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The endothelial nitric-oxide synthase gene is constitutively expressed in endothelial cells. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including a GATA-2 and an Sp-1 binding site. Because they cannot account for the constitutive expression of endothelial nitric-oxide synthase gene in a restricted number of cells, we have searched for other cell-specific regulatory elements. By DNase I hypersensitivity mapping and deletion studies we have identified a 269-base pair activator element located 4.9 kilobases upstream from the transcription start site that acts as an enhancer. DNase I footprinting and linker-scanning experiments showed that several regions within the 269-base pair enhancer are important for transcription factor binding and for full enhancer activity. The endothelial specificity of this activation seems partly due to interaction between this enhancer in its native configuration and the promoter in endothelial cells. EMSA experiments suggested the implication of MZF-like, AP-2, Sp-1-related, and Ets-related factors. Among Ets factors, Erg was the only one able to bind to cognate sites in the enhancer, as found by EMSA and supershift experiments, and to activate the transcriptional activity of the enhancer in cotransfection experiments. Therefore, multiple protein complexes involving Erg, other Ets-related factors, AP-2, Sp-1-related factor, and MZF-like factors are important for the function of this enhancer in endothelial cells. The endothelial nitric-oxide synthase gene is constitutively expressed in endothelial cells. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including a GATA-2 and an Sp-1 binding site. Because they cannot account for the constitutive expression of endothelial nitric-oxide synthase gene in a restricted number of cells, we have searched for other cell-specific regulatory elements. By DNase I hypersensitivity mapping and deletion studies we have identified a 269-base pair activator element located 4.9 kilobases upstream from the transcription start site that acts as an enhancer. DNase I footprinting and linker-scanning experiments showed that several regions within the 269-base pair enhancer are important for transcription factor binding and for full enhancer activity. The endothelial specificity of this activation seems partly due to interaction between this enhancer in its native configuration and the promoter in endothelial cells. EMSA experiments suggested the implication of MZF-like, AP-2, Sp-1-related, and Ets-related factors. Among Ets factors, Erg was the only one able to bind to cognate sites in the enhancer, as found by EMSA and supershift experiments, and to activate the transcriptional activity of the enhancer in cotransfection experiments. Therefore, multiple protein complexes involving Erg, other Ets-related factors, AP-2, Sp-1-related factor, and MZF-like factors are important for the function of this enhancer in endothelial cells. nitric-oxide synthase endothelial NOS human eNOS base pair(s) kilobase pair(s) human microdermal endothelial cell hypersensitive site polymerase chain reaction electromobility shift assay Nitric-oxide synthases (NOS)1 are enzymes that metabolize l-arginine to form NO, and three isoforms have been identified. In the vascular system, the NOS III isoform (eNOS), first identified in endothelial cells, regulates vascular tone (1Palmer R.M.J. Rees D.D. Ashton D.S. Moncada S. Biochem. Biophys. Res. Commun. 1988; 153: 1251-1256Crossref PubMed Scopus (1127) Google Scholar), platelet aggregation (2Alheid U. Frolich J.C. Förstermann U. Thromb. Res. 1987; 47: 561-571Abstract Full Text PDF PubMed Scopus (132) Google Scholar), and smooth muscle cell proliferation (3Garg U.C. Hassid A. J. Clin. Invest. 1989; 83: 1774-1777Crossref PubMed Scopus (1997) Google Scholar). The enzyme is activated by increased intracellular calcium concentration and by translocation from caveolae to cytosol (for review see Ref. 4Fleming I. Busse R. Cardiovasc Res. 1999; 43: 532-541Crossref PubMed Scopus (359) Google Scholar). The eNOS expression is constitutive in endothelial cells, but under several physiological and pathological conditions, transcriptional regulation (5Navarro-Antolin J. Rey-Campos J. Lamas S. J. Biol. Chem. 2000; 275: 3075-3080Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 6Ziegler T. Silacci P. Harrison V.J. Hayoz D. Hypertension. 