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(Circulation Research. 2007;100:1712.)
© 2007 American Heart Association, Inc.

Molecular Medicine

A Three-Kilobase Fragment of the Human Robo4 Promoter Directs Cell Type–Specific Expression in Endothelium

Yoshiaki Okada, Kiichiro Yano, Enjing Jin, Nobuaki Funahashi, Mie Kitayama, Takefumi Doi, Katherine Spokes, David L. Beeler, Shu-Ching Shih, Hitomi Okada, Tatyana A. Danilov, Elizabeth Maynard, Takashi Minami, Peter Oettgen, William C. Aird

From the Center for Vascular Biology Research and Division of Molecular and Vascular Medicine (Y.O., K.Y., E.J., K.S., D.L.B., S.-C.S., H.O. T.A.D., E.M., P.O., W.C.A.), Beth Israel Deaconess Medical Center, Boston, Mass; Graduate School of Pharmaceutical Sciences (N.F., M.K., T.D.), Osaka University, Japan; and Research Center for Advanced Science and Technology (T.M.), the University of Tokyo, Japan.

Correspondence to William C. Aird, MD, Beth Israel Deaconess Medical Center, Molecular and Vascular Medicine; RW-663, 330 Brookline Ave, Boston MA 02215. E-mail waird{at}bidmc.harvard.edu

Robo4, a member of the roundabout family, is expressed exclusively in endothelial cells and has been implicated in endothelial cell migration and angiogenesis. Here we report the cloning and characterization of the human Robo4 promoter. The 3-kb 5'-flanking region directs endothelial cell–specific expression in vitro. Deletion and mutation analyses revealed the functional importance of two 12-bp palindromic DNA sequences at –2528 and –2941, 2 SP1 consensus motifs at –42 and –153, and an ETS consensus motif at –119. In electrophoretic mobility shift assays using supershifting antibodies, the SP1 motifs bound SP1 protein, whereas the ETS site bound a heterodimeric member of the ETS family, GA binding protein (GABP). These DNA–protein interactions were confirmed by chromatin immunoprecipitation assays. Transfection of primary human endothelial cells with small interfering RNA against GABP and SP1 resulted in a significant (50%) reduction in endogenous Robo4 mRNA expression. The 3-kb Robo4 promoter was coupled to LacZ, and the resulting cassette was introduced into the Hprt locus of mice by homologous recombination. Reporter gene activity was observed in the vasculature of adult organs (particularly in microvessels), tumor xenografts, and embryos, where it colocalized with the endothelial cell–specific marker CD31. LacZ mRNA levels in adult tissues and tumors correlated with mRNA levels for endogenous Robo4, CD31, and vascular endothelial cadherin. Moreover, the pattern of reporter gene expression was similar to that observed in mice in which LacZ was knocked into the endogenous Robo4 locus. Together, these data suggest that 3-kb upstream promoter of human Robo4 contains information for cell type–specific expression in the intact endothelium.

Key Words: endothelial cells • Robo4 • gene regulation • transgenic mice

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