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 Blood, 15 September 2008, Vol. 112, No. 6, pp. 2336-2339.
Prepublished online as a Blood First Edition Paper on June 2, 2008; DOI 10.1182/blood-2008-01-135079.

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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

Brief Report

A GABP-binding element in the Robo4 promoter is necessary for endothelial expression in vivo

Yoshiaki Okada1,2,*, Enjing Jin1,*, Vesna Nikolova-Krstevski1, Kiichiro Yano1, Ju Liu1, David Beeler1, Katherine Spokes1, Mie Kitayama2, Nobuaki Funahashi2, Takefumi Doi2, Lauren Janes1, Takashi Minami3, Peter Oettgen1, and William C. Aird1

1 Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA; 2 Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan; and 3 Research Center for Advanced Science and Technology, the University of Tokyo, Tokyo, Japan

We recently demonstrated that the 3-kb 5'-flanking region of the human ROBO4 gene directs endothelial cell–specific expression in vitro and in vivo. Moreover, a GA-binding protein (GABP)–binding motif at –119 was necessary for mediating promoter activity in vitro. The goal of the present study was to confirm the functional relevance of the –119 GABP-binding site in vivo. To that end, the Hprt locus of mice was targeted with a Robo4-LacZ transgenic cassette in which the GABP site was mutated. In other studies, the GABP mutation was introduced into the endogenous mouse Robo4 locus in which LacZ was knocked-in. Compared with their respective controls, the mutant promoters displayed a significant reduction in activity in embryoid bodies, embryos, and adult animals. Together, these data provide strong support for the role of the GABP-binding motif in mediating Robo4 expression in the intact endothelium.


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