(Received for publication, January 31, 1997)
From the Department of Neurology and the Brain
Research Institute, and the ¶ Thyroid Study Unit, Department
of Medicine, The University of Chicago, Chicago, Illinois 60637 and
the ** Department of Neurology, Harvard Medical School,
Boston, Massachusetts 02115
We isolated and characterized the human -Trace
protein (
TP) gene promoter.
TP, also known as prostaglandin
D2 synthase, is a lipocalin secreted from the choroid
plexus and meninges into cerebrospinal fluid. Basal transcription of
the
TP gene is directed from a core promoter found within the first
325 bases of the 5
-flanking sequence. The
TP gene promoter is
responsive to thyroid hormone (3,3
,5-triiodothyronine, T3)
and efficiently repressed by unliganded human thyroid hormone receptor
(TR
). Functional analysis of the
TP promoter in TE671 cells
revealed that responsiveness to T3 occurs in sequences 2.5 kilobase pairs 5
of the start site. Within the hormone-responsive
region we identified a thyroid hormone response element (TRE) located
from
2576 to
2562 base pairs relative to the transcription start
site. The
TP TRE is composed of two directly repeated consensus
half-sites separated by a 3-base pair space (DR3). The
TP TRE forms
specific complexes with TR
. We have shown that a gene active in the
choroid plexus and meninges is responsive to T3.
T3 may play a role in the regulated transport of substances
into the cerebrospinal fluid and ultimately the brain.
-Trace protein (
TP)1 is a
component of human cerebrospinal fluid (CSF) and one of very few
proteins found in CSF not also present in serum. In human CSF,
TP is
present at 2.6 mg/dl, ranking it among the major CSF proteins (1).
TP, identified by Clausen in 1961 (2), is primarily expressed in the
choroid plexus (CP).
TP is also expressed to a lesser extent in
meninges and oligodendrocytes (3, 4). Other than the CNS, the major
site of
TP expression is the epididymis (4, 5).
A protein with similar distribution to TP has been identified as
prostaglandin D2 synthase (PDS) in rats (6, 7). PDS catalyzes the conversion of prostaglandin H2 to
prostaglandin D2 (PGD2). A role for
PGD2 in regulation of sleep induction has been proposed (8,
9). Recently,
TP and PDS were shown to be the same protein (10, 11).
In prior studies we have referred to
TP/PDS as PDS but, in deference
to precedence, we now refer to it as
TP (12).
The human TP message encodes a 180-residue polypeptide that is a
member of the lipocalin superfamily. Lipocalins are secretory proteins
that transport hydrophobic ligands (13, 14). Lipocalin genes appear to
have arisen by gene duplication, with most of them clustered in the q34
region of chromosome 9 in man and in the syntenic b-c region of
chromosome 4 in the mouse (15). In previous work we localized the human
TP gene to the lipocalin gene cluster on 9q34. The
TP gene bears
a striking resemblance to other lipocalin genes, suggesting a role for
TP in transport (12).
CSF, primarily produced by the CP, can be viewed as an ultra filtrate of serum with protein levels approximately 0.5% those in serum. Exchange of proteins and other substances between CSF and the extracellular fluid of the brain is free (16). The CP secretes highly specialized transporters that carry essential substances into the CSF and then to the brain. The primary function of the meninges is the maintenance of the blood-CSF barrier, but it also contributes to CSF and many substances enter into CSF equally well from either the meninges or CP. Cultured meningeal cells secrete many of the same transport proteins as the CP (17). Several CSF transporters have been characterized including transthyretin, transferrin, and ceruloplasmin; they carry thyroxine, iron, and copper, respectively (18, 19).
García-Fernández et al. (20) found that levels
of TP mRNA in the CNS of adult rats decrease following
chemically induced hypothyroidism. The mechanism by which thyroid
hormone (T3) influences
TP gene expression is unknown.
T3 exerts its effects through binding to thyroid hormone
receptors (TR), which are widely distributed in the CNS (21). In the
CP, T3 augments transport function; hypothyroid rats
have reduced Na+-K+-ATPase activity, a marker
for transport processes (22).
To better understand mechanisms of TP gene regulation, we subcloned
the human
TP gene promoter and analyzed its expression in the human
rhabdomyosarcoma cell line TE671. We identify a small core promoter
that directs basal gene transcription at high levels and a distal
element that determines T3 responsiveness.
