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Division of Molecular Endocrinology (C.A.P., M.M., B.M.R., H.H.S.) Departments of Medicine (D.R., M.M., B.M.R., H.H.S.), Cell Biology (D.R.), and Pharmacology (M.M., B.M.R., H.H.S.) Skirball Institute of Biomolecular Medicine (D.R.) New York University Medical Center New York, New York 10016
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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enabled the selective isolation of a
65-kDa protein (p65) from nuclear extracts of rat and human cells.
Binding of p65 to mouse retinoid X receptor- was centered around the
DNA-binding domain. p65 also bound regions encompassing the DNA-binding
domain in estrogen, thyroid hormone, and glucocorticoid receptors. p65
was identified as TLS (translocated-in-liposarcoma), a recently
identified member of the RNP family of nuclear RNA-binding proteins
whose members are thought to function in RNA processing. The N-terminal
half of TLS bound to thyroid hormone receptor with high affinity while
the receptor was bound to appropriate DNA target sites. Functional
studies indicated that the N-terminal half of TLS can interact with
thyroid hormone receptor in vivo. TLS was originally
discovered as part of a fusion protein arising from a chromosomal
translocation causing human myxoid liposarcomas. TLS contains a potent
transactivation domain whose translocation-induced fusion with a
DNA-binding protein (CHOP) yields a powerful transforming oncogene and
transcription factor. The transactivation and RNA-binding properties of
TLS and the nature of its interaction with nuclear receptors
suggest a novel role in nuclear receptor function. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Ligand binding is dependent on a
complex region (domains D, E, and F) that also mediates transcriptional
regulation and receptor dimerization (1, 2, 3, 4, 5, 6). The ligand-binding domain
(LBD) exhibits considerable variability, as befits the structural
diversity of receptor ligands, and contains one of two transactivation
regions (AF-2) of nuclear receptors. AF-2 function is strictly ligand
dependent and does not require other receptor domains (6, 7, 8). Thus,
receptor LBDs fused to the DNA-binding domains (DBDs) of unrelated
transcription factors can elicit ligand-dependent transactivation via
appropriate reporter constructs.
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DNA binding is mediated by an internal domain (C) containing two Cys4 zinc fingers that bind DNA at HREs composed of two or more hexanucleotide half-sites oriented as inverted, everted, or direct repeats (20, 21, 22). The 68-amino acid DBD is the most highly conserved region of the nuclear receptor family and determines HRE preference and target gene selectivity. For some receptors, the DBD may also participate in receptor dimerization and transcriptional regulation (23, 24). With regard to the latter function, specific DBD deletions or mutations of steroid receptors have been identified that do not prevent DNA binding, yet greatly impair transcriptional regulation (9, 10, 11, 12, 23, 24, 25).
Transactivation by nuclear receptors likely involves the recruitment and stabilization of transcription complexes at target gene promoter sites (2, 4). The precise mechanisms by which nuclear receptors elicit such effects have not been fully defined. However, evidence suggests that coregulators may couple receptors to the transcription complex. For example, ligand-dependent activation by the ER can inhibit transactivation by progestin receptors (26). Such "squelching" implies the existence of a limited supply of one or more coregulators mediating receptor effects. Several laboratories have reported interactions between the LBD of receptors and proteins thought to act as coregulators (27, 28, 29, 30, 31). The highly conserved DBD of nuclear receptors could also serve as a site of interactions for coregulator proteins. We now report the identification of the nuclear protein TLS (translocated in liposarcoma) (32) [also known as FUS (33)] as a high-affinity binding protein for the DBD regions of retinoid, steroid, and thyroid hormone receptors. TLS is a member of the RNP family of RNA-binding proteins and was originally identified as part of a fusion protein arising from a reciprocal chromosomal translocation associated with human myxoid liposarcomas (32, 33). In the resulting fusion protein, the N-terminal half of TLS is joined to CHOP, a CAAT/enhancer binding protein (C/EBP)-related transcription factor with site-specific DNA-binding activity (34). This fusion protein appears to act as a potent chimeric transforming oncogene and transcription factor due to the association of a strong transactivation domain from TLS with the DNA-binding activity of CHOP (35). Recent studies indicate that certain RNP proteins may act to couple transcription to RNA processing (36, 37, 38). In addition, TLS, as well as a recently identified putative TAF (hTAFII68) with homology to the TLS RNP domain, have been shown to be associated with a subpopulation of transcription factor IID (TFIID) complexes (39). These properties of TLS, together with the nature of its interaction with nuclear receptors in vitro and in vivo, suggest a role for TLS in nuclear receptor function and raise the possibility of coupled transcription and RNA processing.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Initial experiments used GST fused to mouse retinoid X receptor-1
(mRXR) and unfractionated nuclear extracts from rat pituitary GH4C1
cells or HeLa cells that show high levels of stimulation by nuclear
hormone receptors (5, 6, 40, 41). Although many extracts revealed the
same highly specific interactions with a few select proteins, some
nuclear extracts bound GST-mRXR in an apparently nonspecific
fashion. To reduce nonspecific interactions and improve
reproducibility, GST fusion proteins were constructed containing
discrete portions of mRXR, and nuclear extracts were
fractionated on diethylaminoethyl (DEAE)- and carboxymethyl
(CM)-Sephadex (Fig. 1
). GH4C1 nuclear extracts contained an
RXR-interacting 65-kDa protein (p65) that passed through DEAE-Sephadex
but bound CM-Sephadex at 50 mM KCl and could be eluted from
CM-Sephadex at 250 mM KCl. At 150 mM KCl, p65
specifically bound to GST-mRXR containing the DBD (amino acids
140–205)(Fig. 1B). In particular, GST-mRXR-1–467, 1–205, 1–239,
and 140–240 all reproducibly bound p65, whereas no interaction was
obtained with the LBD (mRXR-206–467) or GST alone.
