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J Biol Chem, Vol. 274, Issue 39, 27439-27447, September 24, 1999
From the Hubrecht Laboratory, Netherlands Institute for
Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
TGF- TGF- TSC-22 has been highly conserved during evolution. The rat and the
mouse genes are 100% identical at the amino acid level, while human
TSC-22 is 98.5% identical (9, 10). In addition, the Drosophila
melanogaster gene shortsighted (shs) or
bunched, which plays an important role in oogenesis, and in
eye, wing, and peripheral nervous system development, is very
homologous (68% identity) in the leucine zipper and adjacent
N-terminal region, which has been designated the TSC box (11, 12).
Another related gene is delta sleep inducing peptide
immunoreactive peptide (DIP), which was
isolated serendipitously and contains the conserved leucine zipper and
TSC box (13, 14). Recently, a synthetic peptide derived from the
porcine DIP gene was shown to homodimerize via this leucine zipper
(15).
Not much is known about the function of TSC-22 or any of its
homologues. Since it contains a leucine zipper, TSC-22 has been hypothesized as being a transcription factor (1). Supporting this,
nuclear localization was reported, although for the homologue shs cytoplasmic localization was observed (1, 11). However, TSC-22 does not belong to any of the known families of leucine zipper
transcription factors, and it does not contain a classical DNA-binding domain such as those in the bZip or bHLH-Zip families. It
has been hypothesized that TSC-22 might act as a repressor, by binding
other leucine zipper-containing transcription factors, such as members
of the AP-1 family, and inhibiting their DNA binding. Another report,
however, showed that TSC-22 could bind to a specific DNA sequence
in vitro (3).
Here, we report that TSC-22 forms homodimers via its leucine zipper
domain. We identify the family member TSC-22 homologous gene-1 (THG-1)
as another TSC-22 interacting partner. Both TSC-22 and THG-1 act as
transcriptional repressors when fused to the DNA-binding domain of
yeast transcription factor GAL-4. At least for TSC-22, this activity
does not reside in the dimerization domain, but it is influenced by the
presence of this domain. These data indicate that TSC-22 belongs to a
homo- and heterodimeric family of leucine zipper-containing factors
that repress transcription when sequestered to DNA.
Cell Culture and Transient Transfections--
Monkey COS-1 cells
were obtained from American Type Culture Collection (Rockville, MD).
Cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's
medium and Ham's F-12 medium (Life Technologies, Inc), buffered with
bicarbonate and supplemented with 7.5% (v/v) fetal calf serum from
Integro (Linz, Austria). For transient transfections, the cells were
cultured in 24-well tissue culture plates. Cells were transfected using
calcium phosphate coprecipitation with indicated amounts of luciferase
reporter, SV2lacZ, and expression plasmids. pBluescript
SK Plasmids--
Fig. 1 shows the TSC-22 and THG-1 fusion
constructs used. Details of the construction of the clones in this
paper can be obtained from the authors upon request. TSC-22 sequences
were obtained in part from American Type Culture Collection (Rockville,
MD) (GenBank accession no. T07973, EST05864; Ref. 17), and in part as a
polymerase chain reaction product from a differential display screen
(7). The entire open reading frame and 5'-untranslated region of both
clones was sequenced. Full-length THG-1 was obtained from American Type
Culture Collection (Rockville, MD; GenBank accession no. AA212193;
I.M.A.G.E. Consortium clone identification no. 725353; Ref. 18) and
sequenced completely. A reporter plasmid containing four NF In Vitro Protein-binding Assay (GST Pull-down)--
Extracts
were made from COS-1 cells cultured in six-well dishes and transfected
with 10 µg of expression plasmid. Cells were transfected by calcium
phosphate coprecipitation as described above, and harvested as
described before (19), in 30 µl 400 mM KCl, 20 mM Tris, pH 7.5, 20% (v/v) glycerol, 2 mM
dithiothreitol, protease inhibitors phenylmethylsulfonyl fluoride (1 mM), leupeptin, aprotinin, pepstatin (all 1 µg/ml), and
chymostatin (10 µg/ml). Alternatively, 35S-labeled
proteins were synthesized in vitro using the TnT coupled rabbit reticulocyte lysate system (Promega, Madison, WI) in the presence of [35S]methionine according to the
manufacturer's description.
Glutathione S-transferase fusion proteins were expressed in
Escherichia coli BL21(plysS). Expression and purification
with glutathione-coated beads (Amersham Pharmacia Biotech) was
performed as described (16). The fusion proteins, loaded on Sepharose beads, were mixed subsequently with in vitro synthesized
proteins or COS-1 extracts in binding buffer (250 mM NaCl,
50 mM Hepes, pH 7.5, 0.5 mM EDTA, 0.1% (v/v)
Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin), heated for 5 min at 42 °C, incubated for 2 h at 4 °C, washed
extensively, resuspended in sample buffer, and analyzed by
SDS-polyacrylamide gel electrophoresis or Western blotting with
anti-tag antibody 12CA5 as described (19).
Electrophoretic Mobility Shift Assay (Gel Shift)--
COS-1
cells were grown in six-well dishes and transfected with in total 10 µg of two expression plasmids and harvested as described above. A
GAL-oligo was end-labeled as described before (21). Whole cell extracts
(5 µl) were incubated with 10,000 cpm of probe (0.1-0.5 ng) and 1 µg of poly(dI-dC) for 30 min at room temperature in a total reaction
mixture of 20 µl containing 20 mM Hepes, pH 7.5, 100 mM KCl, 0.2 mM EDTA, 20% (v/v) glycerol, 1 mM dithiothreitol, 1 µg/µl bovine serum albumin.
Samples were loaded on a 4% polyacrylamide (29:1) gel, containing
0.5× TBE as running buffer.
