-dependent functional interaction between the two AFs has been demonstrated using the two-hybrid system in mammalian cells (6).
One essential element required for the activity of AF2 is a
COOH-terminal region, which is highly conserved in the nuclear receptor
family and has been shown to be essential for transcriptional activation by estrogen and glucocorticoid receptors (7), thyroid hormone receptors (8, 9), and retinoid receptors (10). This sequence
(XE
) has the characteristics of an amphipathic
-helix (7). Determination of the crystal structure of RAR-
, RXR-
, and TR-
ligand-binding domains has revealed that this helix
is repositioned upon ligand-induced conformational transition and
functions as a lid for the ligand pocket (11-13).
The mechanism whereby transcriptional stimulation is achieved remains
unknown, but it is believed that direct or indirect interactions with
target proteins within the transcriptional apparatus could be involved
to allow the assembly of a preinitiation complex (14, 15). A number of
these receptors have been shown to bind directly to the TATA
box-binding protein (TBP) and TFIIB in vitro (16-20). One
of the TBP-associated factors, hTAFII30, also interacts selectively
with the hormone binding domain of the estrogen receptor and appears to
contribute to transcriptional activity in vitro (21).
However, the interaction was unaffected by the binding of either
17-estradiol or antiestrogens such as 4-hydroxytamoxifen and was
mapped to a region that is inactive in mammalian cells (22).
Other factors acting as bridging proteins are probably involved in transcriptional activation by nuclear receptors. Using in vitro protein interaction assays or yeast two-hybrid systems with different nuclear receptor hormone-binding domains as bait, several factors that associate with activated receptors have been characterized. These include hRIP140 (23-26), mTIF1 (27), hTRIP1/mSUG1 (28, 29), several isoforms of SRC-1 (30-33), and CBP/p300 (34-37). Two other factors, namely N-CoR and SMRT, that interact with unliganded thyroid hormone and retinoic acid receptors, have been isolated and shown to act as transcriptional corepressors, which are released upon ligand binding (38, 39). In fact, it seems likely that these intermediary factors (activators or repressors) form a large family of molecules with differential binding sites or affinities for various nuclear receptors.
We report here the cloning of a human factor, which interacts in vitro with transcriptionally active estrogen receptors and appears to be the homolog of mouse TIF1 (27). We have characterized the interaction of hTIF1 with nuclear receptors and shown that a short region is sufficient for hormone-dependent binding to various members of the superfamily. However, it appears that this interaction is selective, since in vitro hormone-dependent interaction with hTIF1 is not observed with receptors belonging to the glucocorticoid receptor subclass.
A random-primed human fetal
liver gt11 expression library (CLONTECH) was
screened using the in vitro 32P-labeled GST-AF2
probe, as described previously (24). A single positive clone (clone 8)
containing a 675-base pair insert was isolated, screened again and
purified. This insert was then randomly labeled with
[32P]dCTP (Megaprime DNA labeling system, Amersham) and
used to screen an oligo(dT)-primed ZR75-1 human breast cancer cells
cDNA library constructed in
ZAPII (24). Several positive clones
were isolated and pBluescript phagemids containing the inserts of
interest were excised in vivo by coinfection with the R408
helper phage. Inserts were then sequenced by the dideoxy chain
termination method using a Sequenase version 2.0 DNA sequencing kit
(U. S. Biochemical Corp.). The 5
end of the cDNA was obtained by
reverse transcriptase-PCR using total RNA from ZR75-1 cells and
specific primers generated from the mTIF1 cDNA sequence (27).
The recombinant vectors allowing expression in Escherichia coli of GST-TFIIB, GST-AF2 (wild-type and mutants), or the plasmid used for in vitro labeling of hRIP140 (pBRIP140) were described previously (24). GST-AF2-AD was constructed by inserting a PCR-generated 96-nucleotide fragment encoding residues 525-556 of mouse ER into the EcoRI site of pGEX-2TK (Pharmacia Biotech Inc.). The nine GST-hTIF1 constructs (wild-type and mut1-mut8) encoding different domains of hTIF1 receptor binding site fused to glutathione S-transferase were constructed by inserting the corresponding DNA fragments produced by PCR from the original clone 8 into the EcoRI site of pGEX-2TK. GST-hRIP140, which encoded a fusion between GST and residues 752-1158 of hRIP140, was generated by inserting a BamHI/BglII fragment from pBRIP3 (24) into the BamHI site of pGEX2TK.
