A Far Upstream, Cell Type-specific Enhancer of the Mouse Thrombospondin 3 Gene Is Located within Intron 6 of the Adjacent Metaxin Gene
1998; Elsevier BV; Volume: 273; Issue: 34 Linguagem: Inglês
10.1074/jbc.273.34.21816
ISSN1083-351X
AutoresMalcolm Collins, Ponlapat Rojnuckarin, Yuhong Zhu, Paul Börnstein,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoThrombospondin 3 (TSP3) is a secreted, pentameric glycoprotein whose regulation of expression and function are not well understood. Mouse Thbs3 is located just downstream from the divergently transcribed metaxin gene (Mtx), which encodes an outer mitochondrial membrane import protein. AlthoughThbs3 and Mtx share a common promoter region, previous studies showed that Mtx is regulated by proximal elements that had little effect on Thbs3 expression. In this study, transient transfection of rat chondrosarcoma cells and NIH-3T3 fibroblasts demonstrated that Thbs3 is regulated in a cell type-specific manner by a position- and orientation-independent far upstream enhancer located within intron 6 of Mtx. Despite its greater proximity to the transcription start site ofMtx, the Thbs3 enhancer did not have a significant effect on Mtx expression. Two DNA-protein complexes, which were both required for activity, were identified when nuclear extracts were assayed with a probe containing the enhancer sequence. The protein in one of these complexes was identified as Sp1, while the other DNA-protein complex remains uncharacterized. A 6-kilobase pair promoter containing the enhancer was able to direct specific expression of the E. coli lacZ gene in transgenic mice, whereas a 2-kilobase pair promoter that lacked the enhancer was inactive. Thus, despite their close proximity, the genes of theMtx/Thbs3 gene cluster are regulated independently. Thrombospondin 3 (TSP3) is a secreted, pentameric glycoprotein whose regulation of expression and function are not well understood. Mouse Thbs3 is located just downstream from the divergently transcribed metaxin gene (Mtx), which encodes an outer mitochondrial membrane import protein. AlthoughThbs3 and Mtx share a common promoter region, previous studies showed that Mtx is regulated by proximal elements that had little effect on Thbs3 expression. In this study, transient transfection of rat chondrosarcoma cells and NIH-3T3 fibroblasts demonstrated that Thbs3 is regulated in a cell type-specific manner by a position- and orientation-independent far upstream enhancer located within intron 6 of Mtx. Despite its greater proximity to the transcription start site ofMtx, the Thbs3 enhancer did not have a significant effect on Mtx expression. Two DNA-protein complexes, which were both required for activity, were identified when nuclear extracts were assayed with a probe containing the enhancer sequence. The protein in one of these complexes was identified as Sp1, while the other DNA-protein complex remains uncharacterized. A 6-kilobase pair promoter containing the enhancer was able to direct specific expression of the E. coli lacZ gene in transgenic mice, whereas a 2-kilobase pair promoter that lacked the enhancer was inactive. Thus, despite their close proximity, the genes of theMtx/Thbs3 gene cluster are regulated independently. The thrombospondins (TSPs) 1The abbreviations used are: TSPthrombospondinRCSrat chondrosarcomaEMSAelectrophoretic mobility shift assaysbpbase pair(s)kbkilobase pair(s)X-gal5-bromo-4-chloro-3-indolyl β-d-galactopyranoside. comprise a family of five secreted matrix glycoproteins that have a characteristic modular structure consisting of a distinct amino-terminal globular domain, three (TSP1 and 2) or four (TSP3 to -5) type II (epidermal growth factor-like) repeats, seven type III (Ca2+-binding) repeats, and a homologous carboxyl-terminal globular domain (for reviews, see Refs. 1Bornstein P. Sage E.H. Methods Enzymol. 