Yeast Two-hybrid--
TSC-2238-144 was cloned in
the yeast expression vector pGBT8 by inserting the
BamHI-SacI fragment from galT38-144 in BamHI-SacI-digested pGBT8, and used as bait in
a yeast two-hybrid screen with the yeast strain YGH-1, which contains a
HIS3 and a lacZ reporter gene. The screen was
performed with a reamplified mouse brain cDNA library as described
(23, 24). Three to 10 days after transformation of yeast with 100 µg
of cDNA library, DNA from His+Gal+ colonies
was isolated and introduced in E. coli strain MH-4. All
clones were sequenced and reintroduced into the yeast cells, together
with the bait or with the empty vector pGBT8. Clones that gave rise to
HIS+LacZ+ colonies only
with bait were considered as true positives.
TSC-22 Forms Homodimers through Its TSC Box Leucine Zipper
Region--
Based on the sequence of the leucine zipper, TSC-22 may
form homodimers (12). To test this, we performed GST pull-down
experiments. In these experiments, we overexpressed a
gstT38-144 fusion construct in E. coli, from
which the protein can be easily purified with glutathione-coated beads.
The gstT38-144-bound beads were incubated with extract
from COS-1-cells transfected with a hemagglutinin-tagged TSC-22
construct (vpT38-144), and checked for
vpT38-144 that remained bound to the beads on Western blot
with an anti-tag antibody. Initially, no bound vpT38-144 was detected, but when the beads and extracts were incubated for 5 min
at 42 °C prior to the 2-h binding step at 4 °C, a specific interaction of vpT38-144 with gstT38-144 (and
not with GST alone), was observed. TSC-22 probably already forms dimers in the COS-1 cells and in E. coli, and these dimers first
have to be disrupted (e.g. by elevated temperature) before
any new dimers can be formed. The specificity control c-Jun (a leucine zipper-containing transcription factor of the AP-1 family) did not bind
specifically to gstT38-144 (Fig. 2A), while the longer constructs gstT7-144 and vpT7-144 gave
similar results as gstT38-144 and
vpT38-144, respectively (data not shown).
We also tested the ability of TSC-22 to form homodimers by means of a
mammalian two-hybrid approach. Different GAL4 DNA-binding domain
(GAL-DBD) constructs containing (parts of) TSC-22 (Fig. 1) were cotransfected with the expression
plasmid vpT38-144 (which contains, apart from the HA tag,
a VP-16 activation domain and amino acids 38-144 of TSC-22) or the
empty expression vector pSG5. Strong activation of a 5×GAL-TATA-luc
reporter construct (five GAL4 DNA-binding sites in front of an E1b TATA
box and luciferase reporter gene) was observed with
vpT38-144 but not with pSG5, indicating that TSC-22 can
form homodimers in vivo (Fig. 2B). When a deletion mutant
was used that only contained the TSC box and leucine zipper
(galT38-102) fused to the GAL-DBD, the reporter was still
strongly activated. With a construct in which the leucine zipper is
partially deleted (galT38-90), the activity was completely
abolished, indicating that this leucine zipper is indeed necessary for
homodimer formation (Fig. 2B).
We studied the ability of TSC-22 to homodimerize in gel shift assays.
In this assay, a radioactively labeled 17-bp oligonucleotide that
contains the GAL4 DNA-binding site (GAL-oligo) is incubated with
GAL-DBD fusion proteins. If this GAL-oligo is bound by a GAL-DBD fusion
protein and separated on a non-denaturing polyacrylamide gel, it runs
slower than an unbound GAL-oligo. After incubation of the GAL-oligo
with extracts from COS-1 cells that were transfected with
galT38-144 we clearly observed shifted bands (lanes 3 and 4) that were specific (competed by 100× excess
unlabeled GAL-oligo and not by non-related oligo; data not shown), and
not present in controls (incubations with extracts of COS-1-cells that
were not transfected with gal constructs; lanes 1 and
2). With extracts of COS-1 cells cotransfected with
galT38-144 and vpT38-144, we also observed a
clearly shifted band (Fig. 2C, lane 5,
band dimer). This band was supershifted upon
adding an antibody against the HA tag present in the
vpT38-144 construct (Fig. 2C, lane
6, band supersh.), while bands from
extracts containing galT38-144 only were not supershifted
(lane 4). This indicates that the supershifted
band must contain vpT38-144 bound to
galT38-144 (the antibody binds the
vpT38-144·galT38-144·GAL-oligo complex
and causes it to migrate slower), again demonstrating that TSC-22 can homodimerize.
Interestingly, we noted additional slowly migrating complexes in the
lanes with the galT38-144-containing extract (Fig. 2C, lanes 3 and 4). These
appear to be competed by vpT38-144, since in
lanes 5 and 6, containing
vpT38-144 in addition to galT38-144, they are
hardly or not visible. They do not arise from endogenous TSC-22 bound
to galT38-144, since this gives rise to faster migrating
complexes (not shown). Possibly, there are other endogenous partners
for TSC-22; some of them probably also bind via the leucine zipper.
Shibanuma et al. (1) suggested that TSC-22 might be able to
bind to members of the AP-1 family. We tried to show interaction with
AP-1 family member by means of direct interaction
(galT38-144 with c-Jun-VP16) or competition
(galT38-144 interaction with vpT38-144
competed by excess amounts of c-Jun, c-Fos, JunD, or JunB) in a
mammalian two-hybrid approach or with GST pull-down experiments (c-Jun
with gstT38-144), but we never found an indication for
interaction (results not shown and Fig. 2A). We conclude
that TSC-22 can homodimerize, and that there are indications that it
can also interact with other proteins, but these do not appear to be
members of the AP-1 family.
TSC-22 Forms Heterodimers with Its Homologue THG-1--
To try to
find such TSC-22 interacting partners, we carried out a yeast
two-hybrid screen. As a bait, we used amino acids 38-144 fused to a
GAL-DBD in a suitable yeast vector, and screened a mouse brain cDNA
library. Upon screening 40 × 106 transformants, we
obtained 36 HIS+ LacZ+
colonies. Upon retransformation in yeast, most of these proved to be
false positives. However, six of these initial candidates were
fragments from a TSC-22 homologue, which we designated
TSC-22 homologous gene-1 (THG-1).
