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• W2099021510 abstract "We isolated and characterized the human β-Trace protein (βTP) gene promoter. βTP, also known as prostaglandin D2 synthase, is a lipocalin secreted from the choroid plexus and meninges into cerebrospinal fluid. Basal transcription of the βTP gene is directed from a core promoter found within the first 325 bases of the 5′-flanking sequence. The βTP gene promoter is responsive to thyroid hormone (3,3′,5-triiodothyronine, T3) and efficiently repressed by unliganded human thyroid hormone receptor β (TRβ). Functional analysis of the βTP 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 βTP TRE is composed of two directly repeated consensus half-sites separated by a 3-base pair space (DR3). The βTP TRE forms specific complexes with TRβ. 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. We isolated and characterized the human β-Trace protein (βTP) gene promoter. βTP, also known as prostaglandin D2 synthase, is a lipocalin secreted from the choroid plexus and meninges into cerebrospinal fluid. Basal transcription of the βTP gene is directed from a core promoter found within the first 325 bases of the 5′-flanking sequence. The βTP gene promoter is responsive to thyroid hormone (3,3′,5-triiodothyronine, T3) and efficiently repressed by unliganded human thyroid hormone receptor β (TRβ). Functional analysis of the βTP 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 βTP TRE is composed of two directly repeated consensus half-sites separated by a 3-base pair space (DR3). The βTP TRE forms specific complexes with TRβ. 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. β-Trace protein (βTP) 1The abbreviations used are: βTPβ-Trace proteinCSFcerebrospinal fluidCNScentral nervous systemCPchoroid plexusCATchloramphenicol acetyltransferaseTRhuman thyroid hormone receptorsTRβhuman thyroid hormone receptor β1RXRαhuman retinoid X receptor αT3thyroid hormone (3,3′,5-triiodothyronine)TREthyroid hormone-responsive element; bp, base pair(s)kbkilobase pair(s)DR3direct repeat with 3-bp spacingIR1inverted repeat with 1-bp spacingrMErat malic enzymeTKthymidine kinasePGD2prostaglandin D2PDSprostaglandin D2 synthaseOLGoligonucleotidesβGALβ-galactosidaseLH-βluteinizing hormone subunit βIGF-IIinsulin-like growth factor II 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, βTP is present at 2.6 mg/dl, ranking it among the major CSF proteins (1Link H. Acta Neurol. Scand. Suppl. 1967; 43: 1-136Crossref Scopus (8) Google Scholar). βTP, identified by Clausen in 1961 (2Clausen J. Proc. Soc. Exp. Biol. Med. 1961; 107: 170-172Crossref PubMed Scopus (185) Google Scholar), is primarily expressed in the choroid plexus (CP). βTP is also expressed to a lesser extent in meninges and oligodendrocytes (3Hochwald G.M. Pepe A.J. Thorbecke G.J. Proc. Soc. Exp. Biol. Med. 1967; 124: 961-966Crossref PubMed Scopus (51) Google Scholar, 4Olsson J.E. Nord L. J. Neurochem. 1973; 21: 625-633Crossref PubMed Scopus (20) Google Scholar). Other than the CNS, the major site of βTP expression is the epididymis (4Olsson J.E. Nord L. J. Neurochem. 1973; 21: 625-633Crossref PubMed Scopus (20) Google Scholar, 5Olsson J.E. Sandberg M. Neurobiology. 1975; 5: 270-276PubMed Google Scholar). β-Trace protein cerebrospinal fluid central nervous system choroid plexus chloramphenicol acetyltransferase human thyroid hormone receptors human thyroid hormone receptor β1 human retinoid X receptor α thyroid hormone (3,3′,5-triiodothyronine) thyroid hormone-responsive element; bp, base pair(s) kilobase pair(s) direct repeat with 3-bp spacing inverted repeat with 1-bp spacing rat malic enzyme thymidine kinase prostaglandin D2 prostaglandin D2 synthase oligonucleotides β-galactosidase luteinizing hormone subunit β insulin-like growth factor II A protein with similar distribution to βTP has been identified as prostaglandin D2 synthase (PDS) in rats (6Urade Y. Fujimoto N. Hayaishi O. J. Biol. Chem. 1985; 260: 12410-12415Abstract Full Text PDF PubMed Google Scholar, 7Nagata A. Suzuki Y. Igarashi M. Eguchi N. Toh H. Urade Y. Hayaishi O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4020-4024Crossref PubMed Scopus (175) Google Scholar). PDS catalyzes the conversion of prostaglandin H2 to prostaglandin D2 (PGD2). A role for PGD2 in regulation of sleep induction has been proposed (8Onoe H. Ueno R. Fujita I. Nishino H. Oomura Y. Hayaishi O. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4082-4086Crossref PubMed Scopus (104) Google Scholar,9Hayaishi O. FASEB J. 1991; 5: 2575-2581Crossref PubMed Scopus (237) Google Scholar). Recently, βTP and PDS were shown to be the same protein (10Zahn M. Mader M. Schmidt B. Bollensen E. Felgenhauer K. Neurosci. Lett. 1993; 154: 93-95Crossref PubMed Scopus (47) Google Scholar, 11Hoffmann A. Conradt H.S. Gross G. Nimtz M. Lottspeich F. Wurster U. J. Neurochem. 1993; 61: 451-456Crossref PubMed Scopus (185) Google Scholar). In prior studies we have referred to βTP/PDS as PDS but, in deference to precedence, we now refer to it as βTP (12White D.M. Mikol D.D. Espinosa R. Weimer B. Le Beau M.M. Stefansson K. J. Biol. Chem. 1992; 267: 23202-23208Abstract Full Text PDF PubMed Google Scholar). The human βTP message encodes a 180-residue polypeptide that is a member of the lipocalin superfamily. Lipocalins are secretory proteins that transport hydrophobic ligands (13Pervaiz S. Brew K. Science. 1985; 228: 335-337Crossref PubMed Scopus (239) Google Scholar, 14Ali S. Clark A.J. J. Mol. Biol. 1988; 199: 415-426Crossref PubMed Scopus (111) Google Scholar). 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 (15Salier J.P. Simon D. Rouet P. Raguenez G. Muscatelli F. Gebhard W. Guenet J.L. Mattei M.G. Genomics. 1992; 14: 83-88Crossref PubMed Scopus (26) Google Scholar). In previous work we localized the human βTP gene to the lipocalin gene cluster on 9q34. The βTP gene bears a striking resemblance to other lipocalin genes, suggesting a role for βTP in transport (12White D.M. Mikol D.D. Espinosa R. Weimer B. Le Beau M.M. Stefansson K. J. Biol. Chem. 1992; 267: 23202-23208Abstract Full Text PDF PubMed Google Scholar). 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 (16Segal M.B. J. Inherited. Metab. Dis. 1993; 16: 617-638Crossref PubMed Scopus (108) Google Scholar). 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 (17Ohe Y. Ishikawa K. Itoh Z. Tatemoto K. J. Neurochem. 1996; 67: 964-971Crossref PubMed Scopus (74) Google Scholar). Several CSF transporters have been characterized including transthyretin, transferrin, and ceruloplasmin; they carry thyroxine, iron, and copper, respectively (18Nilsson C. Lindvall A.M. Owman C. Brain Res. Rev. 1992; 17: 109-138Crossref PubMed Scopus (193) Google Scholar, 19Aldred A.R. Brack C.M. Schreiber G. Comp. Biochem. Physiol. B. 1995; 111: 1-15Crossref PubMed Scopus (103) Google Scholar). Garcı́a-Fernández et al. (20Garcı́a-Fernández L.F. Iniguez M.A. Rodriguez P.A. Munoz A. Bernal J. Biochem. Biophys. Res. Commun. 1993; 196: 396-401Crossref PubMed Scopus (29) Google Scholar) found that levels of βTP mRNA in the CNS of adult rats decrease following chemically induced hypothyroidism. The mechanism by which thyroid hormone (T3) influences βTP gene expression is unknown. T3 exerts its effects through binding to thyroid hormone receptors (TR), which are widely distributed in the CNS (21Bradley D.J. Towle H.C. Young W.S. J. Neurosci. 1992; 12: 2288-2302Crossref PubMed Google Scholar). In the CP, T3 augments transport function; hypothyroid rats have reduced Na+-K+-ATPase activity, a marker for transport processes (22Lindvall-Axelsson M. Hedner P. Owman C. Winbladh B. Acta Physiol. Scand. 1985; 125: 627-632Crossref PubMed Scopus (7) Google Scholar). To better understand mechanisms of βTP gene regulation, we subcloned the human βTP 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-kbXho I-Xba I fragment from the βTP genomic clone pG4CS86 (12White D.M. Mikol D.D. Espinosa R. Weimer B. Le Beau M.M. Stefansson K. J. Biol. Chem. 1992; 267: 23202-23208Abstract Full Text PDF PubMed Google Scholar) was inserted into the Sma I 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 (23Fridovich-Keil J.L. Gudas J.M. Bryan I.B. Pardee A.B. BioTechniques. 