1994; 245: 62-85Crossref PubMed Scopus (155) Google Scholar and 2Adams J. Tucker R.P. Lawler J. The Thrombospondin Gene Family. Springer-Verlag, Heidelberg1995Google Scholar). In addition to these common motifs, TSP1 and TSP2 contain a cysteine-rich α1(I) procollagen homology domain and three type I (TSP or properdin) repeats, separating the amino-terminal domain and the type II repeats. Unlike TSP1 and TSP2, which are trimers, TSP3 to TSP5/COMP (cartilage oligomeric matrix protein) are pentamers (3Malashkevich V.N. Kammerer R.A. Efimov V.P. Schulthess T. Engel J. Science. 1996; 274: 761-765Crossref PubMed Scopus (273) Google Scholar, 4Lawler J. McHenry K. Duquette M. Derick L. J. Biol. Chem. 1995; 270: 2809-2814Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 5Qabar A. Derick L. Lawler J. Dixit V. J. Biol. Chem. 1995; 270: 12725-12729Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Specific domains in TSP1 and TSP2 have been shown to interact with different components of the extracellular matrix, such as types I and V collagen, fibronectin, and laminin, and amino acid sequences within these domains have been implicated in binding to cell surface receptors, including several integrins, CD36, lipoprotein receptor-related protein, and integrin-associated protein (6Bornstein P. J. Cell Biol. 1995; 130: 503-506Crossref PubMed Scopus (587) Google Scholar, 7Gao A.G. Lindberg F.P. Finn M.B. Blystone S.D. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; 271: 21-24Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 8Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 9Lawler J. Hynes R.O. Blood. 1989; 74: 2022-2027Crossref PubMed Google Scholar). thrombospondin rat chondrosarcoma electrophoretic mobility shift assays base pair(s) kilobase pair(s) 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside. Each member of the TSP protein family is expressed in a different temporal and spatial pattern during murine development and in the adult organism (2Adams J. Tucker R.P. Lawler J. The Thrombospondin Gene Family. Springer-Verlag, Heidelberg1995Google Scholar, 10Iruela-Arispe M.L. Liska D. Sage E.H. Bornstein P. Dev. Dyn. 1993; 197: 40-56Crossref PubMed Scopus (188) Google Scholar, 11Lawler J. Duquette M. Whittaker C.A. Adams J.C. McHenry K. DeSimone D.W. J. Cell Biol. 1993; 120: 1059-1067Crossref PubMed Scopus (119) Google Scholar, 12Qabar A.N. Lin Z. Wolf F.W. O'Shea K.S. Lawler J. Dixit V.M. J. Biol. Chem. 1994; 269: 1262-1269Abstract Full Text PDF PubMed Google Scholar, 13DiCesare P. Hauser N. Lehman D. Pasumarti S. Paulsson M. FEBS Lett. 1994; 354: 237-240Crossref PubMed Scopus (220) Google Scholar). In the adult mouse, Thbs3 is expressed predominantly in the lung, in the central nervous system, in cartilage, and in the gastrointestinal tract, with lower levels of expression in other tissues (12Qabar A.N. Lin Z. Wolf F.W. O'Shea K.S. Lawler J. Dixit V.M. J. Biol. Chem. 1994; 269: 1262-1269Abstract Full Text PDF PubMed Google Scholar, 14Vos H.L. Devarayalu S. de Vries Y. Bornstein P. J. Biol. Chem. 1992; 267: 12192-12196Abstract Full Text PDF PubMed Google Scholar). The gene has been mapped to chromosome 3E3-F1 and is located downstream from and transcribed divergently fromMtx, so that 1352 nucleotides separate the translation start sites of the two genes (15Vos H.L. Mockensturm-Wilson M. Rood P.M.L. Maas A.M.C.E. Duhig T. Gendler S.J. Bornstein P. Mamm. Genome. 