All of these clones started at the same amino acid (and therefore were
probably derived from the same cDNA) and included the TSC box and
the leucine zipper. No other leucine zipper proteins were found in the screen.
The observed interaction between TSC-22 and the THG-1 fragment was
confirmed by GST pull-down and gel shift experiments (data not shown).
Furthermore, in some cell lines, both THG-1 and TSC-22 are expressed,
and, judged by immunofluorescence of COS-1 cells transfected with
HA-tagged constructs of these genes, a similar subcellular localization
was observed (mostly nuclear; results not shown). This suggests that
the endogenous proteins are in a position to interact in mammalian
cells; therefore, we cloned full-length THG-1. We identified and
sequenced a human expressed sequence tag (EST; see "Experimental
Procedures" for details) that contained an insert of 1990 bp (Fig.
3A). It contains an open
reading frame of 395 amino acids, which includes the TSC box and
leucine zipper region of THG-1, and which is preceded by a stop codon.
It has a predicted size of 41 kDa, and upon cell-free translation with
an expression construct containing this insert, two protein products of
approximately 45 kDa are formed (Fig. 3B). We do not know
the nature of the difference between these two bands; possibly there
are kinases active in the cell-free extract, or posttranslational
modifications or degradation are occurring. This protein was able to
bind specifically in a GST pull-down to TSC-22 (Fig. 3B). We
also made fusion constructs of full-length THG-1 with the GAL-DBD and
of GAL-DBD with the THG-1 fragment that interacted with TSC-22 in the
yeast two-hybrid screen (gal THG-1 and gal-2h). With these clones we
were able to show a strong activation of the 5×GAL-TATA-luc reporter
after cotransfection with vpT38-144 (the VP16-activation
domain-containing TSC-22 construct, Fig. 3C). This shows
that THG-1 is able to interact with TSC-22 in mammalian cells.
The Dimerization Domain Is Highly Conserved in the TSC-22 Family of
Leucine Zipper Proteins--
We searched GenBank protein data bases
using BLAST software with the TSC-22 and THG-1 protein sequences to
find conserved regions and to identify additional family members. Apart
from the known family members (TSC-22, THG-1, shs from D. melanogaster, human and pig DIP, and its probable mouse ortholog
GILZ; Refs. 11, 13, 14, and 25), we also found the uncharacterized human KIAA0669 protein and a hypothetical protein from
Caenorhabditis elegans, which had high homology in the TSC
box-leucine zipper region. We also searched EST data bases with the TSC
box-leucine zipper region (amino acids 44-122 of TSC-22), but we could
not find evidence for a fifth TSC-22 homologue in mammals. Therefore, at least four mammalian paralogues exist that belong to the TSC-22 family of leucine zipper proteins, and homologues exist even in the
distantly related species D. melanogaster and C. elegans.
An alignment of the TSC box-leucine zipper region of hTSC-22, THG-1,
KIAA0669, hDIP, shs-2, and the hypothetical C. elegans protein is shown in Fig.
4A.4 The central
leucines (and one valine) of the zipper
are all conserved (black residues). Furthermore, the charge
of the amino acids that are important for the dimerization specificity
of the leucine zipper (boxed residues) are conserved for the
mammalian paralogues and shs. This configuration predicts that these
proteins can homodimerize, or heterodimerize with any of the other
family members (12, 26), which we could indeed show for TSC-22 and
THG-1 (Figs. 2 and 3).
The conservation of the domains outside the TSC box-leucine zipper
region is very limited between paralogues. For TSC-22, these domains
are highly homologous in the chick, mouse, rat, and human orthologues,
but they are not conserved in the known paralogues THG-1, KIAA0669,
hDIP, or shs, apart from a few amino acids just C-terminal from the
leucine zipper (Fig. 4A). The N-terminal region of THG-1
does contain two regions with homology to some of its paralogues (Fig.
4, B and C), although we could not find clear
sequence motifs that would give a hint toward the function of these
regions. Apparently, the TSC-22 family is a family of leucine zipper
proteins with a highly conserved dimerization domain, which is coupled
to different N- and C-terminal domains that are only conserved in a
limited manner between paralogues.
TSC-22 and THG-1 Repress Transcription When Sequestered to
DNA--
Ohta et al. (3) showed that TSC-22 can bind
specifically to DNA in vitro. In order to investigate
whether some of the domains of TSC-22 may influence transcription,
i.e. may be activation or repression domains, we tested
constructs containing GAL-DBD with different parts of TSC-22 (see Fig.
1). Upon cotransfection with the reporter construct 5×GAL-TATA-luc in
COS-1, T47D, or 293 cells, we never found a significant transcriptional
activation (results not shown), showing that at least in these cells,
no independent activation domain in TSC-22 is active.
Next we tested whether TSC-22 may have transcriptional repressor
activity. We tested this on luciferase reporters containing four
NF
Many transcription factors, like unliganded retinoic acid receptor
Next we tested whether THG-1 has a similar transcriptional activity as
TSC-22. In cotransfections of galTHG-1 with the reporter construct
5×GAL-TATA-luc in COS-1, we did not find a significant transcriptional
activation (results not shown); however, on the 4i5g reporter, we found
a strong repressor activity of galTHG-1, comparable to that of
galTSC-22 (Fig. 5C). Apparently, both TSC-22 and THG-1 have
repressor activity and may contain independent repression domains.
TSC-22 Contains Independent Repression Domains in the Non-conserved
Regions That Are Enhanced by the Dimerization Domain--
We tested
different galTSC-22 deletion constructs on the 4i5g reporter, to see
whether TSC-22 contains separate repression domains (Fig.