1991; 11: 572-579PubMed Google Scholar) to generate clone pCAT2759 (Fig. 1). Exo- and endonuclease deletions of pCAT2759 produced clones with successive 5′ deletions. Sequence was analyzed as described previously (12White D.M. Mikol D.D. Espinosa R. Weimer B. Le Beau M.M. Stefansson K. J. Biol. Chem. 1992; 267: 23202-23208Abstract Full Text PDF PubMed Google Scholar). Clone pCAT235 was produced by subcloning the region between −2759 and −2080 bp of the βTP promoter into pBSKS+. Small internal deletions were introduced into pCAT235 by digestion with Sty I andEco NI followed by Klenow fill-in or mung bean nuclease digestion to remove one or both of the βTP TRE half-sites, respectively. To liberate the inserts from the pBSKS+ vector, the constructs were opened with Bam HI, made blunt-ended with Klenow, and subsequently digested with Sal I. Gel-purified fragments were subcloned into the pBLCAT2 vector that had been opened first at the Hin dIII site and made blunt-ended with Klenow enzyme followed by digestion with Sal I. The three clones thus produced were as follows: 1) pTK680F, with the 680-bp fragment of pCAT235 in the forward orientation, 2) pTKΔ3′ with a 104-base internal deletion which removes the 3′ half-site of the TRE, and 3) pTKΔ5′ + 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 βTP promoter, was produced by deleting 385 bp of 3′ sequence from clone pTK680F by double digestion with Eco NI and Sal I. Clone pTK680R was produced by subcloning the 680-bpBam HI-Sal I fragment of pCAT235 into the corresponding sites of pBLCAT2. Clone pTKΔDR3 was produced from overlapping oligonucleotides cloned into theHin dIII-Xho I sites of pBLCAT2. Clone pTK100 was produced by PCR amplification of bases −2620 to −2518 bp of the βTP promoter using pCAT2759 as template and oligonucleotides that introduced a 5′ Hin dIII site and a 3′ Xho I site. PCR product was digested with Hin dIII and Xho I 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. (24Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). 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 (12White D.M. Mikol D.D. Espinosa R. Weimer B. Le Beau M.M. Stefansson K. J. Biol. Chem. 1992; 267: 23202-23208Abstract Full Text PDF PubMed Google Scholar). The host cell line used in these studies was the human rhabdomyosarcoma cell line TE671 (ATCC CRL 8805) (25Chen T.R. Dorotinsky C. Macy M. Hay R. Nature. 1989; 340: 106Crossref PubMed Scopus (23) Google Scholar, 26Stratton M.R. Darling J. Pilkington G.J. Lantos P.L. Reeves B.R. Cooper C.S. Carcinogenesis. 1989; 10: 899-905Crossref PubMed Scopus (126) Google Scholar). Cells were passaged in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (BioWhittaker) and 40 βg/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 (27Keown W.A. Campbell C.R. Kucherlapati R.S. Methods Enzymol. 1990; 185: 527-537Crossref PubMed Scopus (95) Google Scholar). For each dish, 1 pmol of CAT construct was co-transfected with 2 βg of β-galactosidase (βGAL) expression vector pRSV-βGAL (28Edlund T. Walker M.D. Barr P.J. Rutter W.J. Science. 1985; 230: 912-916Crossref PubMed Scopus (396) Google Scholar) as an internal control. For TR cotransfections 2 βg of hTRβ1 expression vector was used (29Nakai A. Sakurai A. Macchia E. Fang V. DeGroot L.J. Mol. Cell. Endocrinol. 1990; 72: 143-148Crossref PubMed Scopus (24) Google Scholar). pBSKS+ was added to bring the total DNA in each dish to 12 βg. 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 (30Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar) was used throughout as a positive control vector for CAT expression, and pTK83 was used for T3/TR responses (31Suzuki S. Miyamoto T. Opsahl A. Sakurai A. DeGroot L.J. Mol. Endocrinol. 1994; 8: 305-314PubMed Google Scholar). The negative control for CAT expression was the promoterless CAT vector pJFCAT1 and for T3 responses, pBLCAT2, which contains the TK promoter (32Luckow B. Günther S. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar). To correct for variations in transfection efficiency, cell extracts were assayed for βGAL activity (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 3. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.66Google Scholar). After adjusting for βGAL levels, CAT activity was determined using a variation of the diffusion assay (34Eastman A. BioTechniques. 1987; 5: 731-732Google Scholar). 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-β-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. 