1995; 6: 820-822Crossref PubMed Scopus (13) Google Scholar, 16Collins M. Bornstein P. Nucleic Acids Res. 1996; 24: 3661-3669Crossref PubMed Scopus (12) Google Scholar). Unlike TSP3, metaxin is a ubiquitously expressed gene that encodes an outer mitochondrial membrane import protein and is essential for embryogenesis (17Bornstein P. McKinney C.E. LaMarca M.E. Winfield S. Shingu T. Devarayalu S. Vos H.L. Ginns E.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4547-4551Crossref PubMed Scopus (54) Google Scholar, 18Armstrong L.C. Komiya T. Bergman B.E. Mihara K. Bornstein P. J. Biol. Chem. 1997; 272: 6510-6518Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The divergently transcribed mouse Mtx and Thbs3 genes are also closely linked to the glucocerebrosidase (Gba) and episialin (Muc1) genes (15Vos H.L. Mockensturm-Wilson M. Rood P.M.L. Maas A.M.C.E. Duhig T. Gendler S.J. Bornstein P. Mamm. Genome. 1995; 6: 820-822Crossref PubMed Scopus (13) Google Scholar). The Muc1 gene is located 2.3 kb downstream from the Thbs3 gene and is transcribed in the same direction (15Vos H.L. Mockensturm-Wilson M. Rood P.M.L. Maas A.M.C.E. Duhig T. Gendler S.J. Bornstein P. Mamm. Genome. 1995; 6: 820-822Crossref PubMed Scopus (13) Google Scholar). The Mtx and Gba genes, on the other hand, are transcribed convergently so that their major polyadenylation sites are separated by only 431 nucleotides (17Bornstein P. McKinney C.E. LaMarca M.E. Winfield S. Shingu T. Devarayalu S. Vos H.L. Ginns E.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4547-4551Crossref PubMed Scopus (54) Google Scholar). The human GBA gene is of considerable clinical interest, since mutations in the gene are responsible for the lysosomal storage disorder, Gaucher disease (19Beutler E. Adv. Genet. 1995; 32: 17-49Crossref PubMed Scopus (39) Google Scholar). Although the Thbs3 and Mtx genes share a common promoter region, transient transfection experiments inThbs3- and Mtx-expressing rat chondrosarcoma (RCS) and NIH-3T3 cells demonstrated that the Mtx gene is regulated by elements within a short, unidirectional, proximal promoter region (16Collins M. Bornstein P. Nucleic Acids Res. 1996; 24: 3661-3669Crossref PubMed Scopus (12) Google Scholar). The ubiquitous transcriptional activator, Sp1, and the transcriptional repressor, Sp3, bind to clustered GC boxes within the proximal promoter, but their role in regulation of Mtx is uncertain (16Collins M. Bornstein P. Nucleic Acids Res. 1996; 24: 3661-3669Crossref PubMed Scopus (12) Google Scholar). In this study, we demonstrate by transient transfection experiments in RCS and NIH-3T3 cells that the Thbs3 gene is regulated in a cell type-specific manner by a far upstream orientation- and position-independent enhancer element located within intron 6 of the divergently transcribed Mtx gene. This upstream enhancer, when included in a 6-kb promoter sequence, was able to direct specific expression of the E. coli lacZ gene in transgenic mice. RCS cells (20Choi H.U. Meyer K. Swarm R. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 877-879Crossref PubMed Scopus (83) Google Scholar, 21Mukhopadhyay K. Lefebvre V. Zhou G. Garofalo S. Kimura J.H. de Crombrugghe B. J. Biol. Chem. 1995; 270: 27711-27719Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), a gift from Dr J. Kimura, and NIH-3T3 cells (ATCC CRL-1658) were cultured and transiently transfected using the calcium phosphate-DNA precipitation method, as described previously (16Collins M. Bornstein P. Nucleic Acids Res. 1996; 24: 3661-3669Crossref PubMed Scopus (12) Google Scholar). The cells were co-transfected with a β-galactosidase gene, driven by the SV40 promoter and enhancer (Promega), to control for variation in transfection efficiency. 