The Human, but Not Rat, dio2 Gene Is Stimulated by Thyroid Transcription Factor-1 (TTF-1)
Balázs Gereben1,
Domenico Salvatore1,
John W. Harney,
Helen M. Tu and
P. Reed Larsen
Thyroid Division (B.G., J.W.H., H.M.T., P.R.L.) Department of
Medicine Brigham and Womens Hospital Harvard Medical
School Boston, Massachusetts 02115
Dipartimento di
Endocrinologia ed Oncologia Molecolare e Clinica (D.S.)
Università degli Studi di Napoli "Federico II" Napoli,
Italy 80131
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ABSTRACT
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Types 1 and 2 iodothyronine deiodinases (D1
and D2) catalyze the production of T3 from
T4. D2 mRNA is abundant in the human thyroid
but very low in adult rat thyroid, whereas D1 activity is high in both.
To understand the molecular regulation of these genes in thyroid cells,
the effect of thyroid transcription factor 1 (TTF-1) and the paired
domain-containing protein 8 (Pax-8) on the transcriptional activity of
the deiodinase promoters were studied. Both the approximately 6.5-kb
hdio2 sequence and its most 3' 633 bp were activated
10-fold by transiently expressed TTF-1 in COS-7 cells, but the
hdio1 was unaffected. Surprisingly, the response of the
rdio2 gene to TTF-1 was only 3-fold despite the 73%
identity with the proximal 633-bp region of hdio2 including
complete conservation of a functional cAMP response element at -90.
Neither human nor rat dio2 nor human dio1 was
induced by Pax-8. The binding affinity of four putative TTF-1 binding
sites in hdio2 were compared by a semiquantitative gel
retardation assay using in vitro expressed TTF-1
homeodomain protein. Only two sites, D and C1 (both of which are absent
in rdio2), had significant affinity. Functional analyses
showed that both sites are required for the full response to TTF-1.
These results can explain the differential expression of
dio2 in thyroid and potentially other tissues in humans and
rats.
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INTRODUCTION
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The type 2 iodothyronine deiodinase (D2) catalyzes the first step
in thyroid hormone action, producing T3 from
T4 and regulating the intracellular concentration
of the T3 in the selected tissues in which it is
expressed. This recently cloned selenoenzyme is highly conserved in
sequence during evolution and has been identified in every vertebrate
species examined including fish, tadpoles, chickens, rats, mice, and
humans (1, 2, 3, 4, 5, 6). However, its tissue distribution varies with species.
The pituitary, brain, and brown adipose tissue express D2 mRNA in all
species examined, as expected from earlier activity measurements.
However, D2 mRNA is found in cardiac and skeletal muscle in humans,
which is not the case in the rat (4, 6). In addition, D2 is expressed
in the liver of certain fish and in the adult chicken, in contrast to
its absence in the human and rat (1, 3). An unexpected finding was that
D2 mRNA was highly expressed in human thyroid, with high D2 activity
found in some Graves disease thyroids and in thyroid tissue from a
patient with TSH-induced hyperthyroidism (7). D2 mRNA is also found in
the chicken thyroid (3). Type 1 iodothyronine deiodinase (D1), which
also converts T4 to T3, has
long been known to be expressed in rat and human thyroid (8). However,
neither D2 mRNA nor D2 activity are found in the FRTL-5 rat thyroid
cell line (7, 9).
The transcription factors governing thyroid-specific protein expression
include thyroid transcription factors 1 and 2 (TTF-1, TTF-2) and Pax-8.
TTF-1 (also called T/ebp or Nkx-2.1) cDNA has been cloned and encodes a
homeodomain (HD)-containing protein (10). Its expression is restricted
to the thyroid, lung, and certain regions of the fetal central nervous
system (11). The expression of thyroglobulin (Tg) and thyroperoxidase
(TPO) are stimulated by TTF-1 and Pax-8 (12). TTF-1 also affects the
expression of the TSH receptor and the lung-specific surfactant B
protein (12, 13). An important role for TTF-1 in organogenesis was
shown in the TTF-1 knock-out mouse (14). The homozygous animals are
born dead, without a thyroid or pituitary gland and with atrophic lung
parenchyma. Defects in the ventral forebrain are also present. An
infant with heterozygous deletion of the TTF-1 gene was found to have
thyroid dysfunction, respiratory failure, and delayed mental and motor
development, indicating it has similar importance in humans (15).
On the other hand, the Pax-8 gene encodes a paired domain-containing
protein that is expressed in the thyroid, kidney, and pituitary (16, 17). It binds the "C" site in the 5'-flanking region (FR) of the Tg
gene with TTF-1 in a mutually exclusive fashion (17). Pax-8 is more
important in the transcriptional activation of TPO expression than of
the Tg promoter (12, 18). The core binding site contains a TGCCC motif,
but the binding is also affected by the adjacent 3'-region that
contains an A(G/C)TC sequence in the rat Tg and TPO promoter. However,
mutational studies suggest that important determinants of Pax-8 DNA
binding are located outside of this consensus sequence (17).
TTF-2 has been only recently cloned (19). It is a forkhead-containing
protein involved in thyroid- specific gene expression and necessary
for thyroid morphogenesis. Its specific function, as either a positive
or a negative regulator of thyroid-gene expression, is still under
investigation (12, 19, 20).
