From the § Laboratory of Molecular Embryology, NICHD,
National Institutes of Health, Bethesda, Maryland 20892-5431 and the
Department of Cell Biology, Baylor College of Medicine,
Houston, Texas 77030
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ABSTRACT |
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The thyroid hormone receptor (TR) genes
in Xenopus laevis are regulated by thyroid hormone in all
organs of an animal during metamorphosis. This autoregulation appears
to be critical for systematic transformations of different organs as a
tadpole is transformed into a frog. To understand this autoregulation,
we have previously identified a thyroid hormone response element in the
hormone-dependent promoter of the X. laevis
TR
A gene. We report here the detailed characterization of the
promoter. We have now mapped the transcription start site and
demonstrated the existence of an initiator element at the start site
critical for promoter function. More important, our deletion and
mutational experiments revealed a novel upstream DNA element that is
located 125 base pairs upstream of the start site and that is essential for active transcription from the promoter. Promoter reconstitution experiments showed that this novel element does not function as an
enhancer, but acts as a core promoter element, which, together with the
initiator, directs accurate transcription from the promoter. Finally,
we provide evidence for the existence of a protein(s) that specifically
recognizes this element. Our studies thus demonstrate that the TR
A
promoter has a unique organization consisting of an initiator and a
novel upstream promoter element. Such an organization may be important
for the ubiquitous but tissue-dependent temporal regulation
of the gene by thyroid hormone during amphibian metamorphosis.
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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 TR
A 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
TR
A 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 TR
A promoter into Xenopus oocytes and analyzing the resulting transcript by primer extension and
by PCR cloning of the 5'-end of TR
A mRNA from tadpoles. Deletion and mutational studies demonstrated a unique nature of the TR
A 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.
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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 TR
A 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 TR
A 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 TR
A 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 TR
A and RXR
, equal amounts of TR
A 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 TR
A cDNA clones were obtained that had only a few base changes (different among different clones) compared with the
TR
A 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').
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RESULTS |
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Promoter Analysis in Frog Oocytes Identifies a Minimal Region
Essential for TRA Transcription--
The two X. laevis
TR
genes (TR
A and TR
B) 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 TR
A
gene and identified a strong TRE that mediates the strong autoinduction of the receptor gene (Fig.
1A). However, the start site
of the TR
A 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).
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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.
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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).
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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).
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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 (TR
A and TR
B) 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 TR
A 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.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These two authors contributed equally to this work.
To whom correspondence should be addressed: Lab. of Molecular
Embryology, NICHD, NIH, Bldg. 18T, Rm. 106, Bethesda, MD 20892-5431. Tel.: 301-402-1004; Fax: 301-402-1323; E-mail: Shi{at}helix.nih.gov.
1 The abbreviations used are: T3, thyroid hormone; TR, thyroid hormone receptor; TRE, thyroid hormone response element; bp, base pairs; PCR, polymerase chain reaction; UPE, upstream promoter element; Inr, initiator; RXR, retinoid X or 9-cis-retinoic acid receptor.
2 J. Wong, V. C.-T. Liang, L. M. Sachs, and Y.-B. Shi, unpublished observations.
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REFERENCES |
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