beta -Trace Gene Expression Is Regulated by a Core Promoter and a Distal Thyroid Hormone Response Element*

(Received for publication, January 31, 1997)

David M. White Dagger §, Teiji Takeda par , Leslie J. DeGroot , Kari Stefansson ** and Barry G. W. Arnason Dagger

From the Dagger  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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

We isolated and characterized the human beta -Trace protein (beta TP) gene promoter. beta TP, also known as prostaglandin D2 synthase, is a lipocalin secreted from the choroid plexus and meninges into cerebrospinal fluid. Basal transcription of the beta TP gene is directed from a core promoter found within the first 325 bases of the 5'-flanking sequence. The beta TP gene promoter is responsive to thyroid hormone (3,3',5-triiodothyronine, T3) and efficiently repressed by unliganded human thyroid hormone receptor beta  (TRbeta ). Functional analysis of the beta 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 beta TP TRE is composed of two directly repeated consensus half-sites separated by a 3-base pair space (DR3). The beta TP TRE forms specific complexes with TRbeta . 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.


INTRODUCTION

beta -Trace protein (beta 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, beta TP is present at 2.6 mg/dl, ranking it among the major CSF proteins (1). beta TP, identified by Clausen in 1961 (2), is primarily expressed in the choroid plexus (CP). beta TP is also expressed to a lesser extent in meninges and oligodendrocytes (3, 4). Other than the CNS, the major site of beta TP expression is the epididymis (4, 5).

A protein with similar distribution to beta 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, beta TP and PDS were shown to be the same protein (10, 11). In prior studies we have referred to beta TP/PDS as PDS but, in deference to precedence, we now refer to it as beta TP (12).

The human beta 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 beta TP gene to the lipocalin gene cluster on 9q34. The beta TP gene bears a striking resemblance to other lipocalin genes, suggesting a role for beta 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 beta TP mRNA in the CNS of adult rats decrease following chemically induced hypothyroidism. The mechanism by which thyroid hormone (T3) influences beta 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 beta TP gene regulation, we subcloned the human beta 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.


MATERIALS AND METHODS

Isolation and Sequencing of the beta TP Promoter

The 3.8-kb XhoI-XbaI fragment from the beta 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).


Fig. 1. Deletion analysis of the beta TP promoter. Schematic on the left represents the promoter deletion constructs fused to the CAT gene. Stippling denotes the first 65 bases of the beta TP 5'-untranslated region fused to the CAT gene. The start site of each construct relative to the start site of transcription is shown to the left. pCAT495R has the first 500 bp of the beta TP promoter cloned in the reverse orientation. Restriction sites used to generate clones are marked with down arrows. Bar graphs on the right represent the CAT activity in TE671 cells of the clones shown schematically on the left. Data are expressed as cpm/min/unit beta -GAL activity and are reported as the mean ± S.E. from three separate transfections. The CAT activity of all clones was significantly different from the full-length clone, pCAT2759 (p < 0.05).
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beta TP-Thymidine Kinase (TK) Promoter Fusions

Clone pCAT235 was produced by subcloning the region between -2759 and -2080 bp of the beta 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 beta 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) pTKDelta 3' with a 104-base internal deletion which removes the 3' half-site of the TRE, and 3) pTKDelta 5' + 3', with a 108-base internal deletion which removes both half-sites of the TRE.

Clone pTK300, spanning bases bases-2759 to -2464 of the beta 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 pTKDelta 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 beta 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.

Northern Blot Analysis

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 Culture

The 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 beta -galactosidase (beta GAL) expression vector pRSV-beta GAL (28) as an internal control. For TR cotransfections 2 µg of hTRbeta 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).

Reporter Gene Assays

To correct for variations in transfection efficiency, cell extracts were assayed for beta GAL activity (33). After adjusting for beta 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.

Histochemistry

Transfected TE671 cells were fixed with paraformaldehyde and overlaid with a solution of 0.5 mg/ml 5-bromo-4-chloro-3-indoyl-beta -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.

