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Endocrinology Vol. 139, No. 4 1999-2006
Copyright © 1998 by The Endocrine Society

ARTICLES

Multiple Transcripts Encoded by the Thyroid-Specific Enhancer-Binding Protein (T/EBP)/Thyroid-Specific Transcription Factor-1 (TTF-1) Gene: Evidence of Autoregulation1

Hidenori Oguchi2 and Shioko Kimura

Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Shioko Kimura, Ph.D., Building 37, Room 3E-24, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892. E-mail: shioko{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Multiple transcripts derived from the gene encoding rat thyroid-specific enhancer-binding protein (T/EBP)/thyroid-specific transcription factor-1 (TTIF-1) were identified by complementary DNA cloning and sequencing, and Northern blotting analyses. Six different types of complementary DNAs were identified that differ at their 5' noncoding regions; four contain an intron of different lengths, whereas the other two possess no intron. Ribonuclease protection analyses revealed that multiple promoters are scattered throughout the upstream region, and the usage of these different promoters together with alternative splicing leads to a family of T/EBP messenger RNA (mRNA) species. A similar pattern of expression was also found in the human T/EBP gene expressed in a lung carcinoma cell line. Longer T/EBP mRNAs are more abundant in rat FRTL-5 thyroid cells maintained in the absence of TSH (-TSH) than in cells maintained in the presence of TSH (+TSH). Transfection analyses using the rat T/EBP gene DNA upstream of the ATG initiation codon connected to the luciferase reporter plasmid showed a similar relative activity profile between -TSH and +TSH culture conditions, suggesting that the abundance of longer mRNAs in -TSH conditions may not directly correlate with differences in promoter activities. Rather, TSH status might have a role in maintaining the physiological state of the cells. The upstream DNA of the rat and human T/EBP genes share a cluster of high and low sequence similarities, and both possess respectively 24 and 18 putative T/EBP-binding sites throughout. Cotransfection analyses of the T/EBP promoter-reporter constructs with a T/EBP expression vector into human HepG2 cells, which do not express T/EBP, suggested that autoregulation may be involved in controlling both rat and human T/EBP gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID-SPECIFIC enhancer-binding protein (T/EBP), also called thyroid-specific transcription factor-1 (TTF-1) or NKX-2.1, is a member of the NKX-2 gene family of homeodomain-containing transcription factors (1, 2, 3, 4). T/EBP (TTF-1) regulates thyroid- and lung-specific expression of genes such as those encoding thyroid peroxidase (5, 6, 7), thyroglobulin (8), and TSH receptor (9, 10) in the thyroid, and surfactant proteins (SP) A (11), B (12, 13), and C (14) and Clara cell secretory protein (15) in the lung, respectively (16, 17). T/EBP is also involved in embryonic organogenesis (16, 17). T/EBP expression is first detected on mouse embryonic day 9.5 in the thyroid and lung primordia and restricted areas of the ventral forebrain (18). The T/EBP-null mouse is missing the thyroid and pituitary and has severe defects in the lung and hypothalamus, thus demonstrating an essential role for T/EBP in the genesis of these organs (19).

The T/EBP gene was characterized in rat (20, 21) and human (22) and was found to consist of at least two exons with the presence of a possible additional exon reported in rat (2, 21). Messenger RNAs of different lengths were found on Northern blots using RNAs isolated from rat thyroid-derived FRTL-5 cells, rat thyroid tissue, and lung (1, 2). These messenger RNAs (mRNAs) appeared to correspond to alternatively spliced transcripts, suggesting the presence of a family of T/EBP mRNA species (2). Lonigro et al. (21) have determined three transcription start sites in the rat T/EBP gene around -200 bp relative to the translation start site. However, ribonuclease (RNase) protection assays suggested the presence of another promoter located further upstream.