6A). First we checked whether
size of the fusion protein mattered, but this did not seem to be the
case; the control galRAR
Although the TSC box leucine zipper region, the dimerization domain, is
not sufficient for repression, it apparently plays a role, since adding
this domain to the C-terminal repression domain increases the
repression (galT102-144 versus
galT38-144, Fig. 4C). When we interfere with
the dimerization of galT1-144 by overexpressing the
deletion protein tagT38-102 (which contains the TSC box
and leucine zipper region but not RD1 and RD2 and therefore can
homodimerize but not repress), the repressor activity of
galT1-144 is abolished (Fig. 6B), indicating an
important function for this domain. With full-length TSC-22, the
repressor activity is not influenced at all (Fig. 6B). This suggests that full-length TSC-22 homodimers have repressor activity, but if the repression domains of one of the partners are deleted, this
repressor activity is strongly reduced.
However, constructs in which the repression domains are dimerized
artificially (two copies of RD1 or RD2 in frame behind the GAL-DBD,
designated gal2×T7-90 and gal2×T102-144, respectively) are not or hardly more efficient in repressing the 4i5g
reporter than their single counterparts (Fig. 6C). This
argues against the hypothesis that the enhancing activity of the
dimerization domain on the repressor activity is solely due to
dimerization. Possibly, the TSC box-leucine zipper may have multiple
roles in enhancing the repression domains, which are all necessary to
enhance repressor activity. In conclusion, the repressor activity of
TSC-22 resides in the N- and C-terminally located repression domains, and is enhanced by the centrally located dimerization domain, but this
is probably not mediated solely through its dimerizing properties.
TSC-22 Is a Dimerizing Protein but Does Not Act on AP-1
Transcription Factors--
It has been suggested that TSC-22 might be
a repressor of AP-1 family members (1). Its mode of action would be
similar to that of basic helix-loop-helix protein Id or leucine zipper protein CHOP; TSC-22 might interact with AP-1 family members and inhibit their DNA binding, and in this way repress the function of
these transcription factors (30, 31). However, we did not find any
leucine zipper protein other than TSC-22 or its homologue that binds to
TSC-22, either in directed screening using GST pull-down or in
mammalian two-hybrid screening, or in random screening using the yeast
two-hybrid system. Therefore, we do not find any evidence for this
hypothesis. Instead, we show that TSC-22 dimerizes with its family
members, which strongly suggests that the endogenous protein will do
likewise. These data suggest that TSC-22 acts in an autonomous fashion,
and not by inhibiting DNA binding of AP-1 transcription factors through dimerization.
The Repressor Activity of TSC-22--
The repressor activity of
many repressors, like nuclear hormone receptors Mad or Rb, acts through
histone deacetylase-containing complexes and can be inhibited by the
histone deacetylase inhibitor TSA (27-29, 32-37). The activity of
TSC-22 is not inhibited by this compound, indicating that TSC-22 does
not repress transcription through this machinery. Furthermore, the
TSC-22 repressor activity is remarkable in that it is very sensitive to
promoter architecture, possibly due to distance and orientation effects.
The role of the dimerization domain is quite complex. It has an
enhancing role on the repressor activity, but does not actively repress
itself. Possibly, these repression domains act most strongly in a
dimeric configuration. This also explains a remarkable observation made
in the mammalian two-hybrid assays that the full-length TSC-22 constructs only weakly activate transcription, while deleting a
repression domain in only one of the two partners strongly increases activation.5 Note that an
exogenous activation domain is added to one of the partners in this
assay, hence the activation. However, only dimerization appears not to
be sufficient to enhance the activity of the repression domains.
Possibly, the dimerization domain has a second role, like inducing a
conformational change, that is also needed to enhance repressor activity.
TSC-22 Is a Member of a Family of Interacting Leucine Zipper
Proteins--
In this paper, we show that TSC-22 can dimerize and
repress transcription when sequestered to DNA. Ohta et al.
(3) showed that TSC-22 can bind to a specific DNA-sequence, while
Shibanuma et al. (1) reported a nuclear localization of
TSC-22. Apparently, TSC-22 is a repressive transcription factor. Here
we also show that the homologue THG-1 protein interacts with TSC-22,
has repressor activity, and a truncated THG-1 is detected in the
nucleus in COS-1 cells.4 This therefore suggests that not
only TSC-22 is a repressive transcription factor, but also some of its
family members, including THG-1.
The central region, consisting of the TSC box and leucine zipper, is
highly conserved between TSC-22 and its homologues THG-1, KIAA0669,
DIP, and shs. This region appears to be involved in directing the
protein to the appropriate intracellular compartment (mostly nuclear in
COS-1 cells),5 and is crucial in homodimerization of
TSC-22. The isolation of THG-1 as a TSC-22-interacting protein is
therefore not unexpected. Both TSC-22 and THG-1 can homo- and
heterodimerize with each other. Recently, it was reported that a
peptide derived from porcine DIP was also able to homodimerize (15).
Therefore, at least three of the five family members identified so far
are able to homodimerize, and at least two of them are able to bind to
each other. The residues that are theoretically important for
dimerization specificity are all conserved, and it would not be
surprising if all of the family members interact with one another.
Functional Consequences of TSC-22 Family Member
Interactions--
The repression domains of TSC-22 identified in this
paper are not conserved between family members. Frequently, no clear
sequence similarity exists between repression domains of different
repressors (38). This may be the case for the THG-1 and TSC-22
repression domains, although both contain regions that are rich in
prolines (which is often found in repression domains, 38). For one or
more of the other family members, it is, however, possible that they contain domains with totally different functions, e.g.
transcriptional activation domains. This would add additional
possibilities for regulating transcription, depending on the family
members expressed in a specific cell. In line with this thinking,
overexpression of solely the dimerization domain of TSC-22 severely
reduces the repressor activity, indicating that dimerization partner
may be important for TSC-22 repressor activity. Consequently, it is
possible that, upon binding another partner, TSC-22 changes from a
repressor into the silent partner of a positive acting complex.
Therefore, for determining the function of TSC-22, it is crucial to
investigate the function of the family members, since all of these may
be able to interact with TSC-22 and may therefore influence its activity.
