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• W2056315093 abstract "An evolutionarily conserved vertebrate homologue of the Drosophila NK-3 homeodomain genebagpipe, Nkx3-1, is expressed in vascular and visceral mesoderm-derived muscle tissues and may influence smooth muscle cell differentiation. Nkx3-1 was evaluated for mediating smooth muscle γ-actin (SMGA) gene activity, a specific marker of smooth muscle differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts was unable to elicit SMGA promoter activity but required the coexpression of serum response factor (SRF) to activate robust SMGA transcription. A novel complex element containing a juxtaposed Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal promoter region was found to be necessary for the Nkx3-1/SRF coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF associate through protein-protein interactions and the homeodomain region of Nkx3-1 facilitated SRF binding to the complex NKE·SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain within its C-terminal segment. In addition, mNkx3-1/SRF cooperative activity required an intact Nkx3-1 homeodomain along with the MADS box of SRF, which contains DNA binding and dimerization structural domains, and the contiguous C-terminal SRF activation domain. Thus, SMGA is a novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA promoter is dependent upon SRF. An evolutionarily conserved vertebrate homologue of the Drosophila NK-3 homeodomain genebagpipe, Nkx3-1, is expressed in vascular and visceral mesoderm-derived muscle tissues and may influence smooth muscle cell differentiation. Nkx3-1 was evaluated for mediating smooth muscle γ-actin (SMGA) gene activity, a specific marker of smooth muscle differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts was unable to elicit SMGA promoter activity but required the coexpression of serum response factor (SRF) to activate robust SMGA transcription. A novel complex element containing a juxtaposed Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal promoter region was found to be necessary for the Nkx3-1/SRF coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF associate through protein-protein interactions and the homeodomain region of Nkx3-1 facilitated SRF binding to the complex NKE·SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain within its C-terminal segment. In addition, mNkx3-1/SRF cooperative activity required an intact Nkx3-1 homeodomain along with the MADS box of SRF, which contains DNA binding and dimerization structural domains, and the contiguous C-terminal SRF activation domain. Thus, SMGA is a novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA promoter is dependent upon SRF. smooth muscle γ-actin serum response factor SRF-binding element base pair polymerase chain reaction glutathioneS-transferase phosphate-buffered saline polyacrylamide gel electrophoresis hemagglutinin wild type cytomegalovirus Homeobox proteins are a class of developmentally regulated transcription factors that are important for embryonic patterning and differentiation (reviewed in Ref. 1Gehring W.J. Affolta M. Burglin T. Annu. Rev. Biochem. 1994; 63: 487-536Crossref PubMed Scopus (860) Google Scholar). Although first identified inDrosophila, these regulators have been found in all metazoan species examined to date from fungi to humans. They constitute a family of transcription factors that are recognized by a conserved segment of 60 amino acids, referred to as the homeodomain, that usually recognizes a TTAATT-degenerate DNA consensus binding sequence found within the control elements of target genes (1Gehring W.J. Affolta M. Burglin T. Annu. Rev. Biochem. 1994; 63: 487-536Crossref PubMed Scopus (860) Google Scholar, 2Khorasanizadeh S. Rastinejad F. Curr. Biol. 1999; 9: R456-R458Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 3Mannervik M. BioEssays. 1999; 21: 267-270Crossref PubMed Scopus (33) Google Scholar). Drosophila and vertebrate hox genes are clustered on the genome, exhibit a high degree of structural conservation across species, and are expressed in a temporal and spatial sequence that is also conserved (1Gehring W.J. Affolta M. Burglin T. Annu. Rev. Biochem. 