1998; 32: 351-355Crossref PubMed Scopus (208) Google Scholar), and changes in mRNA stability (7Yoshizumi M. Perrella M.A. Burnett Jr., J.C. Lee M.E. Circ. Res. 1993; 73: 205-209Crossref PubMed Scopus (705) Google Scholar, 8Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (978) Google Scholar) have been described. Several studies were aimed at characterizing the 5′-upstream sequences that drive transcriptional activity of the promoter up to 3500 bp. A major transcriptional effect was identified for Sp-1/Sp-3 and GATA-2 transcription factors, for which binding sites are located respectively at −103 and −230 bp upstream from the major transcription start site (9Zhang R. Min W. Sessa W.C. J. Biol. Chem. 1995; 270: 15320-15326Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 10Wariishi S. Miyahara K. Toda K. Ogoshi S. Doi Y. Ohnishi S. Mitsoui Y. Yui Y. Kawai C. Shizuta Y. Biochem. Biophys. Res. Commun. 1995; 216: 729-735Crossref PubMed Scopus (34) Google Scholar, 11Tang J.-L. Zembowicz A. Xu X.-M. Wu K. Biochem. Biophys. Res. Commun. 1995; 213: 673-680Crossref PubMed Scopus (36) Google Scholar). A second positive regulatory domain was detected between −140/−120 bp in the proximal promoter (12Karantzoulis-Fegaras F. Antoniou H. Lai S.L.M. Kulkarni G. D'Abreo C. Wong G.K.T. Miller T.L. Chan Y. Atkins J. Wang Y. Marsden P.A. J. Biol. Chem. 1999; 274: 3076-3093Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). It was also shown that a 1600-bp human eNOS (heNOS) promoter fragment allows the endothelial expression of a reporter gene in transgenic mice but is not sufficient to observe a full expression of the gene in all endothelial territories and to reproduce the endogenous pattern of expression (13Guillot P.V. Guan J. Liu L. Kuivenhoven J.A. Rosenberg R.D. Sessa W.C. Aird W.C. J. Clin. Invest. 1999; 103: 799-805Crossref PubMed Scopus (56) Google Scholar). In this study, we characterized cis-acting sequences, located 4.9 kb upstream from the major transcription start site, that increase constitutive expression of the heNOS gene in endothelial cells. Analysis of the enhancer sequence by DNase I footprinting and linker-scanning mutants led us to identify five major binding sites, in particular for Erg and other Ets family member proteins, for Sp-1-related factors, and for MZF-like transcription factors. Sonicated salmon sperm DNA, proteinase K, RNase A, poly(dI·dC) were from Roche Molecular Biochemicals. MCDB-131 medium, hydrocortisone and Nonidet P40 (Igepal) from Sigma. Fetal calf serum, penicillin and streptomycin were from Seromed (Berlin, Germany). Epidermal growth factor, human recombinant and polyethyleneimine suspension (EXGEN 500), were from Euromedex (Souffelweyersheim, France). RPMI medium, phosphate-buffered saline, and l-glutamine were from Life Technologies, Inc. DNase I was from Worthington (Lakewood, NJ). HMEC-1 are human dermal microvascular endothelial cells immortalized by transfection with a pBR322-derived plasmid containing the coding region for the simian virus 40 A gene product, large T antigen, and were a gift from Thomas J. Lawley (Emory University, School of Medicine, Atlanta, GA). Human umbilical vein endothelial cells were isolated as described by Jaffe et al.(14Jaffe E.A. Nachman R.L. Becker C.G. Minick C.R. J. Clin. Invest. 1973; 52: 2745-2756Crossref PubMed Scopus (6019) Google Scholar). Both cell types were cultured in MCDB-131 medium supplemented with 20% heat-inactivated fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 ng/ml human recombinant epidermal growth factor, and 1 μg/ml hydrocortisone. HeLa cells were cultured in RPMI medium supplemented by 10% heat-inactivated fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37 °C and 5% CO2 in an humidified incubator. In situ DNase I digestion method was performed as described previously by Stewartet al. (15Stewart A.F. Reik A. Schütz G. Nucleic Acids Res. 1991; 19: 3157Crossref PubMed Scopus (35) Google Scholar), using 0.2% and 0.05%, respectively, of Nonidet P-40 for permeabilization of HMEC-1 or HeLa cells. After phenol extraction, DNA samples (30 μg) were subsequently submitted to Southern blot analysis. DNase I hypersensitivity assay was also performed on isolated nuclei with standard procedures (16Wu C. Nature. 