INTRODUCTION

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Thyroid hormone (T₃)¹ is the causative agent of amphibian metamorphosis, a process that systematically changes most, if not all, organs of a tadpole to prepare the animal for adult terrestrial life (1-3). The hormone is known to regulate the transcription of target genes through their nuclear receptors or thyroid hormone receptors (TRs) (4-11). Thus, it is believed that T₃ induces a cascade of gene regulation in each tissue or organ to effect the metamorphic transition (12). Many T₃ response genes have been isolated from various metamorphosing tadpole tissues, and their developmental expression profiles have implicated potential roles during metamorphosis (12-14).

Among the T₃ response genes are the TR genes themselves. Two TR and two TR genes have been isolated from Xenopus laevis (15, 16), whereas only one TR gene and one TR gene have been cloned from Rana catesbeiana (17, 18). Consistent with their roles in mediating T₃ effects, all TR genes are expressed during metamorphosis and can be up-regulated by T₃ treatment of premetamorphic tadpoles (17-20). In particular, the Xenopus TRA genes have been shown to be directly regulated by T₃ at the transcriptional level (21-24). This T₃ regulation appears to be mediated mostly by a thyroid hormone response element (TRE), consisting of two near-perfect repeats of AGGTCA separated by 4 bp. Interestingly, promoter studies using transient transfection assays in frog tissue culture cells failed to identify any other elements necessary for the TRA promoter due to the lack of information on the transcription start site (22, 23).

Promoters recognized by RNA polymerase II generally contain a TATA box and/or an initiator that directs specific transcription initiation. In many genes, the TATA element is the primary core element responsible for positioning the basal transcription machinery on the promoter (25-27). However, many other genes lack a TATA element and, instead, contain an initiator. The initiator encompasses the transcription start site and is sufficient to position the basal transcription complex. This specific positioning of the basal transcription machinery at a promoter by a TATA and/or initiator element allows basal transcription, which can be enhanced by transcription activators.

To determine the nature of the TRA promoter, we have now mapped its start site by introducing the TRA promoter into Xenopus oocytes and analyzing the resulting transcript by primer extension and by PCR cloning of the 5'-end of TRA mRNA from tadpoles. Deletion and mutational studies demonstrated a unique nature of the TRA promoter, consisting of a novel promoter element and an initiator, thus different from the two major classes of RNA polymerase II promoters mentioned above. We further show that specific proteins exist to recognize the novel promoter element, thus likely allowing specific transcription initiation and activation by TRs.

	MATERIALS AND METHODS

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Plasmid Constructs-- The wild-type pTRA promoter construct was generated by cloning a 1.9-kilobase EcoRI fragment containing 1.6 kilobases of TRA promoter sequence and ~0.3 kilobase of chloramphenicol acetyltransferase gene sequence from plasmid pCAT-WT (22) into pBluescript II KS() (Stratagene). To make a construct of the promoter with a 5'- and/or a 3'-deletion, a 5'-primer (bearing a HindIII restriction site and located at an appropriate position for the desired deletion) and a 3'-primer (bearing a BglII restriction site and located at a downstream position for the desired deletion) were used to PCR-amplify a promoter fragment from the wild-type template. The amplified fragment was then cloned into the wild-type plasmid after removing the promoter sequence by HindIII and BglII digestions.

Microinjection of Xenopus Oocytes-- The preparation of Xenopus stage VI oocytes and the microinjection procedure were essentially as described (29). The TRA promoter plasmid DNA was injected (23 nl/oocyte) either as single-stranded DNA (1.15 ng/oocyte) or double-stranded DNA (2.3 ng/oocyte) into the nuclei (germinal vesicle) of the oocytes, and the indicated amounts of mRNAs for Xenopus TRA and RXR were injected (27 nl/oocyte, 50 ng/µl) into the oocyte cytoplasm. The mRNAs were usually injected 6 h before the injection of DNA. For transcription analysis, ~20 oocytes were injected for each sample to minimize the variations among oocytes and injections. The injected oocytes were incubated at 18 °C overnight in MBSH buffer (29) supplemented with antibiotics (50 units/ml ampicillin and streptomycin) and then collected for transcription analysis.