GST-mRXR-1–239 bound more p65 than either mRXR-1–205 or
140–240, suggesting that the N-terminal A/B domain and a small region
C-terminal to the DBD stabilized the interaction between the DBD and
p65. When the KCl concentration was reduced from 150 mM to
75 mM during protein-binding experiments,
GST-mRXR-1–239 binding to p65 was little changed, whereas
p65-binding by GST-mRXR-140–240 now approached that of
GST-mRXR-1–239 (Fig. 1C). In this experiment with 75 mM
KCl, smaller amounts of 50-kDa (p50) and 105-kDa (p105) proteins also
selectively bound to the GST-RXRs.
The selective binding of p65 to GST-mRXR was reproducibly detected
using CM-Sephadex eluates from four different preparations of GH4C1
nuclear extract. In one experiment, nuclei that had been extracted with
280 mM KCl were extracted a second time with 400
mM KCl (both extracts were processed using DEAE- and
CM-Sephadex). The second extraction at 400 mM KCl yielded
little additional p65 or other proteins capable of interacting with
GST-mRXR-1–239 or GST-mRXR-140–240 (Fig. 1C). Addition of the
RXR ligand 9-cis-retinoic acid (10 µM) during
incubations of GST-mRXR-1–467 with nuclear extract at 100
mM KCl did not alter the binding of p65 and did not elicit
a notable change in the binding of other nuclear proteins (data not
shown). The lack of a ligand effect on p65 binding to mRXR was not
surprising since the interaction is centered on the receptor DBD rather
than the LBD. Protein-binding experiments using GST fusions with human
ER (hER) indicated that the hER DBD (amino acids 185–250) also bound
p65 in GH4C1 nuclear extracts. In buffer containing 150 mM
KCl, GST-hER-1–282 bound p65 whereas GST alone or GST-hER-1–185 or
GST-hER-278–595, which do not contain the DBD, did not (Fig. 1D).
Figure 2 compares studies with extracts
of HeLa cells and rat GH4C1 cells. GST-mRXR proteins containing the
DBD bound p65 as well as two other proteins (p50 and p105) in both
extracts. In multiple experiments the ratio of p65 to p50 or p105
varied suggesting that the binding of p65 to GST-mRXR occurred
independently of the binding of p50 and p105. HeLa-derived p65 migrated
as a slightly larger protein than p65 from rat GH4C1 cells (less than
5-kDa apparent size difference) (Fig. 2). Inclusion of a
5-fold molar excess of a palindromic T3 response
element (TREp) relative to GST-mRXR-140–240 during
incubation with nuclear extract had little ef-fect on
GST-mRXR-140–240 binding to p65, and GST-mRXR-140–240 strongly
binds the TREp under such conditions (data not shown). Thus, DNA
binding does not appear to block the binding of p65 to
GST-mRXR-140–240. Indeed, even a 1000-fold molar excess of the
TREp did not disrupt binding of p65.
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-140–240 is a 36-kDa protein that is well separated in
gels from regions containing target proteins of interest for sequencing
analysis. p65 bound to GST-mRXR-140–240 at 75 mM KCl
was not removed by extensive washing at 100 mM KCl but
could be selectively eluted at 500 mM KCl (Fig. 3A). The eluted material could be
recovered and concentrated by ultrafiltration to yield a highly
purified protein (Fig. 3B), but losses were greater than 50%.
Nonetheless, the marked selectivity of GST-mRXR-140–240 for p65
indicates that this approach can be used for the large-scale isolation
of the protein in its native state from nuclear extracts.
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-140–240. After SDS-PAGE and Coomassie blue staining, gel
slices containing a total of 5–6 µg of p65 were electroeluted in the
presence of endoproteinase Glu-C, and the cleaved peptides were
resolved by HPLC. Two of the peptides were sequenced (Fig. 3C) and were
found to be 100% identical to amino acids 300–310 and 338–348 of
human TLS (32, 33). Human TLS is a 526-amino acid protein with a
predicted mass of 53,370 that migrates anomolously in SDS gels as a 65-
to 68- kDa protein (32), in agreement with the approximate molecular
mass of 65 kDa assigned to the RXR-interacting protein.
Western blot analysis using a monoclonal antibody against the
C-terminal region of human TLS (32) was used to confirm the identity of
p65 as TLS. Using unfractionated GH4C1 nuclear extracts in 150
mM KCl, GST-mRXR-1–467, 1–239, and 140–240 bound a
TLS-immunoreactive protein of 65 kDa, whereas GST alone and
GST-mRXR-206–467 did not (Fig. 4A).
The amount of immunoreactive TLS bound by the various GST-mRXR
proteins paralleled the amounts of p65 bound as detected by Coomassie
blue staining (see Fig. 1B). Similarly, GST-hER-1–287 and GST fused to
chicken thy-roid hormone receptor- (cTR-1–408) bound
immunoreactive TLS from GH4C1 cells while GST-hER-1–185
did not (Fig. 4B). Finally, GSTmRXR-1–239 and GST-hER-1–287
bound immunoreactive TLS from unfractionated HeLa nuclear extracts,
whereas GST-mRXR-206–467 and GST-hER-1–185 did not (Fig. 4C). The
Western blot data confirm that TLS from rat GH4C1 cells and human HeLa
cells strongly interacts with GST-receptor fusions containing the DBD
of either retinoid, thyroid hormone, or estrogen receptors.
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synthesized in vitro (Fig. 5).
TLS-1–274 corresponds to the TLS region that yields a potent
transactivation factor and transforming oncogene when fused to CHOP
(35). This TLS region contains an N-terminal domain rich in glutamine,
serine, and tyrosine [amino acids (aa) 1–165] as well as a
glycine-rich domain containing five poly-Gly tracts and six Arg-Gly-Gly
repeats (aa 166–274)(32). TLS-275–526 contains a putative
RNA-recognition (RNP) motif (aa 287–372) and a second Gly-rich domain
with four poly-Gly tracts and 13 Arg-Gly-Gly repeats.