1980; 286: 854-860Crossref PubMed Scopus (753) Google Scholar). A PCR product extending from 5′-upstream from the DNase I hypersensitive site HS1 to downstream from the initiator methionine codon, was obtained by PCR using a commercially available long PCR kit (Taq+ Precision PCR system, and a human eNOS as S. A. M. F. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). The PCR product was between and sites of the reporter gene of deletion the was by using a kit system, were using and an DNA and containing the bp activator element in were by a product to the promoter fragment of the heNOS a kit was with as and mutants were by were using polyethyleneimine suspension in a commercially available (EXGEN HMEC-1 and HeLa cells were on at 100 and respectively, and with a containing of the reporter gene of gene as transfection and polyethyleneimine suspension for 2 of experiments were performed using expression containing for transcription factors, of J. S. R. Biol. 1995; PubMed Google Scholar), C. A. R. J. 1991; PubMed Scopus Google Scholar), of R. L. M. J. D. D. PubMed Scopus Google Scholar), and of J. procedures were that expression were in polyethyleneimine suspension at with reporter activity was by using as described by the HMEC-1 were as described previously Nucleic Acids Res. PubMed Scopus Google Scholar). cell HeLa cells were on a and with 2 of expression or After 40 the cells were in 40 1 by and in 40 of containing 2 10% Cells were by and cell were by 10 and the cell was and at A fragment of the heNOS gene promoter region to was obtained by PCR using the enhancer. one was with by DNA Technologies, a PCR using 10 of the second DNase I footprinting were performed by of with of for at in 1 and 10% to of DNase I for 2 were performed in the of were by of of proteinase K, 100 μg/ml and and 1 at were phenol and in 10 and with a sequence of the on a linker-scanning were in the the and the were using a method with to the linker-scanning mutants are in I. The was using and is a that to the region of the heNOS promoter and a I site at its to the of the to are base containing a sequence to the promoter sequence to a region at the to the heNOS sequence and containing a site. The was using a and containing a sequence to heNOS promoter and a to the heNOS sequence and containing a site to the After digestion and was to its and the product was submitted to a The product was in of the promoter in obtained were by to all linker-scanning mutants and sequences are and are in a sequences are and are to DNase I regions were to the sequence previously μg) or cell of cells with expression μg) were with of in 10% 1 1 of and 10 of were by on native experiments were performed by or cell with or to of or from J. and were from in EMSA and for to sites are and are and in a sites are and are and sequences were for transcription binding sites using base and K. K. H. T. Nucleic Acids Res. 1995; PubMed Scopus Google Scholar). transfection are expressed as the were by the Analysis of experiments in HMEC-1 and HeLa cells were performed using a of with a of DNase I hypersensitivity experiments were performed on cells as as on isolated obtained with cells are but were obtained with the a located at the of a large fragment located in the region of the heNOS gene 1 three hypersensitive sites were detected by Southern blot experiments in the heNOS gene in HMEC-1 1 the of that sites are located at kb kb and kb upstream from the major transcription start site 1 The hypersensitive site not to previously identified regulatory regions of the heNOS HeLa cells that not as by not of hypersensitive sites detected in experiments 1 A promoter fragment was using a and to a reporter gene After III digestion of this plasmid deletion of the heNOS promoter were obtained and shown in 2 A, of HMEC-1 performed with showed a increase of the promoter with that obtained with the promoter fragment from to not of transcriptional activity. from to transcription to the obtained with the of an activator element located between and of the of the promoter from to not the promoter activity not from to showed a in the promoter activity not the enhancer of the activator