Gel Retardation Assay

Complementary oligonucleotides (OLG) spanning the regions shown in Fig. 7A were used for gel retardation of the beta TP TRE and IR1 elements. The beta TP TRE OLGs were 5'-AGGCAGGGGGATGGCCTTGGTGACCTCTTAGGGTGGA-3' and the complementary strand 5'-TGGCCTCCACCCTAAGAGGTCACCAAGGCCATCCCC-3'. The mutant TRE, Delta DR3, is similar to beta TP TRE but introduces C right-arrow A or C right-arrow 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 [alpha -32P]dCTP. Labeled duplexes, purified on G50 columns, had a specific activity greater than 1.7 × 106 cpm/pmol. Recombinant human TRbeta 1 and RXRalpha were prepared as described by Sakurai et al. (36).


Fig. 7. The TRE from the beta TP promoter binds TRbeta . A, the sequence of the beta TP TRE is presented as is that of the IR1-negative control. Potential TRE half-sites are overlined by arrows. Shown below the beta TP TRE sequence are the nucleotide changes introduced to produce the Delta DR3 element. B and C, gel shift analysis. The beta TP TRE and IR1 regions shown in A were labeled with 32P. The rME promoter TRE probe was similarly labeled and served as the positive control for receptor binding. 10 fmol of labeled probe was incubated with purified recombinant TRbeta , RXRalpha , or both. Protein from a sham receptor preparation (Mock) was used to control for nonspecific protein binding. The rME TRE complexes were resolved on polyacrylamide gels alongside the beta TP TRE and IR1 complexes. H, denotes TRbeta 1/RXRalpha heterodimers, D, denotes TRbeta homodimers, and F denotes unbound probe.
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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 TRbeta 1, and/or 20-100 fmol of RXRalpha . Reactions proceeded at room temperature for 20 min. In supershift experiments, complexes were permitted to form for 20 min. 1 µl of anti-hTRbeta polyclonal antibody beta 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.


RESULTS

Isolation and Identification of the beta TP Core Promoter

The beta TP promoter was isolated from the genomic clone pG4CS86 (12). To localize regions of the promoter important to beta 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 beta TP mRNA at high levels and transfects efficiently (Fig. 2). Parallel transfections of the 10 deletion constructs into TE671 cells revealed the beta TP gene promoter to be highly active, generating CAT activity at a level comparable to the positive control vector pSV2CAT (30). The beta TP core promoter region is small, deletions from -2759 bp to -595 bp had minimal effects on beta TP promoter activity. Deleting the bases between -595 and -325 actually increased beta 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 beta TP gene between -325 and -80 bp of the promoter.


Fig. 2. beta TP mRNA is highly expressed in host cell line TE671. A, Northern blot of total RNA from adult rat brain (lane 1), preconfluent (lane 2), or postconfluent (lane 3) TE671 cells. 10 (lane 1) or 20 µg (lanes 2 and 3) of total RNA was electrophoresed through agarose gels and capillary blotted. The blot was hybridized with a probe generated from a beta TP cDNA template. The beta TP message is below the 18 S band as indicated to the left of the figure. B and C, beta TP promoter constructs transfect efficiently into host cell line TE671. Promoter constructs and a beta -GAL expression vector were co-transfected using the calcium-phosphate method and stained with 5-bromo-4-chloro-3-indoyl-beta -D-galactosidase to reveal transfection efficiency. B, pBSKS control, C, pCAT2759 cotransfected with pRSV-beta GAL.
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Nucleotide Sequence of the beta TP Gene Promoter

The sequence of the core promoter is presented in Fig. 3. The region from -227 to -180 bp of the beta TP promoter has high sequence identity with regions of the human luteinizing hormone subunit beta  promoter (LH-beta ) (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 beta TP promoter is only mildly responsive to forskolin (data not shown).


