| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, July 18, 1995) From the
The thyroid transcription factor 1 (TTF-1) is a
homeodomain-containing protein implicated in the activation of
thyroid-specific gene expression. Here we report that TTF-1 is capable
of activating transcription from thyroglobulin and, to a lesser extent,
thyroperoxidase gene promoters in nonthyroid cells. Full
transcriptional activation of the thyroglobulin promoter by TTF-1
requires the presence of at least two TTF-1 binding sites. TTF-1
activates transcription via two functionally redundant transcriptional
activation domains that as suggested by competition experiments, could
use a common intermediary factor.
Regulation of transcription is mediated by protein factors that
interact with specific DNA sequences located, in promoter and enhancer
elements, at variable distances from the site of assembly of the basal
transcription complex(1, 2) . The recognition of
specific DNA sequences and the ability to stimulate transcription
impinge on structurally distinct domains of transcription
factors(3) . The DNA binding function is determined by one of
several structural motifs such as homeodomains, POU domains, zinc
fingers, basic-leucine zippers, and basic helix-loop-helix
domains(4) . Detailed functional and structural studies have
demonstrated a specific interaction between these motifs and the
cognate DNA sequences(5) . In contrast, protein domains with
transcriptional activating properties are not as well defined. Several
apparently unrelated motifs capable of transcriptional activation have
been identified. Some of them depend on the presence of sequences rich
in a particular amino acid (acidic residues, glutamine-rich,
proline-rich), but many others cannot be grouped in any particular
class(6, 7, 8) . The structures of these
motifs are not known; it has been suggested that the integrity of
either amphipatic The activity of eukaryotic transcription factors can be
regulated by various mechanisms including
phosphorylation(13, 14, 15) , control of the
redox state(16, 17) , and interaction with other
factors(18) . Furthermore, several lines of evidence suggest
that the activity of some transcription factors and the selection of
promoters to be activated by the same factor may depend on a specific
cellular environment. Transcriptional activators contact the basal
transcriptional machinery through intermediates called co-activators
that could be subjected to regulation
themselves(19, 20, 21) . Some of these
co-activators can be relevant in determining the specific action of a
transcription factor(22, 23, 24) . The
thyroid transcription factor 1 (TTF-1) ( In this paper we show
that TTF-1 transactivates the Tg and, albeit to a lesser extent, the
TPO promoter in nonthyroid cells. We demonstrate that two independent
domains, located on either side of the TTF-1 homeodomain, contribute to
the transactivation obtained with the entire protein. Competition
experiments suggest that the two transcriptional activating domains of
TTF-1 could converge on a common pathway.
GAL4-TTF-1 chimeras were
constructed in the plasmid CMV-SG424, which contains the DNA-binding
domain of GAL4 (residues 1-147) under the control of the human
cytomegalovirus enhancer promoter(35) . CMV-SG424 is derived
from pSG424 (36) with the CMV promoter replacing the SV40 early
promoter. DNA fragments, generated by polymerase chain reaction as
described above and encoding different segments of TTF-1, were inserted
between the EcoRI and XbaI restriction sites of
CMV-SG424. The reporter plasmid for the GAL4 fusions, G5E1b, has been
described(20) . To generate C5 E1b, the GAL4 binding sites of
G5E1b were replaced with a pentamer of the TTF-1 binding sequence
(5`-CCCAGTCAAGTGTTCTT-3`) that was inserted between the PstI
and XbaI restriction sites. E1b contains only the E1b TATA box
in front of the CAT coding sequence. The plasmids TPO-Luc,
TPO-Em(26) , pTACAT3, pTACAT11.5, pTACAT13(37) ,
A-core, B-core, C-core, and CBC (25) have been described. The
plasmids CMV-Luc and RSV-CAT used as internal control in transfection
assays were kindly provided by U. Deuschle and G. Morrone,
respectively.
FRTL-5
cells were grown as described by Ambesi-Impiombato and Coon (41) . For the transient expression assay, cells were plated at
5
Figure 1:
TTF-1 activates transcription of Tg and
TPO promoters in HeLa cells. A, the structure of the reported
constructs used is shown. Lettering of the binding sites on each
promoter is the same as in (45) . UFA and UFB indicate the binding sites for ubiquitous factor A and B to the Tg (37) and TPO (26) promoters, respectively. A, B, and C indicate the TTF-1 binding region in both Tg
and TPO promoters. B, 3 µg of either pTACAT3 (
We next asked whether the template requirements for
TTF-1 activation of the Tg promoter were similar between HeLa and
differentiated thyroid cells. To this end, several promoter mutants
were assayed in a co-transfection assay. The minimal Tg promoter
contains three TTF-1 binding sites (Fig. 2, A, B, and C), two of which (A and C) are required for
transcription to occur in thyroid cells. Site A also binds the
ubiquitous factor ubiquitous factor A(25) . However, it could
be shown that, in thyroid cells, this binding is dispensable because a
mutated promoter (CBC in Fig. 2) that abolishes ubiquitous
factor A but maintains TTF-1 binding at A site is even more active than
the wild-type promoter(25) . As shown in Fig. 2, mutants
A-core and C-core have a higly reduced transcriptional activity in
TTF-1-transfected HeLa cells, whereas the B-core mutation does not
significantly interfere with the transactivation by TTF-1. Conversely,
mutant CBC shows a transcriptional activity about 50% higher than the
wild-type Tg promoter. These results go in the same direction as those
obtained in the FRTL-5 cell line ( Fig. 2and (37) ),
suggesting that the template requirements for transcriptional
activation by TTF-1 are the same between thyroid and HeLa cells.
Figure 2:
Effect of mutation at TTF-1 binding sites
on the expression of the Tg promoter in FRTL-5 and in HeLa cells
containing TTF-1. The structure of the promoter used is illustrated in
the bottom part of the figure. Lettering of binding site is as
in Fig. 1. Wild-type and mutated Tg promoters (3 µg) were
transiently transfected in FRTL-5 and HeLa cells (in the latter case 2
µg of CMV-TTF1 were co-transfected). CAT activity was normalized
for transfection efficiency and expressed as the percentage of activity
of pTACAT-3. The values represent the averages of at least three
independent experiments. The error bars show the standard
deviation of the mean.
In
addition to TTF-1 binding sites, at least two other DNA elements are
important for Tg promoter activity in thyroid cells: site K, which is
bound by the thyroid transcription factor 2(25) , and the site
defined by mutation 13(37) . Plasmid pTACAT11.5 contains a
mutated Tg promoter where TTF-2 binding to site K has been
abolished(37) , and in agreement with previous results, the
transcriptional activity of this mutant in FRTL-5 cells is 50% of the
wild-type promoter. The mutation 13, which does not interfere with
TTF-1 binding, results in an even larger decrease of Tg promoter
transcription (Fig. 3A and (37) ). However,
mutations 11.5 and 13 have no effect on Tg promoter transcription when
tested in TTF-1-transfected HeLa cells (Fig. 3A). These
data indicate that both TTF-2 and the protein recognizing the site
defined by mutation 13 are thyroid-specific and that they contribute to
the full activity of the Tg promoter in thyroid cells. Furthermore,
these data suggest that the TTF-1-induced Tg promoter activity, which
we are measuring in the co-transfection assay, is much lower than that
expressed by the same promoter in thyroid cells. Nonetheless, it can be
concluded that TTF-1 can activate transcription in the absence of other
thyroid-specific proteins. To provide further support for this notion,
we constructed C5 E1b (Fig. 3B), a promoter containing
five TTF-1 binding sites, arranged head to tail upstream of the E1b
TATA box. C5 E1b is at least as efficient as the Tg promoter in FRTL-5
cells, and in HeLa cells it is efficiently transcribed only upon
co-transfection of a TTF-1 expression vector.
Figure 3:
Additional thyroid-specific elements
different from TTF-1 are required for full transcriptional activity of
the Tg promoter. A, the mutated Tg promoters (3 µg) were
transiently transfected in FRTL-5 and HeLa cells (in the latter case 2
µg of CMV-TTF1 were co-transfected). In each cell line the activity
of wild-type promoter pTACAT3 was taken as 100%, and the relative
expression of the mutants was assessed. CMV-Luc was co-transfected to
normalize for transfection efficiency. The values represent the
averages of at least three independent determinations. The error
bars show the standard deviation of the mean. B, C5 E1b
or E1b promoters (3 µg) were transiently transfected in FRTL-5 and
HeLa (in the latter case 2 µg of CMV-TTF1 were co-transfected).
Data are presented as described for A.
Figure 4:
Expression and binding activity of TTF-1
deletion mutants. HeLa cells were transfected with expression vectors
encoding either wild-type or deletion mutants of TTF-1. The structure
of TTF-1 mutants are schematically shown in A. Extract from
transfected cells were used for Western blot analysis, with a
TTF-1-specific antibody (B). The positions of molecular weight
markers (
The ability of
various TTF-1 deletions to activate transcription from the Tg promoter
is shown in Fig. 5. The decreased transcriptional activity of
TTF-1 deletions
Figure 5:
Transcriptional activity of TTF-1 deletion
mutants. HeLa cells were transfected with pTACAT3 or C5 E1b (3 µg)
and expression vectors encoding either wild-type or deletion mutants of
TTF-1. The amount of each expression vector was chosen in order to
obtain comparable level of TTF-1 binding activity. CMV-Luc was used as
internal control, and CAT activity was normalized for transfection
efficiency. The activities are expressed as fold of activation
(± S.D.) relative to cells transfected with empty expression
vector. The values represent the averages of at least three independent
experiments.
The decreased
transcriptional activity of mutant TTF-1 deletion mutants
were also tested using the artificial promoter C5 E1b (Fig. 5).
Results comparable with the Tg promoter were obtained, except for
mutant
Figure 6:
Analysis of TTF-1 domains. HeLa and FRTL-5
cells were transfected with the reporter gene G5E1b (3 µg) and
CMV-driven expression vector containing the GAL4 DNA-binding domain
fused to different portions of TTF-1. The amount of expression vector
was chosen as described in the legend to Fig. 5. CMV-Luc was
used as internal control, and CAT activity was normalized for
transfection efficiency. The transactivation activity of the fused
proteins are expressed as fold of activation (± S.D.) relative
to cells transfected with CMV-GAL4 vector containing only the GAL4
DNA-binding domain. The values represent the averages of at least three
independent experiments.
Although the entire COOH-terminal region of TTF-1 (
Figure 7:
Squelching effect of TTF-1 transcriptional
activating domains. A, the structure of activators and
competitors used are indicated together with a selective screening of
TTF-1. HD, homeodomain; N and C, amino- and
carboxyl-terminal activation domain, respectively; I,
inhibitory region). B, HeLa cells were co-transfected with the
reporter gene G5E1b (3 µg), 0.5 µg of each activator, and 3
µg of indicated competitor plasmid (A). CMV-Luc were added
as internal reference, and the results were normalized to the
transfection efficiencies. Results are expressed as the percentage of
activity of each activator co-transfected without competitor. The
results of one representative experiment are
shown.
The present study demonstrates that TTF-1 is capable of
activating transcription from thyroid-specific promoters in nonthyroid
cells, thus providing strong support to the notion that this factor
plays an important role in the differentiation of thyroid follicular
cells. The HeLa cell co-transfection assay used in this paper reflects
some very important functional properties of TTF-1. In thyroid cells
TTF-1 requires at least two properly spaced binding sites in order to
activate transcription from the Tg promoter(45) . Such a
requirement is maintained in HeLa cells, as demonstrated by the
sensitivity of transcriptional activation to Tg promoter mutations.
Furthermore, as in FRTL-5 cells, TTF-1 is capable of activating
transcription in HeLa (and several other) cells from C5 E1b, an
artificial promoter containing a viral TATA box (20) and a
multimerized TTF-1 binding site, thus revealing the intrinsic capacity
of TTF-1 to activate transcription in the absence of other cell
type-specific components. Nonetheless, in defining the regions of TTF-1
necessary for transcriptional activation, we have discovered that this
protein is capable of functioning in a promoter and cell type-specific
manner. The ability of TTF-1 to distinguish between different promoters
and cell types is revealed by the deletion that removes 50 amino acids
at the amino terminus ( In order to define the transcriptional activation
domains of TTF-1, we have analyzed the activity of chimeric proteins
containing different regions of TTF-1 fused to the DNA-binding domain
of GAL4. TTF-1 transcriptional activity appears to be derived from two
activating domains, the N and C domains, that show a functional
redundancy. In fact, TTF-1 containing either N or C shows a
transcriptional activity either on Tg or C5 E1b promoter very similar
to that of the entire protein. A functional redundancy of N and C
transcriptional activating domains has also been demonstrated for Oct-
2(50) . However, such a redundancy is not always appreciable,
because the COOH-terminal domain is only able to activate transcription
from an enhancer type position (51) , and this activity is
B-cell-restricted. In an analogous manner, the TTF-1 transactivating
domains could show redundancy only with certain promoters or
cell-types. A second feature of the transcriptional activating domains
of TTF-1 is that, as is often the case with transcriptional activating
domains, they do not show any sequence homology with activating domains
of other factors previously characterized. In addition, a
glutamine-rich region, located carboxyl-terminal to the homeodomain,
shows an inhibitory effect in all contexts tested. Glutamine-rich
regions are among the better defined transcriptional activating
domains, and some general characteristics of these domains have been
elucidated(46) . However, an inhibitory glutamine-rich region
has never been described. It would be of interest to test whether the
inhibitory domain of TTF-1 interacts with the same specific subset of
TBP-associated factors that has been shown to be required for
transcriptional activation by the glutamine-rich Sp1 (47) . The functional redundancy existing between the NH
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26649-26656
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-helices (VP16 mutants) (9, 10) or 
-sheets (11, 12) could be essential for transcriptional
activity.
)could provide a
useful model to study the regulatory interactions controlling the
functional specificity of a transcription factor. TTF-1 was originally
identified as a protein binding to sequences present in multiple copies
in the promoters of the thyroglobulin (Tg) (25) and
thyroperoxidase (TPO) (26) genes, both of which are exclusively
expressed in the thyroid follicular cells(27) . However, the
presence of TTF-1 in lung(28) , in restricted regions of the
fetal brain, and in thyroid cell precursors (29) , where the Tg
and TPO genes are silent, indicates that the function of TTF-1 is not
restricted to adult thyroid tissue. In support of this notion, TTF-1
has recently been implicated in the transcriptional activation of genes
exclusively transcribed in lung epithelium, such as those encoding for
surfactant proteins A and B(30, 31) . The multiplicity
of roles that TTF-1 plays in vivo suggests that its activity
is subjected to regulation. In this respect we have already shown that
TTF-1 is a phosphoprotein (32) and that its DNA binding
activity is under redox control(33) .
Plasmids
The plasmid CMV-TTF1 was
generated by inserting the entire TTF-1 cistron (28) in the
expression vector Rc-CMV (Invitrogen). The TTF-1 coding sequence
extended for one ATG codon upstream of the sequence published in (28) and was deduced by Lonigro et al. (
)TTF-1 deletion mutants were constructed by polymerase
chain reaction using primers encoding the amino acids at the ends of
each fragment (see below); all deletions were cloned replacing the full
length cDNA between the HindIII and XbaI sites of
CMV-TTF1. The primer always included either HindIII or XbaI restriction sites to facilitate cloning. For all
deletions of TTF-1 amino terminus the sequence 5`-CCACCAATG-3` was
added to provide a ribosome entry site (34) and an ATG codon
for translation initiation. Plasmid M1 was derived from Rc-CMV by
deletion of a BamHI fragment containing the neomycin
resistance and was used as control .Cell Culture and Transfection
HeLa cells
were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. For transient expression
assays, cells were plated at 3 
10
cells/60-mm
tissue culture dish 4-6 h prior to transfection. Transfections
were carried out by the calcium phosphate co-precipitation technique (38) After exposure to calcium phosphate-DNA precipitate for
12-14 h, the cells were washed with phosphate-buffered saline and
grown for 48 h. CAT (39) and luciferase activities (40) on cell extracts were determined as described. 10 cells/60-mm tissue culture dish 48 h prior to
transfection. 3 h prior to transfection, the medium was changed to
Dulbecco's modified Eagle's medium containing 5% calf serum
and growth factors. Transfections were carried out by calcium phosphate
co-precipitation as described(26) .Bandshift Assay
Cellular extracts were
prepared as described(25) . The binding reaction was carried
out in a buffer containing 40 mM Hepes, pH 7.9, 200 mM KCl, 0.5 mM dithiothreitol, and 0.3 mg/ml
poly(dI
dC). After 30 min of incubation at room temperature, free
DNA and DNA-protein complexes were resolved on a 6% polyacrylamide gel
run in 0.5 TBE (2 mM EDTA, 90 mM boric acid,
90 mM Tris-HCl, pH 8.0) for 2-3 h at 4 °C. The gel
was dried and then exposed to an x-ray film at -80 °C.
Oligonucleotide C, used to measure TTF-1 binding activity, has been
described(28) . GAL4 DNA binding activity was measured using a
double-stranded nucleotide with the sequence
5`-GATCGGAGGACAGTACTCCGCTAG-3` (top strand).RNase Protection Assay
Total RNA from
cultured cells was prepared by the acid guanidinium thiocyanate phenol
procedure (42) . The probe for RNase protection was synthesized
by T7 polymerase transcription of XhoI linearized pRTgCat, a
plasmid obtained by cloning the SalI-EcoRI
fragment from pTACAT3 in Bluescript KS (Stratagene). A luciferase
probe, synthesized by T3 polymerase transcription of SacI
linearized pBsrLuc(26) , was used to control for transfection
efficiency. Hybridization and RNase treatment were as
described(43) .Western Blot
Extracts of HeLa transfected
cells, prepared as described previously(44) , were boiled in
Laemmli sample buffer and resolved by 10% SDS-polyacrylamide gel
electrophoresis (43) . After electroblotting onto
nitrocellulose membrane, TTF-1 was localized with a specific antibody (29) using an ECL kit (Amersham Corp.).
TTF-1 Activates Transcription of Tg and TPO
Promoters
Plasmids containing either Tg (pTACAT3) (37) or TPO (TPO-Luc) (26) promoter, fused to CAT or
luciferase (Luc) coding sequences (Fig. 1A),
respectively, were transfected in HeLa cells, which do not contain any
detectable TTF-1. As expected, given the demonstrated thyroid-specific
expression of both promoters(45) , very little transcriptional
activity could be detected from either of them. A dose-dependent
activation of both promoters was observed (Fig. 1B)
upon co-transfection with increasing amounts of a TTF-1 expression
vector containing the TTF-1 cistron under the control of the human
cytomegalovirus promoter (CMV-TTF1). In the case of the Tg promoter,
maximal activation was obtained with 0.25 µg of expression vector,
whereas the TPO promoter was only activated when 4 µg of expression
vector were used. Mutants of Tg (C-core) and TPO (TPO-Em) promoters,
where the TATA proximal TTF-1 binding sites are mutated and do not bind
TTF-1 any longer, are poorly activated, indicating that the
transcriptional activation obtained with wild-type promoters depends on
TTF-1 binding. Transcriptional activation by TTF-1 appears to be
restricted to exogenous, co-transfected promoters, because neither Tg
nor TPO mRNA could ever be detected in TTF-1-producing HeLa cells (data
not shown). Further studies were only carried out on the more
responsive Tg promoter. To test whether Tg transcription in HeLa cells
uses the same basal signals as in thyroid, we carried out the RNase
mapping experiment shown in Fig. 1C, which demonstrates
that the transcripts produced in HeLa cells upon TTF-1 stimulation
initiate at the same site used by the Tg promoter in differentiated
thyroid cells.
) or
TPO-Luc (
) and the respective mutants C-core () and TPO-Em
(
) were introduced into HeLa cells with 0.061, 0.25, 1, and 4
µg of the TTF-1 expression vector CMV-TTF1. The cells were
subsequently lysed and assayed for CAT and luciferase activity. Folds
of activation are considered as the ratio between values obtained with
and without TTF-1 expression vector. CMV-Luc or RSV-CAT were added as
internal reference, and the results were normalized to the relative
transfection efficiencies. Results of one representative experiment are
shown. C, total RNA was extracted from FRTL-5 cells stably
transfected with pTACAT3 (lane 1), HeLa cells transiently
transfected with either pTACAT3 (lane 2), or pTACAT3 and
CMV-TTF-1 (lane 3). In lane 4 total RNA from mock
transfected HeLa cells was used. RNase mapping was carried out with a
pRTgCAT probe (top) to detect CAT transcription originated at
the Tg promoter. A pBsrLuc derived probe reveals luciferase transcripts
derived from CMV luciferase constructs, used to compare transfection
efficiencies in the transient transfection experiments of lanes
2, 3, and 4 (bottom).
Several Parts of TTF-1 Contribute to Its
Transactivating Function
To map TTF-1 sequences involved in
transcriptional activation, expression vectors encoding deletion
mutants of TTF-1 (Fig. 4A) were constructed and
transfected in HeLa cells. Comparable protein levels (Fig. 4B) corresponding to approximately equal DNA
binding activity (Fig. 4C) were obtained for each of
the mutants tested, indicating that differences in transcriptional
activity among TTF-1 deletion mutants are not due to either reduced
protein concentration or impaired DNA binding.
10) are indicated. Aliquots of the
same extracts, known to contain similar amounts of TTF-1 protein, as
shown in B, were used for the mobility shift assay carried out
using an oligonucleotide containing a high affinity TTF-1 binding site
(oligonucleotide C, see ``Materials and Methods'') (C). wt, wild type.
1, 33, 2, and 3 compared with the
wild-type protein indicate the presence of a transcriptional activating
domain (domain N) NH-terminal to the homeodomain. A
critical part of this domain is contained in the amino acids missing in
the 1 mutant, because more extensive deletions, such as those of
mutants 2, 33, and 3, do not decrease the
transcriptional activity any further. Furthermore, the considerable
residual activity of these deletion mutants indicates that other parts
of TTF-1 are capable of transcriptional activation.
6 indicates that an activating
domain (domain C) resides in this region of TTF-1. However, extending
the deletion of mutant 6, such as in mutant 14, a
transcriptional activity comparable with that of the wild-type protein
is obtained. These results are consistent with the presence of an
inhibitory region located between amino acids 221 and 295 (domain I).
The behavior of deletion mutants 26 and 35, where the
putative inhibitory region was removed, supports this notion because
both mutants show a higher activity of the corresponding proteins that
contain this domain (wild-type TTF-1 and mutant 33, respectively).
Interestingly, this inhibitory region is glutamine-rich, even though
glutamine-rich regions are very often found within transcriptional
activating domains(46) . Mutant 36, where both the amino-
and the carboxyl-terminal transactivating domains were disrupted, shows
the least ability to activate transcription, thus confirming the
redundancy of the two activating domains.1 that shows, on this promoter, a transcriptional activity
higher than that of the wild-type protein. This finding suggests that
the amino acid residues 1-50 at the amino terminus of TTF-1 can
function either as an activator or as an inhibitor of transcription in
a promoter-specific manner.The Activation Domains of TTF-1 Are Also Active If
Fused to Heterologous DNA-binding Domain
To obtain an
independent assessment of the functional domain in TTF-1, several
constructs encoding chimeric proteins containing different segments of
TTF-1 fused to the GAL4 DNA-binding domain were prepared. TTF-1/GAL4
fusions were tested on the G5E1b reporter construct (20) containing five GAL4 binding sites upstream to the E1b
TATA box (Fig. 6). The GAL4 DNA-binding domain alone was unable
to activate the expression of the reporter gene in this assay, and its
activity was considered as a reference value for the chimeric
constructs. Consistent with the observations reported in the previous
paragraph, the region of TTF-1 NH-terminal to the
homeodomain contains an autonomous transactivating domain (see
constructs from G7 to G21). The limits of this domain can be
located, at the NH-terminal side, between amino acids 51
and 102 (compare G18 and G19) and, at the COOH terminus,
between amino acids 90 and 123 (compare G1 and G2). In
agreement with the results obtained with the deletion mutants on the C5
E1b promoter, removal of the region spanning between amino acids 1 and
51 determines an increase in transcriptional activity (compare G7
with G18 and G2 with G21). Thus, in two different
contexts, region 1-51 displays an inhibitory effect on TTF-1
transcriptional activation. However, in FRTL-5 cells this domain seems
to have an activating function, because G21 is much less active
than G7. These results suggest that this region of TTF-1 is
capable of functioning in both a cell type- and promoter-dependent
manner.
G11)
is unable to activate transcription when fused to GAL4, deletion of the
glutamine-rich region (amino acids 221-299) demonstrates the
transcriptional activity of the C domain (G13). Moreover, the
glutamine-rich region does not have any transcriptional activity even
when fused to the GAL4 DNA-binding domain (G12). The COOH-terminal
border of the transcriptional activating domain starting from amino
acid 295 is very close to the end of the molecule because a deletion of
the last 20 amino acids results in complete loss in transcriptional
activity (G15).Functional Interplay between the Different Domains of
TTF-1
The functional redundancy of the NH- and
COOH-terminal domains suggests that they might interact with a common
intermediary factor or co-activator. To test this hypothesis, we
verified whether the amino-terminal domain could compete with the
carboxyl-terminal one and vice versa. This inhibition or
``squelching'' effect (47) would result from the
NH- and COOH-terminal domain competing for and thereby
titrating away a common co-activator. Results of these squelching
experiments are shown in Fig. 7. The deletion mutant 14
(containing the NH-terminal domain, up to and including the
homeodomain) is able to inhibit the transcriptional activation induced
by either the NH-terminal domain (G21) or the
COOH-terminal domain (G13) of TTF-1 fused to the DNA-binding
domain of GAL4 on the G5E1b reporter. No reduction was observed in
transcription from the CMV-Luc reporter used to normalize for
transcription efficiency, demonstrating that the construct 14 did
not inhibit transcription in a nonspecific manner. The ability of
14 to inhibit TTF-1-dependent transcription strongly argues for
the existence of a co-factor that is not a component of the basal
transcriptional machinery but it is essential for TTF-1 function. Thus,
both the NH- and COOH-terminal domains could activate the
basal transcriptional machinery through an essential intermediary
factor that is present in the cells at a relatively low concentration,
and it can thereby be titrated away by the NH-terminal
domain over-expressed in a form unable to bind the target promoter.
