title: Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter.
abstract: OBJECTIVE
  The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression.
  
  
  RESULTS
  Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the -42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type-specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell-derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation.
  
  
  CONCLUSIONS
  The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression.
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.). okadabos@phs.osaka-u.ac.jp waird@bidmc.harvard.edu.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Okada
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Funahashi
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Toru
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nishiyama
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Lei
authors.initials: L
authors.lastname: Yuan
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Keisuke
authors.initials: K
authors.lastname: Shirakura
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Alexis S
authors.initials: AS
authors.lastname: Turjman
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kano
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Hiroki
authors.initials: H
authors.lastname: Naruse
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Ayano
authors.initials: A
authors.lastname: Suzuki
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Miki
authors.initials: M
authors.lastname: Sakai
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhixia
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Kenji
authors.initials: K
authors.lastname: Kitajima
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Kenji
authors.initials: K
authors.lastname: Ishimoto
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Nobumasa
authors.initials: N
authors.lastname: Hino
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Masuo
authors.initials: M
authors.lastname: Kondoh
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Yohei
authors.initials: Y
authors.lastname: Mukai
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Nakagawa
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Guillermo
authors.initials: G
authors.lastname: García-Cardeña
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.). okadabos@phs.osaka-u.ac.jp waird@bidmc.harvard.edu.
authors.forename: William C
authors.initials: WC
authors.lastname: Aird
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (Y.O., N.F., T.T., Y.N., K.S., Y.K., H.N., A.S., M.S., J.Z., K.I., N.H., M.K., Y.M., S.N., T.D.); Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA (L.Y., W.C.A.); Department of Pathology, Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA (A.S.T., G.G.-C.); Department of Material Sciences, Massachusetts Institute of Technology, Boston (A.S.T.); and Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan (K.K.).
authors.forename: Takefumi
authors.initials: T
authors.lastname: Doi
mesh_terms: D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D019070:Cell Lineage; D042002:Chromatin Assembly and Disassembly; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044127:Epigenesis, Genetic / Q000187:drug effects; D005347:Fibroblasts / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D057809:HEK293 Cells; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014162:Transfection; D000097905:Roundabout Proteins
chemical_list: D015254:DNA Modification Methylases; D004791:Enzyme Inhibitors; D007041:Hypoxanthine Phosphoribosyltransferase; D011956:Receptors, Cell Surface; D016329:Sp1 Transcription Factor; C462951:ROBO4 protein, human; D000097905:Roundabout Proteins
keywords: DNA methylation; endothelial cells; epigenomics