We thank G .E. Folkers, A. Caricasole, and S. Wissink for discussion and advice, and C. L. Mummery for
critically reading the manuscript.
*
This work was supported by N. V. Organon, Oss, The
Netherlands.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ133115.
2
H. A. Kester and B. van der Burg,
unpublished observations.
4
The accession numbers of proteins are as
follows: human TSC-22, Q15714; human DIP, NP004080; human KIAA0669,
BAA31644; D. melanogaster shs, AAC41608; hypothetical
protein from C. elegans (T18D3.7), Q22544; and chicken
TSC-22, BAA11565.
5
H. A. Kester and B. van der Burg,
unpublished observations.
3
H. A. Kester, C. E. van den Brink, P. T. van der
Saag, and B. van der Burg, manuscript in preparation.
The abbreviations used are:
TSC-22, TGF-
Transforming Growth Factor-
-stimulated Clone-22 Is a
Member of a Family of Leucine Zipper Proteins That Can Homo- and
Heterodimerize and Has Transcriptional Repressor Activity*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-stimulated
clone-22 (TSC-22) encodes a leucine zipper-containing
protein that is highly conserved during evolution. Two homologues are
known that share a similar leucine zipper domain and another conserved
domain (designated the TSC box). Only limited data are available on the
function of TSC-22 and its homologues. TSC-22 is transcriptionally
up-regulated by many different stimuli, including anti-cancer drugs and
growth inhibitors, and recent data suggest that TSC-22 may play a
suppressive role in tumorigenesis. In this paper we show that TSC-22
forms homodimers via its conserved leucine zipper domain. Using a yeast
two-hybrid screen, we identified a TSC-22 homologue (THG-1) as
heterodimeric partner. Furthermore, we report the presence of two more
mammalian family members with highly conserved leucine zippers and TSC
boxes. Interestingly, both TSC-22 and THG-1 have transcriptional
repressor activity when fused to a heterologous DNA-binding domain. The
repressor activity of TSC-22 appears sensitive for promoter
architecture, but not for the histone deacetylase inhibitor
trichostatin A. Mutational analysis showed that this repressor activity
resides in the non-conserved regions of the protein and is enhanced by the conserved dimerization domain. Our results suggest that TSC-22 belongs to a family of leucine zipper-containing transcription factors
that can homodimerize and heterodimerize with other family members and
that at least two TSC-22 family members may be repressors of transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-stimulated clone-22
(TSC-22),1 a gene encoding a
leucine zipper protein, was first isolated as a TGF--responsive gene from a mouse osteoblast cell line (1). TSC-22 is up-regulated by many
stimuli that act via distinct signaling pathways: fibroblast growth
factor 2, epidermal growth factor, dexamethasone,
follicle-stimulating hormone, and different cytokines have all been
shown to induce the expression of the gene (1-4). Kawamata et
al. (5) reported that TSC-22 is up-regulated by an anti-cancer
drug, vesnarinone, in a salivary gland carcinoma cell line. Using an
antisense approach, they showed that TSC-22 has a growth inhibitory
effect on this cell line and that it reduces tumor formation in nude
mice. Moreover, TSC-22 is down-regulated in salivary gland tumors as
compared with normal salivary gland tissue (6). Recently, we carried out a differential display screen to identify progesterone target genes
in mammary carcinoma cells (7). Progestins are used to treat breast
cancer and can induce growth inhibition in the mammary carcinoma cell
line T47D by an unknown mechanism (8). We have found that TSC-22 is
induced by progestins in T47D cells but not in two responsive cell
lines that are not growth-inhibited by progestins
(7).2 Furthermore, we have
found that TSC-22, when overexpressed in a distinct tumor cell line,
also has a growth inhibitory
action.3 These results suggest
that TSC-22 may be a negative regulator of proliferation and may have
tumor suppressor activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
plasmid was added to obtain a total amount of 1.6-2
µg of DNA/well. After 16 h, the medium was refreshed. Cells were
harvested 24 h later and assayed for luciferase activity using the
Luclite luciferase reporter gene assay kit and a TopCount liquid
scintillation counter (Packard, Meriden, CT). Samples were corrected
for transfection efficiency by measuring -galactosidase activity as
described previously (16). Trichostatin A (ICN, Costa Mesa CA)
treatment was done for 24 h, with refreshment of medium and TSA
after 12 h.
B-binding
sites from the ICAM-1 promoter was kindly provided by S. Wissink and
the reporters TATA-luc and 5×GAL-TATA-luc by G. Folkers (19); the
250-bp fragment was cut by digestion with BamHI and
BglII from T7TS2 kindly provided by J. Joore. The pSG5-based
expression plasmid with the c-Jun (obtained from D. Nathans) coding
region inserted were provided by W. Kruijer. The GAL and ICAM sites
containing plasmids were constructed using these plasmids by standard
techniques; cloning details are available upon request. The CMV4
expression vector containing human RelA/p65 has been described
previously (20); all 4×ICAM-containing promoters were activated by
cotransfection of 20 ng of this expression vector, which induces these
reporters 200-, 1000-, 1500-, and 70-fold (for 4i5g, 5g4i, 5g-250-4i,
and 4i5g-250, respectively). The gal fusions were cloned in pSG424, GST
fusions in pGEX2T, tag fusions in pSG5-hemagglutinin tag vector (21,
19, 22), and vpT38-144 fusion in pSG5-hemagglutinin
tag-VP16 vector kindly provided by G. Folkers. galVP16, galRAR-EF,
and galRAR
were described before (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
[in a new window]
Fig. 1.
TSC-22 and THG-1 fusion constructs used.
gal, GAL4 DNA-binding domain; gst, glutathione
S-transferase; tag, hemagglutinin-tagged pSG5
construct; vp, hemagglutinin-tagged VP16-activation
domain-pSG5 construct; LZ, leucine zipper; RD1
and RD2, repression domains 1 and 2.
View larger version (16K):
[in a new window]
Fig. 2.