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protein. GST-TLS-1–274, however, bound
[35S]cTR-1–408 and 1–151 with high affinity. In
contrast, GST-TLS-275–526 exhibited a much lower affinity for these
cTR proteins. Both GST-TLS proteins bound
[35S]cTR1–118 and 51–154 with low affinity (binding
of GST-TLS-275–526 to [35S]cTR1–118 was evident in
autoradiographs but was too weak to detect in photographic
reproductions). Neither GST-TLS protein bound
[35S]cTR-120–408 (which includes the entire LBD)
(Fig. 5B). These data suggest that TLS-1–274 binding to cTR is
centered around the DBD (aa 51–118) with the immediate flanking
regions also participating in the interaction. The finding that both
GST-TLS proteins exhibited a similar low affinity for
[35S]cTR-1–118 and 51–154 indicates that cTR
regions 1–51 and 119–151 do not appear to contribute to TLS-275–526
interactions with [35S]cTR. Overall, the ability of
both TLS-1–274 and 275–526 to bind [35S]cTR, and the
varying strengths of their interactions, suggests a potential role for
the poly-Gly TLS domains in [35S]cTR binding.
T3 did not alter the affinity of [35S]cTR
for either GST-TLS-1–274 or 275–526 at KCl concentrations of 100 or
200 mM (Fig. 6A). The
strength of the interaction of GST-TLS-1–274 with
35S-cTR was further assessed by conducting the binding
reaction for 60 min at 250 mM KCl, followed by an
incubation with varying KCl concentrations for 30 min. KCl
concentrations as high as 1 M at pH 7.9 or 700
mM at pH 6.0 (to partially neutralize acidic residues in
cTR) did not dissociate [35S]cTR bound to
GST-TLS-1–274 (Fig. 6B), indicating a stable high-affinity
complex.
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binding
to GST-TLS-1–274 was compared with that for GST-mRXR
proteins because RXR is known to form a high-affinity interaction with
TR (42, 43, 44) (Fig. 7). Remarkably, the
affinity of cTR for GST-TLS-N-1–274 was greater than for
GST-mRXR-206–467, which comprises the LBD of mRXR and contains
an essential region required for the formation of TR-RXR heterodimers
(43, 44). GST-mRXR-1–239 also bound [35S]cTR, and
the interaction was only slightly weaker than that found with
GST-mRXR-206–467 (Fig. 7). This result was of interest because the
DBD of RXR has been reported to contribute to the formation of TR-RXR
heterodimers on direct repeat DNA elements (45, 46, 47). A comparison of
the binding of GST-mRXR-1–467, GST-mRXR-1–239, and
GST-mRXR-206–467 for cTR indicated that the N-terminal half of
mRXR (aa 1–239) and the LBD (aa 206–467) synergize to enable the
formation of stable, high-affinity cTR-mRXR heterodimers in
solution. The salt stability of cTR-TLS-N complexes was similar to
that of cTR-mRXR-1–467 complexes. Thus, the affinity of TLS for
cTR is similar to mRXR, a known physiological binding partner for
cTR.
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, cTR, and hER are members of the receptor superfamily
that bind to HREs containing consensus AGGTCA half-sites. Binding of
the rGR DBD by TLS was evaluated to determine whether receptors with
differing HRE half-site specificity (AGAACA for the rGR) could also
interact with TLS. Recombinant rGR-440–525 protein (encoding the DBD
and 24 additional residues toward the C terminus) bound GST-TLS-1–274
at 50, 100, or 200 mM KCl. GST alone did not bind
rGR-440–525 (Fig. 8). At 100
mM KCl, 20 pmol (1.5 µg) GST-TLS-1–274 bound
approximately 16 pmol (240 ng by scanning densitometry) of the 1.4-µg
rGR-440–525 input. This is a near-stoichiometric interaction of
rGR-440–525 with GST-TLS-1–274. Thus, TLS appears to display high
affinity for members of the nuclear receptor family with widely varying
ligand and HRE specificities.
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Can Bind DNA Response Elements While Bound to TLS-140–240 suggests
this possibility. To study this more directly, we examined the ability
of cTR to interact with 32P-labeled DNA encoding a DR4
HRE ([32P]DR4) while tethered to GST-TLS-1–274 (Fig. 9). Agarose beads containing GST alone,
GST-TLS-1–274, or GST preincubated with cTR did not bind
significant amounts of [32P]DR4. However, preincubation
of GST-TLS-1–274 with soluble cTR led to a striking 24-fold
increase in the amount of [32P]DR4 bound by
GST-TLS-1–274. Addition of T3 during the incubations did
not alter the amount of [32P]DR4 bound. Thus, it appears
that the interaction of TLS with members of the nuclear receptor family
may enable TLS to be targeted to specific gene promoters in concert
with the nuclear receptor.
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with TLS
and TLS in the cell. Immune precipitation of cell lysates was not
revealing. However, we found that various anti-cTR antibodies did
not immunoprecipitate cTR-TLS complexes in vitro. In
addition, these antibodies blocked the interaction of
[35S]cTR with GST-TLS-N or [35S]TLS with
GST-cTR in vitro, suggesting that TLS interferes with
antibody recognition of the complex. Thus, we used pEBG-cTR-1–408,
a mammalian GST-cTR-1–408 expression vector, to provide evidence
for TLS binding after expression of receptor in cells. 293T cells were
used in these studies since the promoter in the pEBG vector has been
shown to express GST-fusion proteins in these cells (48). In
transfection studies, GST-cTR-1–408 was found to be as active as
wild-type cTR-1–408 in mediating basal repression or
ligand-dependent activation of 
MTV-TREp-CAT. For analysis of TLS
interactions, cells were transfected with pEBG-cTR-1–408 or the
pEBG GST control vector and 40 h later T3 was added to
half of the flasks for 1 h. The cells were then harvested and the
cytosol and nuclear fractions prepared by the method of Dignam et
al. (49). Unfractionated nuclear extracts and cytosol were
incubated with glutathione-agarose for 1 h at 4 C. The beads were
then washed, boiled in SDS loading buffer, and analyzed for GST or TLS
by Western blotting. Figure 10A shows
that nuclear extracts contain similar amounts of GST or
GST-cTR-1–408 after transfection with the indicated pEBG vectors.