Expression of the T/EBP gene is transcriptionally down-regulated by the presence of TSH in rat FRTL-5 thyroid cells (10). It is also shown to be regulated by hepatocyte nuclear factor-3{alpha} and -ß (HNF-3{alpha} and HNF-3ß) in respiratory epithelial cells (23). HNF-3 promoter regulation, which, in turn, involves T/EBP and autoactivation (24). HNF-3 proteins appear to cooperate with T/EBP and other transcription factors for control of lung-specific genes, such as SP-B (25) and Clara cell secretory protein (26, 27), and thyroid peroxidase (28). HNF-3{alpha} and HNF-3ß are known to be expressed in endodermal lineages in the developing foregut before lung bud formation (29, 30). Cross-regulatory mechanisms were suggested to play a role in maintaining the expression of cell-specific transcription factors in the respiratory epithelium (24). Despite the involvement of T/EBP in cross-regulatory mechanisms in the expression of lung-specific genes, little is known about the regulation of the T/EBP gene in the thyroid. Thus, it is important to study how the expression of T/EBP is controlled to understand the signals required for initiating the regulatory cascade that terminates in expression of its target genes in the differentiated thyroid and lung and during embryogenesis. In this report, newly identified T/EBP mRNA species are described that apparently arise from a combination of the usage of multiple transcription start sites and different alternative splicings of the T/EBP primary transcript. A family of T/EBP mRNAs was found in both rat and human T/EBP genes. Similarities were also found in the T/EBP gene upstream sequences between rat and human, and a possible autoregulatory mechanism is discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
FRTL-5 cells (American Type Culture Collection, Rockville, MD; CRL 8305) are a continuous line of functioning epithelial cells derived from normal Fisher rat thyroids (31, 32). The isolation, growth, and basic characteristics of FRTL-5 cells were previously described (10, 32). The cells were grown in Coon’s modified Ham’s F-12 medium supplemented with 5% calf serum, 1 mM nonessential amino acids, and a six-hormone mixture (6H) containing bovine TSH (0.3 mU/ml), insulin (10 µg/ml), hydrocortisone (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (2 ng/ml), and somatostatin (10 ng/ml). The cells were also cultured in 5H medium, which contains all the components of 6H except TSH. Under 5H conditions, FRTL-5 cells become quiescent. Human lung carcinoma NCI-H441 cells (American Type Culture Collection HTB 174) were cultured in RPMI 1640 medium supplemented with 10% FCS.

Screening of complementary DNA (cDNA) and genomic libraries
A cDNA library was constructed using the SuperScript Choice System for cDNA Synthesis (Life Technologies, Grand Island, NY) and polyadenylated RNA isolated from FRTL-5 cells maintained in either 6H or 5H or from NCI-H441 cells. Oligo(deoxythymidine) and random primers were used to construct libraries, which were then combined before screening. The rat and human cDNA libraries were screened using a fragment of about 450 bp of the rat T/EBP cDNA as a probe whose sequence starts at the translation start site ATG (Fig. 1Go, probe b). The libraries were also screened with an upstream fragment of approximately 1 kilobase (kb; -2201 and -1284 bp) prepared by PCR using the rat T/EBP gene (Fig. 1Go, probe f). Rat and human T/EBP genes and the flanking sequences were obtained by screening rat and human genomic libraries (Clontech, Palo Alto, CA) with the rat T/EBP cDNA as a probe and subcloning into pUC 9, pGEM 3Z, or pGEM 7Z vectors. Sequencing of the cDNAs and genomic clones was carried out using fluorescent label-tagged dideoxynucleotides and Taq polymerase, and the products were analyzed with an Applied Biosystems model 373A DNA sequencer (Foster City, CA) and MacVector software (Oxford Molecular Group, Beaverton, OR).



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  		Figure 1. Structure of the 5'-portion of rat and human T/EBP cDNAs. On the top, the genomic structure of the T/EBP gene downstream from the ATG is presented; two exons are shown by a box. The coding sequence is shaded, and the homeobox is indicated in a lightly shaded box inside the coding region. Below the schematic gene, six different types of cDNAs (I–VI) are illustrated in the rat (left) and three are shown in the human (right). Base numbers are based on the ATG as 0. The 5'-end of each type of clone is that of the longest. The numbers of clones isolated from cDNA libraries of rat FRTL-5 cells maintained in 5H or 6H conditions or of human NCI-H441 cells are indicated. The locations of probes used for screening cDNA libraries and Northern blotting are also shown.