There is no information as to the potential role of any of these
transcription factors in the expression of the deiodinase genes. The
present studies were performed to explore the molecular basis for
thyroidal deiodinase expression in human and rat thyroid. We addressed
the role of the two established transcriptional activators of
thyroid-specific gene transcription, TTF-1 and Pax-8. Also, since prior
studies have shown that D2 activity in rat is increased by cAMP, we
also defined the molecular basis for the cAMP responsiveness of
rdio2 (21, 22).
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RESULTS
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D2 mRNA and Activity in the Rat Thyroid Gland
We first determined whether the absence of dio2 mRNA
and activity in FRTL-5 cells was also characteristic of normal or
TSH-stimulated rat thyroid. TSH stimulation was induced by making
animals hypothyroid by methimazole administration. An approximately
7-kb D2 mRNA was barely detectable in the thyroids of normal and
hypothyroid rats after 2 weeks exposure of the blot at -70 C (Fig. 1
). In contrast, the hD2 mRNA is easily
detected after approximately 16 h in human thyroid under similar
conditions (7). D1 mRNA was abundant in the rat thyroid, requiring only
48 h exposure of the same blot (Fig. 1
). The hypothyroid rat
thyroid expressed lower levels of D2 mRNA than did that of normal
animals (Fig. 1
). The D2/ß-actin band density ratios from euthyroid
and hypothyroid rat thyroids were 0.8 and 0.1, respectively, while that
for D1 was 9.6 and 3.2.

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Figure 1. Northern Blot of 30 µg of Thyroid RNA from
Hypothyroid and Euthyroid Rats
The blot was probed as described for D2, and then reprobed for
ß-actin followed by D1. The blot was exposed for 2 weeks, 1 day, and
2 days at -80 C for D2, ß-actin, and D1, respectively.
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We have designed a T4 saturation technique for
quantitation of D2 activity in the presence of high levels of D1 such
as exists in human thyroid (7). The assay takes advantage of the marked
differences in the Michaelis-Menten constant (Km)
of D2 and D1 for T4 (
2 nM and
2
µM, respectively). We have shown that the release of
125I- from 2 nM
125I-T4 by D2 can be
completely blocked by 100 nM T4 while
that from D1 catalyzed 5'-T4 deiodination is
unaffected (7). Such an approach is required because 6-n
propylthiouracil (PTU) cannot completely inhibit D1 activity when
substrate concentration is low (7). Using this approach, we found no
difference in the 125I released at 2 and at 100
nM T4 in the presence of rat thyroid
sonicate, indicating the absence of low Km 5'- T4
deiodinase activity in the adult rat thyroid. This is in contrast to
the 41% inhibition of 125I release by 100
nM T4 using human thyroid homogenate
(Table 1
).
125Iodide release from 100 nM
[125I]T4 is catalyzed by
D1, as shown by its suppression in the presence of 10 µM
rT3 in the human and rat thyroid samples. A
direct D1 activity assay using 1 µM
rT3 as substrate confirmed that D1 activity was
much higher in rat thyroid than in this sample of human thyroid (Table 1
). Thus, there is little D2 mRNA and no detectable D2 activity in rat
thyroid.
Potential TTF-1 but Not Pax-8 Binding Motifs Are Present in the
Human dio2 Gene
We speculated that the much higher D2 mRNA in human than in rat
thyroid might be due to differences in thyroid-specific transcription
factor responsivity of human and rat dio2 genes. A
computer-assisted inspection of the 3' 4 kb of the hdio2
5'-FR was performed to identify possible TTF-1 binding sites in GenBank
clones AF188709 and AC007372. The core of the TTF-1 binding motif is
CAAG but the flanking nucleotide sequence can also influence its
binding properties. A "T" in the 5'-position and a T or G 3' to the
core are required for efficient in vitro binding (12, 23).
We found seven motifs (A, B, C1, D, E, F, G) in the favored
(T)CAAG(G/T) context in the human dio2 gene (Fig. 2
). One motif in poor context, GCAAGA
(C2), was also included because it is located only 6 bp 3' to the C1
site, and we did wish to overlook the possibility of cooperative
effects. The hdio2 5'-FR fragment contains numerous TGCCC
motifs, which are the core of the Pax-8 binding site (17). However,
none of these had the preferred A(G/C)TC sequence in the adjacent
3'-region, such as are found in the Pax-8 binding site of the rat Tg
and TPO promoter (17).

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Figure 2. Schematic Map of the hdio2 5'-FR
This portion of the gene contains seven potential TTF-1 binding sites
in the favored (T)CAAG(G/T) context. (The sites are depicted 5' to 3';
upper strand: A, B, C1, D; lower strand: E, F,
G). The C2 site was investigated because of its proximity to C1.
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The Human but Not the Rat dio2 5'-FR Responds
Transcriptionally to TTF-1
The large number of TTF-1 binding sites suggested that the
hdio2 promoter would respond to coexpressed TTF-1. In fact,
transient coexpression of TTF-1 caused a robust, approximately 11-fold
increase in chloramphenicol acetyltransferase (CAT) expression from the
6.5-kb hdio2 CAT reporter, indicating that one or more of
the TTF-1 sites identified were functional (Fig. 3
). Surprisingly, this response was not
significantly reduced by deletion of approximately 5.9 kb of the 5'-FR,
indicating that sites A, B, E, F, and G were not functional. However,
deletion of either the C1 and C2 (hdio2633
C) or the D binding
sites (hdio2633
D) reduced but did not abolish the
hdio2CAT response (Fig. 3
). The responsiveness to TTF-1 was
reduced to only 3-fold by eliminating all three sites
(hdio2633
CD), suggesting that the functionally critical TTF-1
binding sites were the C and D motifs. The most 3' 83 bp of the
hdio2 promoter (hdio283) showed an approximately 1.8-fold
TTF-1 response (Fig. 3
). With respect to the specificity of the
promoter response to TTF-1 under these conditions, none of the
following promoter CAT constructs were induced more than about
1.7-fold: the
3.7-kb hdio1 5'-FR (24), the
7.5-kb hdio3 gene, the 109-bp thymidine kinase promoter,
or the empty pOCAT2 vector (25). There was a 2.4-fold induction of a
137-bp rGH promoter CAT construct (26), which is of unknown
significance.