Fig. 3. The 5'-flanking region of the human beta TP gene. The nucleotide sequence and putative regulatory elements of the beta TP core promoter are presented. Down arrows mark the start sites of the beta TP promoter deletion constructs. TATA element is boxed. Other potential regulatory elements are overlined. The region of the core promoter with homology to human LHbeta and IGF II promoters is underlined. Two palindromic regions are overlined with double arrows. Lines with single arrows denote directly repeated sequences. The transcriptional start site is marked as +1.
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The beta TP Gene Promoter Is Responsive to Thyroid Hormone (T3) and Human Thyroid Hormone Receptor beta  (TRbeta )

In vivo analysis has shown that beta TP mRNA expression is regulated by T3. To determine if the human promoter mounted transcriptional responses to T3 and TRbeta , we studied T3 effects on the beta 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 beta TP gene responds to T3 and TRbeta , the full-length clone, pCAT2759, was cotransfected with a TRbeta expression vector (29) into TE671 cells and cultured in the presence or absence of 100 nM T3. The results reveal that the beta TP promoter is strongly regulated by TRbeta in a T3-dependent manner (Fig. 4). beta TP transcription is elevated 4-fold over basal levels in the presence of T3 and TRbeta . Unliganded TRbeta (no T3) represses the activity of the beta TP promoter 12-fold compared with basal levels. When both effects are considered, beta TP promoter activity is stimulated 45-fold by T3 over the level observed with unliganded TRbeta alone. Fig. 4 also shows that the response of pCAT2759 to T3 and TRbeta is in the range observed with the strong TRE of the rME-positive control, pTK83 (31). Varying the amount of TRbeta 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 TRbeta and T3 different from those observed for the beta TP promoter, indicating that beta TP promoter responses do not result from effects on cell viability or transcriptional competence.


Fig. 4. The beta TP promoter is positively responsive to T3 and TRbeta and negatively responsive to TRbeta alone. Activities of the full-length clone, pCAT2759, and heterologous promoters either in the presence or absence of T3 and TRbeta (TRB in the figure) are shown. Transfected TE671 cells were cultured in the presence of 100 nM T3 or with hormone vehicle alone. Basal activity (open bars), activity in the presence of 100 nM T3 alone (horizontal striped bars), of TRbeta alone (vertical striped bars), and in the presence of both TRbeta and T3 (solid bars) are shown. pTK83 contains the TRE from the rME promoter upstream of the TK promoter, and pBLCAT2 contains the TK minimal promoter. Data are expressed as CAT activity divided by the CAT activity of pCAT2759 in the absence of T3 and TR (basal activity) and reported as the mean ± S.E. from three separate transfections.
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T3-responsive Region of the beta TP Gene Promoter

To identify the T3-responsive region of the beta TP promoter, the deletion constructs (Fig. 1) were cotransfected with a TRbeta expression vector or pBSKS sham control and cultured with 100 nM T3. Only the full-length clone, pCAT2759, shows activation by T3 and TRbeta , 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 TRbeta , the activity of the deletion constructs in the presence of T3 and TRbeta was compared with that from TRbeta alone. The results, shown in Fig. 5B, again demonstrate that only the -2759-bp clone possesses major responses to T3.


Fig. 5. Delineation of the thyroid hormone-responsive region of the beta TP promoter. A, deletion constructs were cotransfected with either a human TRbeta expression vector or pBSKS and cultured with 100 nM T3. Data are expressed as CAT activity in the presence of TRbeta expression vector and T3 divided by CAT activity in the presence of pBSKS and T3. B, pTK83 and beta TP promoter deletion constructs were cotransfected with a TRbeta expression vector and cultured with 100 nM T3 or with hormone vehicle alone. Data are expressed as CAT activity in the presence of TRbeta and T3 divided by CAT activity in the presence of TRbeta alone. C, cells were transfected as in A but were not supplemented with T3. Repression is calculated as CAT activity generated by the deletion construct alone divided by CAT activity in the presence of TRbeta . Data are reported as the mean ± S.E. from a representative transfection.
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Similar experiments were performed to identify the region responsible for TRbeta -mediated repression. Deletion of the region responsible for T3 activation (-2759 to -2018 bp) barely altered repression by unliganded TRbeta (Fig. 5C). Repression by unliganded TRbeta was alleviated by deletions inward from -1423 bp. Thus, repression occurs at alternative or additional sites to those responsible for T3-mediated activation.

Identification of a Thyroid Hormone-responsive Element (TRE) in the beta TP Gene Promoter

To characterize further the T3-responsive region of the beta 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 beta 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 beta 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.