5′-fragments and internal deletion fragments of the Thbs3 promoter (see Figs. 1, 3, and 5) were obtained from genomic clones containing the Mtx gene (22Bornstein P. Devarayalu S. Edelhoff S. Disteche C.M. Genomics. 1993; 15: 607-613Crossref PubMed Scopus (32) Google Scholar) or the 3′-end of the Gba gene (23Tybulewicz V.L.J. Tremblay M.L. LaMarca M.E. Willemsen R. Stubblefield B.K. Winfield S. Zablocka B. Westphal H. Mulligan R.C. Ginns E.I. Nature. 1992; 357: 407-410Crossref PubMed Scopus (243) Google Scholar) and were subcloned into the pGL2-Basic luciferase vector (Promega). The Thbs3 enhancer was also subcloned, in both orientations, into the previously described −83 and −377 bp Mtx promoter-luciferase reporter gene constructs, either upstream from the Mtx promoter fragment or downstream from the luciferase gene (16Collins M. Bornstein P. Nucleic Acids Res. 1996; 24: 3661-3669Crossref PubMed Scopus (12) Google Scholar). Cells lysates were prepared from transfected cells by the freeze-thaw method (24Rosenthal N. Methods Enzymol. 1987; 152: 704-720Crossref PubMed Scopus (403) Google Scholar), and the luciferase activity in extracts was measured using the Luciferase Assay System (Promega), as described by the manufacturer. The β-galactosidase activity in RCS and NIH-3T3 cell extracts was measured using the Galacto-Light Chemiluminescent Reporter Assay (Tropix), or withO-nitrophenyl-β-d-galactopyranoside as a substrate (24Rosenthal N. Methods Enzymol. 1987; 152: 704-720Crossref PubMed Scopus (403) Google Scholar).Figure 3Analysis of the Thbs3 far upstream enhancer element. The upper part of the figure shows a schematic diagram of the mouseThbs3 promoter with the terminal exons of the Gbagene, the Mtx gene, the Thbs3-Mtxintergenic region, and exon A of the Thbs3 gene. Translated and untranslated sequences are represented as filled andopen boxes, respectively. Transcription start sites and the direction of transcription are indicated byarrows. Thbs3 promoter-LUC constructs are represented in relation to the above genetic map. Large deletions in these constructs are indicated with dotted lines, and small deletions within the SacI site are indicated with ×. A CelII (C) site, situated immediately upstream from the Thbs3 translation start site, served as the 3′-boundary of the Thbs3 promoter. The percentage of luciferase activity is expressed relative to the activity of the 6.0-kb construct, and S.D. values for each promoter construct and the number of determinations (n) are indicated. A,ApaI; S, SmaI; P,PstI; St, StyI; Sa,SacI; X, Xmn I; Sn,SnaBI.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Mutational analysis of cis-acting elements within the Thbs3 far upstream enhancer. Top, schematic diagram of the mouse Thbs3promoter showing the terminal three exons of the Mtx gene and exon A of the Thbs3 gene. The translated sequences and untranslated sequences are represented as filled andopen boxes, respectively. The indicated restriction enzymes were used to clone two internal deletion mutations (shown below). A CelII (C) site situated immediately upstream from the Thbs3 translation start site served as the 3′-boundary of the Thbs3 promoter fragment. A, ApaI; N, Nar I; Sa, SacI; St, StyI. Middle, the indicated Thbs3 promoter-luciferase constructs were transfected into RCS cells. The percentage of luciferase activity, expressed relative to the 6.0-kb construct, S.D. values for each promoter construct, and the number of determinations (n) are indicated. Bottom, nucleotide sequence of the StyI–Nar I fragment from theThbs3 enhancer. The bases deleted within the two internal deletion constructs are in lowercase type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 10–20 μg of RCS or NIH-3T3 cell RNA was fractionated