Preparation of mRNA in Vitro-- The pSP64(A)-xTRA, pSP64(A)-xRXR, and pSP64(A)-Gal4-VP16 plasmids (30, 31) were linearized with EcoRI, and in vitro transcription was performed using an SP6 Message Machine kit (Ambion) as described by the manufacturer. A typical reaction with ~0.5 µg of linearized template in a 20-µl reaction yielded 10-15 µg of capped mRNA. The mRNAs were resuspended in diethyl pyrocarbonate-treated water at a final concentration of 50 ng/µl and injected into the cytoplasm of groups of oocytes (27 nl/oocyte). After incubation overnight, the oocytes were collected. We examined the relative levels of expression of the TRA and RXR receptors in the oocytes by coinjection of [³⁵S]methionine and mRNA (31), which indicated that similar amounts of receptors were produced when equal concentrations of mRNAs were injected. Therefore, in all experiments with the injection of TRA and RXR, equal amounts of TRA and RXR mRNAs were mixed to give a final concentration as indicated for each.

Transcription Analysis-- Transcription analysis by primer extension from the injected oocytes were performed essentially as described (29). Briefly, ~20 injected oocytes were collected for each sample, rinsed with 400 µl of MBSH buffer, and then homogenized in 300 µl of 0.25 M Tris (pH 8.0). To isolate RNA, 50 µl of of RNazol reagent/oocyte was added to the sample, vortexed, and then incubated on ice for 15 min before centrifugation. The clean supernatant was transferred to a new tube and extracted once with an equal volume of phenol/chloroform. The RNA was then precipitated with 0.7 volume of isopropyl alcohol, rinsed with 70% ethanol, and dissolved in diethyl pyrocarbonate-treated water. For primer extension analysis, RNA from 1 or 2 oocyte eq was annealed with either the end-labeled chloramphenicol acetyltransferase primer (5'-GGTGGTATATCCAGTGATTTTTTTCTCCAT-3', located just downstream of the TR promoter sequence) or primer I (5'-ATCCTTATAAACGGTGAGTAGTGATGTACT-3', located at +109 to +80) in 10 µl of 0.4 M KCl at 65 °C for 10 min, 55 °C for 25 min, and 42 °C for 5 min. Thirty microliters of reverse transcription mixture (67 mM Tris-HCl (pH 8.3), 8 mM MgCl₂, 5 mM dithiothreitol, 1 mM dNTP mixture, 1 unit of RNasin, and 10 units of Superscript II) were then added. The reaction was incubated at 42 °C for 1 h and then stopped by ethanol precipitation. The products were analyzed on a 6% sequencing gel and visualized by autoradiography. As an internal control, a histone H4 antisense primer (5'-GGCTTGGTGATGCCCTGGATGTTATCC-3') was included in the primer extension reaction to quantify the endogenous H4 mRNA level.

PCR Cloning of the 5'-End of the TRA mRNA-- The anchor PCR cloning procedure was performed according to Frohman et al. (32) with slight modifications. Ten micrograms of stage 64 tadpole RNA were reverse-transcribed as described above with primer II (5'-AAAAGCCATGAATATCCTGTA-3', +136 to +116). The cDNAs were isolated by phenol extraction, phenol/chloroform extraction, and ethanol precipitation and were resuspended in 1× Tris/EDTA. One-fifth of the cDNAs were precipitated again with ethanol in an ammonium acetate buffer and resuspended into 20.6 µl of 1× Tris/EDTA. For tailing, 2.4 µl of 2.5 mM dATP, 6 µl of 5× tailing buffer (Life Technologies, Inc.), and 15 units of terminal deoxynucleotidyltransferase (Life Technologies, Inc.) were added, and the mixture was incubated for 12 min at 37 °C and heated for 15 min at 65 °C. The reaction mixture was diluted to 500 µl in 1× Tris/EDTA, and 5-µl aliquots were used for amplification in 50 µl of PCR mixture (5 µl of 10× Taq polymerase buffer with MgCl₂ (Promega), 0.8 µl of 25 mM dNTP, 0.65 µl of 0.1 µg/µl (dT)₁₇ adapter, 0.8 µl of 0.1 µg/µl adapter, 0.8 µl of 0.1 µg/µl primer I, and 2.5 units of Taq DNA polymerase (Promega)) (for adapter primer sequence, see Ref. 32). Using a DNA thermal cycler (Perkin-Elmer), the mixture was denatured at 94 °C for 4 min, annealed at 42 °C for 2 min, and extended at 72 °C for 30 min before 40 cycles of amplification using a step program (94 °C, 40 s; 55 °C, 2 min; and 72 °C, 3 min), followed by a 30-min final extension at 72 °C. PCR products were cloned with the Original TA cloning kit (Invitrogen). Individual clones were isolated and sequenced with a T7 Sequenase Version 2.0 DNA sequencing kit (Amersham Pharmacia Biotech). Seven independent TRA cDNA clones were obtained that had only a few base changes (different among different clones) compared with the TRA genomic clone. These changes were likely derived from PCR errors due to the use of Taq polymerase and/or sequence polymorphisms. However, all had their 5'-end at position +1 or +3.