However, GSH-agarose binding only detected immunoreactive TLS in the
nuclear extracts of cells expressing GST-cTR-1–408 (Fig. 10B). As
with the in vitro binding studies, T3 did not
alter the TLS-cTR interaction. GST immunoreactivity was also present
in the cytosolic fraction. However, GST-cTR-1–408 was not detected,
and no TLS was identified in the binding assay (not shown). Although
these studies support the notion that cTR and TLS can interact
in vivo, we cannot exclude the possibility that TLS and
GST-cTR-1–408 associate in vitro after the nuclear
extraction.
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containing the DBD in vitro, we sought to determine whether
this interaction could occur in the intact cell. Transfection studies
were performed with pcDNA vectors expressing wild-type TLS, TLS-N, and
TLS-C using CaPO4 coprecipitation. These studies were
performed in an effort to identify a dominant negative effect or other
evidence for a functional interaction of cTR with TLS. Because
TLS is a highly abundant protein, we used 30 µg of these expression
vectors to transfect HeLa cells or 293T cells to express these proteins
at levels higher than the endogenously expressed TLS. Although we could
not express TLS at levels higher than endogenous TLS, we were able to
express TLS-N or TLS-C at levels that were at least 10-fold greater
than endogenous TLS (established by Western blotting). In those
experiments (in the absence of cTR), TLS expression mediated a
modest reduction in basal gene activity, TLS-C had no effect, and TLS-N
markedly increased expression (
20-fold) of MTV-TREp-CAT or
SV-DR4-CAT. Similar results were also found with Rat2 fibroblasts.
The mechanism of promoter stimulation mediated by TLS-N was not
established but may reflect its association with basal transcription
factors when expressed at very high levels. This high level of promoter
activity evoked by TLS-N precluded its use as a dominant negative probe
for analysis of TLS-cTR interactions in vivo. Expression
of TLS-C did not alter T3-dependent stimulation by
cTR.
Because of the marked stimulation mediated by expression of high levels
of TLS-N, we used a mammalian two-hybrid approach to provide evidence
for a TLS-receptor interaction (Table 1).
GAL4-TLS-N is known to be a potent transactivator (35). An amount of
GAL4-TLS-N vector was used (0.2 µg) that gives moderate stimulation
to assess whether the various cTR proteins could interact with
GAL4-TLS-N in vivo and, thus, alter its activity.
Transfection of HeLa cells with GAL4-TLS-N, but not the GAL4-DBD,
activated gene expression from pMC110, a GAL4-CAT reporter gene.
Expression of cTR(120–408), containing only the LBD, did
not alter the extent of stimulation by GAL4-TLS-N (Table 1). In
contrast, wild-type cTR(1–408) repressed activation by GAL4-TLS-N,
and this repression was largely reversed by T3. This
finding is consistent with the notion that cTR interacts with TLS
via its DBD and that a transcriptional inhibitor(s) binds to the LBD of
TR and is released by T3 to unmask the TLS-dependent
transactivation (50). Two recently cloned factors (SMRT and NcoR) are
candidates for this corepressor activity (51, 52). Additional evidence
to support an interaction of the N-terminal region of cTR containing
the DBD with the N terminus of TLS comes from studies with
cTR(1–221)VP16. This chimera lacks most of the LBD and, therefore,
would not be expected to associate with transcriptional repressor
proteins. Thus, an association of cTR(1–221)VP16 with GAL4-TLS-N
would not result in repression but would be expected to lead to further
activation via the VP16 activation domain. Table 1 shows that
expression of cTR(1–221)VP16 does not alter the activity of the
GAL4-DBD but further enhances activation by GAL4-TLS-N, providing
further evidence that cTR and TLS can interact in the cell.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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deletion mutants to GST-TLS, involves the receptor DBD with
closely flanking regions contributing to the stability of the complex.
TLS binds to nuclear receptors primarily through its N-terminal half.
Experiments with full-length [35S]cTR indicated that
TLS binding is stable in 1 M KCl (Fig. 6); thus, TLS
binding is unlikely to reflect a simple ionic interaction with acidic
elements of receptor DBDs. Indeed, the C-terminal half of TLS,
containing more than 75% of its basic residues and its most
hydrophilic domains, displayed low affinity for cTR. TLS binding to
nuclear receptors also displayed high specificity. Only two other
proteins (p50 and p105) exhibited binding to GST-mRXRß approaching
that of TLS. Studies are in progress to identify and/or clone p50 and
p105, which do not react with TLS-monoclonal antibody (Fig. 4) or
polyclonal antibody (not shown). Finally, TLS from either rat or human
cell lines bound nuclear receptors from diverse species (mouse,
chicken, human, and rat). Thus, the interaction between TLS and nuclear
receptors appears to have been highly conserved among
vertebrates. TLS is one of only a few proteins identified to interact with the DNA-binding region of either steroid, thyroid, or retinoid receptors without interfering with DNA binding. Calreticulin has been reported to interact with the DBD of the GR and interfere with receptor activity when expressed in vivo (53). c-Jun and the p53 tumor suppressor have been reported to decrease TR DNA binding and transactivation via interaction with the DBD (54, 55). In contrast, like TLS, the human immunodeficiency virus type 1 (HIV-1) tat transactivator binds the TR DBD without altering DNA binding (56), and this interaction results in enhanced T3 stimulation of the HIV-1 long terminal repeat (56, 57). The structural conservation of the DBD of nuclear receptors may allow for an interaction of diverse members of the receptor family with a common binding protein (i.e. TLS). However, the regions flanking the DBD in the TRs and RXRs also appeared to contribute to TLS binding. Unlike the DBD, these flanking regions are quite variable among nuclear receptors but may nonetheless possess common structural features affecting DBD conformation and TLS binding.