The 3.8-kb
XhoI-XbaI fragment from the TP genomic clone
pG4CS86 (12) was inserted into the SmaI site of pBSKS+, and
approximately 1 kb of 3
sequence was excised using the Exo III/mung
bean nuclease system (Stratagene). The resulting fragment, spanning
from
2759 to +65 bp, was subcloned into the CAT vector pJFCAT1 (23)
to generate clone pCAT2759 (Fig. 1). Exo- and endonuclease deletions of
pCAT2759 produced clones with successive 5
deletions. Sequence was
analyzed as described previously (12).
Clone pCAT235
was produced by subcloning the region between 2759 and
2080 bp of
the
TP promoter into pBSKS+. Small internal deletions were
introduced into pCAT235 by digestion with StyI and
EcoNI followed by Klenow fill-in or mung bean nuclease
digestion to remove one or both of the
TP TRE half-sites,
respectively. To liberate the inserts from the pBSKS+ vector, the
constructs were opened with BamHI, made blunt-ended with
Klenow, and subsequently digested with SalI. Gel-purified
fragments were subcloned into the pBLCAT2 vector that had been
opened first at the HindIII site and made blunt-ended with
Klenow enzyme followed by digestion with SalI. The three
clones thus produced were as follows: 1) pTK680F, with the 680-bp
fragment of pCAT235 in the forward orientation, 2) pTK
3
with a
104-base internal deletion which removes the 3
half-site of the TRE,
and 3) pTK
5
+ 3
, with a 108-base internal deletion which
removes both half-sites of the TRE.
Clone pTK300, spanning bases bases2759 to
2464 of the
TP
promoter, was produced by deleting 385 bp of 3
sequence from clone
pTK680F by double digestion with EcoNI and SalI.
Clone pTK680R was produced by subcloning the 680-bp
BamHI-SalI fragment of pCAT235 into the
corresponding sites of pBLCAT2. Clone pTK
DR3 was produced from
overlapping oligonucleotides cloned into the
HindIII-XhoI sites of pBLCAT2. Clone pTK100 was
produced by PCR amplification of bases
2620 to
2518 bp of the
TP
promoter using pCAT2759 as template and oligonucleotides that
introduced a 5
HindIII site and a 3
XhoI site.
PCR product was digested with HindIII and XhoI
and subcloned into the corresponding sites in pBLCAT2.
Total RNA was isolated from adult rat brain or TE671 cells using the method of Chomczynski et al. (24). Total RNA was electrophoresed through 1% agarose gels containing 3% formaldehyde, capillary blotted onto a GeneScreen nylon membrane (DuPont NEN), and probed as described previously (12).
Transient Transfections and Cell CultureThe host cell line used in these studies was the human rhabdomyosarcoma cell line TE671 (ATCC CRL 8805) (25, 26). Cells were passaged in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (BioWhittaker) and 40 µg/ml gentamicin (Life Technologies, Inc.). For TR cotransfections, Dulbecco's modified Eagle's medium with 10% charcoal/dextran-treated fetal bovine serum (Hyclone) was used.
On the day preceding transfection, 4 × 105 cells were
seeded into 60-mm culture dishes. Plasmid DNA was transfected into
cells using the calcium phosphate co-precipitation method (27). For each dish, 1 pmol of CAT construct was co-transfected with 2 µg of
-galactosidase (
GAL) expression vector pRSV-
GAL (28) as an
internal control. For TR cotransfections 2 µg of hTR
1 expression vector was used (29). pBSKS+ was added to bring the total DNA in each
dish to 12 µg. Medium was replaced 18 h after transfection. Where necessary, T3 or hormone vehicle were introduced into
the fresh medium at a final concentration of 100 nM. After
48 h the cells were harvested. pSV2CAT (30) was used
throughout as a positive control vector for CAT expression, and pTK83
was used for T3/TR responses (31). The negative control for
CAT expression was the promoterless CAT vector pJFCAT1 and for
T3 responses, pBLCAT2, which contains the TK promoter (32).
To correct for variations in
transfection efficiency, cell extracts were assayed for GAL activity
(33). After adjusting for
GAL levels, CAT activity was determined
using a variation of the diffusion assay (34). All transfections were
repeated at least four times. Data, reported as mean ± S.E.,
except where noted, are from three separate transfections.