7 A were used for gel retardation of the βTP TRE and IR1 elements. The βTP TRE OLGs were 5′-AGGCAGGGGGATGGCCTTGGTGACCTCTTAGGGTGGA-3′ and the complementary strand 5′-TGGCCTCCACCCTAAGAGGTCACCAAGGCCATCCCC-3′. The mutant TRE, ΔDR3, is similar to βTP 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 (35Miyamoto T. Suzuki S. DeGroot L.J. Mol. Endocrinol. 1993; 7: 224-231PubMed Google Scholar). Complementary OLGs were hybridized and 5′ overhangs filled in with Klenow polymerase and [α-32P]dCTP. Labeled duplexes, purified on G50 columns, had a specific activity greater than 1.7 × 106 cpm/pmol. Recombinant human TRβ1 and RXRα were prepared as described by Sakurai et al. (36Sakurai A. Suzuki S. Katai M. Miyamoto T. Kobayashi H. Nakajima K. Ichikawa K. DeGroot L.J. Hashizume K. Mol. Cell. Endocrinol. 1995; 110: 103-112Crossref PubMed Scopus (6) Google Scholar). 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/βl poly(dI-dC), 20–100 fmol of TRβ1, and/or 20–100 fmol of RXRα. Reactions proceeded at room temperature for 20 min. In supershift experiments, complexes were permitted to form for 20 min. 1 βl of anti-hTRβ polyclonal antibody β62 (37Falcone M. Miyamoto T. Fierro-Renoy F. Macchia E. DeGroot L.J. Endocrinology. 1992; 131: 2419-2429Crossref PubMed Scopus (79) Google Scholar) 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 βTP promoter was isolated from the genomic clone pG4CS86 (12White D.M. Mikol D.D. Espinosa R. Weimer B. Le Beau M.M. Stefansson K. J. Biol. Chem. 1992; 267: 23202-23208Abstract Full Text PDF PubMed Google Scholar). To localize regions of the promoter important to βTP 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 (25Chen T.R. Dorotinsky C. Macy M. Hay R. Nature. 1989; 340: 106Crossref PubMed Scopus (23) Google Scholar, 26Stratton M.R. Darling J. Pilkington G.J. Lantos P.L. Reeves B.R. Cooper C.S. Carcinogenesis. 1989; 10: 899-905Crossref PubMed Scopus (126) Google Scholar) expresses βTP mRNA at high levels and transfects efficiently (Fig. 2). Parallel transfections of the 10 deletion constructs into TE671 cells revealed the βTP gene promoter to be highly active, generating CAT activity at a level comparable to the positive control vector pSV2CAT (30Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar). The βTP core promoter region is small, deletions from −2759 bp to −595 bp had minimal effects on βTP promoter activity. Deleting the bases between −595 and −325 actually increased βTP 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 βTP gene between −325 and −80 bp of the promoter. The sequence of the core promoter is presented in Fig. 3. The region from −227 to −180 bp of the βTP promoter has high sequence identity with regions of the human luteinizing hormone subunit β promoter (LH-β) (68% identity, bases −239 to −192) (38Jameson J.L. Lindell C.M. Habener J.F. DNA ( N. Y. ). 1986; 5: 227-234Crossref PubMed Scopus (31) Google Scholar) and the human insulin-like growth factor II P4 promoter (80% identity, bases −318 to −287) (IGF-II) (39de Pagter-Holthuizen P. Jansen M. van der Kammen R.A. van Schaik F.M.A. Sussenbach J.S. Biochim. Biophys. Acta. 1988; 950: 282-295Crossref PubMed Scopus (135) Google Scholar). The IGF-II P4 promoter is active in the CP (40Ohlsson R. Hedborg F. Holmgren L. Walsh C. Ekstrom T.J. Development. 1994; 120: 361-368Crossref PubMed Google Scholar). 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 (41Mermod N. Williams T.J. Tjian R. Nature. 1988; 332: 557-561Crossref PubMed Scopus (226) Google Scholar) and to the cAMP response element, ENKCRE-2, found within the proenkephalin gene promoter (42Comb M. Birnberg N.C. Seasholtz A. Herbert E. Goodman H.M. Nature. 1986; 323: 353-356Crossref PubMed Scopus (525) Google Scholar). However, the βTP promoter is only mildly responsive to forskolin (data not shown). In vivo analysis has shown that βTP mRNA expression is regulated by T3. To determine if the human promoter mounted transcriptional responses to T3 and TRβ, we studied T3 effects on the βTP 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 βTP gene responds to T3 and TRβ, the full-length clone, pCAT2759, was cotransfected with a TRβ expression vector (29Nakai A. Sakurai A. Macchia E. Fang V. DeGroot L.J. Mol. Cell. Endocrinol. 