Human receptor cDNAs and their cognate ligands used in this study
correspond to ER (estradiol), RAR (retinoic acid; RA), RXR
(9-cis-RA), VDR (vitamin D3), TR
1
(T3), GR (dexamethasone), PR-A and B (R5020), AR (DHT), MR
(aldosterone), COUP-TFI, COUP-TFII, and HNF4 (EMBL data bank). All of
them were in pSG5 vector (except MR, which is in pGEM4) under the
control of the bacterial T7 polymerase promoter.
In vitro binding assays were performed essentially as described (24). Briefly, 35S-labeled proteins (receptors or interacting proteins) were cell-free-synthesized using the TNT lysate system (Promega) and incubated overnight at 4 °C with bacterially expressed and purified GST, GST-AF2, GST-AF2-AD, GST-hRIP140, or GST-hTIF1 fusion proteins in the presence of the cognate ligands at micromolar concentrations. Protein interactions were analyzed by SDS-PAGE followed by fluorography (Amplify; Amersham) and quantified using a PhosphorImager (Fujix BAS1000). In some cases, the gel was stained with Coomassie Brilliant Blue (Bio-Rad) prior to fluorography, to visualize the GST fusion proteins present in each track.
Gel Shift AssayThe double-stranded oligonucleotide corresponding to the vitellogenin A2 estrogen response element (ERE) (40) was 32P-labeled using Klenow enzyme. Binding reactions (20 µl) were performed in the presence or absence of ligands (1 µM) on ice for 1 h using 4 µl of ER-primed reticulocyte lysate and 30 µg of purified GST or GST fusion proteins in 10 mM Tris, pH 7.5, 75 mM KCl, 5% glycerol, 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.1 µg/µl poly(dI-dC), plus protease inhibitors. The labeled ERE (50 fmol) was then added, incubated for 20 min at room temperature, and analyzed on a 5% polyacrylamide gel.
To
isolate cDNAs encoding putative mediators of the
ligand-dependent activation function of nuclear receptors,
we have applied the strategy previously used to clone hRIP140 cDNA
(24). We have screened a human fetal liver random-primed cDNA
expression library with the 32P-labeled probe containing
the mouse ER hormone binding domain fused to glutathione
S-transferase (GST-AF2 probe). We have isolated a single
positive clone (clone 8) encoding a 225-amino acid peptide, which
interacted directly with the wild-type GST-AF2 probe in an
estrogen-dependent manner (Fig.
1A). The binding characteristics of the
protein encoded by this clone were similar to those previously observed
for hRIP140 and hRIP160 (23), i.e. lack of interaction (i)
in the absence of ligand (ii) in the presence of the antiestrogen 4-hydroxytamoxifen (iii) with transcriptionally defective mutants in the conserved amphipathic -helix (Fig. 1A).
The cDNA clone 8 contained a 675-base pair insert, which did not
cross-hybridize with hRIP140. When used as a probe in Northern blot
analysis of poly(A)+ RNA isolated from MCF7 breast cancer
cells, this fragment detected a major band of about 4.5 kilobases (data
not shown). Sequence analysis confirmed that this cDNA was
different from hRIP140 and from sequences present in the data bases at
the time of the cloning. We then isolated longer cDNA clones from
an oligo(dT)-primed ZR75-1 human breast cancer cell cDNA library
and sequenced the 5 end of the longest clone (clone 16). This revealed
strong homology with the sequence of mouse TIF1, which was present in
the data base as the NH2-terminal moiety of the mouse T18
oncoprotein (41), and indicated that we have isolated the human TIF1
cDNA. As shown in Fig. 2A, the overall
conservation of TIF1 amino acid sequence between mouse (27) and human
species appears relatively high (more than 92%; see also Fig.