TLS (32) [also known as FUS (33)] was originally identified as part of a fusion protein arising from a chromosomal translocation in human myxoid liposarcomas (32, 33). In this protein, the N-terminal half of TLS is fused with the open reading frame of CHOP, a member of the C/EBP family of transcription factors. In certain human myeloid leukemias, the N-terminal half of TLS is fused with the DBD of ERG (a member of the ets family of transcription factors) (58). Human TLS is a 526-residue protein that is closely related to EWS (656 residues, 55.6% identity) (32). The N-terminal half of EWS has been identified in translocation-induced fusions with different transcription factors in Ewing sarcomas (FLI-1, ERG, ETV-1) (59, 60, 61), a C/EBP family member (ATFI) in malignant melanoma of soft parts (62), the Wilm’s tumor gene product (WT1) in desmoplastic small round cell tumors (63), and an orphan member of the steroid/thyroid receptor family (CHN) in myxoid chondrosarcoma (64). A feature of all tumor-derived TLS and EWS fusion proteins is the fusion of the N-terminal half of TLS or EWS with transcription factor domains containing site-specific DNA-binding activity.
The functional role(s) of wild-type TLS or EWS remains to be
determined. However, analysis of TLS and EWS fusion proteins indicate
that their N-terminal regions possess a potent transactivation domain
rich in Gln, Tyr, and Ser residues (35, 65, 66, 67). It appears that fusion
of such domains with transcription factor DBDs leads to aberrant
transcriptional regulation and cell transformation. In view of such
findings, the identification of TLS as a high-affinity binding protein
for the nuclear receptor family of transcription factors suggests a
role for TLS in nuclear receptor function. In particular, in
vitro studies with GST-TLS-1–274 revealed that TLS-1–274 lacks
intrinsic affinity for a DR4 HRE but can associate with the HRE
via a TLS-cTR complex. Thus, TLS binding to nuclear
receptors may tether its transactivation domain to specific gene
promoters where further interactions may occur relevant to
transcriptional regulation. Consistent with this model are studies
indicating that TLS and cTR may physically and functionally interact
after expression in vivo (Fig. 10 and Table 1). Because of
the high abundance of TLS, a more definitive analysis of functional
TLS-cTR interactions will require the use of TLS-deficient cell
lines and/or TLS knockout mice.
TLS Is a Member of a Nuclear RNA-Binding Protein Family That May
Act to Couple Transcriptional Activation with RNA Processing
TLS and EWS appear to comprise a distinct subfamily of RNP
proteins (32, 33, 35, 59) that has been highly conserved through
evolution because a closely related gene (SARFH) is expressed in
Drosophila (68, 69). Like other RNP proteins (70), TLS and
EWS contain a RNP domain and two regions with multiple ArgGlyGly
repeats and bind RNA in vitro (32, 68, 71). The RNA-binding
activity of TLS is primarily contributed by its C-terminal half
containing the RNP domain and numerous ArgGlyGly repeats (32). Thus,
nuclear receptor binding and RNA binding appear to be mediated by
separate domains. In view of TLS and EWS similarities, it is notable
that Western blot analysis did not reveal EWS binding to GST-receptor
fusions even though substantial EWS was present in nuclear extracts
(data not shown). GST-receptor fusions also did not bind hnRNP-Al,
an abundant RNP protein with features reminiscent of TLS. Thus, TLS may
be unique among known RNP proteins in its high affinity for nuclear
receptors.
TLS and SARFH can be detected in intimate association with transcriptionally active chromatin and have been suggested to participate in transcriptional regulation and/or hnRNA processing in concert with other RNP proteins (35, 68, 72). In addition, TLS, as well as a recently identified putative TAF (hTAFII68) with homology to the TLS RNP domain, have been shown to be associated with a subpopulation of TFIID complexes (39). These findings suggest that TLS binding to nuclear receptors might serve as a priming mechanism to recruit TLS to the TFIID complex or to accelerate the subsequent processing of primary transcripts generated in response to hormone-induced transcription. This role would be consistent with recent studies indicating that certain RNP proteins may act to couple transcription to RNA processing (36, 37, 38).
Interplay of the A/B domain, DBD, and the LBD in Ligand-Mediated
Transcriptional Activation
The present studies were undertaken to identify potential
coregulators that may participate in the transcriptional actions of
nuclear receptors. The identification of TLS as a potential coregulator
is promising in view of the evidence indicating that TLS is localized
at transcriptionally active genes (68) and a subset of TFIID complexes
(39) and generates a potent transcription factor when fused with
disparate DNA-binding proteins. However, our identification of the
receptor DNA-binding region as the site of interaction was somewhat
unexpected because numerous studies have indicated a role for the LBD
in hormone-induced transactivation. Thus, the LBDs of nuclear receptors
can effectively mediate ligand-dependent transactivation when coupled
to heterologous DBDs derived from transcriptional activators such as
GAL4 (6, 8). Nonetheless, N-terminal domains of many nuclear receptors
possess a transactivation region (AF-1) distinct from that in the LBD
(AF-2). The GR, ER, and androgen receptor become constitutive
transactivators after removal of their LBDs, suggesting that the LBD
may mask the transcriptional effects of AF-1 in a ligand-dependent
manner (9, 10, 11, 12, 14, 15). Many of these studies also suggested a role for
the DBD in transcriptional regulation distinct from DNA binding
per se. Indeed, studies in yeast have identified mutations
in the GR DBD that interrupt transactivation without altering DNA
binding (23, 24, 25). Thus, it has been suggested that the DBD and the
N-terminal AF-1 domain may interact to mediate some of the
transactivation produced by nuclear receptors. In this regard, it is of
note that zinc finger DBDs of some other transcription factors have
been implicated in a transcriptional role distinct from DNA binding
(73, 74, 75).