1990; 72: 143-148Crossref PubMed Scopus (24) Google Scholar) into TE671 cells and cultured in the presence or absence of 100 nm T3. The results reveal that the βTP promoter is strongly regulated by TRβ in a T3-dependent manner (Fig. 4). βTP transcription is elevated 4-fold over basal levels in the presence of T3 and TRβ. Unliganded TRβ (no T3) represses the activity of the βTP promoter 12-fold compared with basal levels. When both effects are considered, βTP promoter activity is stimulated 45-fold by T3 over the level observed with unliganded TRβ alone. Fig. 4 also shows that the response of pCAT2759 to T3 and TRβ is in the range observed with the strong TRE of the rME-positive control, pTK83 (31Suzuki S. Miyamoto T. Opsahl A. Sakurai A. DeGroot L.J. Mol. Endocrinol. 1994; 8: 305-314PubMed Google Scholar). Varying the amount of TRβ 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 TRβ and T3 different from those observed for the βTP promoter, indicating that βTP promoter responses do not result from effects on cell viability or transcriptional competence. To identify the T3-responsive region of the βTP promoter, the deletion constructs (Fig. 1) were cotransfected with a TRβ expression vector or pBSKS sham control and cultured with 100 nm T3. Only the full-length clone, pCAT2759, shows activation by T3 and TRβ, localizing the T3 responsive region to the sequence between −2759 and −2018 bp (Fig. 5 A). 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 TRβ, the activity of the deletion constructs in the presence of T3 and TRβ was compared with that from TRβ alone. The results, shown in Fig.5 B, again demonstrate that only the −2759-bp clone possesses major responses to T3. Similar experiments were performed to identify the region responsible for TRβ-mediated repression. Deletion of the region responsible for T3 activation (−2759 to −2018 bp) barely altered repression by unliganded TRβ (Fig. 5 C). Repression by unliganded TRβ 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 βTP 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 βTP promoter T3-responsive region also confers strong T3induction 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 βTP 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.7 A), which is composed of two directly repeated half-sites separated by 3 bp (DR3). To test the role of the βTP 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 T3and TRβ from 8.0- to 2.9-fold (pTKΔ3′ in Fig.6). A similar deletion of both half-sites of the βTP TRE results in the loss of T3 induction (pTKΔ5′+3′ in Fig. 6). Thus deletion of the βTP TRE results in the loss of the T3 responses identified in the upstream fragment of the βTP promoter. Gel shift assays were used to determine if the βTP TRE formed specific complexes with TRβ. The binding of TRβ to the βTP TRE was compared with that of the DR4 type TRE from the rME promoter (43Petty K.J. Desvergne B. Mitsuhashi T. Nikodem V.M. J. Biol. Chem. 1990; 265: 7395-7400Abstract Full Text PDF PubMed Google Scholar). 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. 7 A). The βTP TRE binds and shifts with TRβ homodimers and more intensely when the RXRα accessory protein is present (Fig. 7 B). 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 βTP TRE and TRβ/RXRα. As expected, the IR1 element failed to bind TRβ and shifted only faintly in the presence of TRβ/RXRα (Fig. 7 C). To further characterize the βTP TRE, cold competitions were performed using the βTP TRE element itself or the rME element. As expected, unlabeled βTP TRE competes with labeled βTP TRE (Fig.8 A). The rME element competes effectively with the βTP TRE element for binding to TRβ indicating that βTP TRE forms complexes in the same fashion as rME (Fig.8 B). Within TRE half-sites, loss of one or both of the two conserved G nucleotides substantially reduces TR binding and TRE function (44Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1497) Google Scholar, 45Farsetti A. Desvergne B. Hallenbeck P. Robbins J. Nikodem V.M. J. Biol. Chem. 1992; 267: 15784-15788Abstract Full Text PDF PubMed Google Scholar). To test the role of these nucleotides, a mutant βTP