on 1.2% agarose gels, transferred to Zetabind® Transfer Membranes (CUNO), hybridized with cDNA probes for Thbs3 or β-actin, and washed and stripped according to standard procedures. The Thbs3 probe was a 1-kb XhoI fragment from the 5′-region of the cDNA, while a 1.1-kb mouse β-actin DECA probe, spanning nucleotides 762–1837 (Ambion), was used to control for loading and transfer of RNA. Double-stranded Thbs3promoter-luciferase reporter gene plasmid DNA was sequenced using the dideoxy chain-terminating method and the Dye Terminator Cycle Sequencing Ready Reaction DNA Sequencing Kit with AmpliTaq® FS DNA polymerase (Perkin-Elmer). The luciferase vector primer, GL primer 1 (Promega) or oligonucleotides corresponding to known sequences of the Mtx gene (17Bornstein P. McKinney C.E. LaMarca M.E. Winfield S. Shingu T. Devarayalu S. Vos H.L. Ginns E.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4547-4551Crossref PubMed Scopus (54) Google Scholar) were synthesized by IDT® and used as sequencing primers. The samples were analyzed with an Applied Biosystems PRISM™ 377 DNA sequencer, and the resulting sequences were analyzed with the Genetics Computer Group (GCG) or GENEPRO (Riverside Scientific, Seattle) programs. Nuclear proteins were isolated from RCS and NIH-3T3 cells using the method of Lee and Green (25Lee K.A.W. Green M.R. Methods Enzymol. 1990; 181: 20-30Crossref PubMed Scopus (63) Google Scholar), and their concentrations were determined by the Bradford method (26Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (218585) Google Scholar), with chymotrypsin as a standard. Various DNA fragments of theThbs3 enhancer (see Fig. 4) were radiolabeled with the Klenow fragment of DNA polymerase or T4 DNA kinase and were used as probes in electrophoretic mobility shift (EMSA), competition, and supershift assays, as described previously (16Collins M. Bornstein P. Nucleic Acids Res. 1996; 24: 3661-3669Crossref PubMed Scopus (12) Google Scholar). The 6.0- and 2.0-kbThbs3 promoter-β-galactosidase reporter gene constructs were prepared by cloning the ApaI–CelII andHindIII–CelII Thbs3 promoter fragments, respectively, upstream from the translation start site of the placF vector (see Fig. 1 B; Ref. 27Mercer E.H. Hoyle G.W. Kapur R.P. Brinster R.L. Palmiter R.D. Neuron. 1991; 7: 703-716Abstract Full Text PDF PubMed Scopus (235) Google Scholar). The 6.0- and 2.0-kbThbs3-β-gal inserts were released from the plasmid vector sequences, gel-purified with a Prep-A-Gene® DNA Purification Matrix Kit (Bio-Rad), resuspended in 10 mmTris (pH 7.6) and 0.25 mm EDTA, and clarified through Ultrafree®-MC 0.45-μm filter units (Millipore Corp.). The purified DNA was microinjected into fertilized C57BL/6 × C3H F1 mouse oocytes, and the oocytes were transplanted into the oviducts of pseudopregnant female mice (Jackson Laboratories) using standard procedures (28Hogan B. Constantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). Founder mice were identified by slot blot analysis of tail DNA. Briefly, DNA was prepared from tail biopsies (29Laird P.W. Zijderveld A. Linders K. Rudnicki M.A. Jaenisch R. Berns A. Nucleic Acids Res. 1991; 19: 4293Crossref PubMed Scopus (1304) Google Scholar), and 5 μg was transferred to Zetabind® Transfer Membranes (CUNO). After UV cross-linking and prehybridization, the membranes were hybridized with a β-galactosidase gene probe (825-bpNcoI–ClaI fragment of placF), washed, and exposed to x-ray film. Transgenic lines were established by breeding positive founders with negative siblings. The transgene copy number in each transgenic line was determined by probing a Southern blot ofNcoI-digested DNA with a radioactively labeled 310-bpEco47III–CelII DNA fragment of theThbs3 proximal promoter (see Fig. 2). Tissues were stained for β-galactosidase activity as described previously, with some modifications (27Mercer E.H. Hoyle G.W. Kapur R.P. Brinster R.L. Palmiter R.D. Neuron. 