TSC-22 forms homodimers. A,
GST pull-down assay with GST and gstT38-144 to pull-down
HA-tagged-T38-144 construct (vpT38-144) with
35S-labeled c-Jun as specificity control. Bound
vpT38-144 was detected by Western blotting with -tag
antibody 12CA5. 35S-Labeled c-Jun was detected by
autoradiography. B, mammalian two-hybrid assay with
gal-TSC-22 constructs (200 ng) and TSC-22 construct
vpT38-144 or empty vector pSG5 (200 ng) in COS-cells.
Values from three experiments (in duplicate) were normalized and
averaged, and gal-DBD activity set at 1, indicated is also the standard
error. C, gel shift assay with different COS-1 extracts, cotransfected with the
indicated expression plasmids or empty vector, using a GAL-oligo. The
positions of a nonspecific band (nonsp.), the dimer bound to
the oligo, the dimer supershifted (supersh.) with the
-tag antibody 12CA5, and the additional complexes mentioned in the
text (add.) are indicated.
View larger version (47K):
[in a new window]
Fig. 3.
TSC-22 forms heterodimers with its homologue
THG-1. A, cDNA and protein sequence of THG-1.
B, GST pull-down assay with GST and gstT38-144
on 35S-labeled TSC-22 and THG-1. C, mammalian
two-hybrid assay with gal-THG-1 constructs (200 ng) and TSC-22
construct vpT38-144 or empty vector pSG5 (200 ng) in COS
cells. Values from three experiments (in duplicate) were averaged, and
fold induction is expressed compared with combinations gal-pSG5,
gal-2h-pSG5, or galTHG-1-pSG5. The standard error is also
indicated.
View larger version (23K):
[in a new window]
Fig. 4.
Conserved regions of TSC-22 family
members. A, alignment of TSC box-leucine zipper region
of (top to bottom): human THG-1, human TSC-22,
human DIP, human KIAA0669, D. melanogaster shs, and
hypothetical protein from C. elegans (T18D3.7) (see Footnote
3). Shaded residues are residues conserved between THG-1 and
family members, black residues are the central leucine
zipper residues, and boxed residues are the amino acids
important for the dimerization specificity (29).The number
at left indicates the amino acid at which the alignment
starts for the protein of that line. B, alignment of
homologous region amino acids 5-19 of THG-1 of (top to
bottom): human THG-1, human KIAA0669, and chicken TSC-22.
Shaded residues are conserved residues between THG-1 and
family members. C, alignment of homologous region amino
acids 62-99 of THG-1 of (top to bottom): human
THG-1, human KIAA0669, chicken TSC-22, and D. melanogaster
shs. Shaded residues are conserved residues between THG-1
and family members.
B-binding sites from the ICAM-promoter (which can thus be
activated by the NFB transcription factor p65) and 5 GAL binding sites (which can thus bind GAL-DBD constructs) in different
configurations, since this may influence repressor activity. On the
reporter 4×ICAM-5×GAL-TATA-luc (4i5g), we observed a strong
repressive effect when galTSC-22 was cotransfected in COS-1 cells (Fig.
5A; note that repressor activity is expressed as fold repression, i.e. high values
mean strong repressor activity), as well as in T47D and 293 cells (data not shown). A TSC-22 expression plasmid lacking the GAL-DBD did not
repress this reporter, nor was a 4×ICAM-TATA-luc reporter lacking GAL
binding sites repressed by the galTSC-22 (data not shown), indicating
that the repression is mediated via the GAL sites in the reporter. On a
luciferase reporter in which the distance between the GAL sites and the
TATA box was increased by 250 bp compared with 4i5g (4i5g-250), we
noticed that galTSC-22 still repressed, but to a lesser extent than on
4i5g (Fig. 4A). When we reversed orientation between GAL and
ICAM sites, 5×GAL-4×ICAM-TATA-luc (5g4i), repression decreased even
more, but was still present (Fig. 4A). When we additionally
increased the distance between the GAL sites and ICAM-TATA box,
5×GAL-250 bp-4×ICAM-TATA-luc (5g-250-4i), the repressive effect of
galTSC-22 was gone (Fig. 4A). Apparently, TSC-22 does
contain repressor activity when sequestered to DNA, but it is sensitive
to promoter architecture, and both distance between repressor and
activator/TATA box and orientation of activator and repressor seem
to play a role.
View larger version (22K):
[in a new window]
Fig. 5.
Repressor activity of TSC-22 and THG-1.
A, fold repression (activity of gal-DBD alone divided by
galTSC-22) of galTSC-22 (200 ng) on different promoters in COS-1 cells,
with gal-DBD alone set at 1. Values from three independent experiments
(in duplicate) were normalized and averaged; the standard error is also
indicated. At left is a schematic of the organization of the
promoter elements in the different reporters used (with size in bp of
the elements in italics). B, effect of TSA on
repressor activity of galTSC-22 and full-length galRAR in COS-1
cells on the 4i5g reporter. Values from three experiments (in
duplicate) were normalized and averaged, and gal-DBD alone was set at 1 for each concentration TSA. C, fold repression of galTHG-1
and galTSC-22 constructs (200 ng) on the 4×ICAM-5×GAL-TATA-luc
reporter. Values from three experiments (in duplicate) were normalized
and averaged, and gal-DBD alone was set at 1.
(RAR), repress transcription through histone deacetylase-containing complexes, which can be inhibited by trichostatin A (TSA; Refs. 27-29). We wanted to test whether TSC-22-mediated repression also acts
through such a complex, and could be inhibited by TSA. As a positive
control, we tested a GAL-DBD construct containing full-length RAR
(galRAR), which strongly represses the 4i5g reporter. This repression is suppressed by the histone deacetylase inhibitor TSA, in
contrast to galTSC-22-mediated repression (Fig. 5B). These data therefore suggest that TSC-22 does not repress through histone deacetylase-containing complexes, but instead uses a distinct mechanism.