TRE was constructed (ΔDR3) in which the G residues at bases −2573–74 and −2564–63 were changed to T or A, respectively (Fig. 7 A). The reconfigured element, ΔDR3, failed to bind TRβ homodimers and bound TRβ/RXRα heterodimers only faintly (Fig. 8 C) proving that residues known to be crucial for TR binding in other TREs are also necessary for TR binding to the βTP TRE. Confirming the results from gel shift, placement of the mutant βTP 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 pTKΔDR3 in Fig. 6). Thus, the intact sequence of the βTP TRE is necessary to bind TRβ and to activate transcription in response to T3 and TRβ. To probe the composition of the DNA-protein complexes, anti-hTRβ antibody was used to super-shift DNA-protein complexes that had formed with TRβ. βTP TRE-protein complexes, formed in the presence of TRβ or TRβ/RXRα, were shifted by antibody, demonstrating that the complexes with βTP TRE were formed by TRβ binding (Fig.8 D). Basal transcription of the β-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 (39de Pagter-Holthuizen P. Jansen M. van der Kammen R.A. van Schaik F.M.A. Sussenbach J.S. Biochim. Biophys. Acta. 1988; 950: 282-295Crossref PubMed Scopus (135) Google Scholar), which is active in the choroid plexus (40Ohlsson R. Hedborg F. Holmgren L. Walsh C. Ekstrom T.J. Development. 1994; 120: 361-368Crossref PubMed Google Scholar), and to the β-LH gene, which is active in the CNS (38Jameson J.L. Lindell C.M. Habener J.F. DNA ( N. Y. ). 1986; 5: 227-234Crossref PubMed Scopus (31) Google Scholar). The human βTP gene is regulated by TRβ in a T3-dependent manner. T3 and TRβ substantially elevate βTP promoter activity, whereas unliganded TRβ effectively represses the promoter. The level of T3-dependent activation observed is comparable to that observed using a classical TRE from the rME promoter (43Petty K.J. Desvergne B. Mitsuhashi T. Nikodem V.M. J. Biol. Chem. 1990; 265: 7395-7400Abstract Full Text PDF PubMed Google Scholar), indicating that the overall response of the βTP promoter to T3 is strong (Fig. 4). Deletion analyses indicate that the βTP 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 βTP-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 βTP TRE is lost with mutation of the two conserved G nucleotides within each half-site. Gel shift experiments demonstrate that the βTP TRE forms specific complexes with both TRβ homodimers and TRβ·RXRα heterodimers. We used cold competitions with the rME TRE, mutagenesis of the βTP TRE, and super-shifts with anti-TRβ specific antibodies to demonstrate that βTP TRE binds to and interacts with TRβ in a manner consistent with other TRE sequences. The βTP TRE is well upstream of the core promoter. This organization places the βTP 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 (46Clark A.R. Wilson M.E. London N.J. James R.F. Docherty K. Biochem. J. 1995; 309: 863-870Crossref PubMed Scopus (31) Google Scholar), the rat S14 gene which has multiple TREs located in a 200-bp region around −2.6 kb (47Liu H.C. Towle H.C. Mol. Endocrinol. 1994; 8: 1021-1037PubMed Google Scholar), and the rat ucp gene which has two TREs located in the region around −2.3 kb (48Rabelo R. Schifman A. Rubio A. Sheng X. Silva J.E. Endocrinology. 1995; 136: 1003-1013Crossref PubMed Scopus (91) Google Scholar, 49Cassard-Doulcier A.-M. Larose M. Matamala J.C. Champigny O. Bouillaud F. Ricquier D. J. Biol. Chem. 1994; 269: 24335-24342Abstract Full Text PDF PubMed Google Scholar). Both the βTP 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 (44Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1497) Google Scholar, 50Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar). 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 (48Rabelo R. Schifman A. Rubio A. Sheng X. Silva J.E. Endocrinology. 1995; 136: 1003-1013Crossref PubMed Scopus (91) Google Scholar). 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 (51Miyamoto T. Suzuki S. DeGroot L.J. Mol. Cell. Endocrinol. 1994; 102: 111-117Crossref PubMed Scopus (9) Google Scholar). The sequences flanking the half-sites may prove to be more important in honing the T3 response of the βTP TRE than the spacings between the half-sites. Koenig et al. (52Katz R.W. Subauste J.S. Koenig R.J. J. Biol. Chem. 1995; 270: 5238-5242Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) 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 (53Mader S. Leroy P. Chen J.