Transfected TE671 cells were fixed with
paraformaldehyde and overlaid with a solution of 0.5 mg/ml
5-bromo-4-chloro-3-indoyl--D-galactosidase, 2.5 mM ferri/ferrocyanide, 1 mM MgCl2,
15 mM NaCl, and 50 mM Tris-HCl, pH 7.5. The
reaction proceeded overnight in the dark at 37 °C.
Complementary oligonucleotides (OLG)
spanning the regions shown in Fig. 7A were used for gel
retardation of the TP TRE and IR1 elements. The
TP TRE OLGs were
5
-AGGCAGGGGGATGGCCTTGGTGACCTCTTAGGGTGGA-3
and the complementary
strand 5
-TGGCCTCCACCCTAAGAGGTCACCAAGGCCATCCCC-3
. The mutant TRE,
DR3, is similar to
TP TRE but introduces C
A or C
T
mutations at bases
2573,
2572,
2564, and
2563. The IR1 OLGs
were 5
-TTGACCACAGGGACTGAGGAGTCCGTCCTGA-3
and the complementary strand
5
-TCGGTTCAGGACGGACTCCTCAGTCCCTGTG-3
. The positive control probe for
TR binding was the rat malic enzyme promoter (rME) TRE (35).
Complementary OLGs were hybridized and 5
overhangs filled in with
Klenow polymerase and [
-32P]dCTP. Labeled duplexes,
purified on G50 columns, had a specific activity greater than 1.7 × 106 cpm/pmol. Recombinant human TR
1 and RXR
were
prepared as described by Sakurai et al. (36).
Binding reactions contained 10 fmol of labeled TRE (approximately
17,000 cpm), 20 mM HEPES, pH 8.0, 50 mM KCl,
0.1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 28 ng/µl poly(dI-dC), 20-100 fmol of TR1, and/or 20-100 fmol of
RXR
. Reactions proceeded at room temperature for 20 min. In
supershift experiments, complexes were permitted to form for 20 min. 1 µl of anti-hTR
polyclonal antibody
62 (37) was then added
followed by incubation for a further 20 min at room temperature.
DNA-protein complexes were resolved on non-denaturing 5% PAGE gels run
at room temperature.
The
TP promoter was isolated from the genomic clone pG4CS86 (12). To
localize regions of the promoter important to
TP transcription, a
set of 10 promoter-CAT gene fusion constructs with increasing 5
deletions was produced, the 5
termini ranging from
2759 to + 16 bp
in the untranslated region (Fig. 1). The human
rhabdomyosarcoma cell line TE671 (25, 26) expresses
TP mRNA at
high levels and transfects efficiently (Fig. 2).
Parallel transfections of the 10 deletion constructs into TE671 cells
revealed the
TP gene promoter to be highly active, generating CAT
activity at a level comparable to the positive control vector
pSV2CAT (30). The
TP core promoter region is small,
deletions from
2759 bp to
595 bp had minimal effects on
TP
promoter activity. Deleting the bases between
595 and
325 actually
increased
TP promoter activity 1.8-fold. Deletions within the 325-bp
core promoter region results in major loss of activity (Fig. 1). The
80-bp clone is inactive, which localizes the sequences necessary for
maximal basal activation of the
TP gene between
325 and
80 bp of
the promoter.
Nucleotide Sequence of the
The sequence
of the core promoter is presented in Fig. 3. The region
from 227 to
180 bp of the
TP promoter has high sequence identity
with regions of the human luteinizing hormone subunit
promoter
(LH-
) (68% identity, bases
239 to
192) (38) and the human
insulin-like growth factor II P4 promoter (80% identity, bases
318
to
287) (IGF-II) (39). The IGF-II P4 promoter is active in the CP
(40). A 20-bp near-perfect palindrome (PAL I, bases
176 to
157)
bears extended homology to the AP4 site originally identified in the
SV40 enhancer (41) and to the cAMP response element, ENKCRE-2, found
within the proenkephalin gene promoter (42). However, the
TP
promoter is only mildly responsive to forskolin (data not shown).
The
In vivo analysis has shown that TP mRNA
expression is regulated by T3. To determine if the human
promoter mounted transcriptional responses to T3 and TR
,
we studied T3 effects on the
TP promoter in TE671 cells.