1994; 63: 487-536Crossref PubMed Scopus (860) Google Scholar). Genome and expression analyses have now firmly established that there are a variety of homeodomain-containing proteins in vertebrates including a class of unique genes that are dispersed throughout the genome (1Gehring W.J. Affolta M. Burglin T. Annu. Rev. Biochem. 1994; 63: 487-536Crossref PubMed Scopus (860) Google Scholar, 4Doevendans P.A. Van Bilsen M. Int. J. Biochem. Cell Biol. 1996; 28: 387-403Crossref PubMed Scopus (45) Google Scholar, 5Holland P.W.H. Garcia-Fernande J. Dev. Biol. 1996; 173: 382-395Crossref PubMed Scopus (361) Google Scholar). Among the dispersed class of homeodomain proteins is the NK family, which was first defined by four genes (nk-1 through nk-4) identified inDrosophila. This family exhibits specific homology within the homeodomain and shares other regions of conserved sequence outside the homeodomain (4Doevendans P.A. Van Bilsen M. Int. J. Biochem. Cell Biol. 1996; 28: 387-403Crossref PubMed Scopus (45) Google Scholar, 5Holland P.W.H. Garcia-Fernande J. Dev. Biol. 1996; 173: 382-395Crossref PubMed Scopus (361) Google Scholar, 6Harvey R.P. Dev. Biol. 1996; 178: 203-216Crossref PubMed Scopus (496) Google Scholar, 7Kim Y. Nirenberg M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7716-7720Crossref PubMed Scopus (250) Google Scholar). In Drosophila, two nkgenes, tinman and bagpipe, have been found to be closely linked and are expressed and participate in the specialization of mesoderm-derived heart and visceral organs (8Azpiazu N. Frasch M. Genes Dev. 1993; 7: 1325-1340Crossref PubMed Scopus (635) Google Scholar, 9Bodmer R. Development. 1993; 118: 719-729Crossref PubMed Google Scholar). Mutations intinman block dorsal vessel (Drosophila heart equivalent) and visceral musculature formation; however,bagpipe mutations lead to only midgut visceral musculature abnormalities. Thus, bagpipe may be a downstream target oftinman (8Azpiazu N. Frasch M. Genes Dev. 1993; 7: 1325-1340Crossref PubMed Scopus (635) Google Scholar). nkx3-1 and nkx3-2, two murine homologues of the bagpipe gene, have recently been identified (10Kos L. Chiang C. Mahon K.A. Mech. Dev. 1998; 70: 25-34Crossref PubMed Scopus (43) Google Scholar, 11Sciavolino P.J. Abrams E.W. Yang L. Austenberg L.B. Shen M.M. Abate-Shen C. Dev. Dyn. 1997; 209: 127-138Crossref PubMed Scopus (145) Google Scholar, 12Tribioli C. Frasch M. Lufkin T. Mech. Dev. 1997; 65: 145-162Crossref PubMed Scopus (92) Google Scholar, 13Yoshiura K.I. Murry J.C. Genomics. 1997; 45: 425-428Crossref PubMed Scopus (14) Google Scholar). These genes demonstrate overlapping expression patterns in somites of early embryos (∼8.5–9 embryonic days); however, only thenkx3-2 gene was expressed in the lateral and splanchnic mesoderm (10Kos L. Chiang C. Mahon K.A. Mech. Dev. 1998; 70: 25-34Crossref PubMed Scopus (43) Google Scholar, 12Tribioli C. Frasch M. Lufkin T. Mech. Dev. 1997; 65: 145-162Crossref PubMed Scopus (92) Google Scholar). In later stage embryos and in adults nkx3genes are differentially expressed. Nkx3-1 is predominantly expressed in brain, kidney, blood vessels, and the male reproductive system (10Kos L. Chiang C. Mahon K.A. Mech. Dev. 1998; 70: 25-34Crossref PubMed Scopus (43) Google Scholar, 11Sciavolino P.J. Abrams E.W. Yang L. Austenberg L.B. Shen M.M. Abate-Shen C. Dev. Dyn. 1997; 209: 127-138Crossref PubMed Scopus (145) Google Scholar, 14Tanaka M. Kashara H. Bartunkova S. Schinke M. Komuro I. Inagaki H. Lee Y. Lyons G.E. Izumo S. Dev. Genet. 1998; 22: 239-249Crossref PubMed Scopus (70) Google Scholar), whereas Nkx3-2 is found in the lateral plate mesoderm surrounding the mid- and hindgut and within cartilagenous condensations (12Tribioli C. Frasch M. Lufkin T. Mech. Dev. 1997; 65: 145-162Crossref PubMed Scopus (92) Google Scholar, 13Yoshiura K.I. Murry J.C. Genomics. 