2B for sequence comparison of the nuclear receptor binding
site). Two clusters of amino acids in the NH2 terminus of
the molecule (residues 14-25 and 107-111 in hTIF1) are, however,
highly divergent. By contrast, we found in the ZR75-1 cDNA library
a clone coding for an hTIF1 isoform with the same insertion of 34 amino
acids already described for the mouse protein (27) suggesting that this
variant form, perfectly conserved between the two species, could be of
physiological importance.
Using GST pull-down experiments with a 90-kDa peptide in
vitro expressed from clone 16, we confirmed that hTIF1 interaction with wild-type GST-AF2 was strongly increased (~14-fold) by estradiol (Fig. 1, panel B, lanes 3 and 4, and
panel C) but not by the antiestrogens 4-hydroxytamoxifen or
ICI164,384 (lanes 5 and 6). This association was
also dramatically reduced by various mutations in the conserved amphipathic -helix that abolish AF2 transcriptional activity (7).
When either of two pairs of hydrophobic residues (547/548 or 543/544)
or the negatively charged glutamic acid residue at position 546 were
mutated, the effect of estradiol on hTIF1 binding was reduced to less
than 2-fold (Fig. 1C). In these GST pull-down assays, hTIF1
binding to wild-type GST-AF2 in the presence of estradiol (57% of
input; 14-fold increase versus control; Fig. 1C)
was very similar to that of hRIP140 (56% of input; 12-fold increase
versus control; data not shown).
The initial hTIF1 cDNA
clone 8 isolated from the library encoded a 225-amino acid peptide
(Fig. 2B). This fragment overlapped the region between
residues 539 and 750 in the mouse protein, which was shown to be the
minimal domain sufficient to interact with RXR in the yeast
two-hybrid system (27). To define more accurately the border of the
nuclear receptor binding site (NRBS) on the hTIF1 molecule, we
generated by PCR a GST-hTIF1 vector and several deletion mutants
(mut1-mut8; Fig. 2C). We then tested their ability to
interact with 35S-labeled in vitro translated ER
in GST pull-down experiments. As shown in Fig. 2C, strong
estradiol-dependent interaction (19-fold increase over
control) was observed with GST-hTIF1. Deletion of 102 residues from the
COOH terminus (mut1) affected only moderately this interaction (10-fold
induction in the presence of estradiol). By contrast, when the hTIF1
fragment was reduced to 96 (mut2) or 73 amino acids (mut3),
hormone-dependent binding was totally abolished, indicating
that an important motif was localized in the last 27 residues of mut1.
Starting from mut1, we then generated five other mutants (mut4-mut8).
In GST pull-down assays, hormone-inducible binding of ER was retained
with mut4-mut7 but not with mut8. Together, these results indicate
that the minimal NRBS on hTIF1 is localized between residues 716 and
741 and that the 11 amino acids at the NH2 terminus of mut7
are necessary for ER binding. As shown in Fig. 2D, this
sequence exhibits some homologies (conservation of Ser and Leu
residues) with sequences in the two NRBS of RIP140 (25).
Since the integrity of the AF2 activation domain core plays a crucial
role in the binding of hTIF1 to ER (Fig. 1, A and
C), we have examined whether the region of the receptor
encompassing this domain was sufficient to support in vitro
interaction with hTIF1. The region from amino acid 525 to 556 was fused
to GST and used to precipitate labeled hTIF1. As shown in Fig.
3 (lane 5), no specific binding was obtained,
even though the level of GST-AF2-AD was slightly higher than those of
GST-AF2 wild-type or mut. This suggests that other regions of the ER
ligand binding domain are probably required to generate the complete
binding site.
Since in vitro binding characteristics of hTIF1
(i.e. the requirement for hormone and for an intact AF2
activating domain) were similar to those obtained for hRIP140 (23-25),
it was possible that these two potential transcriptional coactivators
contact the same region of the hormone binding domain. To investigate this possibility, we performed pull-down experiments to test the ability of ER to contact simultaneously hTIF1 and hRIP140. We used
GST-hTIF1 or GST-hRIP140 to precipitate, respectively, labeled hRIP140
or hTIF1 bound to ER. As shown in Fig. 4A,
labeled ER efficiently bound (almost 50% of input) to GST-hTIF1 in an
hormone dependent manner (compare lanes 3 and 4).