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Figure 3. Activity of Human and Rat dio2
Constructs after Cotransfection with a Rat TTF-1-Expressing or Empty
Vector in COS-7 Cells
The hdio26.5 construct contains 6.5 kb of the human
dio2 5'-FR. The rdio2#1 contains approximately 3.8 kb of
the rat dio2 5'-FR. Data are shown as the mean ±
SEM of the CAT/hGH ratios of at least five separate
experiments; *, P < 0.01; **,
P < 0.001 vs hdio2633 by ANOVA
followed by Newman-Keuls.
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To determine whether the lack of a TTF-1 response of the
rdio2 promoter could explain its low expression in rat
thyroid, we cloned the promoter and approximately 12 kbp of the 5'-FR
of the rdio2 gene from a Lambda Dash II genomic
library.2 A 3.8-kb
rdio2 5'-FR fragment was subcloned into the CAT expression
vector pOCAT2 (rdio2#1) and evaluated for a TTF-1 response (Fig. 3
). In
contrast to hdio2, the 3.8-kb rdio2 CAT showed
only a 3-fold response to coexpression of TTF-1.
Comparison of the Human and Rat dio2 5'-FR
Sequences
Since the TTF-1 response of hdio2 was localized to the
most 3' 633 bp of the 5'-FR, we compared these sequences to the
corresponding region of rdio2 (21). These were 73%
identical, including complete conservation of the cAMP response element
(CRE) at about -90 (Fig. 4
). However,
the C1 and C2 TTF-1 binding sites are absent in the rat sequence, and
the D site is eliminated by a G-to-A exchange in the CAAG core of the
motif (Fig. 4
). The most 3'
120-bp region is virtually identical to
that of rdio2 and contains the CRE, the TATA box, and the
most 5'-hdio2 TSS.
Analysis of the Putative TTF-1 Binding Sites in the
hdio2 Gene by Gel Retardation Assay Using a Rat TTF-1 HD
Protein
To correlate a functional response of the putative TTF-1 binding
sites in hdio2 with their affinities for TTF-1 and to
establish that mutations in these eliminate binding, we performed
electromobility shift assays (EMSA) using purified, bacterially
expressed rat TTF-1 HD. The high-affinity TTF-1 binding site of the rat
thyroglobulin (Tg) promoter sequence was used as positive control
(oligo "C" in Ref. 17). The sequences of the wild-type and mutated
oligonucleotides are illustrated in Fig. 5
. The binding affinity of a given
hdio2 site was estimated by comparing its capacity to
inhibit binding of labeled rat Tg oligo "C" to the in
vitro expressed rat TTF-1 HD protein (Fig. 6
). The densitometric analysis of that
experiment is depicted graphically in Fig. 7
. Only two of the four consensus
sequences, C1/C2 and D, were effective inhibitors of the binding of the
Tg site "C" sequence to the rat TTF-1 HD. The D site was the most
effective inhibitor, approximately equivalent to the rat Tg oligo
"C." Mutation of the two adenine nucleotides in the core of the D
site abolished its potency (Figs. 6
and 7
). Mutation of the C1 site
reduced the competitive potency of the C1, C2 oligo to that of the
negative control while mutation of the C2 site had no effect. However,
the C1/C2 fragment was approximately 10-fold less potent than the
positive control or the D oligo (Fig. 7
). To confirm the validity of
the results of these competition studies, we demonstrated that the
binding of the labeled D site to the rat TTF-1 HD is saturable (Fig. 8
).

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Figure 5. Hdio2 Oligonucleotides Used for the
TTF-1 EMSA
The wild-type and mutated sense oligonucleotides are shown. *, The
control was oligo "C" as described by Zannini et al.
(17 ). The sequence of the E, F, and G sites is also indicated.
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Figure 6. Semiquantitative EMSA for the Comparison of the
Affinities of Rat TTF-1 HD Binding for the A, B, C1, C2, and D Sites of
the hdio2 5'-FR to that of the rat Tg TTF-1 Binding Site
"C" Indicated as a Positive Control (see Fig. 5 )
The "Free probe" lane contains only tracer-labeled rat Tg "C"
oligo. "No competitor" controls contained the probe and the TTF-1
HD protein, but no unlabeled competitor oligo. The numbers above
each lane are the unlabeled probe concentrations. Retarded
protein/DNA complex and free probe are indicated with
solid and open arrowheads, respectively.
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Figure 7. Densitometric Analysis of EMSA Results from Fig. 6
Estimating the Relative Binding Affinity of the Various Potential
hdio2 TTF-1 Binding Sites
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Figure 8. Direct Confirmation of Saturable Binding of oligo D
to the TTF-1 HD by EMSA
The "Free D probe" lane contains only the tracer labeled
hdio2 D oligo. The "No competitor" lane contains the
tracer-labeled D oligo and the TTF-1 HD protein. The "Dwt" lane
contains tracer oligo D, the same quantity of HD protein, and 1080
nM unlabeled oligo D. Retarded protein/DNA complex and free
probe are indicated with solid and open
arrowheads, respectively.