Results from reporter constructs (Fig. 4,
top) and Western blot analysis (data not shown) indicate
that TE671 cells do not express thyroid hormone receptors. Thus to
determine if the human
TP gene responds to T3 and TR
,
the full-length clone, pCAT2759, was cotransfected with a TR
expression vector (29) into TE671 cells and cultured in the presence or
absence of 100 nM T3. The results reveal that
the
TP promoter is strongly regulated by TR
in a
T3-dependent manner (Fig. 4).
TP
transcription is elevated 4-fold over basal levels in the presence of
T3 and TR
. Unliganded TR
(no T3)
represses the activity of the
TP promoter 12-fold compared with
basal levels. When both effects are considered,
TP promoter activity
is stimulated 45-fold by T3 over the level observed with
unliganded TR
alone. Fig. 4 also shows that the response of pCAT2759
to T3 and TR
is in the range observed with the strong
TRE of the rME-positive control, pTK83 (31). Varying the amount of
TR
expression vector cotransfected with CAT constructs did not alter
the result (data not shown). As shown in Fig. 4, two heterologous
promoter-CAT constructs, pSV2CAT and pBLCAT2, have
responses to TR
and T3 different from those observed for
the
TP promoter, indicating that
TP promoter responses do not
result from effects on cell viability or transcriptional competence.
T3-responsive Region of the
To identify the T3-responsive region of the
TP promoter, the deletion constructs (Fig. 1) were cotransfected
with a TR
expression vector or pBSKS sham control and cultured with
100 nM T3. Only the full-length clone,
pCAT2759, shows activation by T3 and TR
, localizing the
T3 responsive region to the sequence between
2759 and
2018 bp (Fig. 5A). There was no significant
activation by T3 alone (pBSKS sham) over basal levels for
any of the deletion constructs examined (data not shown). To account
for the repressive effects from unliganded TR
, the activity of the
deletion constructs in the presence of T3 and TR
was
compared with that from TR
alone. The results, shown in Fig.
5B, again demonstrate that only the
2759-bp clone
possesses major responses to T3.
Similar experiments were performed to identify the region responsible
for TR-mediated repression. Deletion of the region responsible for
T3 activation (
2759 to
2018 bp) barely altered repression by unliganded TR
(Fig. 5C). Repression by
unliganded TR
was alleviated by deletions inward from
1423 bp.
Thus, repression occurs at alternative or additional sites to those
responsible for T3-mediated activation.
To characterize further the
T3-responsive region of the TP promoter, 680 bp of
upstream sequence (
2759 bp to
2080 bp) was cloned upstream of the
minimal thymidine kinase (TK) promoter fused to the CAT gene as
contained in the pBLCAT2 vector. T3 stimulates an 8-fold
increase of CAT activity when the 680-bp fragment was cloned in the
forward direction and 9.5-fold when cloned in the reverse orientation
(Fig. 6). Thus the
TP promoter
T3-responsive region also confers strong T3
induction on the heterologous TK promoter in an orientation independent
manner. To delineate the T3-responsive region of the
upstream fragment, two constructs were produced that successively
removed 5
and 3
sequence. In the first construct a deletion was
introduced on the 3
end of the 680-bp fragment, leaving the 295 bp of
5
sequence (pTK300 in Fig. 6). The construct, pTK300, is
nearly as active as the original 680-bp fragment (7.6- versus 8-fold activation), indicating that the 3
sequence
makes a negligible contribution to the T3 response. The
second construct further narrowed the sequence on both 5
and 3
ends
of the 300-bp construct, encompassing 102 bp from
2620 to
2518 bp
of the
TP promoter (pTK100 in Fig. 6). The 102-bp
construct is as effective as the 295-bp construct (7.5- versus 7.6-fold) and nearly as effective as the 680-bp
construct, indicating that the T3-responsive region is
located within sequence spanning
2620 to
2518 bp.
The sequence between 2620 and
2518 bp was searched for half-sites
that conformed to the general consensus 5
-PuGG(A/T)CPu-3
(where Pu
indicates a purine nucleoside) and that possessed the number and
spacing of half-sites consistent with known TREs. Using this approach a
TRE was identified between bases
2576 and
2562 bp (Fig.
7A), which is composed of two directly
repeated half-sites separated by 3 bp (DR3). To test the role of the
TP TRE in directing the T3 responses, deletion analysis
was used to remove the 3
half-site and subsequently both half-sites of
the TRE from the 680-bp fragment. Deletion of the 3
site and
3
-flanking sequence results in a drop in activation by T3
and TR
from 8.0- to 2.9-fold (pTK
3
in Fig.
