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 Women’s 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 1Go). The hypothyroid rat thyroid expressed lower levels of D2 mRNA than did that of normal animals (Fig. 1Go). 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.

 
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 1Go). 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 1Go). Thus, there is little D2 mRNA and no detectable D2 activity in rat thyroid.


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Table 1. Deiodination in Rat and Human 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. 2Go). 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.

 
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. 3Go). 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 (hdio2–633{Delta}C) or the D binding sites (hdio2–633{Delta}D) reduced but did not abolish the hdio2CAT response (Fig. 3Go). The responsiveness to TTF-1 was reduced to only 3-fold by eliminating all three sites (hdio2–633{Delta}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 (hdio2–83) showed an approximately 1.8-fold TTF-1 response (Fig. 3Go). 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 hdio2–6.5 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 hdio2–633 by ANOVA followed by Newman-Keuls.

 
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. 3Go). 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. 4Go). 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. 4Go). 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.



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Figure 4. Comparison of the Promoter and 5'-FRs of the Rat and Human dio2 Genes

The C and D TTF-1 binding sites are indicated. The region containing the CRE and the TATA box is highly conserved. The position of an artificial SacI site used to create the hdio2–633{Delta} constructs is also shown, as are the HindIII and BglII sites used for the constructs in Figs. 3Go and 10Go.

 
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. 5Go. 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. 6Go). The densitometric analysis of that experiment is depicted graphically in Fig. 7Go. 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. 6Go and 7Go). 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. 7Go). 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. 8Go).



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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. 5Go)

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. 6Go 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.

 
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. 3Go). 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. 4Go). Therefore, we introduced the specific C1 and D site mutations already shown to eliminate the TTF-1 binding by EMSA into hdio2–633 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 hdio2–633, and mutation in both were required to reduce the response to TTF-1 to that of the 3.8-kb rat dio2 (Fig. 9Go). 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. hdio2–633 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.

 
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. 4Go) (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. 10Go).



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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 {alpha} 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.

 
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 hdio2–6.5 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. 4Go). 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 hdio2–633 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)].


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. (3–4 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 1Go) 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. 4Go 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. 6Go, 7Go, and 9Go) 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 (A–C) 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 {alpha}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 300–546 in GenBank clone U53505) obtained by the rD2/d1s and rD2/d2r oligos (see Table 2Go) generated from a rat D2 cDNA clone kindly provided by Drs. V. Galton and D. St. Germain and labeled by {alpha}[32P]dCTP. Four positive clones were isolated and two (~12–15 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).


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Table 2. PCR Primers Used for Generation of Fragments for CAT Constructs and for Library Screening

 
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 (hdio2–6.5) or its most 3' 633 bp (hdio2–633) 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 (hdio2–633?C) (Fig. 4Go). A BglII deletion removed the region between -492 and -84, including the D site and the CRE (hdio2–633?D). The hdio2–633?CD construct was obtained by digesting hdio2–633?C by BglII combining the two internal deletions in the same plasmid. A SacI-BglII deletion removed the region between -633 and -84 (hdio2–83).

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 2Go) 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 2Go) 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 hdio2–633 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. 10Go). 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 {alpha}-catalytic subunit of PKA was tested in HEK-293 cells using 10 µg of rdio2CAT plasmid and 100 ng PKA {alpha}-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. 5Go). For probe generation, fragment D and the positive control were labeled by T4 polynucleotide kinase using {gamma}[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 Women’s Hospital (Boston, MA). Thyroids were collected at the operating room immediately after removal from the patients and placed on ice. After the pathologist’s 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 {alpha}[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 5.6/2xDenhardt’s 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 (12–14 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 {alpha} or {gamma} [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 Women’s 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. Back

2 The sequence reported in this paper has been deposited in the GenBank under accession no. AF249274. Back

Received for publication August 2, 2000. Revision received September 27, 2000. Accepted for publication October 3, 2000.


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