Gel Mobility Shift Assay-- Two nanograms of ³²P-labeled double-stranded oligonucleotides were mixed with 9 µg of cell extract, made from X. laevis tissue culture cell line XL58 as described (22), in 20 µl of 1× binding buffer (20 mM Tris-HCl (pH 7.5), 40 mM NaCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.1% Triton X-100, and 10% glycerol) containing 1 µg of poly(dI·dC). After a 20-min incubation at room temperature, the reaction mixture was analyzed directly on a 5% polyacrylamide gel (0.5× Tris borate/EDTA, running at 4 °C for ~4.5 h with 1× Tris/EDTA as the running buffer). The double-stranded oligonucleotides used included UPE, mUPE, Inr (made of 5'-TAA TTA ATA AAG TAC CCC CAG TTG TAA AAT-3' and 5'-ATT TTA CAA CTG GGG GTA CTT TAT TAA TTA-3'), and mInr (made of 5'-TGT ATT ATA ATT AAT ACC CAG TTG TAA AAT-3' and 5'-ATT TTA CAA CTG GGT ATT AAT TAT AAT ACA-3').

	RESULTS

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Promoter Analysis in Frog Oocytes Identifies a Minimal Region Essential for TRA Transcription-- The two X. laevis TR genes (TRA and TRB) are regulated identically during development and by thyroid hormone (16, 33). Genomic structure analyses have revealed that each TR gene produces mRNAs with two alternative 5'-ends, i.e. having two different 5'-exons (exons a and b, respectively) (33). Although the relative locations of the two exons, i.e. whether exon b is located upstream of exon a or vice versa, have yet to be determined, both exons are independently transcribed (21, 33). The expression from the promoter upstream of exon a is maintained at low but constitutive levels. In contrast, the promoter upstream of exon b is repressed in the absence of T₃, but is activated to high levels when T₃ is present. We (22) and others (23) have previously analyzed this T₃-inducible promoter (upstream of exon b) of the TRA gene and identified a strong TRE that mediates the strong autoinduction of the receptor gene (Fig. 1A). However, the start site of the TRA promoter had not been determined, possibly due to the low abundance of the mRNA in vivo or generated from transfection (22). Thus, it has not be possible to identify any elements other than the TRE that are necessary for reporter gene expression in transient transfection assays (22).