-Y. Chambon P. J. Biol. Chem. 1993; 268: 591-600Abstract Full Text PDF PubMed Google Scholar). The βTP promoter shows considerable T3-independent repression by TRβ (54Damm K. Thompson C.C. Evans R.M. Nature. 1989; 339: 593-597Crossref PubMed Scopus (469) Google Scholar). Repression appears to be specific for the βTP promoter as two heterologous promoters used in this study (SV2 and TK) are only mildly affected by unliganded TRβ. 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 (55Carr F.E. Wong N.C.W. J. Biol. Chem. 1994; 269: 4175-4179Abstract Full Text PDF PubMed Google Scholar, 56Cohen O. Flynn T.R. Wondisford F.E. J. Biol. Chem. 1995; 270: 13899-13905Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Additionally, unliganded TR might disrupt protein-protein interactions necessary for transcription as has been observed in the human glycoprotein hormone α gene (57Madison L.D. Ahlquist J.A. Rogers S.D. Jameson J.L. Mol. Cell. Endocrinol. 1993; 94: 129-136Crossref PubMed Scopus (32) Google Scholar). TE671 cells express βTP but not TRβ. These properties permitted the examination of basal βTP promoter activity in the absence of the potentially confounding effects of TRβ. Subsequent expression of TRβ in TE671 cells allowed dissection of the repressive effects of TRβ on βTP basal transcription from the more familiar role of TRβ in activation. The basal activity of the βTP promoter observed in TE671 cells in the absence of TRβ may be physiologically relevant. Although TR expression is widespread it is not universal (37Falcone M. Miyamoto T. Fierro-Renoy F. Macchia E. DeGroot L.J. Endocrinology. 1992; 131: 2419-2429Crossref PubMed Scopus (79) Google Scholar, 58Jannini E.A. Dolci S. Ulisse S. Nikodem V.M. Mol. Endocrinol. 1994; 8: 89-96Crossref PubMed Scopus (92) Google Scholar). The increase in the activity of the βTP promoter by T3and TRβ and its repression by TRβ alone, as shown here, agrees with and provides a dual mechanism for the reduced βTP mRNA levels observed in thyroidectomized rats (20Garcı́a-Fernández L.F. Iniguez M.A. Rodriguez P.A. Munoz A. Bernal J. Biochem. Biophys. Res. Commun. 1993; 196: 396-401Crossref PubMed Scopus (29) Google Scholar). The thyroid hormone responsiveness of the βTP promoter may also imply T3control of PGD2 synthesis. However, the high levels of βTP and βTP promoter activity observed should be contrasted to the low levels of PGD2 observed in human CSF (59Westcott J.Y. Murphy R.C. Stenmark K. Prostaglandins. 1987; 34: 877-887Crossref PubMed Scopus (36) Google Scholar). Perhaps βTP 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 βTP and other lipocalin transporters provides indirect support for a role in lipid transport. Further support for the role of βTP in transport processes has recently been provided by the work of Hoffmann et al. (60Hoffmann A. Bachner D. Betat N. Lauber J. Gross G. Dev. Dyn. 1996; 207: 332-343Crossref PubMed Scopus (52) Google Scholar) who, in a careful in situ analysis of βTP expression in the developing mouse, have observed βTP expression at or near a number of blood-tissue barriers, hinting at a role for βTP in transport across these barriers. The present study indicates that T3 exerts a measure of control over βTP gene expression. If βTP 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 βTP transcription remain to be addressed. Foremost among them is whether the expression of βTP is regulated in a tissue-specific manner, implying the use of different enhancer elements within the βTP promoter or different combinations of tissue-specific transcription factors. Additionally, the role of T3 on βTP expression must be examined in the context of the other tissues in which it is expressed. We thank Dr. Mark A. Jensen for critically reading the manuscript." @default.
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• W2099021510 title "β-Trace Gene Expression Is Regulated by a Core Promoter and a Distal Thyroid Hormone Response Element" @default.
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