1997; 45: 425-428Crossref PubMed Scopus (14) Google Scholar). In adult tissues, nkx3 genes retain expression within the mesoderm-derived structures surrounding vascular (Nkx3-1 in the blood vessels) and visceral (Nkx3-2 in the mid- and hindgut mesoderm) organs and may influence smooth muscle cell differentiation. At present, potential target genes regulated by the Nkx3 family have yet to be identified. Smooth muscle cells are integral cellular components of most organs through their role in controlling vascular tone, gastrointestinal motility, fluids movement, and airway resistance. Differentiated smooth muscle cells are characterized by their expression of a unique subset of contractile protein isoforms including smooth muscle α-actin (15Carroll S.L. Bergsma D.J. Schwartz R.J. J. Biol. Chem. 1986; 261: 8965-8976Abstract Full Text PDF PubMed Google Scholar, 16Gabbiani G. Kocher O. Bloom W.S. Vandekerchove J. Weber K. J. Clin. Invest. 1994; 73: 148-152Crossref Scopus (264) Google Scholar, 17McHugh K.M. Dev. Dyn. 1995; 204: 278-290Crossref PubMed Scopus (123) Google Scholar, 18McHugh K.M. Crawford K. Lessard J.L. Dev. Biol. 1991; 148: 442-458Crossref PubMed Scopus (141) Google Scholar), smooth muscle γ-actin (17McHugh K.M. Dev. Dyn. 1995; 204: 278-290Crossref PubMed Scopus (123) Google Scholar, 18McHugh K.M. Crawford K. Lessard J.L. Dev. Biol. 1991; 148: 442-458Crossref PubMed Scopus (141) Google Scholar, 19Miwa T. Manabe Y. Kurokawa K. Kamada S. Kandu N. Burns G. Ueyama H. Kakunaga T. Mol. Cell. Biol. 1991; 11: 3296-3306Crossref PubMed Google Scholar, 20Kovacs A.M. Zimmer W.E. Cell Motil. Cytoskeleton. 1993; 24: 67-81Crossref PubMed Scopus (13) Google Scholar, 21Kovacs A.M. Zimmer W.E. Gene Expr. 1998; 7: 115-129PubMed Google Scholar), smooth muscle myosin heavy and light chains (22Babij P. Periasamy M. J. Mol. Biol. 1989; 210: 673-679Crossref PubMed Scopus (154) Google Scholar, 23Kallmeier R.C. Somasundaram C. Babij P. J. Biol. 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Biol. 1992; 12: 2359-2371Crossref PubMed Scopus (48) Google Scholar, 30Gallagher P.J. Herring B.P. J. Biol. Chem. 1991; 266: 23945-23952Abstract Full Text PDF PubMed Google Scholar, 31Herring B.P. Smith A.F. Am. J. Physiol. 1996; 270: C1656-C1665Crossref PubMed Google Scholar). Furthermore, these cells possess the capability to express appropriate levels of these characteristic smooth muscle proteins even though they are derived from diverse embryonic origins (32Topouzis S. Majesky M.W. Dev. Biol. 1996; 178: 430-445Crossref Scopus (252) Google Scholar). In contrast to skeletal and cardiac muscle cells, smooth muscle cells retain their capacity to modulate reversibly their phenotype during postnatal development (33Mosse P.R. Campbell G.R. Campbell J.H. Arteriosclerosis. 1986; 6: 664-669Crossref PubMed Google Scholar, 34Owens G. Physiol. Rev. 1995; 75: 487-517Crossref PubMed Scopus (1404) Google Scholar). This phenotypic modulation includes a reentry into the cell cycle and an altered expression of the characteristic proteins of the differentiated cell, effects that have been implicated in the pathogenesis of certain cardiovascular (35Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9999) Google Scholar) and gastrointestinal (36MacDermott R.P. Inflammatory Bowel Disease: Current Status and Future Approaches. Elsevier Scientific Publishing Co., Inc., New York1988: 21-42Google Scholar) disease states. Thus, a knowledge of the molecular mechanisms that control smooth muscle development and cell-specific gene expression will provide insights into cellular differentiation and the physiological responses of these cells to injury and disease states. We have examined the expression of the smooth muscle γ-actin (SMGA)1 gene as a model to understand the molecular mechanisms that regulate genes during smooth muscle differentiation. In aves (20Kovacs A.M. Zimmer W.E. Cell Motil. Cytoskeleton. 