-EF (which misses binding sites for
coactivators and corepressors and is therefore transcriptionally
inactive) contains many more amino acids (204 amino acids fused to
GAL-DBD) than galT1-144 or its deletion constructs (at
most 144 amino acids fused to GAL-DBD) but galRAR-EF does not
repress. Furthermore, we verified in gel shifts whether all constructs
were expressed properly, and therefore differences in repressor
activity could not be explained by differences in expression levels
(data not shown). In COS-cells, only galTSC-22 deletion constructs
galT38-102 and galT38-90, which lack the N-
and C-terminal domains, showed no repression. It should be noted that
the leucine zipper in galT38-102 is still intact. Mutation
of either the N- or the C-terminal domain also significantly reduces
repressor activity (galT38-144 versus galT38-102 and galT1-144 versus
galT38-144 or galT7-144, in which the
deletion of the first six amino acids already interferes with the
function of the first repression domain). Since these two regions
contain repressing activity, we designated them repression domain 1 and
2 (RD1 and RD2).
View larger version (16K):
[in a new window]
Fig. 6.
Role of different domains in repressor
activity of TSC-22 in COS-cells. A, fold repression of
different galTSC-22 deletion constructs (20 ng) on the 4i5g reporter in
COS-1 cells. gal-DBD alone was set at 1. Values are the mean of at
least four independent experiments (in duplicate), the standard error
is also indicated. dbd, gal-DBD alone; R,
galRAR -EF (transcriptionally inactive control; GAL-DBD fused to
truncated RAR); numbers on the X axis
designate the different galTSC-22 deletion clones used. B,
effect of overexpression of TSC-22 and tagT38-102 expression plasmids (200 ng) on repressor activity of galTSC-22 (200 ng) on the 4i5g reporter in
COS-1 cells. Values from five experiments (in duplicate) were
normalized and averaged, and activity of gal-DBD alone with pSG5 was
set at 1. C, effect of artificial dimerization of RD1 and
RD2 on repressor activity on the 4i5g reporter in COS-1 cells. 200 ng
of gal-DBD expression vector used, values from four experiments (in
duplicate) were normalized and averaged, and gal-DBD alone was set at
1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 31-30-2510211;
Fax: 31-30-2516464; E-mail: bvdb@niob.knaw.nl.
ABBREVIATIONS
-stimulated clone-22;
TGF-, transforming growth factor-;
shs, shortsighted;
DIP, delta sleep inducing peptide;
GST, glutathione
S-transferase;
DBD, DNA-binding domain;
HA tag, hemagglutinin tag;
RD1 and RD2, repression domains 1 and 2;
THG-1 and
THG-2, TSC-22 homologous genes 1 and 2;
EST, expressed sequence tag;
4i5g, 4×ICAM-5×GAL-TATA-luc reporter;
4i5g-250, 4×ICAM-5×GAL-250
bp-TATA-luc reporter;
5g4i, 5×GAL-4×ICAM-TATA-luc reporter;
5g-250-4i, 5×GAL-250 bp- 4×ICAM-TATA-luc reporter;
RAR, retinoic
acid receptor ;
TSA, trichostatin A;
gal-2h, fusion construct of the
THG-1 fragment cloned in the yeast two-hybrid screen with the GAL-DBD;
bp, base pair(s);
oligo, oligonucleotide;
ICAM, intercellular
adhesion molecule. GAL, DNA-binding site of yeast transcription factor
GAL4;
gal, DNA-binding domain of GAL4.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Shibanuma, M.,
Kuroki, T.,
and Nose, K.
(1992)
J. Biol. Chem.
267,
10219-10224 2.
Kawa-uchi, T.,
Nose, K.,
and Noda, M.
(1995)
Endocrine
3,
833-837
3.
Ohta, S.,
Shimekake, Y.,
and Nagata, K.
(1996)
Eur. J. Biochem.
242,
460-466[Medline]
[Order article via Infotrieve]
4.
Trenkle, T.,
Welsh, J.,
Jung, B.,
Mathieu-Daude, F.,
and McClelland, M.
(1998)
Nucleic Acids Res.
26,
3883-3891 5.
Kawamata, H.,
Nakashiro, K.,
Uchida, D.,
Hino, S.,
Omotehara, F.,
Yoshida, H.,
and Sato, M.
(1998)
Br. J. Cancer
77,
71-78[Medline]
[Order article via Infotrieve]
6.
Nakashiro, K.,
Kawamata, H.,
Hino, S.,
Uchida, D.,
Miwa, Y.,
Hamano, H.,
Omotehara, F.,
Yoshida, H.,
and Sato, M.
(1998)
Cancer Res.
58,
549-555 7.
Kester, H. A.,
Van der Leede, B. M.,
Van der Saag, P. T.,
and Van der Burg, B.
(1997)
J. Biol. Chem.
272,
16637-16643 8.
Clarke, C. L.,
and Sutherland, R. L.
(1990)
Endocr. Rev.
11,
266-301[CrossRef][Medline]
[Order article via Infotrieve]
9.
Hamil, K. G.,
and Hall, S. H.
(1994)
Endocrinology
134,
1205-1212[Abstract]
10.
Jay, P.,
Ji, J. W.,
Marsollier, C.,
Taviaux, S.,
Bergelefranc, J. L.,
and Berta, P.
(1996)
Biochem. Biophys. Res. Commun.
222,
821-826[CrossRef][Medline]
[Order article via Infotrieve]
11.
Treisman, J. E.,
Lai, Z. C.,
and Rubin, G. M.
(1995)
Development
121,
2835-2845[Abstract]
12.
Dobens, L. L.,
Hsu, T.,
Twombly, Y.,
Gelbart, W. M.,
Raftery, L. A.,
and Kafatos, F. C.
(1997)
Mech. Dev.
65,
197-208[CrossRef][Medline]
[Order article via Infotrieve]
13.
Sillard, R.,
Schulzknapp, E. P.,
Vogel, P.,
Raida, M.,
Bensch, K. W.,
Forssmann, W. G.,
and Mutt, V.