6). A similar deletion of both half-sites of the
TP TRE results in
the loss of T3 induction
(pTK
5
+3
in Fig. 6). Thus deletion
of the
TP TRE results in the loss of the T3 responses
identified in the upstream fragment of the
TP promoter.
Gel shift assays were used to determine if the TP TRE formed
specific complexes with TR
. The binding of TR
to the
TP TRE was compared with that of the DR4 type TRE from the rME promoter (43).
An IR1 type element, between bases
2110 and
2088 bp, which was
determined not to contribute to T3 responses (data not shown), was used as a negative control (Fig. 7A). The
TP
TRE binds and shifts with TR
homodimers and more intensely when the RXR
accessory protein is present (Fig. 7B). The shifted
bands are at levels of intensity similar to those obtained when the rME
element is used, indicating formation of high affinity complexes between the
TP TRE and TR
/RXR
. As expected, the IR1 element failed to bind TR
and shifted only faintly in the presence of TR
/RXR
(Fig. 7C).
To further characterize the TP TRE, cold competitions were performed
using the
TP TRE element itself or the rME element. As expected,
unlabeled
TP TRE competes with labeled
TP TRE (Fig. 8A). The rME element competes effectively
with the
TP TRE element for binding to TR
indicating that
TP
TRE forms complexes in the same fashion as rME (Fig.
8B).
Within TRE half-sites, loss of one or both of the two conserved G
nucleotides substantially reduces TR binding and TRE function (44, 45).
To test the role of these nucleotides, a mutant TP TRE was
constructed (
DR3) in which the G residues at bases
2573-74 and
2564-63 were changed to T or A, respectively (Fig. 7A).
The reconfigured element,
DR3, failed to bind TR
homodimers and
bound TR
/RXR
heterodimers only faintly (Fig. 8C)
proving that residues known to be crucial for TR binding in other TREs are also necessary for TR binding to the
TP TRE. Confirming the results from gel shift, placement of the mutant
TP TRE element upstream of the TK promoter within the pBLCAT2 vector failed to confer
T3 responses to the TK-CAT construct in TE671 cells
(construct pTK
DR3 in Fig. 6). Thus, the intact
sequence of the
TP TRE is necessary to bind TR
and to activate
transcription in response to T3 and TR
.
To probe the composition of the DNA-protein complexes, anti-hTR
antibody was used to super-shift DNA-protein complexes that had formed
with TR
.
TP TRE-protein complexes, formed in the presence of
TR
or TR
/RXR
, were shifted by antibody, demonstrating that the
complexes with
TP TRE were formed by TR
binding (Fig. 8D).
Basal transcription of the -Trace gene is directed from a small
and highly active core promoter. The core promoter is found within the
first 325 bp of upstream sequence and directs CAT gene expression in
TE671 cells at a level similar to the pSV2CAT-positive control vector. Regions of the core promoter bear striking sequence identity to the P4 promoter of the IGF-II gene (39), which is active in
the choroid plexus (40), and to the
-LH gene, which is active in the
CNS (38).
The human TP gene is regulated by TR
in a
T3-dependent manner. T3 and TR
substantially elevate
TP promoter activity, whereas unliganded TR
effectively represses the promoter. The level of T3-dependent activation observed is comparable
to that observed using a classical TRE from the rME promoter (43),
indicating that the overall response of the
TP promoter to
T3 is strong (Fig. 4).
Deletion analyses indicate that the TP thyroid hormone-responsive
region lies between
2759 and
2018 bp. When placed upstream of the
TK minimal promoter in either orientation, this region confers
T3 regulation on the heterologous TK promoter. Further deletion analysis of
TP-TK promoter fusions allowed the
T3-responsive region to be localized to the sequence
between
2620 and
2518 bp, a region in which we have identified a
TRE composed of two consensus half-sites separated by a 3-bp spacer
(DR3-type). The 3
half-site of the TRE exactly matches the general
consensus half-site, and its deletion results in substantial although
not complete loss of T3 induction. Deletion of both
half-sites completely abolishes T3 induction. As with other
TREs, T3 induction from the
TP TRE is lost with mutation
of the two conserved G nucleotides within each half-site.