1993; 24: 67-81Crossref PubMed Scopus (13) Google Scholar, 21Kovacs A.M. Zimmer W.E. Gene Expr. 1998; 7: 115-129PubMed Google Scholar, 37Zimmer W.E. Browning C.L. Kovacs A.M. Dev. Biol. 1996; 175: 399Google Scholar) as in mammals (17McHugh K.M. Dev. Dyn. 1995; 204: 278-290Crossref PubMed Scopus (123) Google Scholar, 18McHugh K.M. Crawford K. Lessard J.L. Dev. Biol. 1991; 148: 442-458Crossref PubMed Scopus (141) Google Scholar, 19Miwa T. Manabe Y. Kurokawa K. Kamada S. Kandu N. Burns G. Ueyama H. Kakunaga T. Mol. Cell. Biol. 1991; 11: 3296-3306Crossref PubMed Google Scholar), SMGA expression is restricted to smooth muscle tissues with the exception of the post-meiotic spermatocyte (38Kim E. Walters S.H. Hale L.E. Hect N.B. Mol. Cell. Biol. 1989; 9: 1875-1881Crossref PubMed Scopus (60) Google Scholar); thus SMGA provides an excellent marker for the smooth muscle phenotype. In addition, tissue-restricted expression of SMGA arises during early embryonic development of the vasculature and gastrointestinal tract (17McHugh K.M. Dev. Dyn. 1995; 204: 278-290Crossref PubMed Scopus (123) Google Scholar, 18McHugh K.M. Crawford K. Lessard J.L. Dev. Biol. 1991; 148: 442-458Crossref PubMed Scopus (141) Google Scholar, 20Kovacs A.M. Zimmer W.E. Cell Motil. Cytoskeleton. 1993; 24: 67-81Crossref PubMed Scopus (13) Google Scholar, 21Kovacs A.M. Zimmer W.E. Gene Expr. 1998; 7: 115-129PubMed Google Scholar, 39Landerholm T.E. Dong X.R. Lu J. Belaguli N.S. Schwartz R.J. Majesky M.W. Development. 1999; 126: 2053-2062Crossref PubMed Google Scholar), suggesting that the activation of SMGA transcription may require factors unique to the differentiating smooth muscle cell. Transcriptional regulation of the SMGA gene appears to require the interplay of positive- and negative-acting cis elements within the promoter. Two regions displaying positive acting transcriptional activity were mapped on the SMGA promoter, referred to as the specifier and modulator domains (21Kovacs A.M. Zimmer W.E. Gene Expr. 1998; 7: 115-129PubMed Google Scholar). A key ciselement for smooth muscle-specific SMGA transcription found in both of these domains is the CArG/SRE (CC(A/T)6GG) motif. The positive acting transcriptional activity of the specifier and modulator domains is derived from the binding of SRF to the SRE sites within these domains, and we have demonstrated that SRF-containing complexes play a prominent role in the developmental activation of the SMGA gene (40Browning C.L. Culberson D.E. Aragon I.V. Fillmore R.A. Croissant J.D. Schwartz R.J. Zimmer W.E. Dev. Biol. 1998; 194: 18-37Crossref PubMed Scopus (76) Google Scholar). Based upon our studies and the many examples from SRF-dependent regulation of cardiac (6Harvey R.P. Dev. Biol. 1996; 178: 203-216Crossref PubMed Scopus (496) Google Scholar, 14Tanaka M. Kashara H. Bartunkova S. Schinke M. Komuro I. Inagaki H. Lee Y. Lyons G.E. Izumo S. Dev. Genet. 1998; 22: 239-249Crossref PubMed Scopus (70) Google Scholar, 41Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 42Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar, 43Chen C.Y. Croissant J.D. Majesky M. Topouzis S. McQuinn T. Frankovsky M.J. Schwartz R.J. Dev. Genet. 1996; 19: 119-130Crossref PubMed Scopus (110) Google Scholar) and skeletal (44Belaguli N. Schildmeyer L.A. Schwartz R.J. J. Biol. Chem. 1997; 272: 18222-18231Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 45Boxer L.M. Prywes R. Roeder R.G. Kedes L. Mol. Cell. Biol. 1989; 9: 515-527Crossref PubMed Scopus (117) Google Scholar, 46Croissant J.D. Kim J.H. Eichele G. Goering L. Lough J. Prywes R. Schwartz R.J. Dev. Biol. 1996; 177: 250-264Crossref PubMed Scopus (157) Google Scholar, 47Groisman R. Masutani H. Libovitch M. Robin P. Soudant I. Trouche D. Harel-Bellan A. J. Biol. Chem. 1996; 271: 5258-5264Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 48Grueneberg D.A. Natesan S. Alexandre C. Gilman M.Z. Science. 