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Both C1 and D Sites Are Required for a Complete Functional Response
of hdio2 to TTF-1
The studies reported above localized the functional TTF-1 response
to sequences in the most proximal 633 bp of the hdio2 5'-FR
consistent with the low affinity of the A and B sites in the EMSAs.
However, elimination of the high-affinity D site via a BglII
deletion reduced, but did not eliminate, the response to TTF-1 despite
its high affinity (Fig. 3
). A potential explanation for this
paradoxical result was that the BglII deletion moved the C1
TTF-1 binding site to a more proximal position in the promoter, thereby
increasing its effectiveness (see Fig. 4
). Therefore, we introduced the
specific C1 and D site mutations already shown to eliminate the TTF-1
binding by EMSA into hdio2633 CAT and transfected these
constructs into a human glioblastoma cell line U87. This line was
selected since, in transient transfection analyses with TTF-1, we found
it to have reduced nonspecific TTF-1-stimulated activity when compared
with COS cells. Furthermore, D2 is normally expressed in astroglial
cells (27) although it is not present in the U87 cells (data not
shown). A 10-fold response of the 633-bp hdio2 promoter
occurred with 60 ng cotransfected TTF-1 plasmid but the
rdio2 5'-FR construct was again only 3-fold stimulated.
Surprisingly, given the results of the EMSA studies, mutations of C1 or
D were equally effective in reducing the transcriptional response of
the hdio2633, and mutation in both were required to reduce
the response to TTF-1 to that of the 3.8-kb rat dio2 (Fig. 9
). Qualitatively similar results were
obtained in COS-7 cells (data not shown).

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Figure 9. Effect of Mutations in the C1 and D TTF-1 Binding
Sites of the hdio2 Gene on the Functional Response to
Transiently Expressed Rat TTF-1 in U87 Human Glioma Cells
The C1 (hdio2C1Mut), D (hdio2DMut), or C1 and D (hdio2C1Dmut) TTF-1
binding sites were mutated by site-directed mutagenesis as described in
Materials and Methods. Data are shown as the mean
± SEM of the CAT/hGH ratios of at least four separate
experiments based on CAT/hGH ratios; *, P < 0.01;
**, P < 0.001 vs. hdio2633 by
ANOVA followed by Newman-Keuls. Data are shown as the mean of
response ± SEM of at least four separate experiments
based on CAT/hGH ratios.
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The CRE of rdio2 Responds to Protein Kinase A
The hdio2 gene is regulated by cAMP via a canonical
CRE, and this site is completely conserved in the rdio2
5'-FR (Fig. 4
) (21). As expected, the response of rat dio2
to the catalytic subunit of protein kinase A (PKA) was abolished only
when the region containing this CRE is removed (rdio2#4). This confirms
that rdio2 responds to cAMP via a single canonical CRE (Fig. 10
).

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Figure 10. Response of the rdio2 Promoter to
Cotransfected a Catalytic Subunit of PKA
An 3.8 kb rdio2 5'FR (rdio2#1) and its truncated
derivatives were transfected into HEK-293 cells with 100 ng of vector
expressing the catalytic subunit of PKA or an empty vector. Data
are shown as the mean of response ± SEM of three
separate experiments based on CAT/hGH ratios; *, P
< 0.05 vs rdio2#1; **, P < 0.001
vs. rdio2#1 and rdio2#2 by ANOVA followed by
Newman-Keuls.
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The Human and Rat dio2 and hdio1 Genes Are
Not Affected by Pax-8, whereas hdio2 Is Down-Regulated via
an AP-1 Site
The role of Pax-8, another major transcription factor of
importance for thyroid-specific gene expression, was addressed with
respect to regulation of the dio2 as well as the
dio1 gene. The hdio26.5 CAT plasmid containing the 6.5-kb
hdio2 5'-FR, the
3.8 kb rdio2#1 CAT construct, and a
promoter reporter plasmid containing the
3.7 kb long
hdio1 5'-FR were also transfected into COS-7 to evaluate a
functional response along with 0.5 µg rat Pax-8-expressing plasmid
(17, 24). None of these were induced more than approximately 2.3-fold
while a 5- to 10-fold induction was found with the positive control,
CP5-CAT (28).
The hdio2 5'-FR contains a consensus AP-1 site at -524 to
-518 but its function has not been evaluated (Fig. 4
). This site was
mutated by introducing an AGACCTC replacement
for TGACTCA in the 633-bp hdio2 5'-FR (hdio2-AP1Mut). The
mutant and the wild-type hdio2633 constructs were transfected into
COS-7 cells in three separate experiments along with an hGH plasmid to
monitor transfection efficiency. Inactivation of the AP-1 site
increased the hdio2 basal promoter activity approximately
2-fold, suggesting that the hdio2 promoter is down-regulated
via the AP-1 site (data not shown). There was no response of either the
wild-type hdio2 or the AP-1 mutant to phorbol ester
[12-O-tetradecanoylphorbol 13-acetate (TPA)].
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DISCUSSION
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The effects of thyroid-specific transcription factors TTF-1 and
Pax-8 on the dio2 gene were examined to address their
potential role in the regulation of dio2 expression in
different species. The present studies were initiated to explain the
very high levels of D2 mRNA in the human thyroid gland but no
detectable D2 mRNA expression in the rat FRTL-5 thyroid cell line (7).
We speculated that if the FRTL-5 line was representative of the rat
thyroid in general that differential responses to thyroid-specific
transcription factors might explain this.