Fig. 6. Deletion analysis of the thyroid hormone-responsive region of the beta TP promoter. Bases -2759 to -2080 of the T3 responsive region of the beta TP promoter were cloned upstream of the heterologous TK promoter fused to the CAT gene. Schematic on the left represents the promoter constructs fused to the TK-CAT gene of the pBLCAT2 vector. The two squares represent each half-site of the TRE element, where the solid square represents the 3' half-site with perfect match to consensus. The hatched squares represent mutated half-sites as described under "Results." The oval represents an inverted repeat of consensus half-sites identified in the upstream region. Gaps within the schematic represent internal deletions introduced into the sequence. TK fusion constructs were cotransfected with a TRbeta expression vector and cultured with 100 nM T3 or with hormone vehicle alone. Bar graph on the right represents the fold activation of CAT activity in TE671 cells of the clones shown schematically on the left. Data are expressed as CAT activity in the presence of TRbeta and T3 divided by CAT activity in the presence of TRbeta alone. Data are reported as the mean ± S.E. from three separate transfections.
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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 beta 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 TRbeta from 8.0- to 2.9-fold (pTKDelta 3' in Fig. 6). A similar deletion of both half-sites of the beta TP TRE results in the loss of T3 induction (pTKDelta 5'+3' in Fig. 6). Thus deletion of the beta TP TRE results in the loss of the T3 responses identified in the upstream fragment of the beta TP promoter.

Gel shift assays were used to determine if the beta TP TRE formed specific complexes with TRbeta . The binding of TRbeta to the beta 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 beta TP TRE binds and shifts with TRbeta homodimers and more intensely when the RXRalpha 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 beta TP TRE and TRbeta /RXRalpha . As expected, the IR1 element failed to bind TRbeta and shifted only faintly in the presence of TRbeta /RXRalpha (Fig. 7C).

To further characterize the beta TP TRE, cold competitions were performed using the beta TP TRE element itself or the rME element. As expected, unlabeled beta TP TRE competes with labeled beta TP TRE (Fig. 8A). The rME element competes effectively with the beta TP TRE element for binding to TRbeta indicating that beta TP TRE forms complexes in the same fashion as rME (Fig. 8B).


Fig. 8. Gel shift analysis of the DR3 element from the beta TP promoter. A and B, cold competitions were performed using unlabeled probe at the molar excesses indicated at the top of the figure. Lanes marked C identify labeled rME-positive controls, and lanes marked M identify proteins isolated from a sham receptor preparation. A gives the result of competing cold beta TP TRE against labeled beta TP TRE; B, the result of competing cold rME TRE against labeled beta TP TRE. C, binding of TRbeta and RXRalpha to the beta TP TRE probe is compared with a mutated beta TP TRE probe (Delta DR3) in which two G nucleotides within each of the TRE half-sites have been changed to T or A. The location of the mutated bases are as shown below the sequence of the beta TP TRE element in Fig. 7A. D, Super-shifts. Homo- or heterodimeric complexes of TRbeta and RXRalpha were formed with the beta TP TRE probe and super-shifted with antibody specific to TRbeta . Antibody shifted complexes are marked with SS. In all panels, B marks probe-protein complexes, and F marks unbound probe. Gel shifts were performed as described in Fig. 7 using 10 fmol of 32P-labeled beta TP TRE probe.
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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 beta TP TRE was constructed (Delta 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, Delta DR3, failed to bind TRbeta homodimers and bound TRbeta /RXRalpha 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 beta TP TRE. Confirming the results from gel shift, placement of the mutant beta 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 pTKDelta DR3 in Fig. 6). Thus, the intact sequence of the beta TP TRE is necessary to bind TRbeta and to activate transcription in response to T3 and TRbeta .

To probe the composition of the DNA-protein complexes, anti-hTRbeta antibody was used to super-shift DNA-protein complexes that had formed with TRbeta . beta TP TRE-protein complexes, formed in the presence of TRbeta or TRbeta /RXRalpha , were shifted by antibody, demonstrating that the complexes with beta TP TRE were formed by TRbeta binding (Fig. 8D).


DISCUSSION

Basal transcription of the beta -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 beta -LH gene, which is active in the CNS (38).

The human beta TP gene is regulated by TRbeta in a T3-dependent manner. T3 and TRbeta substantially elevate beta TP promoter activity, whereas unliganded TRbeta 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 beta TP promoter to T3 is strong (Fig. 4).