element was in to the promoter and or to an promoter experiments performed in HMEC-1 with and showed a transcriptional activity to 2 transfection experiments using showed an transcriptional activity as with the promoter 2 A pattern of transcriptional activation was with the in human umbilical vein endothelial cells 2 of activation in the endothelial cell In transfection experiments using HeLa cells showed transcriptional activation of the native promoter by the heNOS enhancer, in transcriptional activity was between and 2 the showed a increase in transcriptional activity in HeLa cells with the promoter 2 A large increase of transcriptional activity was also using the with in HeLa 2 DNase I experiments were performed to identify regulatory elements in the heNOS enhancer activity. to the activator element of the promoter was as a in experiments with HMEC-1 shown in the in A, and The element A bp and is in its by a DNase site. and element bp. bp and an hypersensitive site an we elements A, and but element was not the of DNase I a was by bp of element linker-scanning studies binding sites to identified the DNase I elements. linker-scanning mutants and showed a in transcriptional activity with the in HMEC-1 The mutants and of the enhancer and an enhancer In the linker-scanning of the enhancer and the linker-scanning effect on enhancer activity. the of five regions of the enhancer in the regulation of heNOS of the linker-scanning mutants in were in by bp in the regions by DNase I footprinting DNase I elements linker-scanning of are described in I. of enhancer and of the linker-scanning mutants were with that of are expressed as the of five experiments to A was to on element A, bp of Several complexes were using a in EMSA experiments 2 and of of all the complexes and of the sequence binding sites for several transcription factors located in element A, in particular for transcription factors and for transcription factors family were and for the A site or for the sequence to for complexes and a major effect of this element in the protein in the sequence of all complexes and a binding site with sequences, the of I The with a in the binding site to and that MZF-like factors bind element A, factors the that HMEC-1 cells to with and blot not and blot not we were to observe supershift using the or an the domain of not of expression with plasmid effect on the enhancer transcriptional activation not element was restricted to base by linker-scanning mutants and A containing the element sequence multiple complexes in the of 2 and which several were by of and of region binding sites for AP-2, and experiments performed using an showed for In an Sp-1 for complexes of the sequences of and Sp-1 Sp-1 binding sites within the the Sp-1 binding site for that the of for in Therefore, the of and Sp-1 for a for I the binding of an MZF-like an a in the site all complexes with to that of the an implication of Sp-1-related and MZF-like transcription factors in the of complexes in element but not of Ets family of the on the region with HMEC-1 in a which was with of a site in the we a using a of a an for the site to the Erg transcription factors or factors, we not observe supershift on complexes by HMEC-1 and not that Erg transcription factors were in The performed with an on the element in the of one major and several complexes of also an site. showed a for for the site to Therefore, the binding of Ets-related to the sites located in and experiments performed with using an factors not to not In an Erg transcription factors of a with and were obtained with HeLa cells with several expression Ets-related transcription factors. cell of HeLa cells with and expression led to complexes in EMSA experiments using 2 and or and experiments in HMEC-1 showed a effect of Erg transcription factor on transcriptional activity of the enhancer with an led to a of transcriptional activity at a with but effect at a not experiments using and in HeLa cells showed a effect of Erg on sites located in the first bp of the promoter sequence 1 and It is to the effect of on the enhancer the effect of Erg, as by a of was in HMEC-1 and HeLa cells and the site in cotransfection experiments of HeLa cells with the Erg expression transcription to the with the promoter not that Erg or other