1991; 7: 703-716Abstract Full Text PDF PubMed Scopus (235) Google Scholar). Briefly, tissues were fixed at 4 °C for 30 to 45 min in fresh ice-cold 2% formaldehyde, 0.2% glutaraldehyde, 2 mm MgCl2, 5 mmEGTA, 0.1 mm NaH2PO4 (pH 7.3). The fixed tissues were then rinsed three times for 30 min each in 0.1 mm NaH2PO4 (pH 7.3), 2 mm MgCl2, 0.2% Nonidet P-40, 0.1% sodium deoxycholate and stained in the rinse buffer containing 1 mg/ml X-Gal, 5 mm K3Fe(CN)6, and 5 mm K4Fe(CN)6, in the dark at room temperature for 48 h. The stained tissues were rinsed three times in phosphate-buffered saline and stored in 70% ethanol. Although the mouseThbs3 and Mtx genes share a common promoter region, we previously showed that Mtx is regulated by a short, unidirectional, proximal promoter region containing clustered Sp1-binding motifs (16Collins M. Bornstein P. Nucleic Acids Res. 1996; 24: 3661-3669Crossref PubMed Scopus (12) Google Scholar). The Mtx-Thbs3 intergenic region, on the other hand, was virtually inactive when assayed in theThbs3 direction in Thbs3-expressing RCS cells. Northern blot analysis showed that the endogenous Thbs3 gene was expressed at relatively high levels in these cells, while the gene was expressed at much lower levels in NIH-3T3 cells (Fig.1 A). These findings suggest that additional element(s), not contained in the intergenic region, contribute to the expression of the Thbs3 gene. Some eukaryotic genes encoding extracellular matrix components contain enhancers within their first introns. A 2.2-kb BamHI fragment, extending from the 5′-region of intron A into exon D of theThbs3 gene (22Bornstein P. Devarayalu S. Edelhoff S. Disteche C.M. Genomics. 1993; 15: 607-613Crossref PubMed Scopus (32) Google Scholar), was therefore cloned upstream from the −2034 bp Thbs3 promoter-luciferase gene in the negative orientation and assayed in both RCS and NIH-3T3 cells. There was no significant difference in luciferase activity when the activity of this construct was compared with that of the promoter construct lacking the intragenic segment (data not shown), suggesting that sequences within the 5′ portion of the Thbs3 gene do not contribute to its expression in these cells. Similar results were obtained when intron A of the human THBS3 gene was assayed for enhancer activity inTHBS3-expressing small cell carcinoma cells. 2J. Silver and P. Bornstein, unpublished observations. To test whether far upstream sequences might contribute to the expression of the mouse Thbs3 gene, a 6.0-kbThbs3 promoter and several of its 5′-deletion fragments were cloned upstream from the luciferase reporter gene, and each was co-transfected with the β-galactosidase reporter gene into RCS and NIH-3T3 cells. The 6.0-kb Thbs3 promoter includes the 1.4-kb intergenic region and most of the Mtx gene. As shown in Fig.1 B, the 6.0-kb Thbs3 promoter was 10 times more active than the 2.0-kb promoter in RCS cells, but the two constructs were equally active in NIH 3T3 cells. Estimates of the relative expression of −6.0 Thbs-LUC in RCS and NIH-3T3 cells also indicated a 10-fold higher expression in RCS cells (data not shown), a finding consistent with the relative expression of the endogenousThbs3 gene in the two cell lines (Fig. 1 A). There was a 5-fold decrease in luciferase activity in RCS cells when the activity of the 5.4-kb (SacI) Thbs3 promoter construct was compared with that of the 5.5-kb (PstI) construct. In contrast, similar luciferase activities were obtained