In the ER, activity of the N-terminal AF-1 domain is ligand-dependent
(see Introduction), and DNA binding by retinoic acid
receptors can modulate LBD interactions with putative corepressor
proteins (76). Such findings imply reciprocal interactions between the
LBD, DBD, and N-terminal A/B domains. The interaction of TR and
retinoic acid receptors with large corepressor proteins (N-CoR, 270
kDa; SMRT, 168 kDa) (51, 52) involves a region of the LBD that closely
flanks the DBD. Ligand binding leads to dissociation of corepressors
from these receptors (50, 51, 52), which may permit various coregulators to
participate in transactivation. Thus, corepressors may suppress the
activity of the receptor-TLS complex, and ligand-evoked dissociation of
corepressors from the LBD may relieve this repression. Indeed, actions
of wild-type cTR to block transactivation mediated by GAL4-TLS-N,
and the reversal of this repression by T3 (Table 1), are
consistent with this model. In some respects, it might be anticipated
that the most highly conserved region of the nuclear receptor family
(the DBD) may play a role in transcriptional regulation distinct from
its role in DNA binding. The structural conservation of the DBD may
enable a single coregulator targeted to this domain to couple a wide
array of receptors with differing ligands to identical components of
the transcription complex. The structural variability among receptor
LBDs and N-terminal A/B domains, on the other hand, suggests that a
number of coregulators may be required to mediate such transcriptional
roles. Identification of TLS as a high-affinity binding protein for
nuclear receptors may facilitate efforts to understand the interplay
between the DBD and other receptor domains in transcriptional
regulation by nuclear receptors.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
, which expresses a fusion protein between GST and
wild-type mRXR-1–467, was provided by Paul T. van der Saag (28).
GST fusions with deletion mutants of mRXR (mRXR-1–205,
mRXR-1–239, mRXR-140–240, and mRXR-206–467) were
constructed by PCR amplification of mRXR using 5'-primers containing
a BglII site linked to the first codon of mRXR in-frame
with pGEX2T, and a 3'-primer ending with an in-frame stop codon
extended with an EcoRI site.
BglII-EcoRI digests of PCR products were cloned
into the corresponding sites of
BamHI-EcoRI-digested pGEX2T.
pGEX2T-cTR-1–408 expresses GST fused with wild-type chicken thyroid
hormone receptor (cTR)(41). pGEX vectors expressing GST fusions
with regions of the human estrogen receptor (hER) (GST-hER-1–185;
GST-hER-1–287; GST-hER-278–595) were provided by Peter J. Kushner
(77) or Myles Brown (27). GST linked to the N-terminal and C-terminal
regions of human TLS were constructed by David Ron (manuscript
submitted). GST-TLS-1–274 was cloned using an artificially created
BamHI site in the 5'-untranslated region of TLS cDNA (32)
and a unique internal BspHI site in the coding sequence.
GST-TLS-275–526 was cloned using the same BspHI site in the
coding sequence and an XhoI site at the 3'-end of the TLS
cDNA.
pEBG is a vector regulated by the human elongation factor 1-
promoter that expresses GST in mammalian cells and has been used to
express GST-fusion proteins in 293T cells (48). The entire
cTR-1–408 was excised from pGEX2T-cTR-1–408 with
BamHI and was cloned into the analogous BamHI
site of pEBG to form pEBG-cTR-1–408.
pEXPRESS vectors containing the Rous sarcoma virus long terminal repeat
linked to a phage T7 RNA polymerase promoter (78) were used for
in vitro transcription and translation of cTR-1–408,
cTR-1–118, cTR-1–151, cTR-51–154, and cTR-120–408 (41).
cTR(1–392)VP16 was constructed from wild-type cTR in pEXPRESS by
excising the DNA encoding the last 16 amino acids of cTR with
SacI and AfI III and replacing the DNA with the
SacI-AfIIII fragment from GAL4-VP16 (50). This
links amino acid 392 of cTR in-frame with the 90-amino acid
transactivation domain of the Herpes simplex virus VP16 activator.
cTR(1–221)VP16 was constructed by blunt-end ligation after the
NaeI and SacI fragment were deleted from
cTR(1–392)VP16. This removes most of the LBD of cTR. pSG424
expressing the yeast GAL4 DBD and pMC110, a GAL4 chloramphenicol
acetyltransferase (CAT) reporter gene, were described previously (79).
GAL4-TLS-N contains the N-terminal region (aa 1–267) of TLS linked
in-frame to the GAL4-DBD in pSG424 (35).
Experiments with the rGR DBD (rGR-440–500) used a recombinant peptide prepared as previously described from a plasmid encoding the rGR region 440–525 (80).
Preparation of Nuclear Extracts
HeLa or GH4C1 cells were grown to 70–80% confluence in DHAP
medium (50, 56) containing 10% calf serum. Cells were harvested using
1.5 mM EDTA in Dulbecco’s PBS. After cell detachment, the
EDTA was neutralized with an equal volume of serum-free DHAP medium.
