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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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Submitted January 22, 2008
Accepted May 13, 2008

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

Yoshiaki Okada, Enjing Jin, Vesna Nikolova-Krstevski, Kiichiro Yano, Ju Liu, David Beeler, Katherine Spokes, Mie Kitayama, Nobuaki Funahashi, Takefumi Doi, Lauren Janes, Takashi Minami, Peter Oettgen, and William C Aird*

CVBR, Beth Israel Deaconess Medical Center, Boston, United States
Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
RCAST, The University of Tokyo, Tokyo, Japan

* Corresponding author; email: waird{at}bidmc.harvard.edu.

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 to 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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