We then preincubated increasing amounts of labeled hRIP140 with ER
before precipitation with GST-hTIF1 (lanes 5-9) and looked
for the formation of a ternary complex GST-hTIF1·ER·hRIP140.
Whereas binding of ER was still clearly observable, specific
precipitation of hRIP140 in these conditions was almost undetectable
since for the highest amount of hRIP140 used (lane 9), less
than 2% of input material was bound. Moreover, most of this binding
was not ER-mediated since a similar amount of hRIP140 was precipitated
by GST-hTIF1 in the absence of ER (lane 10). Identical
results were obtained in the converse experiment using GST-hRIP140 to
pull-down hTIF1 (Fig. 4B). Together these results indicated
that, in our pull-down conditions, simultaneous binding of hTIF1 and
hRIP140 on ER is not possible.
Interaction of hTIF1 with DNA-bound ER
The electromobility
shift assay was used to examine the association of GST-hTIF1 with ER
bound to DNA. In vitro expressed ER was incubated with
purified GST or GST-hTIF1 before addition of 32P-labeled
ERE. As shown in Fig. 5, the retardation profile
obtained in the presence of GST was in agreement with previously
published results (42). Binding of ER to DNA was not dependent on the presence of hormone, but the mobility of the complexes varied according
to the ligand. In the presence of estradiol, the ER·ERE complexes
migrated faster than in the absence of ligand or in the presence of
4-hydroxytamoxifen (lanes 1-3), probably reflecting a
different conformation of the receptor. Addition of purified GST-hTIF1
did not modify the position or intensity of the retarded bands in the
absence of ligand or in the presence of 4-hydroxytamoxifen (compare
lanes 1 and 3 to lanes 4 and
6). By contrast, in the presence of estradiol, we observed a
shift in the position of the ER·ERE complex and a slight decrease in
the intensity of the band (compare lanes 2 and
5). This shift was reproducibly obtained even when ER was
produced in COS-1 cells or in baculovirus-infected insect cells (data
not shown), and it probably corresponds to the formation of an
heteromeric complex of GST-hTIF1 and ER on DNA. We have checked that
this altered migration was not due to a modification in the estradiol
binding capacity of ER or in the dissociation kinetics of hormone after
binding of GST-hTIF1 (data not shown). Moreover, similar results were
obtained when we used GST-hRIP140 fusion protein (Fig. 5, lane
8), thus confirming previous results showing that, in
DNA-dependent assays for protein-protein interaction, the
binding of hRIP140 to ER (25) or to RAR/RXR heterodimers (43) was also
ligand-dependent. By contrast, no retardation was observed
with GST-TFIIB (Fig. 5, lane 10), emphasizing the
specificity of this effect.
Interaction of hTIF1 with Other Nuclear Receptors
To study
the specificity of hTIF1 interaction with nuclear receptors, we have
tested whether other members of the superfamily were able to interact
with GST-hTIF1 in the presence of their cognate ligands. As shown in
Table I, vitamin D3 (VDR), thyroid hormone
(TR1), retinoid X (RXR
), and retinoic acid (RAR
) receptors bound to GST-hTIF1 in the presence of their cognate ligand (more than
12% of input). Specific binding (which represents less than 2.5% of
input in the absence of ligand) is increased by more than 8-fold in the
presence of hormone. In addition, as observed for ER in the presence of
antiestrogens, the interaction of RAR
was not significantly
increased in the presence of the antagonist Ro 41-5253 (44). Moreover,
we have checked that chimeric proteins found in acute promyelocytic
leukemia and resulting from the fusion of RAR
to promyelocytic
leukemia protein (PML) in t(15;17) or promyelocytic leukemia zinc
finger protein (PLZF) in t(11;17) (45) also retained a strong retinoic
acid-dependent interaction with GST-hTIF1 (data not
shown).