We found extremely low concentrations of dio2 mRNA in rat
thyroid, and D2 activity was undetectable consistent with earlier
results in FRTL-5 cells (7, 9). The absence of significant D2 activity
differs from results reported by Bates et al. (34
pmol/h/mg protein) (29). However, these authors found only 14%
inhibition of 1 nM
[125I]rT3 deiodination by
100 nM unlabeled T4 in rat
thyroid homogenate. This is similar to our results (Table 1
) and
indicates that rT3 deiodination by D2 is minimal
in rat as opposed to human thyroid. The methodological description
provided is not sufficiently detailed to resolve this apparent internal
discrepancy (29).
A functional 5'-FR-CAT transient expression assay showed that the
hdio2 gene was highly responsive (
10 fold) to coexpressed
rat TTF-1 HD. We isolated and subcloned a
3.8-kb rat dio2
5'-FR/promoter clone to compare with the human gene. Consistent with
our hypothesis, the rat dio2 gene responded only
approximately 3- fold to coexpressed TTF-1. Neither human nor rat
dio2 was induced by Pax-8, and both genes contain one
functional CRE about 90 bp 5' to the transcriptional start site (Fig. 4
and Ref. 21). These results suggest that the lack of response to TTF-1
could explain the much higher expression of D2 mRNA in human than in
rat thyroid, although the alternative possibility of a rat
thyroid-specific transcriptional repressor cannot be completely ruled
out. The human dio1 gene is unresponsive to Pax-8 or
TTF-1.
A number of studies have evaluated the DNA sequence requirements for
TTF-1 binding. Detailed in vitro analysis of binding of
randomly generated Tg "C" site sequences to the rat TTF-1 HD
protein showed that the binding site consensus was CCCAGTCAAGTGTTCTT
(23). The consensus derived from analysis of the TTF-1 binding sites of
the rat, human, bovine, and dog Tg and TPO promoters (ACTCAAGTNNNN)
(12) or only from the rat Tg promoter (...
NNNAC(G)TCAAGTA(G)NNNN ... ) (30) were more flexible. These studies
indicate that a CAAG core is usually present but not sufficient for
efficient TTF-1 HD binding. Seven TCAAG(G/T) motifs were found in the
3'
4 kb hdio2 5'-FR. However, the functional studies of
the hdio2 gene indicated that only the most proximal two
sites, C1 (
620 bp 5' to the major TSS) and D (
230 bp 5' to this
site), were required for a complete response to TTF-1. In comparison,
footprinting and sequence homology search-based approaches identified
three TTF-1 binding sites in the rat and human Tg promoter, three in
the rat and five in the human TPO gene, and one site in the rat TSH
receptor promoter (12).
The order of magnitude of the response of hdio2 to TTF-1
(
10 fold) in both COS-7 and U87 cells is similar to that conferred
by the rat Tg "A" and "C" TTF-1 binding sites in FRTL-5 cells
(30). This transcriptional response is quite large compared with the
less than 2-fold response of the sodium iodine symporter promoter in
FRT cells (31) or to the approximately 2.5-fold response of the TSH
receptor in COS-7 cells (32).
In vitro determination of the TTF-1 binding affinity of the
hdio2 sites by EMSA showed that the C1 and D
hdio2 TTF-1 binding sites that were important in the
functional studies showed the highest HD binding affinity. However,
despite the fact that the D site binding affinity was approximately
10-fold higher than that of the C1, C1 was as active as the D site
(Figs. 6
, 7
, and 9
) even though it is about 390 bp farther away from
the TSS.
TTF-1 binds its recognition sequence via a HD, that is highly similar
to the Drosophila NK2 HD (33). The rat TTF-1 HD can
reproduce the binding of the entire protein (10). It is well known that
the DNA binding specificity of HDs is promiscuous since the same HD can
recognize different sequences without a clear consensus (34, 35, 36). For
example, the functionally important TTF-1 binding sites in the bovine
Tg gene upstream enhancer element do not contain a CAAG motif (37).
However, the TTF-1 binding motifs in most of the known functional TTF-1
sites do contain the CAAG core as in hdio2 in contrast to
the typical TAAT core recognition sequence of the known HDs (12,
34, 38).
The TTF-1 HD binding to the core is significantly affected by the
flanking nucleotides. On the basis of the comparison of the
characterized TTF-1 binding sites with the available in
vitro TTF-1 HD binding data, the presence of a TCAAG(G/T) motif is
generally required for efficient TTF-1-governed transcription (12, 23).
However, this rule must be tempered by functional studies on the rat Tg
promoter. Three TTF-1-protected regions (AC) were found by DNAse
footprinting each containing a TCAAGT motif. However, only two are
functional since studies in FRTL-5 cells show that mutation of the B
motif has a limited influence on promoter activity (12, 30, 39). This
discrepancy between in vitro TTF-1 binding data and
functional relevance was also demonstrated in the bovine Tg upstream
enhancer element, where one of the three sites footprinted by TTF-1 is
not functional (37).
In the case of hdio2, the flanking nucleotides might explain
the difference between the TTF-1 HD binding of the C1 and D sites. The
strong in vitro binding of the D site may be due to the G in
position 7 (1 T CAAG G/T G 7), which increases the TTF-1 HD binding
in vitro (23) since only the D site fulfills this criterion
among the A, B, C, and D hdio2 promoter TTF-1 binding sites.
However, the discrepancy between the in vitro binding and
in vivo function of the C1 and D sites is in accordance with
the data showing that the TTF1-HD affinity of a site does not
necessarily parallel its function. Clearly, the latter criterion is the
most specific requirement.