 
RNase protection assay
RNase protection assay was carried out using total RNA isolated from rat FRTL-5 cells maintained in the absence of TSH (5H conditions) and the PRA II RNase protection assay kit (Ambion, Austin, TX). A fragment of DNA between -1946 and -1558 bp with XhoI and EcoRI sites at its 5'- and 3'-ends, respectively, was prepared by PCR, followed by subcloning into the pGEM 7Z vector. This sequence flanks the 5'-end of the type III clone. A riboprobe was prepared by using XhoI-digested plasmid, SP-6 RNA polymerase, and [{alpha}-32P]CTP (800 Ci/mmol; New England Nuclear-DuPont, Boston, MA). Reaction products were run on 5% polyacrylamide-8 M urea gel.

Northern blot analyses
mRNA was isolated from rat FRTL-5 cells maintained in the presence (6H) or absence (5H) of TSH by using a FastTrack kit (Invitrogen, San Diego, CA). RNA was electrophoresed on a 2.2 M formaldehyde-1% agarose gel, blotted to a GeneScreen Plus membrane (New England Nuclear-DuPont), and hybridized with various probes (Fig. 1Go) by the method of Church and Gilbert (33). The filters were washed with 2 x SSC (2 x SSC is 0.3 M NaCl and 30 mM sodium citrate, pH 7.0) containing 0.5% SDS at 65 167 C.

Transfection experiments
Fragments containing various lengths of the rat and human T/EBP gene upstream DNAs were generated by PCR using synthetic oligonucleotide primers and the rat or human T/EBP genomic clones as a template. PCR fragments were inserted into the HindIII site of the promoterless luciferase vector pSV0AL-A{Delta}5' (34). HindIII sites were incorporated into the PCR fragments in the case of human gene, whereas for the rat T/EBP gene, due to the internal HindIII site present in the upstream sequence, blunt end ligation was carried out at the HindIII site. An expression plasmid, pCMV4-T/EBP-1, which contains the rat T/EBP cDNA, has been described previously (2) and was used for cotransfection assays. Ten micrograms of rat or human T/EBP-promoter luciferase constructs were transfected into rat FRTL-5 cells by the diethylaminoethyl-dextran method (35) or into human NCI-H441 cells by the calcium phosphate method (36), respectively. Two micrograms of pSV2CAT were used to normalize transfection efficiency. In the case of cotransfection analyses, 2 or 10 µg pCMV4-T/EBP-1 plasmid were respectively added to DNA mixtures containing rat or human constructs and transfected into human hepatoma HepG2 cells by the calcium phosphate method. For transfection of FRTL-5 cells maintained in 5H medium, cells were grown in 6H until 70–80% confluence was obtained. Cells were then switched to 5H conditions for 5 days, followed by culture in 6H for several hours before transfection. After transfection, the cells were maintained in 5H conditions. All of the cells transfected were harvested 2 days later, and luciferase activity was measured as described previously (5), using a Monolight 2010 luminometer (Analytical Luminescence Laboratories, San Diego, CA). Activity was obtained as an integrated value of luminescence for 30 sec. Chloramphenicol acetyltransferase activity was measured as previously described (5, 37).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of rat T/EBP cDNAs
In a previous study (2), several T/EBP cDNA clones were isolated from a rat FRTL-5 thyroid cell cDNA library that contained, among different sequences, what appeared to be an intron. By comparing the sequences of these cDNAs with those of the rat T/EBP gene and the flanking region, it became clear that they were derived from an alternately spliced primary transcript of the gene. These and Northern blotting results suggested that the T/EBP gene may have multiple alternately spliced introns, which leads to a family of T/EBP mRNA species (2). Lonigro et al. reported multiple transcription start sites and suggested the presence of a family of T/EBP mRNAs (21). To better understand the nature of the gene, cDNA libraries were constructed from rat FRTL-5 thyroid cells maintained in either 6H (+TSH) or 5H (-TSH) conditioned medium and screened with the 5'-portion of the rat T/EBP cDNA (~450 bp) as a probe (Fig. 1Go, probe b). Among the cDNA clones isolated, approximately 40 clones contained DNA around and upstream of the translation start site ATG of the gene in addition to many others containing DNA only downstream of the ATG. These approximately 40 clones were grouped into 6 different types by comparison with the genomic sequence (Fig. 1Go). Type II contains the most clones (13 from the 5H library and 8 from the 6H library); the longest has a continuous sequence from the translation start site up to -178 bp, assuming the ATG as 0. The second most abundant class of clones (eight from 5H library and two from 6H library) are found in type I, the longest of which possesses a continuous sequence from the ATG up to -963 bp. The 5'-end of the remaining nine clones in the type I class resides between -362 and -963 bp. All clones belonging to types III–VI contain an intron that appears to be alternately spliced. All of the introns have consensus GT and AG sequences at their 5'- and 3'-ends, respectively (Table 1Go). Types III and IV share the same intron donor site at -1248 bp, whereas the intron acceptor site resides at -362 and -13 bp for types III and IV, respectively. The intron donor site for types IV, V, and VI clones are present at -1248, -747, and -111 bp, whereas they share the same intron acceptor site at -13 bp. The type V sequence encodes an extra 30 amino acids in-frame with the T/EBP-coding sequence. Three additional cDNAs were isolated by using a 1-kb fragment of the rat T/EBP gene sequence between -2201 and -1284 bp (Fig. 1Go, probe f). One clone covers sequence between -1714 and -243 bp, and the other two overlapping clones have sequence between -2539 and -1186 bp. Interestingly, the majority of clones containing DNA upstream of the gene’s translation start site were isolated from the 5H library. These results suggest that multiple promoters may exist, especially around -200, -1000, and -1700 bp upstream, and that there may be another promoter further upstream of -1700 bp. The data also suggest that the upstream promoters may be more active under 5H conditions.


