FIG 9.

GATA2 directly activates Aqp2 expression through the evolutionary conserved GATA sites in the promoter region. (A) BirA/FLBio system for chromatin pulldown assay. Flag and biotinylation tags are fused to the N terminus of GATA2 protein. FLBio-GATA2 and E. coli BirA biotin ligase are stably cotransfected into mIMCD cells. (B) Confirmation of FLBio-GATA2 expression in the BirA/FLBio GATA2 mIMCD cells by immunoblotting analysis. The mIMCD cell line transfected only with BirA is used for control. Immunoblotting using GATA2 antibody detected the FLBio-GATA2 and endogenous GATA2 proteins in the FLBio-GATA2-transfected cells, while the BirA-only transfected cells express only the endogenous GATA2 protein (top panel). Biotinylated GATA2 is detected by streptavidin-HRP and is absent from the BirA-only transfected cells (middle panel). Lamin B antibody is used as loading control (bottom panel). (C) Chromatin pulldown experiments show that FLBio-GATA2 binds to the Aqp2 promoter in the mIMCD cell. Two genomic DNA regions around Gata1 locus (G1-3.8 kb and G1-14.4 kb) are used as negative controls. Data are presented as means ± SD from three independent experiments. Statistical significance of differences is indicated (*, P < 0.05; Student's unpaired t test). (D) Construction of 1.5-kb wild-type (Aqp2-WT-LUC) or GATA site-mutated (Aqp2-mut-LUC) Aqp2 promoter-luciferase reporter. Asterisks indicate nucleotides that are conserved between human and mouse Aqp2 genes. (E) Cotransfection of GATA2-expressing plasmid (pEF-GATA2) in the mIMCD cells activates the Aqp2-WT-LUC reporter but not the Aqp2-WT-LUC reporter. