(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 -helices (VP16 mutants) (9, 10) or
-sheets (11, 12) could be essential for transcriptional
activity.
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) ()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) .
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 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) .
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 () 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).
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 (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.
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 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.
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 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.
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 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.
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 (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).
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 (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) .
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- 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.