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  		Table 1. Sequence of the exon-intron junctions of the rat T/EBP type III, IV, V, and VI cDNAs

 
Isolation of human T/EBP cDNAs
To compare the sequences of the rat and human T/EBP mRNAs, a cDNA library was constructed from human lung NCI-H441 cells. The library was screened with two of the same probes (Fig. 1Go, probes b and f) that were used for screening the rat FRTL-5 cDNA library. A total of 19 clones were isolated, with sequence upstream of the ATG (Fig. 1Go). In contrast to 6 different types of rat T/EBP cDNAs, only 3 of the cDNAs were found in the human library. Six overlapping clones could be grouped in type I, the longest of which had a sequence starting at -968 bp. Nine clones were categorized in the type II class. The longest clone contained a continuous sequence up to -187 bp from the ATG. Three clones possessed an intron of type VI. The nucleotide sequence of the junction is identical to that of the rat type VI clones. Among the human cDNAs, this is the only intron identified, and none of the other introns were found. Similar to the 2 overlapping clones identified in the rat, 1 cDNA clone was found with DNA between -2740 and -721 bp. These results suggest the presence of multiple promoters, localized especially around -200 and -1000 bp, and possibly a third promoter further upstream of the human T/EBP gene.

Analyses of transcription start sites
RNase protection assay was carried out using a DNA fragment between -1946 and -1558 bp upstream of the translation start site of the rat T/EBP gene (Fig. 2Go). This sequence flanks the 5'-end of the type III clones. Multiple protected fragments with different intensities were obtained. Four bands are found clustered around -1700 bp upstream, one of which, however, was very faint. Another band was identified at -1850 bp with intensity comparable to those of the three bands found around -1700 bp. The main band with the highest intensity, however, was obtained at -1898 bp. The fully protected band of 388 bp with a relatively high intensity was also found, which clearly suggests the presence of another transcription start site(s) further upstream of -1946 bp. Similar results of multiple transcription start sites were obtained when a fragment flanking around -1000 bp upstream of the translation start site was used (data not shown). These results suggest that no definitive promoters are present in the upstream region of the rat T/EBP gene; rather, multiple promoters may be scattered throughout.