All subsequent steps were conducted at 0–5 C using a procedure similar
to that of Dignam et al. (49). Cells were pelleted at
500 x g for 10 min and washed once in PBS and once
with 20 volumes of hypotonic lysis buffer (10 mM Tris-HCl,
pH 7.8, 10 mM KCl, 1 mM MgCl2). The
cells were resuspended in four packed-cell volumes of hypotonic lysis
buffer and disrupted in a Dounce homogenizer with a loose-fitting
("B") pestle. Nuclei were pelleted at 2000 x g
and, after the supernatant was removed, the nuclear pellet was
recentrifuged at 10,000 x g for 15 min. The nuclei
were resuspended in 2 volumes of nuclear extraction buffer (10
mM Tris-HCl, pH 7.8, 420 mM KCl, 25% glycerol,
1 mM dithiothreitol (DTT), 1 mM PMSF) to yield
a final KCl concentration of about 280 mM. Nuclei were then
homogenized with a Dounce B-pestle, and the homogenate was gently
stirred for 30 min to release soluble proteins from nuclei. After
centrifugation at 15,000 x g for 25 min, the
supernatant was dialyzed for 3–5 h against 20 mM Tris-HCl,
pH 7.8, 100 mM KCl, 25% glycerol, 1 mM DTT,
0.5 mM PMSF. The buffer was changed once. The dialyzed
nuclear extract was centrifuged for 15 min at 13,000 x
g to remove any precipitate, and the final supernatant was
stored as aliquots at -70 C until use. Protein concentrations of
nuclear extracts ranged from 3–5 mg/ml for HeLa cells and 6–9 mg/ml
for GH4C1 cells.
Nuclear extracts were usually fractionated on DEAE-Sephadex and CM-Sephadex before use in protein-binding assays using GST-fusion proteins. The extracts were diluted in 20 mM Tris-HCl (pH 7.8) containing 10% glycerol and 2 mM DTT to give a final KCl concentration of about 50 mM. After centrifugation (15 min at 13,000 x g in a microfuge), diluted extracts (3–4 mg total protein in 4–5 ml) were applied to microcolumns containing 0.5–1.0 ml of packed DEAE-Sephadex A-50 equilibrated in elution buffer (EB) containing 20 mM Tris-HCl, pH 7.8, 50 mM KCl, 2 mM DTT, and 10% glycerol. The flow-through fraction was collected and combined with a second elution with one column volume of EB. This material was then applied to 0.5- to 1.0-ml columns of CM-Sephadex C-50 equilibrated in EB. After a wash with three to four column volumes of EB, bound proteins were eluted with three column volumes of EB containing 250 mM KCl. This fraction was then diluted with buffer containing 20 mM HEPES (pH 7.9), 10% glycerol, and 2 mM DTT to give the indicated concentration of KCl and adjusted to contain 1 mM MgCl2, 10 µM ZnCl2, 0.05% Triton X-100, and 40 µg/ml leupeptin.
Preparation of GST Fusion Proteins
Escherichia coli expressing GST-fusion proteins
was incubated with 1 mM
isopropyl-ß-D-thiogalactopyranoside for 25 min at 37 C.
Cultures were then chilled in ice for 10 min and centrifuged at
2000 x g for 10 min at 5 C, and the supernatant was
discarded. All subsequent procedures were conducted at 0–5 C. Cell
pellets from each 500-ml culture were resuspended in 25 ml PBS
containing 40 µg/ml leupeptin, 20 µg/ml pepstatin, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 40 mM EDTA. Cells
were incubated with 400 µg/ml lysozyme for 10 min on ice and then
disrupted using a cell sonicator (125 watts for 10–15 sec, repeated
three times). The samples were then centrifuged at 10,000 x
g for 15 min, and the supernatant was then incubated with
300–500 µl of a 1:1 slurry of GSH-agarose beads in PBS for 20 min.
The GSH beads were then washed three times in 50 ml PBS: the last wash
included 2 mM DTT and 50 µM ZnCl2
to reform any denatured zinc finger domains present in the GST-receptor
fusion proteins. The samples were resuspended in an equal volume of
glycerol and stored at -20 C until use. Storage in 50% glycerol at
-20 C does not alter GST-fusion protein binding to GSH-agarose and
prevents proteolytic degradation of fusion proteins that may occur upon
storage at 5 C.
Protein-Binding Assays
Nuclear extracts, chromatography fractions, or in
vitro transcription and translation products were diluted in
protein-binding buffer (PBB) (20 mM HEPES, pH 7.9, 100
mM KCl, 1 mM MgCl2, 10
µM ZnCl2, 2 mM DTT, 10%
glycerol, 0.05% Triton X-100, 40 µg/ml leupeptin). In some
experiments, KCl concentrations were varied as noted. In
protein-binding assays using nuclear extracts or purified rGR-DBD, 1–2
µg of GST-fusion protein bound to GSH-agarose (7.5–15 µl) were
used. In experiments comparing the protein-binding efficiencies of
different GST-proteins, GSH-agarose was added as needed to yield
equivalent pellet volumes. The samples were rotated for 1 h at 5 C
with 1 ml of nuclear extract (300–750 µg protein/ml). Pellets were
centrifuged at 1000 x g for 4 min, washed two or three
times with 1 ml cold PBB, and then stored at -20 C until analysis by
SDS-PAGE. The samples were then suspended in 2–3 volumes of Laemmli
sample buffer containing 100 mM DTT, heated for 5 min at
100 C, and electrophoresed in 10% or 12% SDS-polyacrylamide gels as
appropriate. In some experiments, nuclear extracts were incubated with
GST-RXR proteins with a palindromic HRE (TREp) for TRs and RXRs
(5'AGGTCA TGACCT-3') with HindIII cohesive
ends.
Protein-binding experiments with 35S-labeled proteins
labeled in reticulocyte lysates used 400–500 ng GST-fusion protein in
a packed agarose volume of 5–6 µl.
