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J. Biol. Chem., Vol. 279, Issue 33, 35087-35100, August 13, 2004

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The Cell-specific Expression of Endothelial Nitric-oxide Synthase

A ROLE FOR DNA METHYLATION^*

Yvonne Chan, Jason E. Fish¶||, Cheryl D'Abreo, Steven Lin, G. Brett Robb¶, Anouk-Martine Teichert, Fotula Karantzoulis-Fegaras, Angela Keightley, Brent M. Steer, and Philip A. Marsden, Recipient of a Career Investigator Award from the Heart and Stroke Foundation of Canada and supported by Canadian Institutes of Health Research Grant MOP 36381¶**

From the Renal Division and Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, Ontario M5S 1A8, Canada and the ^¶Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

The basis for the endothelial cell-restricted expression of endothelial nitric-oxide synthase (eNOS) is not known. While transgenic promoter/reporter mice demonstrated endothelium cell-specific eNOS expression, we found robust expression of episomal eNOS promoter/reporter constructs in cell types that do not express the native eNOS transcript. To explore the mechanism underlying this differential activity pattern of chromatin-versus episome-based eNOS promoters, we examined the methylation status of 5'-regulatory sequences of the human eNOS gene. DNA methylation differed dramatically between endothelial and nonendothelial cell types, including vascular smooth muscle cells. This same cell type-specific methylation pattern was observed in vivo in endothelial and vascular smooth muscle cells of the mouse aorta at the native murine eNOS promoter. We addressed the functional consequences of methylation on eNOS transcription using transient transfection of in vitro methylated promoter/reporter constructs and found that methylated constructs exhibited a marked decrease in the synergistic action of Sp1, Sp3, and Ets1 on eNOS promoter activity. The addition of methyl-CpG-binding protein 2 further reduced the transcriptional activity of methylated eNOS constructs. Importantly, chromatin immunoprecipitation demonstrated the presence of Sp1, Sp3, and Ets1 at the native eNOS promoter in endothelial cells but not in vascular smooth muscle cells. Finally, robust expression of eNOS mRNA was induced in nonendothelial cell types following inhibition of DNA methyltransferase activity with 5-azacytidine, demonstrating the importance of DNA methylation-mediated repression. This report is the first to show that promoter DNA methylation plays an important role in the cell-specific expression of a constitutively expressed gene in the vascular endothelium.

Received for publication, May 6, 2004

^* The Laser Capture Microdissection Facility (University of Toronto) is generously supported by a Heart and Stroke group grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains two additional figures.

These two authors contributed equally to this work.

^|| Recipient of a Natural Sciences and Engineering Research Council of Canada Graduate Scholarship.

^** To whom correspondence should be addressed: Medical Sciences Bldg., Rm. 7358, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-2441; Fax: 416-978-8765; E-mail: p.marsden{at}utoronto.ca.

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