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  		Figure 2. RNase protection assay for determination of the transcription start site of the rat T/EBP gene. Upper panel: Lane 1, Yeast transfer RNA without RNase treatment; lane 2, yeast transfer RNA with RNase treatment; lane 3, 3.2 µg 5H total RNA; lane 4, 6.4 µg 5H total RNA; lane 5, 12.8 µg 5H total RNA. The mol wt standard (Std) shown on the left is HinfI-cut 0X174. RNase-protected fragments are indicated on the right. Lower panel: Schematization of the results. Sequence of the probe used for RNase protection assay spans between -1946 and -1558 bp and flanks the 5'-end of the type III clones (shown in shaded box). The protected fragments are shown by arrows of different sizes depending on the intensity of the band. The sizes of the fragments (base pairs) and their corresponding locations in the upstream of the rat T/EBP gene are indicated below and above, respectively.

 
Sequences of the rat and human T/EBP gene upstream regions
To compare the upstream structures of the rat and human T/EBP genes, the genomic DNAs were cloned and sequenced. In the 3-kb upstream sequences of the two genes, there were clusters of high and low similarities (Fig. 3Go). The 2 sequences were almost identical (98% similarity) within 300 bp upstream from the ATG. The next approximately 700 bp of the upstream DNA had 81% sequence similarity, which decreased to 60% between -1 and -1.4 kb. The cluster of high similarity (94%) appeared again further upstream of -250 bp. After this and up to about -2.4 kb, a similarity of 75% was found, and in further upstream DNA of the 2 genes, similarity became as low as 56%. Throughout 3 kb of the rat and human T/EBP gene upstream sequences, respectively, 24 and 18 putative T/EBP binding sites were found. Eight of them were found at the exact locations when 2 sequences were aligned. These results suggest that T/EBP gene expression may be at least in part autoregulated.



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  		Figure 3. Comparison of rat and human T/EBP gene upstream sequences. Dot matrix analysis was carried out using -3 kb of the rat and human T/EBP gene upstream sequences on MacVector program. A cluster of high and low sequence similarities is shown with the percent similarities. The putative T/EBP-binding sites that are found at the exact locations when two sequences are aligned are shown by a closed box for the coding strand and an open box for the noncoding strand.

 
Presence of multiple mRNA species
In a previous study, Northern blotting analyses using the rat T/EBP cDNA homeodomain sequence as a probe revealed the presence of at least two T/EBP mRNA species of approximately 2.6 and 3.6 kb in rat FRTL-5 cells (2). These two mRNA bands were broad and appeared to consist of several discrete mRNA species. When an intron sequence downstream of ATG was used as a probe (Fig. 1Go, probe a), only the 3.6-kb mRNA band was obtained (2). In the present study, Northern blotting analyses using various 5'-portions of the T/EBP mRNA sequences as probes (Fig. 1Go, b–e) revealed the presence of a third band of approximately 4.2 kb in addition to broad 2.6- and 3.6-kb bands (Fig. 4Go). The intensities of all bands were higher in cells maintained in 5H conditions than in those maintained with 6H. This agrees with the previously documented phenomenon that TSH down-regulates T/EBP mRNA levels in FRTL-5 cells (10). The intensity of the 4.2-kb band became higher as the probe contained more upstream sequence relative to the ATG (in the order of b, c, and d shown in Fig. 1Go). Similarly, the intensity of the 3.6-kb band increased with the more upstream sequence contained in a probe. This is more evident with 5H mRNAs than 6H mRNAs, suggesting that in FRTL-5 cells maintained under 5H conditions, there is a greater population of mRNAs containing more sequence upstream of the translation start site. These results agree very well with those presented in Fig. 1Go, in which more cDNAs containing DNA upstream of the ATG were obtained from the 5H library. When probe e was used, however, the 3.6- and 4.2-kb bands were very faint. Although overexposure of the film did reveal the presence of the 4.2-kb band in 5H mRNAs, the major bands were found at approximately 3.9 and 2.8 kb. These could be the result of cross-hybridization or could represent a minor population of the T/EBP gene transcripts. A weak band of approximately 4.9 kb was also observed under 5H conditions with the probes d and e, and many weak, but discrete, bands (<3.6 kb) were found in 6H-derived mRNAs hybridized with probes c and d. These results clearly demonstrate that the T/EBP gene encodes a family of T/EBP mRNA species and suggest that the upstream promoter may be more active in 5H conditions. Alternatively, an unknown mechanism may be more active in 6H conditions, which results in many T/EBP mRNA species that are missing the 5'-end of the mRNA.