Deletion analyses indicate that the beta 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 beta 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 beta TP TRE is lost with mutation of the two conserved G nucleotides within each half-site.

Gel shift experiments demonstrate that the beta TP TRE forms specific complexes with both TRbeta homodimers and TRbeta ·RXRalpha heterodimers. We used cold competitions with the rME TRE, mutagenesis of the beta TP TRE, and super-shifts with anti-TRbeta specific antibodies to demonstrate that beta TP TRE binds to and interacts with TRbeta in a manner consistent with other TRE sequences.

The beta TP TRE is well upstream of the core promoter. This organization places the beta 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 beta 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 beta 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 beta TP promoter shows considerable T3-independent repression by TRbeta (54). Repression appears to be specific for the beta TP promoter as two heterologous promoters used in this study (SV2 and TK) are only mildly affected by unliganded TRbeta . 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 alpha  gene (57).

TE671 cells express beta TP but not TRbeta . These properties permitted the examination of basal beta TP promoter activity in the absence of the potentially confounding effects of TRbeta . Subsequent expression of TRbeta in TE671 cells allowed dissection of the repressive effects of TRbeta on beta TP basal transcription from the more familiar role of TRbeta in activation. The basal activity of the beta TP promoter observed in TE671 cells in the absence of TRbeta may be physiologically relevant. Although TR expression is widespread it is not universal (37, 58).

The increase in the activity of the beta TP promoter by T3 and TRbeta and its repression by TRbeta alone, as shown here, agrees with and provides a dual mechanism for the reduced beta TP mRNA levels observed in thyroidectomized rats (20). The thyroid hormone responsiveness of the beta TP promoter may also imply T3 control of PGD2 synthesis. However, the high levels of beta TP and beta TP promoter activity observed should be contrasted to the low levels of PGD2 observed in human CSF (59). Perhaps beta 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 beta TP and other lipocalin transporters provides indirect support for a role in lipid transport. Further support for the role of beta TP in transport processes has recently been provided by the work of Hoffmann et al. (60) who, in a careful in situ analysis of beta TP expression in the developing mouse, have observed beta TP expression at or near a number of blood-tissue barriers, hinting at a role for beta TP in transport across these barriers.

The present study indicates that T3 exerts a measure of control over beta TP gene expression. If beta 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 beta TP transcription remain to be addressed. Foremost among them is whether the expression of beta TP is regulated in a tissue-specific manner, implying the use of different enhancer elements within the beta TP promoter or different combinations of tissue-specific transcription factors. Additionally, the role of T3 on beta TP expression must be examined in the context of the other tissues in which it is expressed.


FOOTNOTES

*   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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M98537[GenBank].


§   Supported in part by NINDS Grant PHS PO1 NS 24575. To whom correspondence should be addressed: The University of Chicago, Dept. of Neurology, 5841 S. Maryland Ave., MC2030, Chicago, IL 60637. Tel.: 773-702-6541; Fax: 773-702-9076; E-mail: dwhite{at}drugs.bsd.uchicago.edu.
par    Supported by United States Public Health Service Grant DK-27384 and the David Wiener Research Fund.
1   The abbreviations used are: beta TP, beta -Trace protein; CSF, cerebrospinal fluid; CNS, central nervous system; CP, choroid plexus; CAT, chloramphenicol acetyltransferase; TR, human thyroid hormone receptors; TRbeta , human thyroid hormone receptor beta 1; RXRalpha , human retinoid X receptor alpha ; T3, thyroid hormone (3,3',5-triiodothyronine); TRE, thyroid hormone-responsive element; bp, base pair(s); kb, kilobase pair(s); DR3, direct repeat with 3-bp spacing; IR1, inverted repeat with 1-bp spacing; rME, rat malic enzyme; TK, thymidine kinase; PGD2, prostaglandin D2; PDS, prostaglandin D2 synthase; OLG, oligonucleotides; beta GAL, beta -galactosidase; LH-beta , luteinizing hormone subunit beta ; IGF-II, insulin-like growth factor II.

ACKNOWLEDGEMENT

We thank Dr. Mark A. Jensen for critically reading the manuscript.


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