(1993)
Eur. J. Biochem.
216,
429-436[Medline]
[Order article via Infotrieve]
14.
Vogel, P.,
Magert, H. J.,
Cieslak, A.,
Adermann, K.,
and Forssmann, W. G.
(1996)
Biochim. Biophys. Acta
1309,
200-204[Medline]
[Order article via Infotrieve]
15.
Seidel, G.,
Adermann, K.,
Schindler, T.,
Ejchart, A.,
Jaenicke, R.,
Forssmann, W. G.,
and Rosch, P.
(1997)
J. Biol. Chem.
272,
30918-30927 16.
Kalkhoven, E.,
Wissink, S.,
Van der Saag, P. T.,
and Van der Burg, B.
(1996)
J. Biol. Chem.
271,
6217-6224 17.
Adams, M. D.,
Soares, M. B.,
Kerlavage, A. R.,
Fields, C.,
and Venter, J. C.
(1993)
Nat. Genet.
4,
373-386[CrossRef][Medline]
[Order article via Infotrieve]
18.
Lennon, G. G.,
Auffray, C.,
Polymeropoulos, M.,
and Soares, M. B.
(1996)
Genomics
33,
151-152[CrossRef][Medline]
[Order article via Infotrieve]
19.
Folkers, G. E.,
and Van der Saag, P. T.
(1995)
Mol. Cell. Biol.
15,
5868-5878[Abstract]
20.
Caldenhoven, E.,
Liden, J.,
Wissink, S.,
Van de Stolpe, A.,
Raaijmakers, J.,
Koenderman, L.,
Okret, S.,
Gustafsson, J. A.,
and Van der Saag, P. T.
(1995)
Mol. Endocrinol.
9,
401-412[Abstract]
21.
Folkers, G. E.,
Van der Leede, B. M.,
and Van der Saag, P. T.
(1993)
Mol. Endocrinol.
7,
616-627[Abstract]
22.
Folkers, G. E.,
Van Heerde, E. C.,
and Van der Saag, P. T.
(1995)
J. Biol. Chem.
270,
23552-23559 23.
Spaargaren, M.,
and Bischoff, J. R.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12609-12613 24.
Molenaar, M.,
Van de Wetering, M.,
Oosterwegel, M.,
Peterson-Maduro, J.,
Godsave, S.,
Korinek, V.,
Roose, J.,
Destree, O.,
and Clevers, H.
(1996)
Cell
86,
391-399[CrossRef][Medline]
[Order article via Infotrieve]
25.
D'Adamio, F.,
Zollo, O.,
Moraca, R.,
Ayroldi, E.,
Bruscoli, S.,
Bartoli, A.,
Cannarile, L.,
Migliorati, G.,
and Riccardi, C.
(1997)
Immunity
7,
803-812[CrossRef][Medline]
[Order article via Infotrieve]
26.
Vinson, C. R.,
Hai, T. W.,
and Boyd, S. M.
(1993)
Genes Dev.
7,
1047-1058 27.
Yoshida, M.,
Kijima, M.,
Akita, M.,
and Beppu, T.
(1990)
J. Biol. Chem.
265,
17174-17179 28.
Taunton, J.,
Hassig, C. A.,
and Schreiber, S. L.
(1996)
Science
272,
408-411[Abstract]
29.
Nagy, L.,
Kao, H. Y.,
Chakravarti, D.,
Lin, R. J.,
Hassig, C. A.,
Ayer, D. E.,
Schreiber, S. L.,
and Evans, R. M.
(1997)
Cell
89,
373-380[CrossRef][Medline]
[Order article via Infotrieve]
30.
Benezra, R.,
Davis, R. L.,
Lockshon, D.,
Turner, D. L.,
and Weintraub, H.
(1990)
Cell
61,
49-59[CrossRef][Medline]
[Order article via Infotrieve]
31.
Ron, D.,
and Habener, J. F.
(1992)
Genes Dev.
6,
439-453 32.
Heinzel, T.,
Lavinsky, R. M.,
Mullen, T. M.,
Soderstrom, M.,
Laherty, C. D.,
Torchia, J.,
Yang, W. M.,
Brard, G.,
Ngo, S. D.,
Davie, J. R.,
Seto, E.,
Eisenman, R. N.,
Rose, D. W.,
Glass, C. K.,
and Rosenfeld, M. G.
(1997)
Nature
387,
43-48[CrossRef][Medline]
[Order article via Infotrieve]
33.
Hassig, C. A.,
Fleischer, T. C.,
Billin, A. N.,
Schreiber, S. L.,
and Ayer, D. E.
(1997)
Cell
89,
341-347[CrossRef][Medline]
[Order article via Infotrieve]
34.
Laherty, C. D.,
Yang, W. M.,
Sun, J. M.,
Davie, J. R.,
Seto, E.,
and Eisenman, R. N.
(1997)
Cell
89,
349-356[CrossRef][Medline]
[Order article via Infotrieve]
35.
Brehm, A.,
Miska, E. A.,
McCance, D. J.,
Reid, J. L.,
Bannister, A. J.,
and Kouzarides, T.
(1998)
Nature
391,
597-601[CrossRef][Medline]
[Order article via Infotrieve]
36.
Magnaghi-Jaulin, L.,
Groisman, R.,
Naguibneva, I.,
Robin, P.,
Lorain, S.,
Le Villain, J. P.,
Troalen, F.,
Trouche, D.,
and Harel-Bellan, A.
(1998)
Nature
391,
601-605[CrossRef][Medline]
[Order article via Infotrieve]
37.
Luo, R. X.,
Postigo, A. A.,
and Dean, D. C.
(1998)
Cell
92,
463-473[CrossRef][Medline]
[Order article via Infotrieve]
38.
Cowell, I. G.
(1994)
Trends Biochem. Sci.
19,
38-42[CrossRef][Medline]
[Order article via Infotrieve]
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