Surprisingly, 3, which contains the COOH-terminal region of TTF-1
up to and including the homeodomain is unable to inhibit either
G21 or G13 transcriptional activity. Even though the level of
expression of 3 in the cells is comparable with that of 14 as
measured by DNA binding assays (data not shown), it is still
conceivable that the interaction of 3 with the intermediary factor
is subordinate to a limiting step (e.g. interaction with
another protein, phosphorylation, etc.) that does not allow the
attainment of sufficient concentration of the 3 species
``competent'' for squelching.
1). When the activity of 1 is compared
with that of wild-type TTF-1 in HeLa cells, a 2-fold reduction in the
ability to activate the Tg promoter but a 3-fold improvement in
transcription from C5E1b are observed, suggesting that TTF-1 is
sensitive to promoter structure and adjusts the activity of its
amino-terminal region accordingly. The ability of a transcription
factor to function as activator or repressor, depending on the promoter
context, has been previously reported for c-ErbA (48) and
Rap1(49) . The sensitivity of TTF-1 to promoter structure is
also suggested by the much higher transactivation observed with the Tg
than with the TPO promoter. Even though the number and spacing of
binding sites is quite similar in the regulatory regions of both genes,
the orientation of the binding sites and their affinity toward TTF-1
are not identical between the two promoters(45) , suggesting
that these features could be important modulators of TTF-1 activity.
Interestingly, this also appears to be the case for the
thyroid-specific transcriptional factor Pax-8, which binds a single
site in both Tg and TPO promoters. In both cases the Pax-8 binding site
overlaps with the TTF-1 binding site proximal to the TATA box. However,
Pax-8 activates the TPO promoter more efficiently of the Tg promoter (44) .- and
COOH-terminal activating domains of TTF-1 suggests that they could
converge on the same functional pathway or intermediary factor.
Transcriptional co-factors are, of course, relevant for all
transcription factors, but they could play an essential role in the
cell type- and promoter-specific transcriptional activation. The best
example is the B-cell-specific activation of immunoglobulin promoters
by the ubiquitous factor Oct-1, which is mediated by B-cell-specific
transcriptional co-factors(22, 23, 24) .
TTF-1 presents a similar different problem, because even though it
shows a very restricted tissue distribution, it nonetheless has
different functions in thyroid and lung, two of the three cell types
where it is expressed. Unfortunately, no information is available on
putative targets of TTF-1 in developing diencephalic neurons, the third
cell type known to contain TTF-1 proteins. TTF-1 is able to activate
transcription of Tg and TPO genes in thyroid but not in lung tissue
and, conversely, it is able to activate transcription of the SPB gene
in lung but not in thyroid tissue. HeLa cells seem to be a neutral
environment, because TTF-1 is able to activate both thyroid (this
study) and lung targets (30, 31) in this cellular
context. It is conceivable that this loss of specificity could be due
to the transient transfection assay, as it has been suggested that at
high levels of protein some cell type-specific requirements could be
lost(52) . Experiments are in progress to test whether some of
the specificity could be obtained in stable transformants expressing
lower levels of TTF-1. Artificial promoters containing only TTF-1
binding sites are being constructed, and some of them do show a better
transcription in lung or thyroid, suggesting that promoter architecture
is an important component in cell type-specific activation by TTF-1. (
)Nonetheless, the squelching data presented in this paper
suggest the presence of TTF-1-specific co-activators. Thyroid- and
lung-specific TTF-1 co-activators could be responsible for activation
of different promoters by TTF-1 in the two tissues. The isolation of
such co-factors from both tissues is in progress and may reveal
interesting mechanisms behind in the differentiation of endodermal
derivatives.