It is of interest that despite the 73% identity between the 3'-portion
of the rat and human dio2 promoters, the C1 and D TTF-1
binding sites of the hdio2 are not present in
rdio2. We conclude that this is responsible for the very
different TTF-1 responsiveness of these two genes and is likely to
explain the striking difference in the expression levels of the D2 mRNA
in the human and rat thyroid. It may also be noted that, in the
recently published mouse dio2 promoter region (40), both the
C1 and D TTF-1 binding sites are not present, suggesting this is
characteristic of the rodent dio2 gene. These TTF-1 binding
sites may also be relevant to expression of D2 in other human
TTF-1-expressing tissues such as the developing brain.
It should also be noted that D2 activity is lower in human thyroid than
might be expected from the high level of D2 mRNA. There are several
potential reasons for this. The 5'-untranslated region (UTR) of the D2
mRNA contains three short open reading frames, which may reduce
translation efficiency (21). It is also known that the proteasomal
destruction of D2 is markedly accelerated by T4
deiodination (41, 42). If the T4 in human thyroid
cells is deiodinated by D2, this would also reduce the ratio of D2
protein to D2 mRNA.
TTF-1 is present in a variety of thyroid and lung tumors (43). TTF-1
expression is reduced in certain human thyroid carcinoma cell lines,
and its expression can be restored by demethylating agents (44). On the
other hand, the nuclear extract from toxic human thyroid adenomas
contains elevated levels of TTF-1 while phosphorylated CREB is reduced
(45). Thyroid adenomas also express high D2 mRNA levels (7). These
results suggest that it is TTF-1, not cAMP, that accounts for the high
levels of D2 mRNA in human thyroid cells. Since TTF-1 is reduced in
many thyroid carcinomas, one might expect that D2 as well would be
reduced and could serve as an additional marker for
differentiation.
The expression of TTF-1 in the human central nervous system has not
been localized, and there is no direct comparison of D2 expression in
the human and rat brain and pituitary. However, D2, but not D1,
activity is found in the adult human central nervous system (46). We
also found no D1, but high D2, activity in two
-glycoprotein
pituitary tumors (7). Further studies are required to investigate
whether TTF-1 expression increases D2 expression in the human pituitary
and brain.
With respect to other potential influences on D2 expression, we have
shown that the hdio2 gene responds to PKA via a single
canonical CRE located 90 bp 5' to the TSS (21). In vivo D2
activity and mRNA are increased in rat brown fat by a cAMP-mediated
pathway linked to the sympathetic nervous system, and D2 activity is
induced in cultured astrocytes by (Bu)2cAMP (4, 22, 27, 47). This response can be explained by the single canonical CRE
in the rdio2 promoter in the same position as that in
hdio2.
While the hdio2 contains a consensus AP-1 site, we could not
induce the hdio2 promoter in HEK-293 cells by a combination
of the PKC activator, phorbol ester, and A23187 (21). On the other
hand, in primary cultures of human thyroid cells, TPA causes a 50%
decrease in D2 activity (48). While neither the wild-type nor AP-1
mutant hdio2 constructs responded to phorbol ester in our
transient expression system, mutation of the AP-1 site caused a 50%
increase in basal promoter activity, suggesting a constitutive negative
effect due to this site.
In conclusion, the present findings indicate that D2 expression in the
human thyroid is positively controlled by TTF-1 via two DNA binding
sites, C1 and D, which are not present in the rdio2 gene.
The lack of the C1 and D sites in the rat D2 promoter can explain the
very low D2 mRNA levels in the rat thyroid. The human dio1
gene does not respond to TTF-1 or Pax-8, but both human and rat
dio2 genes respond to cAMP. These results, together with the
presence of D2 mRNA in the human, but not rodent, myocardium and
skeletal muscle, indicate that species-specific differences in
dio2 genes may cause significant phenotypic differences in
tissue D2 expression.
 |
MATERIALS AND METHODS
|
---|
Rat Genomic Library Screening and Subcloning
The rdio2 5'-FR was isolated from a rat genomic
Lambda Dash II library (Stratagene, La Jolla, CA). The
plating and screening were performed using standard techniques (49).
Clones were screened with a rat D2 5'-UTR PCR fragment (bp 300546 in
GenBank clone U53505) obtained by the rD2/d1s and rD2/d2r oligos (see
Table 2
) generated from a rat D2 cDNA clone kindly
provided by Drs. V. Galton and D. St. Germain and labeled by
[32P]dCTP. Four positive clones were
isolated and two (
1215 kb each) were subcloned into Bluescript KS.
One of these was digested 5' by NotI and 3' by
BamHI; the latter was in the 5'-UTR 160 bp 5' to the
initiator ATG of the rat D2 cDNA previously reported (GenBank U53505,
position 404). The approximately 4.4-kb fragment, which hybridized to
the labeled rD2/d1s, was subcloned into the pOCAT2 vector to form
rdio2#1. The 5'-transcriptional start site was identified by comparison
to the hdio2 gene. The rdio2 5'-FR has been
entered in GenBank (accession no. AF249274).