|
By contrast, when we evaluated the interaction of the other steroid receptors, namely glucocorticoid (GR), progesterone (PR form A and B), androgen (AR), and mineralocorticoid (MR) receptors, we observed a weak interaction (less than 5% of input) with GST-hTIF1 even in the presence of the cognate hormone. A slight increase in binding to GST-hTIF1 was, however, detectable with MR in the presence of aldosterone. In addition, we analyzed the association of GST-hTIF1 with two orphan receptors that bind DNA as homodimers and behave as transcriptional repressor (COUP-TFII) or activator (hepatocyte nuclear factor 4; HNF4) and observed very low specific interaction representing less than 3% of input (data not shown). Other transcription factors such as c-Fos, c-Jun, CREB, E1A, or MyoD, shown to bind CBP (46), also failed to interact specifically with hTIF1 in vitro (data not shown). Together these results indicate that, in our experimental conditions, hTIF1 selectively interacts in a ligand-dependent fashion only with specific members of the nuclear receptor superfamily.
Using a protein-protein interaction assay, we have searched for
human transcription intermediary factors able to bind the hormone-dependent activation domain of the estrogen
receptor. We have isolated a single cDNA clone, which appears to be
the human counterpart of mTIF1, a murine factor isolated using a yeast genetic screen based on the capacity to stimulate RXR activity (27).
This factor, initially characterized as a fusion protein with the
equivalent of the human proto-oncogene B-RAF (41), exhibits
interesting structural features. Several domains (a RING finger, B box
fingers, a coiled-coil domain, a plant homeodomain finger, and a
bromodomain) potentially involved in protein-DNA or protein-protein
interactions (47-49) have been identified. Most of the consensus
residues in these domains are conserved between mouse and human, and
the overall conservation between the two species is around 93% (for
comparison, another potential coactivator for nuclear receptor,
hTRIP1/mSUG1 is 99.3% conserved) (29). The region of hTIF1 located in
the middle of the molecule is one of the most divergent from mTIF1, and
it also contains the potential alternative splicing site generating an
isoform with a 34-amino acid insertion perfectly conserved between the
two species.
We have shown that a short sequence of 26 residues is sufficient for
the in vitro binding of hTIF1 to the estrogen receptor in
the presence of estradiol. This domain presents significant homologies
with the two receptor binding sites characterized on hRIP140 (25),
suggesting that they could contact the same interface on nuclear
receptors. On the other hand, our results also indicate that the
-helix of ER, which contains the AF2 activation domain, is required
but not sufficient for the interaction between the receptor and hTIF1.
This suggests that other parts of the hormone binding domain
participate in the formation of the binding interface and is apparently
not in accord with several reports, which have shown that the
amphipathic
-helix from mouse RXR
or
(10, 50) and chicken
TR
(9) can autonomously transactivate when fused to a DNA-binding
domain. However, it should be noted that, in all three cases,
transcriptional activation was lower than that obtained with the
full-length hormone binding domain, suggesting that only suboptimal
configurations of the activation domain are generated in such chimeric
proteins and thus possibly explaining the undetectable in
vitro association with hTIF1. Moreover, it has been shown in the
case of mSUG1 that the AF2-AD, while essential, was also not sufficient
for an efficient interaction in yeast two hybrid experiments (29).
Recent studies based on the determination of the crystal structure of
different nuclear receptor hormone binding domains (11-13) have
demonstrated that in the unliganded RXR, the amphipathic helix that
contains the AF2 activation domain points away from the core of the
molecule. In the liganded RAR
, this helix (H12) is folded back and
residues in the core of AF2-AD establish contacts both with the ligand
and other part of the HBD such as helix 4. It seems therefore likely
that helix 12 alone, while necessary, is insufficient for binding
proteins such as hTIF1 and that the interface is rather a
tridimensional structure generated upon conformational change of the
HBD (involving the swing of H12). In agreement with this hypothesis, it
has been shown that a point mutation in H4 of RAR
(K264A) or RXR
(R307A) decreases the in vitro interaction of mTIF1
(51).