Ets transcription factors in the regulation of the heNOS gene expression by binding to the sites identified in and D. this of site located in or were by a and enhancer transcriptional activity was by 10 that of Ets site in or Ets site located in led to a of activity with the enhancer. The of were the linker-scanning for and for which enhancer activity. that Ets-related transcription factors and Erg in the activity of the enhancer of the of an endothelial specificity of transcription factors in the enhancer we performed EMSA experiments using HeLa and the EMSA and we the with obtained using HMEC-1 using the and the and not the using was not using HeLa cell at identify elements of the heNOS we kb of the promoter region for the of DNase sites that are with transcriptionally active J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). hypersensitive sites and were in HMEC-1 cells and were in HeLa cells, which not that they endothelial regulation of heNOS gene HS1 is located kb upstream from the major transcription start site, and deletion mutants the that this hypersensitive site to a major transcriptional regulatory the obtained with the deletion mutants that a transcription activator is between and The site, located at not to a regulatory region identified we not observe of the transcriptional activity of the promoter this region was or The site, located at to a previously identified region of the promoter, located between and bp (9Zhang R. Min W. Sessa W.C. J. Biol. Chem. 1995; 270: 15320-15326Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 10Wariishi S. Miyahara K. Toda K. Ogoshi S. Doi Y. Ohnishi S. Mitsoui Y. Yui Y. Kawai C. Shizuta Y. Biochem. Biophys. Res. Commun. 1995; 216: 729-735Crossref PubMed Scopus (34) Google Scholar, 11Tang J.-L. Zembowicz A. Xu X.-M. Wu K. Biochem. Biophys. Res. Commun. 1995; 213: 673-680Crossref PubMed Scopus (36) Google Scholar) and also detected using deletion mutants between and not the positive regulatory domain of the promoter, located bp and involving an Sp-1 binding site (9Zhang R. Min W. Sessa W.C. J. Biol. Chem. 1995; 270: 15320-15326Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, F. Antoniou H. Lai S.L.M. Kulkarni G. D'Abreo C. Wong G.K.T. Miller T.L. Chan Y. Atkins J. Wang Y. Marsden P.A. J. Biol. Chem. 1999; 274: 3076-3093Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) was not detected by DNase I hypersensitivity experiments. studies us to identify a activator region from to with to the transcription start site. activator as a enhancer of its and and also upstream from an the fragment showed a enhancer activity in HMEC-1 to the promoter with its enhancer activity to the heNOS promoter, between cis-acting sequences located in the enhancer and the regulatory sequences by the obtained in HeLa cells showed an endothelial specificity of between the enhancer and the promoter region in transcriptional activity was detected the enhancer was between and transcriptional activity of the enhancer, upstream from the heNOS promoter or in of the promoter, is in HeLa cells. that between transcription factors in HeLa cells and the enhancer in its It also that the endothelial specificity of the enhancer in its native due to the between and of sequences active in cells or of a important the in endothelial cells. identified sites and of by DNase I footprinting experiments within the with A and sites the Among the linker-scanning mutants to sites, the enhancer activity from to A, and and and the transcriptional the not the transcriptional activity of the the of regulatory domain in this that the region multiple transcriptionally active elements in a and that sites for transcription factors in the activator in with studies that transcription factors M. Xu D. D. R. 1995; Google Scholar, M. M. M. H. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). we identified a binding sequence in A and in region binding specificity were by in EMSA experiments using an containing an binding site R. Biol. PubMed Scopus Google Scholar). It is also that the linker-scanning mutants in the enhancer and a in transcriptional that the full activation an The long between is with a for transcription factor binding sites are by bp H. W. G. C. K. J.C. D. J. 1999; PubMed Scopus Google Scholar) and also with the in the promoter, of active sites bp M. M. M. H. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). using we not observe supershift or of the complexes with A. In of an expression not a effect of on the enhancer activity not Therefore, the implication of an MZF-like factor, on sequence and experiments, they not a for or in the enhancer activity. we that an factor is at as an an transcription factor, which was described in the number implication to is is to bind Sp-1-related factor, complexes are with an Sp-1 Sp-1 binding site is also a major regulatory element of the proximal heNOS promoter for constitutive (9Zhang R. Min W. Sessa W.C. J. Biol. Chem. 1995; 270: 15320-15326Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 10Wariishi S. Miyahara K. Toda K. Ogoshi S. Doi Y. Ohnishi S. Mitsoui Y. Yui Y. Kawai C. Shizuta Y. Biochem. Biophys. Res. Commun. 1995; 216: 729-735Crossref PubMed Scopus (34) Google Scholar, 11Tang J.-L. Zembowicz A. Xu X.-M. Wu K. Biochem. Biophys. Res. Commun. 1995; 213: 673-680Crossref PubMed Scopus (36) Google Scholar, F. Antoniou H. Lai S.L.M. Kulkarni G. D'Abreo C. Wong G.K.T. Miller T.L. Chan Y. Atkins J. Wang Y. Marsden P.A. J. Biol. Chem. 1999; 274: 3076-3093Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) and as with K. Zembowicz K. J.-L. Wu J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the enhancer sites, located at and were identified by linker-scanning and of the cells several of the Ets family of transcription factors under or activated L. M. J. D. D. PubMed Scopus Google Scholar, G. M. S. M. D. Google Scholar, A. Y. P. Circ. Res. 1999; PubMed Scopus Google Scholar, A. K. D. P. M.A. P. J. Biol. 1998; Google Scholar). Among all the Ets factors that we have Erg is the only Ets factor for which we found of EMSA experiments using from HeLa cells that only Erg and but not or are able to bind and element binding site. In an the binding of Erg to element we were not able to supershift or in experiments using a to element obtained by in HMEC-1 with Ets expression showed that only Erg the enhancer activity. activation was also in HeLa cells, but in this was to the effect of Erg on the enhancer of its effect on the cotransfection experiments, the enhancer activity large of were or not effect at cotransfection The seems to due to the expression of of in EMSA which that is the of a of for native HMEC-1 Ets-related factors that bind or Among the other Ets-related factors and only the proximal promoter of heNOS not as was shown in studies (12Karantzoulis-Fegaras F. Antoniou H. Lai S.L.M. Kulkarni G. D'Abreo C. Wong G.K.T. Miller T.L. Chan Y. Atkins J. Wang Y. Marsden P.A. J. Biol. Chem. 1999; 274: 3076-3093Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). the that factors in the heNOS enhancer other Ets factors are to also experiments using element showed that a was by an several other complexes were not complexes factors they are by an the Ets of detected in the complexes by using an several Ets factors. In an Ets factor not to in region an Ets obtained using are restricted to the Ets site are with obtained using linker-scanning mutants that and the transcriptional effect of the It is that linker-scanning the binding of to the Ets site. using a fragment of the eNOS promoter the et Miller T.L. Wang Y. Marsden P.A. J. Circ. 2000; PubMed Google Scholar) obtained transgenic mice in which the expression pattern was to that of the endogenous regulatory regions of the eNOS gene are between mice and this that the enhancer we have identified in the of the and the restricted pattern of The of this enhancer in the human promoter by al. (13Guillot P.V. Guan J. Liu L. Kuivenhoven J.A. Rosenberg R.D. Sessa W.C. Aird W.C. J. Clin. Invest. 1999; 103: 799-805Crossref PubMed Scopus (56) Google Scholar) the pattern of expression in this In an enhancer was identified at from the transcription start site. between several elements located in the activator region from Among all transcription factors Erg was the transcription factor able to bind the enhancer and the important transcription factors in the activity and to the and Ets family to identified. for and Erg expression for and expression of for and expression for expression and for and for
Laumonnier et al. (Fri,) studied this question.