)))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  P. Minoo, L. Hu, N. Zhu, Z. Borok, S. Bellusci, J. Groffen, D. Kardassis, and C. Li SMAD3 prevents binding of NKX2.1 and FOXA1 to the SpB promoter through its MH1 and MH2 domains Nucleic Acids Res., January 17, 2008; 36(1): 179 - 188. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Riesco-Eizaguirre and P. Santisteban A perspective view of sodium iodide symporter research and its clinical implications. Eur. J. Endocrinol., October 1, 2006; 155(4): 495 - 512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. D'Andrea, R. Iacone, T. Di Palma, R. Nitsch, M. G. Baratta, L. Nitsch, R. Di Lauro, and M. Zannini Functional Inactivation of the Transcription Factor Pax8 through Oligomerization Chain Reaction Mol. Endocrinol., August 1, 2006; 20(8): 1810 - 1824. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Velez, E. Costamagna, E. T. Kimura, L. Fozzatti, C. G. Pellizas, M. M. Montesinos, A. M. Lucero, A. H. Coleoni, P. Santisteban, and A. M. Masini-Repiso Bacterial Lipopolysaccharide Stimulates the Thyrotropin-Dependent Thyroglobulin Gene Expression at the Transcriptional Level by Involving the Transcription Factors Thyroid Transcription Factor-1 and Paired Box Domain Transcription Factor 8 Endocrinology, July 1, 2006; 147(7): 3260 - 3275. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Moya, G. Perez de Nanclares, L. Castano, N. Potau, J. R. Bilbao, A. Carrascosa, M. Bargada, R. Coya, P. Martul, E. Vicens-Calvet, et al. Functional Study of a Novel Single Deletion in the TITF1/NKX2.1 Homeobox Gene That Produces Congenital Hypothyroidism and Benign Chorea But Not Pulmonary Distress J. Clin. Endocrinol. Metab., May 1, 2006; 91(5): 1832 - 1841. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Maeda, T. C. Hunter, D. E. Loudy, V. Dave, V. Schreiber, and J. A. Whitsett PARP-2 Interacts with TTF-1 and Regulates Expression of Surfactant Protein-B J. Biol. Chem., April 7, 2006; 281(14): 9600 - 9606. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Amendola, P. De Luca, P. E. Macchia, D. Terracciano, A. Rosica, G. Chiappetta, S. Kimura, A. Mansouri, A. Affuso, C. Arra, et al. A Mouse Model Demonstrates a Multigenic Origin of Congenital Hypothyroidism Endocrinology, December 1, 2005; 146(12): 5038 - 5047. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Dentice, C. Luongo, A. Elefante, R. Ambrosio, S. Salzano, M. Zannini, R. Nitsch, R. Di Lauro, G. Rossi, G. Fenzi, et al. Pendrin Is a Novel In Vivo Downstream Target Gene of the TTF-1/Nkx-2.1 Homeodomain Transcription Factor in Differentiated Thyroid Cells Mol. Cell. Biol., November 15, 2005; 25(22): 10171 - 10182. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Altmann, R. B. Schulz, G. Glensch, H. Eskerski, S. Zitzmann, M. Eisenhut, and U. Haberkorn Effects of Pax8 and TTF-1 Thyroid Transcription Factor Gene Transfer in Hepatoma Cells: Imaging of Functional Protein-Protein Interaction and Iodide Uptake J. Nucl. Med., May 1, 2005; 46(5): 831 - 839. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S M Park and V K K Chatterjee Genetics of congenital hypothyroidism J. Med. Genet., May 1, 2005; 42(5): 379 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Wei, W. K. Miskimins, and R. Miskimins Stage-specific Expression of Myelin Basic Protein in Oligodendrocytes Involves Nkx2.2-mediated Repression That Is Relieved by the Sp1 Transcription Factor J. Biol. Chem., April 22, 2005; 280(16): 16284 - 16294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Garcia-Barcelo, R. W. Ganster, V. C.H. Lui, T. Y.Y. Leon, M.-T. So, A. M.F. Lau, M. Fu, M.-H. Sham, J. Knight, M. S. Zannini, et al. TTF-1 and RET promoter SNPs: regulation of RET transcription in Hirschsprung's disease Hum. Mol. Genet., January 15, 2005; 14(2): 191 - 204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Dentice, C. Luongo, A. Elefante, R. Romino, R. Ambrosio, M. Vitale, G. Rossi, G. Fenzi, and D. Salvatore Transcription Factor Nkx-2.5 Induces Sodium/Iodide Symporter Gene Expression and Participates in Retinoic Acid- and Lactation-Induced Transcription in Mammary Cells Mol. Cell. Biol., September 15, 2004; 24(18): 7863 - 7877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Stepchenko and M. Nirenberg Mapping activation and repression domains of the vnd/NK-2 homeodomain protein PNAS, September 7, 2004; 101(36): 13180 - 13185. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Rivas, B. Mellstrom, J. R. Naranjo, and P. Santisteban Transcriptional Repressor DREAM Interacts with Thyroid Transcription Factor-1 and Regulates Thyroglobulin Gene Expression J. Biol. Chem., August 6, 2004; 279(32): 33114 - 33122. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Yang, X. Lian, A. Cowen, H. Xu, H. Du, and C. Yan Synergy between Signal Transducer and Activator of Transcription 3 and Retinoic Acid Receptor-{alpha} in Regulation of the Surfactant Protein B Gene in the Lung Mol. Endocrinol., June 1, 2004; 18(6): 1520 - 1532. |
ABSTRACT