1992; 257: 1089-1095Crossref PubMed Scopus (256) Google Scholar) muscle genes, we predicted that SRF requires association with other factors to regulate the SMGA gene. Here we demonstrate the cooperative interaction of Nkx3-1 with SRF-activating SMGA transcription. The conservation of structure and activity of actin gene transcriptional machinery permits examining these components utilizing a heterologous cotransfection assay. Transcriptional synergy is supported by a complex of regulatory elements that is composed of immediately adjacent NKE-SREcis elements within the SMGA proximal promoter. Our studies demonstrate that the SMGA gene promoter serves as a target for the NK3 family of transcription factors, specifically the Nkx3-1 factor, and supports the hypothesis that appropriate transcriptional regulation of smooth muscle specific genes requires the combinatorial interactions of SRF with coaccessory trans-acting factors expressed within the smooth muscle cell. Approximately 2300 bp of the avian smooth muscle γ-actin promoter and 5′ deletions have been cloned into pGL-3 basic plasmid driving the expression of the reporter gene luciferase (21Kovacs A.M. Zimmer W.E. Gene Expr. 1998; 7: 115-129PubMed Google Scholar, 37Zimmer W.E. Browning C.L. Kovacs A.M. Dev. Biol. 1996; 175: 399Google Scholar, 40Browning C.L. Culberson D.E. Aragon I.V. Fillmore R.A. Croissant J.D. Schwartz R.J. Zimmer W.E. Dev. Biol. 1998; 194: 18-37Crossref PubMed Scopus (76) Google Scholar). The −1224, −176, and −108 5′ deletions of the smooth muscle γ-actin promoter were used in transfection experiments in the current study. Mutations were made to γ-actin promoter elements SRE1, SRE2, and NKE1 using multiple primer polymerase chain reaction (PCR) and verified by DNA sequencing. The SMGA SRE1 was disrupted by changing the wild type element from 5′-CCTATTTAGG-3′ to 5′-CCTATCCCGG-3′ and the SRE2 sequence (5′-CCTATATGG-3′) mutated to 5′-CCTTATGTTT-3′. The SMGA NKE1 was disrupted by changing the wild type element from 5′-CACTTAGCCT-3′ to 5′-CACCCCCCCT-3′). The NKE1/SRE1 double mutant contained the mutations to both sites. PCR was used to isolate the 862-bp Nkx3-1 cDNA from 6-day embryoid bodies with forward primer 5′-GCTCTAGAATGCTTAGGGTAGCGGAGCC-3′ and reverse primer 5′-TTGGATCCAGAGACCCCCAGGGAAGACAG. The isolated cDNA was cloned into pCRII (Invitrogen, TA Cloning Kit) and verified by sequencing. The Nkx3-1 fragment was excised from pCRII withXbaI and BamHI and cloned into identical sites of the pCGN vector downstream of the cytomegalovirus promoter and HA epitope tag. Mutations of the Nkx3-1 sequence were made using PCR-based techniques (Stratagene, Excite PCR Mutagenesis Kit). The pCGN-Nkx3-1 cDNA clone was the template for these experiments, and all mutants were cloned into the same vector maintaining an HA epitope tag for each of the mutant proteins. All mutants were confirmed by DNA sequencing and Western analyses of lysates derived from transfected cells. The −330-bp cardiac α-actin promoter cloned in front of a luciferase reporter gene has been previously described. The construction of the consensus NKE reporter construct A20 has also been described previously (41Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Briefly, the 3× (NKE)-tata-luciferase construct was constructed by inserting a linker containing three copies of an intermediate strength NKE in front of the α-cardiac actin minimal TATA box driving the expression of luciferase. Human SRF expression vectors (pCGN-SRF, pCGN-SRFΔC, and pCGN-SRFpm1) driven by the cytomegalovirus promoter have been previously described (41Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 42Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar, 43Chen C.Y. Croissant J.D. Majesky M. Topouzis S. McQuinn T. Frankovsky M.J. Schwartz R.J. Dev. Genet. 1996; 19: 119-130Crossref PubMed Scopus (110) Google Scholar) and were generously supplied by Ron Prywes. The construction and use of the pCGN-Nkx2.5 expression vector has been previously described (41Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 42Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar, 43Chen C.Y. Croissant J.D. Majesky M. Topouzis S. McQuinn T. Frankovsky M.J. Schwartz R.J. Dev. Genet. 