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  		Figure 4. Northern blotting analyses of mRNAs isolated from rat FRTL-5 cells. Cells were maintained in 5H or 6H conditions, and hybridization was carried out using four different probes, as shown in Fig. 1Go. The sizes of the four main bands obtained are indicated on the left. The 4.9-kb band, which is faint, but clearly seen with probes d and e, is marked by a closed arrowhead. The 4.2-kb band is very weakly seen with probe e, as pointed out by an open arrowhead. The two additional bands of 2.8 and 3.9 kb that were obtained with probe e are shown by an open box. Many discrete bands are observed in 6H mRNAs with probes c and d, which are marked by a closed box. To show the amount of mRNAs in each lane and that the mRNAs are intact, actin hybridization is shown on the bottom.

 
Analyses of rat T/EBP gene promoter activities
To understand the mechanism of T/EBP gene transcription, several luciferase-reporter constructs of the rat T/EBP gene were prepared that contained different lengths of DNA upstream from the ATG. These constructs were transfected into FRTL-5 cells maintained in either 6H or 5H conditions. Similar profiles of luciferase activity were obtained under both 6H and 5H culture conditions. The -1.15-kb construct showed a severalfold increase in luciferase activity relative to that in the vector only (Fig. 5Go). The -1.94-kb construct exhibited at most a 2-fold increase in luciferase activity compared with that of the -1.15-kb construct in the absence of TSH (5H). The -5.18-kb construct showed decreased luciferase activity, which may be due to the large size of the plasmid used in the transfection assays (5) or the presence of a negative regulatory element. These results demonstrate that there is no difference between two culture conditions in the expression profile of the gene as judged by luciferase activities.



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  		Figure 5. Transfection analyses of the rat T/EBP gene upstream sequence. Four different lengths of the upstream DNA from the translation start site of the rat T/EBP gene were connected to the luciferase reporter plasmid, which were then transfected into rat FRTL-5 cells cultured in 5H or 6H conditions, as described in Materials and Methods. Luciferase activity obtained with the vector only was arbitrarily set as 1, and the relative activities are used to show the activities of others. The value shown is the mean of three transfection assays.

 
Possibility of autoregulation of T/EBP gene expression
To determine whether T/EBP gene expression is autoregulated, rat and human luciferase-reporter constructs were cotransfected with the T/EBP expression vector (2). The same rat reporter constructs as those used in Fig. 5Go and similarly prepared human reporter constructs were cotransfected into human hepatoma HepG2 cells, which do not express T/EBP, with a vector expressing rat T/EBP at ratios of T/EBP vs. reporter of 5:1 and 1:1, respectively (Fig. 6Go). With the rat T/EBP gene, a maximum 5-fold increase was obtained with the construct -4.11 kb, whereas the construct -1.94 kb did not show a significant increase by cotransfection of the T/EBP expression vector. In the case of the human gene, a 5- to 10-fold increase was found in all of the constructs examined. The larger increase obtained in the human gene compared with the rat could be due to a 5-fold greater amount of cotransfected T/EBP expression vector relative to that in the reporter constructs used in the experiments. Alternatively, it could be due to the differences between rat FRTL-5 cells and human HepG2 cells, but not between human NCI-H441 cells and HepG2 cells, in the level of other transcription factors such as HNF-3s, which cooperatively regulate the T/EBP gene. In fact, the activity profile of the human T/EBP gene promoter constructs cotransfected with T/EBP expression vector to HepG2 cells was similar to that obtained when these constructs were transfected into human lung NCI-H441 cells (data not shown). These results suggest that the T/EBP gene may be autoregulated.