View larger version (42K): [in this window] [in a new window]   Fig. 1.   A, organization of the thyroid hormone-inducible promoter of the X. laevis TRA gene. The transcription start site is at position +1. An UPE is present at 151 to 125, and sequence of the Inr region is also shown. A TRE, consisting of two near-perfect repeats of AGGTCA separated by 4 bp, is present at 262 bp downstream of the start site. The sequences are from Ranjan et al. (22). The vertical arrows at +1 and +3 indicate the positions of the 5'-ends of the PCR clones of TRA cDNA from stage 64 tadpole RNA, with three and four independent clones, respectively. B, transcription start site mapping. A single-stranded wild-type TR promoter construct was injected into oocytes with or without prior injection of the mRNAs for TR and RXR. The promoter plasmid was replicated in vivo and assembled into chromatin. After overnight incubation in the presence or absence of 50 nM T3, the RNA transcribed from the chromatinized template was analyzed by primer extension using the pCAT primer. A sequencing ladder of the promoter plasmid with the same primer was used to determine the start site.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Deletion analyses reveal that sequences upstream of 172 and downstream of +8 are not necessary for promoter function. Double-stranded promoter constructs containing the indicated regions of the promoter were microinjected into oocytes. After overnight incubation, the RNA was isolated and analyzed by primer extension with 32P-labeled pCAT primer. Lanes 1-10 correspond to control without DNA and the constructs from top (wild-type (WT)) to bottom (pTRp11). The products derived from the TR promoter are indicated by small arrowheads. The large arrowhead indicates the product generated from an endogenous oocyte RNA due to cross-hybridization by the pCAT primer, which served as a control for RNA isolation and primer extension (24). Promoter activities in oocytes were normally analyzed at least twice, and similar results were obtained (data not shown).

Deletion and Mutational Analyses Reveal the Existence of a Novel DNA Element and an Initiator in the TRA Promoter-- To further characterize the DNA sequences necessary for the basal promoter activity, additional 5'- and 3'-deletion constructs were made and injected into oocytes in the double-stranded form. Primer extension analysis showed that 5'-deletion up to 154 (pTRp5'-7) still yielded an active promoter, whereas a further deletion of 13 bp (pTRp5'-6) or more resulted in an inactive promoter (Fig. 3A). Thus, the region from 154 to 141 is an essential element for the activity.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Deletion analyses identify a minimal promoter region located at 154 to +7. 5'-Deletion (A) and 3'-deletion (B) constructs were microinjected into oocytes in double-stranded form, which allowed efficient transcription of the wild-type (WT) promoter in the absence of both T3 and TR/RXR. After overnight incubation, the RNA was isolated and analyzed by primer extension with primer I for the TR transcript and with the H4 primer for the endogenous histone H4 mRNA, which served as an internal control (indicated by arrowheads).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Mutational studies further define the UPE and the Inr. Site-directed mutagenesis was performed to generate the mutant constructs. The promoter constructs were injected into oocytes, and transcripts were analyzed as described in the legend Fig. 3. The arrowhead indicates the internal control. Note that the mutations at 148 to 143 (lane 3) and 144 to 140 (lane 4) in the UPE and at 3 to +2 (lane 6) and +6 to +1 (lane 5) in the Inr abolished the promoter function, whereas the mutation at 130 to 125 (lane 2) in the UPE had no effect. The double mutation at 144 to 140 and at 3 to +2 (lane 7), which restored the partial complementarity between the UPE and Inr, was also inactive, indicating that the sequences, not the complementarity, of the UPE and Inr are important for promoter function. WT, wild-type.

The Novel DNA Element (UPE) Is Not an Enhancer, but Constitutes an Essential Part of the Basal Promoter-- The inability of the UPE deletion and mutational constructs to support transcription suggests that the UPE may function as either a basal promoter element or an enhancer. In the latter case, the activity of the basal promoter may be too weak to be detected by the primer extension assay (once the UPE is mutated) and should be rescued by adding a different enhancer. To distinguish between these two possibilities, we reintroduced the UPE or a truncated UPE in both orientations into a deletion construct that had no UPE and tested the transcriptional activity of the resulting constructs (Fig. 5). The results showed that the truncated UPE failed to rescue the promoter function (Fig. 5, constructs 5 and 6), as expected. Interestingly, the UPE was able to rescue the promoter when placed in the same orientation as in the wild-type promoter (Fig. 5, construct 4), but it failed to do so when placed in the opposite orientation (construct 3). Thus, the UPE functions in an orientation-dependent manner, thus most likely as a promoter element, not as an enhancer. Consistently, when we placed the UPE in either orientation into a construct containing the enhancerless SV40 early promoter, we found that it failed to alter the promoter activity (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Promoter reconstitution suggests that the UPE functions as a basal promoter element. The truncated promoter (construct 1, 154 to +316) containing the minimal promoter sequence was transcriptionally active (lane 1), whereas a short construct (construct 2, 63 to +316) was inactive (lane 2). Placing the UPE back into construct 2 in the wild-type orientation (construct 4), but not in the opposite orientation (construct 3), restored the promoter activity (lanes 4 and 3, respectively). In contrast, placing a truncated UPE back into construct 2 in either orientation (constructs 5 and 6) did not rescue the promoter (lanes 5 and 6, respectively). Oocyte injection and transcript analyses were done as described in the legend to Fig. 3. The arrowhead indicates the internal control.

