The cell-specific expression of endothelial nitric-oxide synthase: a role for DNA methylation.
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.
Chan
Yvonne
Y
Renal Division and Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Fish
Jason E
JE
D'Abreo
Cheryl
C
Lin
Steven
S
Robb
G Brett
GB
Teichert
Anouk-Martine
AM
Karantzoulis-Fegaras
Fotula
F
Keightley
Angela
A
Steer
Brent M
BM
Marsden
Philip A
PA
D000818:Animals; D001011:Aorta / Q000473:pathology; D001374:Azacitidine / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D013447:Sulfites / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D002843:Chromatin; D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; C494042:Ets1 protein, mouse; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D013447:Sulfites; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D008156:Luciferases; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D012260:Ribonucleases; D001374:Azacitidine; C009279:sodium bisulfite