Gel shift experiments demonstrate that the TP TRE forms specific
complexes with both TR
homodimers and TR
·RXR
heterodimers. We used cold competitions with the rME TRE, mutagenesis of the
TP
TRE, and super-shifts with anti-TR
specific antibodies to demonstrate that
TP TRE binds to and interacts with TR
in a manner consistent with other TRE sequences.
The TP TRE is well upstream of the core promoter. This organization
places the
TP promoter in a growing family of genes distinguished by
TRE elements distal to the core promoter. These include the human
insulin gene where the TRE is located at
1 kb (46), the rat
S14 gene which has multiple TREs located in a 200-bp region
around
2.6 kb (47), and the rat ucp gene which has two
TREs located in the region around
2.3 kb (48, 49). Both the
TP TRE
and one of the ucp TREs, the downstream TRE, are composed of
two directly repeated half-sites separated by a 3-bp spacer. Directly
repeated half-sites with three base pair separations are commonly
associated with vitamin D receptors in accordance with the 3,4,5 rule
(44, 50). However, the rat ucp downstream TRE is
unresponsive to induction by vitamin D receptors, indicating that
DR3-type TREs are capable of T3-specific responses (48).
Additionally, in vitro analyses have shown that DR3 elements can bind TR and direct T3 responses at or near the level
observed with the more common DR4 spacing (51). The sequences flanking the half-sites may prove to be more important in honing the
T3 response of the
TP TRE than the spacings between the
half-sites. Koenig et al. (52) have identified an extended
consensus half-site sequence which functions as an equally strong TRE
regardless of whether the spacing between the half-sites is 3, 4, or 5 bp. A similar dependence on half-site and flanking sequence,
independent of half-site spacing, has been noted for the closely
related retinoic acid receptor elements (53).
The TP promoter shows considerable T3-independent
repression by TR
(54). Repression appears to be specific for the
TP promoter as two heterologous promoters used in this study
(SV2 and TK) are only mildly affected by unliganded TR
.
Deletion of the sequences responsible for T3 activation
does not alleviate repression. Repression may result from TR binding to
TRE half-sites located elsewhere in the promoter. TRE half-sites can
comprise a functional element in TRE-mediated repression (55, 56). Additionally, unliganded TR might disrupt protein-protein interactions necessary for transcription as has been observed in the human glycoprotein hormone
gene (57).
TE671 cells express TP but not TR
. These properties permitted the
examination of basal
TP promoter activity in the absence of the
potentially confounding effects of TR
. Subsequent expression of
TR
in TE671 cells allowed dissection of the repressive effects of
TR
on
TP basal transcription from the more familiar role of TR
in activation. The basal activity of the
TP promoter observed in
TE671 cells in the absence of TR
may be physiologically relevant. Although TR expression is widespread it is not universal (37, 58).
The increase in the activity of the TP promoter by T3
and TR
and its repression by TR
alone, as shown here, agrees with and provides a dual mechanism for the reduced
TP mRNA levels observed in thyroidectomized rats (20). The thyroid hormone responsiveness of the
TP promoter may also imply T3
control of PGD2 synthesis. However, the high levels of
TP and
TP promoter activity observed should be contrasted to the
low levels of PGD2 observed in human CSF (59). Perhaps
TP functions as a ligand transporter within the CNS, as is the case
for other proteins secreted by the CP and meninges. The structural
similarity between
TP and other lipocalin transporters provides
indirect support for a role in lipid transport. Further support for the
role of
TP in transport processes has recently been provided by the
work of Hoffmann et al. (60) who, in a careful in
situ analysis of
TP expression in the developing mouse, have
observed
TP expression at or near a number of blood-tissue barriers,
hinting at a role for
TP in transport across these barriers.
The present study indicates that T3 exerts a measure of
control over TP gene expression. If
TP functions to transport a specific ligand into CSF then T3 potentially exerts a
general level of control over the availability of the ligand in the
CNS. Important questions regarding the regulation of
TP
transcription remain to be addressed. Foremost among them is whether
the expression of
TP is regulated in a tissue-specific manner,
implying the use of different enhancer elements within the
TP
promoter or different combinations of tissue-specific transcription
factors. Additionally, the role of T3 on
TP expression
must be examined in the context of the other tissues in which it is
expressed.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M98537[GenBank].
We thank Dr. Mark A. Jensen for critically reading the manuscript.