1996; 19: 119-130Crossref PubMed Scopus (110) Google Scholar). The Nkx3-1 homeodomain cDNA was isolated by PCR on the full-length cDNA using forward primer 5′-TCACCAAGCAGCCACAGAAG-3′ (359–379 bp) and reverse primer 5′-CTGCTTTCGCTTGGTCTTATAGC-3′ (529–552 bp). The Nkx3-1 homeodomain protein consisted of 193 bp, including 13 bp 5′ to the homeodomain. To express Nkx3-1 in bacteria, the cDNAs for the full-length and homeodomain-only region of Nkx3-1 were isolated by PCR, verified by DNA sequencing, and cloned into pRSET B (Invitrogen) vector. The full-length and homeodomain-only Nkx3-1 cDNA PCR products were cloned into pCRII (Invitrogen, TA Cloning Kit). The cDNA for the Nkx3-1 homeodomain region was excised from pCRII withHindIII and XhoI, sites internal to the PCR primers, and ligated into the HindIII/XhoI sites of pRSET B, downstream of a 6× histidine tag. These constructs were used to transform BL21 (DE3) cells (Novagen). Freshly transformed cells were grown in 500 ml of LB broth containing 100 μg/ml ampicillin at 37 °C to A600 = 0.8. Isopropyl-β-d-thiogalactopyranoside at a final concentration of 1 mm was then added, and growth continued for another 2 h. Cells were harvested and suspended in 20 ml of column buffer (20 mm Tris, pH 7.4, 200 mm NaCl, 1 mm EDTA), sonicated, and cellular debris removed by centrifugation. The fusion protein was purified by binding to a metal affinity column (CLONTECH), washed extensively, and then eluted with fractions of column buffer containing 10–200 mm imidazole. The concentration of the protein was determined by Bradford protein assay, and purity was determined by Coomassie staining after SDS-polyacrylamide gel electrophoresis. Full-length human SRF was expressed and purified as a glutathioneS-transferase (GST) fusion protein as described previously (41Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 42Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar, 43Chen C.Y. Croissant J.D. Majesky M. Topouzis S. McQuinn T. Frankovsky M.J. Schwartz R.J. Dev. Genet. 1996; 19: 119-130Crossref PubMed Scopus (110) Google Scholar). The concentration of the protein was determined by Bradford protein assay and purity determined by Coomassie staining after SDS-polyacrylamide gel electrophoresis. Monkey CV-1 fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were plated at an approximate density of 5 × 105 cells per 6-cm plate. Cells were transfected 24 h post-plating using LipofectAMINE (Life Technologies, Inc.) as described previously (49Reecy J.M. Li X. Yamada M. DeMayo F.J. Newman C.S. Harvey R.P. Schwartz R.J. Development. 1999; 126: 839-849Crossref PubMed Google Scholar). Briefly, each transfection reaction contained 1 μg of luciferase reporter plasmid (γ-actin, A20, or α-CA) and various amounts of transactivator plasmids (pCGN-Nkx3.1 and/or pCGN-SRF). All transfections were balanced to 2 μg of DNA with empty vector in order to keep the level of DNA and CMV promoter constant in all transfection reactions. Cells were transfected 16–18 h after which the transfection media were removed and replaced with Dulbecco's modified Eagle's medium supplemented with 2% horse serum and 10 μg/ml insulin for an additional 48 h. Cells were then harvested by washing with PBS and then scraped in 400 μl of 1× Reporter Lysis Buffer (Promega). Cellular debris was removed by centrifugation, and 30 μl of supernatant was analyzed for luciferase activity by mixing with 100 μl of luciferase substrate (20 mm Tris-HCl, pH 8.0; 4 mm MgSO4, 0.1 mm EDTA, 30 mm dithiothreitol, 0.5 mm ATP, 0.5 mmd-luciferin, 0.25 mm coenzyme A). Emitted luminescence was measured for 10 s. Protein concentrations were measured by the Bradford assay (Bio-Rad) and used to normalize luciferase activity. Interactions between Nkx3-1 and SRF were examined byin vivo immunoprecipitation using previously described methodology (42Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar). Briefly, 3T3 cells plated on 100-mm dishes were transfected with 2 μg of either pCGN-Nkx3.1 or pCGN-SRF plasmid DNA using LipofectAMINE (Life Technologies, Inc.). Forty eight hours post-transfection the cells were washed twice in ice-cold PBS, harvested in 1 ml of ice-cold PBS, and collected by centrifugation at 4 °C. Cells were resuspended in EBC buffer (50 mm Tris, pH 8.0; 120 mm NaCl; 0.5% Nonidet P-40; 2 μg ml leupeptin; 2 μg/ml