A GABP-binding element in the Robo4 promoter is necessary for endothelial expression in vivo.
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.
Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
Yoshiaki
Y
Okada
Enjing
E
Jin
Vesna
V
Nikolova-Krstevski
Kiichiro
K
Yano
Ju
J
Liu
David
D
Beeler
Katherine
K
Spokes
Mie
M
Kitayama
Nobuaki
N
Funahashi
Takefumi
T
Doi
Lauren
L
Janes
Takashi
T
Minami
Peter
P
Oettgen
William C
WC
Aird
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004622:Embryo, Mammalian; D004727:Endothelium / Q000378:metabolism*; D051268:GA-Binding Protein Transcription Factor / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D009374:Neoplasms, Experimental; D011401:Promoter Regions, Genetic; D011956:Receptors, Cell Surface / Q000235:genetics*; D014018:Tissue Distribution; D014183:Transplantation, Heterologous; D000097905:Roundabout Proteins
D051268:GA-Binding Protein Transcription Factor; D011956:Receptors, Cell Surface; C462951:ROBO4 protein, human; D000097905:Roundabout Proteins