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  		Figure 6. Cotransfection analyses of the rat and human T/EBP gene upstream sequence-reporter plasmid with T/EBP expression vector. Four of the rat and human T/EBP gene upstream sequence-luciferase reporter plasmids were transfected into human HepG2 cells, which do not express T/EBP, together with the T/EBP expression vector, pCMV4-T/EBP-1 (2). Luciferase activities without cotransfection are shown in the shaded bar and those with cotransfection are shown in the open bar. The activity of vector only without cotransfection was arbitrarily set as 1, and the relative activities are used to show the activities of others. The values are the mean of three transfection assays. Note the difference in the amount of T/EBP expression vector used between rat and human transfection assays, as described in the text and Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies suggested that multiple transcription start sites may exist throughout upstream of the rat T/EBP gene. Primary transcripts from each promoter may be differentially subjected to posttranscriptional modification. Indeed, Lonigro et al. has shown that three transcription start sites are found at -200, -181, and -130 bp within the 340-bp upstream sequence from the ATG of the rat T/EBP gene, and a type VI intron is present (21). Evidence from the present studies indicates that these multiple upstream promoters may be more active under 5H conditions, in the absence of TSH. Alternatively, there may be only one promoter present further upstream, and a yet unknown mechanism might be present that makes the primary transcripts susceptible to degradation at their 5'-ends. This machinery may be more active in 6H conditions, resulting in very few long mRNAs in the presence of TSH. The unknown mechanism might also be responsible for the apparent presence of multiple promoters throughout the upstream of the rat T/EBP gene. RNase protection analyses revealed the presence of another promoter(s) further upstream of -1946 bp. Two overlapping cDNA clones isolated that contain DNA between -2539 and -1186 bp suggest that the 5'-most promoter may be present as upstream as -2539 bp. The location of the most upstream promoter of the rat T/EBP gene is yet to be determined. Transcripts from this promoter and/or promoters located between -2539 and -1946 bp could account for the faint bands of 2.8, 3.9, and/or 4.9 kb band observed with probes d and e in Northern blotting analyses.

The human T/EBP cDNAs can be grouped into only three types compared with rat DNA. Interestingly, the pattern and the number of clones obtained from a human lung cancer cell line are very similar to those of the rat T/EBP cDNAs obtained from the 6H library. The only exception was the length of the longest type VI clone, which extended up to -1001 bp. The presence of an intron upstream of ATG has not been reported in the human. A clone with sequence from -2740 to -721 bp was also isolated. These results suggest that the human T/EBP gene may also have multiple scattered promoters, and the most upstream promoter could be located as upstream as -2740 bp. There seem to be similar transcriptional and posttranscriptional mechanisms present in the T/EBP gene regulation between rat FRTL-5 cells maintained under 6H conditions and human NCI-H441 cells.

Northern blotting results revealed the presence of multiple T/EBP mRNA species in rat FRTL-5 cells. Higher levels of mRNAs are found using 5H conditions, which agrees with the TSH down-regulation of T/EBP mRNA levels reported using this cell line (10). When dog thyrocytes in primary culture were used, however, the T/EBP mRNA level was not affected by forskolin treatment (38). The reason for this discrepancy is not clear at this time. Similar contradictory findings have been reported for the effect of TSH on the mRNA level of thyroid peroxidase. In the case of dog thyroid cells and slices, the TSH-induced increase in the thyroid peroxidase mRNA level is transcriptional (39), whereas in FRTL-5 cells, it is posttranscriptional (40). Whether these results are due to differences between systems (primary or in vivo culture vs. cell line) or species (dog vs. rat) remains to be determined.