pepstatin; and 1 mmphenylmethylsulfonyl fluoride), rocked at 4 °C for 15 min, and centrifuged at 4 °C. The supernatant was transferred to a new tube and protein concentration determined (Bio-Rad), and 500 μg of protein extracts containing SRF and Nkx3-1 were incubated for 2 h at 4 °C with 4 μg of anti-SRF antibody (Santa Cruz Biotechnology) in a total of 500 μl of NETN buffer (20 mm Tris, pH 8.0; 100 mm NaCl; 1 mm EDTA; 5 mm MgCl; 1 mm dithiothreitol; 0.05% Nonidet P-40; 1 mmphenylmethylsulfonyl fluoride). Non-transfected 3T3 cell extracts served as controls. Thirty microliters of protein G PLUS/protein A-agarose beads (Oncogene Science) equilibrated in NETN buffer were then added, and the incubation was continued for an additional 2 h. The beads were collected by brief centrifugation, washed three times with 1 ml of NETN buffer, and suspended in 30 μl of 2× SDS sample buffer. Samples were then boiled 5 min, briefly centrifuged, and the supernatant separated by 10% SDS-PAGE. Proteins were visualized by Western blot analysis using anti-HA antibody and ECL (Amersham Pharmacia Biotech). Double-stranded oligonucleotides corresponding to the smooth muscle γ-actin NKE1/SRE1 elements were constructed consisting of −100 to −74 bp of the promoter (5′-CCATCACTTAGCCTATTTAGGGTCTT-3′). The oligonucleotide was end-labeled using the polynucleotide kinase reaction, and band shift assays were performed as described previously (40Browning C.L. Culberson D.E. Aragon I.V. Fillmore R.A. Croissant J.D. Schwartz R.J. Zimmer W.E. Dev. Biol. 1998; 194: 18-37Crossref PubMed Scopus (76) Google Scholar, 41Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 42Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar, 43Chen C.Y. Croissant J.D. Majesky M. Topouzis S. McQuinn T. Frankovsky M.J. Schwartz R.J. Dev. Genet. 1996; 19: 119-130Crossref PubMed Scopus (110) Google Scholar). Briefly, 20 μl containing 1 μg of either poly(dI-dC) or poly(dG-dC) binding buffer (10 mm Tris, pH 8.0; 1 mmdithiothreitol; 1 mm sodium phosphate; 5% glycerol; 50 mm sodium chloride) and nanogram quantities of purified bacterially expressed proteins were incubated 10 min at room temperature, and then the probe was added (20,000 cpm/reaction) and incubated for 15 min at room temperature. Binding complexes were then run on a 5% polyacrylamide gel in 0.5× TBE buffer. The gel was prerun for 20 min, followed by sample electrophoreses for 2 h at 180 V. Binding complexes were visualized by autoradiography. DNase I footprinting was performed on a fragment of the SMGA gene from −108 to +15 using purified SRF and Nkx3-1 proteins. This fragment was cloned into pGL-3 vector as described previously (21Kovacs A.M. Zimmer W.E. Gene Expr. 1998; 7: 115-129PubMed Google Scholar, 40Browning C.L. Culberson D.E. Aragon I.V. Fillmore R.A. Croissant J.D. Schwartz R.J. Zimmer W.E. Dev. Biol. 1998; 194: 18-37Crossref PubMed Scopus (76) Google Scholar). The DNA was digested with enzymes present in the vector multiple cloning site that were unique within the gene promoter/luciferase vector (NheI for coding strand and HindIII for non-coding strand), and the DNA construct was then labeled with 32P using Klenow enzyme. Single end-labeled DNA was then digested with the second enzyme and the fragment purified by electrophoresis on polyacrylamide gels as described previously. Each mapping assay contained ∼25 fmol of fragment (25,000 cpm) in 50 μl of gel shift assay buffer described above. Proteins were added to the assay tubes on ice, and the reaction was continued on ice for 1 h, after which DNase I was added and the reaction changed to room temperature for 1 min. Subsequent steps were carried out essentially as described previously (41Chen C.Y. Schwartz R.J. J. Biol. Chem. 1995; 270: 15628-15633Abstract Full Text Full Text" @default.
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• W2056315093 title "The Smooth Muscle γ-Actin Gene Promoter Is a Molecular Target for the Mouse bagpipe Homologue, mNkx3-1, and Serum Response Factor" @default.
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