for all 5′ deletions of the 6.0-kb construct, up to an RsaI site at −1346 bp, when these constructs were transfected into NIH-3T3 cells (Fig. 1 B and Table I). A gradual decrease in luciferase activity was noted when additional 5′-deletion constructs of the −1346 bp Thbs3 promoter were assayed in the latter cells (Table I; Ref. 16Collins M. Bornstein P. Nucleic Acids Res. 1996; 24: 3661-3669Crossref PubMed Scopus (12) Google Scholar). Further analysis of the 6.0-kb Thbs3 promoter demonstrated that additional factors bound within other regions of the promoter, but apparently they play only a minor role in the constitutive regulation of the gene. These findings suggest that a cis-acting element(s), located between −5.4 and −5.5 kb in the 3′-region of intron 6 of theMtx gene, together with its associated transcription factor(s), serves as a cell-specific enhancer of Thbs3 in RCS cells and that its activity correlates with the cellular expression of the endogenous Thbs3 gene (Fig. 1 B). Additional evidence that the Thbs3 enhancer functions in a cell-specific manner was provided by the finding that the −5.7 (−5.1/0.5)Thbs3-LUC construct containing theThbs3 enhancer (see Fig. 3) was unable to activate theThbs3 basal promoter when transfected into NIH-3T3 cells (data not shown).Table IDeletion analysis of the −2 kb Thbs3 promoterConstructLuciferase activityaAdjusted to a value of 100% for the 6-kbThbs3 promoter (see Fig. 1 B). The number of determinations (n) is shown in parentheses.RCS cellsNIH-3T3 cells%−2034Thbs3-LUC9.9 ± 1.7 (7)94.9 ± 18.2 (6)−1346Thbs3-LUC13.1 ± 2.6 (4)bThese data are from Collins and Bornstein (16) and are also adjusted to a value of 100% for the 6-kb promoter.118.7 ± 23.0 (6)bThese data are from Collins and Bornstein (16) and are also adjusted to a value of 100% for the 6-kb promoter.−1150 Thbs3-LUC8.7 ± 1.1 (4)bThese data are from Collins and Bornstein (16) and are also adjusted to a value of 100% for the 6-kb promoter.69.7 ± 9.6 (4)bThese data are from Collins and Bornstein (16) and are also adjusted to a value of 100% for the 6-kb promoter.−976Thbs3-LUC8.3 ± 0.7 (4)bThese data are from Collins and Bornstein (16) and are also adjusted to a value of 100% for the 6-kb promoter.57.2 ± 16.4 (6)bThese data are from Collins and Bornstein (16) and are also adjusted to a value of 100% for the 6-kb promoter.−463 Thbs3-LUC5.6 ± 1.5 (12)24.9 ± 6.7 (5)−311Thbs3-LUC7.1 ± 1.3 (3)bThese data are from Collins and Bornstein (16) and are also adjusted to a value of 100% for the 6-kb promoter.27.7 ± 6.8 (3)bThese data are from Collins and Bornstein (16) and are also adjusted to a value of 100% for the 6-kb promoter.LUC1.8 ± 1.3 (6)bThese data are from Collins and Bornstein (16) and are also adjusted to a value of 100% for the 6-kb promoter.5.2 ± 1.6 (12)bThese data are from Collins and Bornstein (16) and are also adjusted to a value of 100% for the 6-kb promoter.a Adjusted to a value of 100% for the 6-kbThbs3 promoter (see Fig. 1 B). The number of determinations (n) is shown in parentheses.b These data are from Collins and Bornstein (16Collins M. Bornstein P. Nucleic Acids Res. 1996; 24: 3661-3669Crossref PubMed Scopus (12) Google Scholar) and are also adjusted to a value of 100% for the 6-kb promoter. Open table in a new tab The major polyadenylation sites of the convergently transcribedMtx and glucocerebrosidase (Gba) genes are only 431 bp apart (17Bornstein P. McKinney C.E. LaMarca M.E. Winfield S. Shingu T. Devarayalu S. Vos H.L. Ginns E.