In the Northern blotting results, longer T/EBP mRNAs are more abundant in cells maintained in 5H conditions than in those maintained with 6H. This finding is supported by the results presented in Fig. 1Go, in which longer cDNA clones were mainly obtained from the 5H library. The high levels of longer mRNA species under 5H conditions, however, do not seem to correlate with higher luciferase activity in transfection assays, and a similar profile of relative luciferase activities were obtained between 5H and 6H conditions with all of the constructs examined. Regardless of which promoter is used, maximal promoter activity appears to require a -1.94-kb upstream sequence in both the 5H or 6H conditions. Although no difference was obtained in relative luciferase activity between 5H and 6H conditions, this does not exclude the possibility that selective usage of some promoter(s) and/or a splicing machinery may be present that are different between the 5H and 6H conditions. There is also a possibility that translation efficiency may be affected by different sized mRNAs, resulting in no apparent correlation between levels of mRNAs and promoter activity. How the sizes and levels of the T/EBP mRNA correlate with the levels and functions of the protein in different culture conditions remains to be determined. In this respect, it is noteworthy that both FRTL-5 cells in 6H conditions and NCI-H441 cells are actively growing and appear to have similar transcriptional and posttranscriptional regulatory mechanisms of T/EBP gene expression, whereas FRTL-5 cells in 5H conditions are quiescent and regulate the expression of the T/EBP gene differently from proliferating cells. This may suggest a correlation between the physiological state of the cells and the two different T/EBP gene regulatory mechanisms. Additional experiments are required to answer these questions.

A stretch of high sequence similarity is present, interspersed with low similarity, within -2.4 kb of the upstream sequences of the rat and human T/EBP genes, where many putative T/EBP-binding sites are found. Eight of the binding sites are located at the exact locations where the two upstream sequences share high similarity, suggesting that the T/EBP gene may be autoregulated. Indeed, cotransfection experiments demonstrated that this may be the case in both rat and human T/EBP genes. Other transcription factors, such as C/EBP-{alpha} (CCAAT enhancer-binding protein) (41) and Pit-1 (pituitary-specific transcription factor) (42) are known to be autoregulated in their own expressions. The regulation of HNF-3{alpha} and HNF-3ß gene promoters also involves autoregulation (24, 43). HNF-3{alpha} and HNF-3ß are expressed during early embryogenesis in the endodermal lineages, including the thyroid and lung around or just before the time when T/EBP expression starts (18, 29, 30). HNF-3s activate the expression of lung-specific (25, 26, 27) and thyroid-specific (28) genes as well as the T/EBP gene (23). Thus, we found many putative HNF-3-binding sites in both rat and human T/EBP gene upstream sequences between -3 kb and the translation start site. Cross-regulatory mechanisms between T/EBP, HNF-3s, and other lung-specific transcription factors have been suggested to play a role in maintaining cell-specific gene expression in the lung (24). It is reasonable to think that this may also be the case in the thyroid.

In summary, we report a pattern of T/EBP gene expression in both rat and human indicative of the use of multiple upstream promoters and alternative splicing, resulting in mRNAs with variable lengths and sequences of 5'-untranslated regions. The structures of the mRNAs differed mainly upstream of the translation start site together with the presence or absence of the downstream intron, leading to a family of T/EBP mRNA species. Longer mRNAs are present at higher levels in FRTL-5 cells maintained under 5H conditions than in cells maintained with 6H, suggesting that they might have a role in maintaining the physiological state of the cells. These studies also suggest that the expression of the T/EBP gene may be autoregulated.


   Acknowledgments
 
We thank Frank Gonzalez for helpful discussions and critical review of the manuscript.


   Footnotes
 
1 The sequences upstream of the T/EBP gene translation start site that are analyzed in this paper were submitted to GenBank under accession no. AF027332 for the human and AF027333 for the rat. Back

2 Current address: Department of Obstetrics and Gynecology, Toyota Memorial Hospital, Toyota, Aichi 471, Japan. Back

Received October 7, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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