View larger version (54K): [in this window] [in a new window]   Fig. 6.   Promoter reconstitution experiments confirm that the UPE is essential for basal promoter function. One or three copies of the Gal4 DNA-binding sites were inserted upstream of an active promoter (pTRp5'-7, 154 to +316) or inactive promoter (pTRp5'-1, 63 to +316), both containing the TRE at +262. The original and new constructs were microinjected in the double-stranded form into oocytes with or without prior injection of TR/RXR in the presence of T3 or Gal4-VP16 mRNA. After overnight incubation, the RNA was isolated and analyzed as in the legend to Fig. 3. The arrowhead and arrow indicate the internal control and TR products, respectively. The TR product migrated to a similar location as a product derived from nonspecific priming of endogenous RNA. Due to the use of 32P-labeled primer I with a lower specific activity and lower levels of TR promoter DNA used in this experiment, the signal for the TR decreased relative to the nonspecific product. Thus, a signal derived from nonspecific endogenous RNA was observed for the inactive promoter (pTRp5'-1, lanes 3-8; compared with Fig. 3A, lane 8). The inactive promoter and its derivatives were not affected by either TR/RXR or Gal4-VP16 despite the presence of their binding sites. On the other hand, TR/RXR (lanes 1 and 2) and Gal4-VP16 (lanes 9 and 10) were able to activate the functional promoter containing their binding sites.

Specific Recognition of the UPE by Xenopus Proteins-- We used gel mobility shift assay to determine whether proteins exist to recognize the UPE. We isolated protein extracts from Xenopus oocytes and from a Xenopus tissue culture cell line that is known to be able to regulate endogenous TR gene expression in a thyroid hormone-dependent manner, just like in tadpoles.² When a ³²P-labeled UPE oligonucleotide was mixed with the extract, several complexes were formed (Fig. 7). The same results were obtained when oocyte extract was used (data not shown). All of them could be competed away by the unlabeled UPE itself, but the major one could not be competed by the truncated UPE (mUPE) (Fig. 7) or by a mutant UPE bearing the mutations that inactivate its promoter activity (data not shown).

View larger version (122K): [in this window] [in a new window]   Fig. 7.   The UPE forms specific complexes with Xenopus proteins. Two nanograms of 32P-labeled double-stranded oligonucleotide containing the UPE were mixed with protein extract from Xenopus tissue culture cells in the presence of 5, 10, 25, and 100 ng of the unlabeled UPE itself, a truncated UPE (mUPE), the Inr, or a mutated Inr (mInr). The resulting complexes were analyzed by gel mobility shift assay. The arrowhead points to the specific complex that was competed by UPE, but not by mUPE or mInr. The Inr was able to compete due its sequence similarity to UPE.

	DISCUSSION

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Thyroid hormone receptors mediate the biological effects of thyroid hormone. Perhaps the most dramatic T₃-dependent process is amphibian metamorphosis, during which an aquatic tadpole is transformed into a terrestrial frog. As expected, the TR genes are highly expressed during amphibian metamorphosis (17-20, 31, 35-38). Interestingly, the expression of TR genes has been shown to be regulated by T₃ temporally in a tissue-dependent manner that correlates with tissue-specific changes during development, even though they are regulated by T₃ ubiquitously in all organs (31, 37, 39-42), and this regulation is directly mediated by TRs themselves (22, 23). We have shown here that this spatial and temporal autoregulation appears to involve, in addition to a TRE, an initiator and a novel promoter element located ~140 bp upstream of the start site.