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4547-4551Crossref PubMed Scopus (54) Google Scholar). Because of the close proximity of theThbs3 enhancer to the Gba gene, it was possible that elements within the body of the Gba gene could also participate in regulation of the Thbs3 gene. To test this hypothesis, a KpnI site within exon 7 of the Gbagene was used to clone a 9.0-kb Thbs3-promoter luciferase construct (Fig. 1 C). As shown in Fig. 1 C, there was a 35% decrease in luciferase activity when the 9-kb promoter was assayed in RCS cells and compared with the −6.0 kb promoter construct. This finding indicates that no net positive regulatory elements for theThbs3 gene are located within the 3′ two-thirds of theGba gene. All of the elements responsible for Thbs3 gene expression in NIH-3T3 fibroblasts were located within theMtx-Thbs3 intergenic region (Table I; Ref. 16Collins M. Bornstein P. Nucleic Acids Res. 1996; 24: 3661-3669Crossref PubMed Scopus (12) Google Scholar). Three positive regulatory regions, each of which contributed equally to the luciferase activity in the fibroblasts, were identified. The first region contains the TATA-less 311-bp proximal Thbs3promoter. Since a unique transcription start site for theThbs3 gene has not been determined and several sites may exist, the promoter is numbered from the translation start site. Two INR sequences, 5′-YYANWYY-3′, were identified upstream from the translation start site, generating 5′-untranslated regions of 57 and 96 nucleotides, respectively (Fig. 2B; 30Javahery R. Khachi A. Lo K. Zenzie-Gregory B. Smale S.T. Mol. Cell. Biol. 1994; 14: 116-127Crossref PubMed Scopus (598) Google Scholar). The lengths of both potential 5′-untranslated regions are within the average length of between 20 and 100 nucleotides for most eukaryotic mRNAs (31Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4181) Google Scholar). As shown in Fig. 2 B, the 311-bp promoter also contains an inverted CCAAT box and two inverted GC boxes. A 513-bp sequence between nucleotides −976 and −463 formed the second region, while the third region, extending from −1346 to −1150, contained the 196-bp GC-rich Mtx minimal promoter. These data confirm our initial findings that the Mtx minimal promoter, which is located upstream from −1150, does not play a major role in the regulation of the Thbs3 gene. To test whether the enhancer element located in intron 6 of the metaxin gene could function in a position-independent manner, a 344-bp SmaI–SacI fragment, which was expected to contain the enhancer based on the experiments described in Fig. 1 B, was cloned upstream from the SnaBI site at −463 bp to produce the −5.7 (−5.4/−0.5)Thbs3-promoter luciferase construct. To our surprise, only basal luciferase activity was detected when this construct was assayed in RCS cells (Fig.3). There are at least three possible explanations for this finding: 1) the 463-bp basal Thbs3promoter is incomplete; 2) the Thbs3 enhancer functions in a position-dependent manner so that part or all of the intervening sequences are required, or specific sequences in the intervening region function coordinately with the enhancer; or 3) theSacI site and its flanking sequences form part of the enhancer element. To test the third possibility, a 4-bp internal deletion was generated within the SacI site of the −6.0 kbThbs3 promoter by SacI cleavage, blunt-ending with DNA polymerase I, and ligation. The deletion was confirmed by DNA sequencing. There was a 75% decrease in luciferase activity when the 4-bp deletion construct was assayed (Fig. 3; −6.0 (ΔSacI, 4bp)). The activity of the 4-bp deletion construct, 23.9 ± 1.4%, was similar to that of the −5.4 kb (SacI) 5′ deletion construct, 17.0 ± 4.7% (Fig. 1 B). To test whether the sequences flanking the SacI site could activate the Thbs3 promoter, several fragments c
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