The hypothesis that different parts of the HBD participate in the
formation of an active binding site for putative transcriptional coactivators could explain our observation showing that only a subgroup
of nuclear receptors (ER, TR, RAR, RXR, and VDR) exhibit hormone-dependent interaction with hTIF1. The structure of
the AF2-AD core is so well conserved among all members of the
superfamily that the binding specificity probably involves other
regions of the HBD. It is striking to note for instance that the
important position corresponding to the above-mentioned Lys-264 in
RAR is occupied by a charged residue only in receptors that bind
hTIF1 (51). From a more general point of view, the subfamily of steroid receptors that do not bind hTIF1 (GR, PR, AR, and MR) have strong sequence homologies both in the DNA and hormone binding domains (52,
53). A parallel could be made between the discriminatory residues of
the P-box in the DNA-binding domain, which select the specific
half-site of the response element (54, 55), and some amino acids in the
HBD yet unidentified, which allow the interaction with hTIF1. The
significance of this binding specificity is unknown, but it should be
noted that the other potential coactivators (TRIP/SUG1 and hRIP140)
that interact with ER also bind TR, RAR, and RXR but not GR (25, 28,
29).2 The opposite specificity seems to
take place for the effects of PML on nuclear receptors transactivation
since PR, MR, AR, and GR are preferentially stimulated by PML
cotransfection (56). Similarly, RSP5 potentiates GR and PR but not ER
transcriptional activities (57). By contrast, SRC-1/GRIP1/TIF2
(30-32), CBP (37), and hbrm/hSNF2 (58, 59) modulate the activity of
receptors from the two subgroups, and RAP46 (60) also binds to
receptors without such a discrimination. On the other hand, ARA70
appears to be a specific coactivator for the androgen receptor
(61).
Our results and those from Chambon's laboratory (27, 29) have provided several lines of evidence supporting the idea that TIF1 can mediate at least in part the nuclear receptor hormone dependent activation function AF2. However, the mechanisms involved remain to be elucidated. We have confirmed that hTIF1 as its mouse homolog is not able to interact in vitro with either TBP or TFIIB (data not shown), and it is possible that TIF1 does not function as a bridging protein between receptors and basal transcription machinery but rather as a mediator of chromatin remodeling. For instance, hTIF1 could facilitate nuclear receptor function by countering transcriptional repression mediated by histones or other chromatin-associated proteins as suggested in the case of the SWI-SNF complex (62).
Several factors have been shown to interact in a liganddependent manner with nuclear receptors hormone binding domains and therefore could play a role of mediator in transcriptional activation as already suggested for SRC-1 (30-32), RIP140 (25, 26), or CBP/p300 (34-37). It will be now very important to find out what are their respective roles and whether their activities are essential or not, additive or synergistic. Concerning hTIF1 and hRIP140, our data suggest that their binding on the receptor is mutually exclusive at least in vitro. However, it is obvious that, in the nucleus, additional factors could allow simultaneous binding of these two proteins. It is also possible that the recruitment of these receptor interacting proteins is sequential, one acting after the other. Depending on the cell type and/or the promoter context, specific binding of only one type of intermediary factor could take place on the receptor, and such a mechanism could be involved, at least partly, in tissue-specific regulation of gene expression. Further studies such as gene inactivation will be necessary to define the exact role of these factors in transcriptional control by nuclear hormone receptors.
We thank M. G. Parker, P. Chambon, R. Evans,
N. Poujol, S. Roux, A. Bonnieu, and F. Schaufele for expression
plasmids; M. Klaus for Ro41-5253; D. Mathieu and R. White for cDNA
libraries; and J. Seeler and A. Marchio for cloning the 5 end of hTIF1
cDNA. We are also grateful to J. Y. Cance for photographs, to J. C. Nicolas for discussions, and to people from the laboratory for critical
reading of the manuscript.
While this manuscript was under review, Le Douarin et al. reported the mapping of the minimal NRBS of mTIF1 to the same region and demonstrated that mTIF1 exerts a transcriptional repressing activity in mammalian cells (Le Douarin, B., Nielsen, A. D., Garnier, J. M., Ichinose, H., Jeanmougin, F., Losson, R., and Chambon, P. (1996) EMBO J. 15, 6701-6715).