Eukaryotic CAT Expression Vectors and Constructs
The D2 5'-FR fragments were inserted into polylinker sites of a
CAT reporter vector, pOCAT2 (25). The hdio2 CAT construct
containing the
6.5 kb hdio2 5'-FR (hdio26.5) or its
most 3' 633 bp (hdio2633) has been described (21). The latter
contains an artificial SacI site just 5' to -633 so that a
SacI/HindIII deletion was used to remove the
fragment between -633 and -610, which contained the two putative
TTF-1 binding motifs, C1 and C2 (hdio2633?C) (Fig. 4
). A
BglII deletion removed the region between -492 and -84,
including the D site and the CRE (hdio2633?D). The hdio2633?CD
construct was obtained by digesting hdio2633?C by BglII
combining the two internal deletions in the same plasmid. A
SacI-BglII deletion removed the region between
-633 and -84 (hdio283).
PCR-based mutagenesis was used to introduce point mutations into the C1
and D TTF-1 binding sites, changing the CAAG core to CgtG in both. To
prepare the C1 mutant (mut), D wild-type (wt) construct (hdio2C1Mut), a
mut sense (Bp56, Table 2
) and a wt antisense (hD2g12r) oligonucleotide
were used. The SacI/Pst (+7; blunt) digested fragment was
inserted between the SacI and HincII sites of
pOCAT2. For the C1 wt, D mut construct (hdio2DMut) overlap-extension
Vent PCR was used. In brief, Tp12 (wt sense) and Bp57 (mut antisense,
Table 2
) fragment and the hD2DS1Mut (mut sense) and hD2g12r (wt
antisense) fragment were combined by PCR using Tp12 and hD2g12r as the
outside oligonucleotides, and the 3'-blunt-ended fragment inserted
into pOCAT2 at a SacI and blunt-ended PstI site.
The C1, D double mutant (hdio2C1DMut) was constructed by exchanging the
BglII fragment of the hdio2DMut with that in the
hdio2C1Mut.
To construct the AP-1 mutated 633-bp hdio2 construct
(hdio2-AP1Mut), the Tp12-Bp110 Vent fragment was cut by
HindIII and AvrII and inserted between the
corresponding sites of the wild-type hdio2633 CAT, mutating the
TGACTCA site to aGACctc.
The rdio2#1 constructs contained a NotI and a
BamHI site, the latter located in the 5'-UTR. The fragment
was blunted and subcloned into the HincII site of pOCAT2 and
contained
3.8 kb rdio2 5'-FR and
600 bp rD2 5'-UTR.
This 3'- end was 160 bp 5' to the initiator ATG of the rD2 coding
region (GenBank U53505). Further 5'-truncations were performed starting
with the rdio2#1, removing an EcorI-HindIII
(resulting in rdio2#3) or EcorI-BglII fragment
(resulting in rdio2#4) (Fig. 10
). The two truncated rdio2
constructs contain 658 or 83 bp 5' to the putative rdio2
TSS, respectively, as well as a portion of the 5'-UTR sequence. All
constructs were sequenced to confirm the accuracy of the constructions
and the mutations.
DNA Transfection and CAT Expression Assays
The reporter CAT plasmids were transfected into U87 or COS-7
cells as previously described using calcium phosphate precipitation
(26). For each transfection, 10 µg pOCAT2-based vector were
cotransfected with 0.06 or 0.1 µg rat TTF-1 for U87 or COS-7 cells,
respectively (10). The Pax-8 responsiveness was studied in COS-7 cells
using 0.5 µg Pax-8 coding expression vector (17). The CP5-CAT was
used as positive control (28). The TTF-1, Pax-8, and CP5-CAT clones
were kindly provided by Dr. R. Di Lauro. The rat dio2
response to the
-catalytic subunit of PKA was tested in HEK-293
cells using 10 µg of rdio2CAT plasmid and 100 ng PKA
-catalytic
subunit expressing plasmids. The PKA- expressing plasmid was a gift
of Dr. R. Maurer (50). For the uninduced control, a cytomegalovirus
(CMV)-ßGal or an empty CDM-8 vector was transfected in the same
quantity as the TTF-1- or Pax-8-expressing vector. The cells were
sonicated and assayed for CAT activity as described by Seed and
Sheen (51). The transfection efficiency was monitored by the
cotransfection of 3 µg TKhGH, as described previously (26). In the
Pax-8 experiments, the pXGH5 plasmid was used (52). The results are
expressed as the mean of CAT activity/hGH ±
SEM. Each construct was studied in duplicate in
at least three separate transfections.
Semiquantitative EMSA
Sense and antisense oligonucleotides containing hdio2
putative TTF-1 binding sites were annealed to produce double-stranded
DNA. As a positive control, we used the high-affinity TTF-1 binding
site (site "C") of the rat Tg promoter sequence (see Fig. 5
). For
probe generation, fragment D and the positive control were labeled by
T4 polynucleotide kinase using
[32P]dATP.
The binding buffer contained 20 mM Tris-HCl (pH
7.5), 200 mM KCl, 1 mM
dithiothreitol, 10% glycerol, 1 mg BSA per ml, 1 mg poly(dI-dC) per
ml, and varying concentrations of the unlabeled DNA fragment. The
protein was purified rat TTF-1 HD protein expressed in bacteria, kindly
provided by Dr. R. Di Lauro (34). The unlabeled competitor was
incubated with HD for 10 min at room temperature. Then
30,000 cpm
probe was added, and the mixture was incubated for an additional 30 min
at room temperature. Bound and free probe were separated in a
nondenaturing PAGE (7%) at 4 C in 1xTBE running buffer. After drying,
autoradiography was performed at -80 C for approximately 3 h. For
the semiquantitative analysis of competitive potency, samples were run
at least twice. The density was determined using a Computing
Densitometer (software v.3.22, Molecular Dynamics, Inc.
Sunnyvale, CA). The density of the shifted band was expressed as a
fraction of the sum of the densities of the upper and lower bands for
each lane. This was then expressed as a fraction of that found without
competitor.