Each of the TR genes (TRA and TRB) in X. laevis is transcribed from two promoters based on transcript analysis (21, 33). One of the promoters is constitutively expressed at low levels, and the other is thyroid hormone-inducible. We (22, 33) and others (23) have shown previously by transient transfection that this T₃-inducible promoter of the TRA gene contains a strong TRE (Fig. 1A), which mediates transcriptional repression by unliganded TR/RXR and activation by T₃-bound TR/RXR. However, these earlier studies failed to reveal any other DNA elements critical for promoter function due to the use of an indirect reporter assay and a lack of information on the transcription start site. By directly analyzing the transcripts derived from the promoter both in the absence and presence of TR/RXR and/or T₃, we have shown here that the basal and T₃-induced transcription starts at 262 bp upstream of the TRE. We have further defined a minimal promoter for accurate transcription that includes the sequence from 154 to +7 relative to the start site. Within this promoter region, no TATA box is present, suggesting that the promoter belongs to the class of TATA-less promoters. Consistently, we have found that the promoter contains an essential initiator, a key feature of TATA-less promoters (26, 27).

Sequence comparison reveals no obvious resemblance of the initiator to any groups of initiators identified so far (25, 43, 44). While transcription from TATA-less promoters usually starts from an A residue, primer extension analysis clearly indicate that a G residue is used. Thus, the TR initiator is a novel element.

In addition to the initiator, our deletion and mutational analysis also revealed the existence of an important, novel UPE. The activity of the promoter depends on the presence of both the initiator and the UPE since it is abolished when either one is mutated or deleted. Several lines of evidence argue that the UPE functions as a novel core promoter element, but not as an enhancer. First, the UPE cannot be substituted by single or multiple TRE- or Gal4-binding sites, which, however, can mediate the strong transcriptional activation by liganded TR/RXR or Gal4-VP16, respectively, when the UPE is also present. Second, the function of UPE is orientation-dependent. In general, transcription enhancers function in an orientation-independent manner. The absolute requirement for both the initiator and UPE is exceptional since, in other TATA-less promoters, the initiator is sufficient to direct low levels of basal transcription, although the presence of binding sites for transcription factors like SP-1 can augment promoter activity.

The Xenopus TRA promoter bears some similarities to the human TR promoter (45). Both promoters are autoregulated by TRs themselves, and TREs have been identified in the promoters. In addition, binding sites for transcription factor SP-1 or related factors are present in both genes and are important for human promoter function (46, 47). Although the SP-1 sites are dispensable for Xenopus promoter function in tissue culture cells or oocytes (Fig. 2) (22), it cannot be ruled out that they may play a role in development.

Distinct differences, however, exist between the human and Xenopus TR promoters. The human promoter has a TATA-like motif and Oct-1 elements (45-47). The Xenopus promoter lacks such elements. Instead, it contains an initiator element and an upstream promoter element.

A surprising feature of the Xenopus TR promoter comes from the sequence similarity between the UPE and initiator (Fig. 1) (22). The UPE and initiator are orientated in opposite directions in the TR promoters and thus could potentially form a heteroduplex. Interestingly, no transcription initiated from the UPE region, but in an opposite direction could be detected using a primer located upstream (5' to 3' direction is toward the UPE) of the UPE (data not shown), indicating that the sequence differences between the UPE and initiator and/or other sequences in the minimal promoter are important for the directionality of the promoter.

We have also tested whether the potential secondary structure formed due to the complementarity of the initiator and UPE is involved in promoter function. Reciprocal mutations that maintain the potential secondary structure and its GC content or stability failed to produce an active promoter, suggesting that the sequences, but not the potential secondary structures, are important for promoter function. Consistent with this, gel mobility shift experiments revealed the existence of a protein(s) that binds specifically to the UPE and initiator. The mutations/deletions that inactivate the UPE or initiator also abolish the ability to compete for binding. Thus, the binding by this protein(s) correlates with the transcription function of the UPE and initiator. The identity and nature of this protein(s) will be of particular interest for future studies on TRA promoter regulation.