L-[35S]cysteine-labeled cTR proteins were
prepared using 2 µg of appropriate pEXPRESS vectors and TNT
reticulocyte lysates (Promega, Madison, WI)(41). Approximately 1 µl
of 35S-labeled reaction product (from a total of 50
µl) was used in each binding assay. The incubations with reticulocyte
lysate-labeled proteins also contained 20 µg/ml of RNase A.
The formation of TLS-cTR-DNA complexes was studied using a DR4
oligonucleotide HRE (5'-AGGTCAcaggAGGTCA-3')
(flanked by HindIII cohesive ends), which was labeled to
high specific activity with [32P]CTP using Klenow
polymerase (81). GST alone or GST-TLS-1–274 (100–200 ng in 6 µl of
slurry of GSH-agarose) was rotated for 60 min at 5 C with PBB alone or
PBB containing an excess of purified cTR prepared as described
previously (81). After the beads were washed three times with 1 ml PBB,
the agarose beads were rotated for 30 min at 25 C with
[32P]DR4 and washed twice with PBB at 5 C, and the
pellets were counted for 32P.
Protein Sequencing
Protein bands of interest were cut from gels that had been
stained with Coomassie blue-R250. Gel slices were stored at -20 C
until about 5–6 µg of protein were collected. Protein bands were
then electroeluted from the gel slices in the presence of
endoproteinase Glu-C, and peptide fragments were resolved by HPLC and
subjected to automated sequence analysis as previously described (82, 83).
Western Blot Analysis
Gels were electroblotted to nitrocellulose membranes using a
semidry transfer apparatus, and the membranes were blocked and
incubated with primary antibody as previously described (84). Membranes
were probed for TLS immunoreactivity using monoclonal antibody IG10
(hybridoma supernatant, 1:10) directed against the C terminus of human
TLS (32). Immunoreactive bands were detected using rabbit anti-mouse
IgG-peroxidase and enhanced chemiluminescence (Amersham, Arlington
Heights, IL). Expression of GST or GST-cTR-1–408 in mammalian cells
using pEBG vectors was assessed by probing blots with anti-GST antibody
(Santa Cruz Biotechnology, Santa Cruz, CA).
Cell Culture and Transfection
HeLa cells were cultured and transfected by electroporation (41, 50, 81) with 5 µg of pMC110, a GAL4 reporter gene expressing
chloramphenicol acetyltransferase (CAT) alone or with the following
expression vectors as listed in Table 1; GAL4-DBD, GAL4-TLS-N,
cTR(1–408), cTR(120–408), or cTR(1–221)VP16. After
incubation for 48 h with or without T3, cells were
harvested for assay of CAT activity (41, 50, 81). The amount of protein
used in the assays was adjusted to keep the percent conversion of
[14C]chloramphenicol below 40%, which is in the linear
range. CAT activity values were normalized to represent the percentage
of [14C]chloramphenicol acetylated by a specific amount
of cell protein in 16 h at 37 C. All experiments were performed
using duplicate or triplicate flasks and were repeated at least three
times. Variation among duplicate or triplicate flasks was less than
10%. Human 293T cells were transfected with pEBG or pEBG-cTR-1–408
by CaPO4 coprecipitation as previously described (48).
Forty hours later, half of the flasks received 1 µM
T3 for 1 h, and the cells were then harvested for the
preparation of cytosol and nuclear extracts by the method of Dignam
et al. (49) as described earlier. Equal amounts of cell
protein (1 mg) were incubated with GSH-agarose for 1 h at 4 C,
and the GSH-agarose bound proteins were then analyzed by Western
blotting as described above.
|
ACKNOWLEDGMENTS |
|---|
, Peter Kushner and
Gabriela Lopez for GST-hER-1–185 and 1–282, Myles Brown for
GST-hER-278–595, Leonard Freedman for rGR-440–525, and Bruce
Mayer for pEBG. We thank Ron Beavis at the Seaver Mass Spectrometry and
Protein Chemistry Laboratory at the Skirball Institute of Biomolecular
Medicine at the New York University Medical Center (NYUMC) for protein
sequencing. |
FOOTNOTES |
|---|
This research was supported by NIH Grant DK-16636 to H.H.S. and a Senior Fellowship Award DK-09211 to C.A.P. D.R. is supported by NIH Grant CA-60945 and is a Pew Scholar in Biomedical Sciences and a Stephen Birnbaum Scholar of the Leukemia Society of America. Oligonucleotide synthesis was provided by the NYUMC General Clinical Research Center (NIH, NCRR, Grant M01RR00096). H.H.S. and D.R. are members of the NYUMC Cancer Center (Grant CA-16087). Sequence analysis and database searches were through the NYUMC Research Computing Resource which received support from the National Science Foundation (Grant DIR-8908095).
1 On sabbatical leave from the Department of Pharmacology, New York
Medical College, Valhalla, NY. 
Received for publication September 4, 1997. Accepted for publication October 3, 1997.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
is essential for
transcriptional activation and interaction with the general
transcription factor TFIIB. Mol Cell Biol 15:4507–4517[Abstract]
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|
|
|
|
|
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|
|
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|
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|
|
|
|
 |
|
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|
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|
|
|
|
|
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|
|
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|
|
|
|
|
|
A. Lerga, M. Hallier, L. Delva, C. Orvain, I. Gallais, J. Marie, and F. Moreau-Gachelin Identification of an RNA Binding Specificity for the Potential Splicing Factor TLS J. Biol. Chem., February 23, 2001; 276(9): 6807 - 6816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hakli, U. Karvonen, O. A. Janne, and J. J. Palvimo The RING Finger Protein SNURF Is a Bifunctional Protein Possessing DNA Binding Activity J. Biol. Chem., June 22, 2001; 276(26): 23653 - 23660. [Abstract] [Full Text] [PDF]
|
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