Northern Blots
Male Sprague Dawley rats were made hypothyroid using 0.05%
methimazole for 3 weeks under a protocol approved by the Animal Use
Committee. Hypothyroidism was verified by the absence of
T4 in serum by T4 RIA. All
human tissues were obtained under protocols approved by the
Institutional Review Board at the Brigham and Womens Hospital
(Boston, MA). Thyroids were collected at the operating room immediately
after removal from the patients and placed on ice. After the
pathologists inspection and removal of relevant samples, the
remaining tissues were immediately frozen in liquid nitrogen. This was
within approximately 15 min of removal from the patient. After this,
they were transferred to -80 C and kept at that temperature until
aliquots were processed for either RNA extraction or deiodinase assays.
RNA was isolated according to the single-step RNA isolation method of
Chomczynski (53). Various amounts were precipitated with 0.3
M sodium acetate, washed in 75% ethanol, resuspended in
1x RNA loading dye, and electrophoresed at room temperature according
to the procedure of Lehrach et al. (54). Thirty micrograms
of rat thyroid gland RNA of hypothyroid and euthyroid rats were loaded
on a 1.2% agarose/1x3-(N-morpholino)propanesulfonic
acid/1.85% formaldehyde gel containing 0.5 µg/ml ethidium bromide.
The RNA was transferred to Genescreen Plus (NEN Life Science Products, Boston, MA) by the capillary method against 10xSSC
and probed first with the
1.1-kb EcoRI fragment from rD2
in pBS-KS (4). The cDNA probe was labeled by the random primer method
using Prime-It kit (Stratagene, La Jolla, CA) and
[32P]dCTP. One million counts/min per ml of
hybridization solution was applied. Blots were hybridized at 42 C for
16 h in 40% formamide/0.64 M NaCl/0.8
mM EDTA, pH 7.4/0.04 M
PO4 buffer, pH 5.6/2xDenhardts solution/1.6%
SDS/7% dextran sulfate/80 µg/ml sonicated denatured thymus DNA.
After hybridization, blots were sequentially washed three times with
2xSSC/0.1% SDS at room temperature, 20 min; once at 42 C, 20 min;
0.5xSSC/0.1% SDS at 42 C for 20 min, 0.2xSSC/0.1% SDS at 42 C for
20 min; and then at 50 C, 55 C, 60 C. Blots were exposed to film for 2
weeks. They were stripped of residual signal, and then probed with a
mouse ß-actin probe (450 bp, KpnI/SacI),
random-primed, and washed as above. Blots were exposed to film for 1
day. Blots were then stripped and checked to ensure that there was no
residual signal, and then probed with rD1 probe [G21-KS
(XhoI/HindIII), 775 bp fragment]. Hybridization
was as for rD2 except that 100 µg/ml of denatured, sonicated thymus
DNA was used. The blots were washed up to 0.2xSSC/0.1% SDS at 45 C
for 30 min and exposed for 2 days. Densitometry was performed using a
computing densitometer (see above). The rD2 and rD1 signals were
normalized to that of ß-actin.
D2 Activity
The thyroids of three adult (1214 weeks) male Sprague Dawley
rats and a patient with TSH-induced hyperthyroidism were sonicated in
0.1 M potassium phosphate buffer (pH 6.9), 1 mM
EDTA (PE) buffer containing 0.25 M sucrose and 10
mM dithiothreitol (DTT). The animal and the human samples
were obtained under approved of animal and IRB protocols.
Sonicated protein (100 µg) was assayed in duplicate for 5'-deiodinase
activity for 120 min at 37 C in a final volume of 300 µl PE buffer
containing 20 mM DTT and 2 or 100 nM
T4 or 10 µM
rT3 in the presence of approximately 90,000 cpm
of [125I]T4 purified by
chromatography on an LH-20 column (7). Five and 20 µg of sonicate
protein were assayed in duplicate for D1 activity for 60 min at 37 C in
300 µl PE buffer containing 1 µM rT3, 120,000 cpm of
[125I]rT3, and 10 mM DTT. The
125I-
and substrate were separated by
trichloroacetic acid (TCA) after the addition of horse serum as
described previously (55). Quantities of sonicate were adjusted to
consume less than 30% of the substrate. The background for the assays
was 1.8% of total T4 and 1.5% of total
rT3.
Sequencing
The rdio2 5'-FR fragment and all constructs were
sequenced in the ABI Prism 377 automated sequencer using dye
terminators.
Reagents
[125I]T4 or
rT3 and
or
[32P]
were purchased from NEN Life Science Products
(Boston, MA). Other chemicals were of molecular biology or reagent
grade. All primers were synthesized by Life Technologies, Inc. (Gaithersburg, MD).
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. V. Galton and D. St. Germain for the kind gift of
the rD2 cDNA and Dr. R. Di Lauro for the TTF-1, Pax-8, and CP5-CAT
plasmids and for the in vitro-expressed TTF-1 homeodomain
protein.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. P. Reed Larsen, Thyroid Division, Department of Medicine, Brigham and Womens Hospital, Harvard Medical School, Room 560, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail: larsen{at}rascal.med.harvard.edu
This work was supported by NIH Grants DK-36256 and T-32-DK-07529.
1 These authors contributed equally to this paper. 
2 The sequence reported in this paper has been
deposited in the GenBank under accession no. AF249274. 
Received for publication August 2, 2000.
Revision received September 27, 2000.
Accepted for publication October 3, 2000.
 |
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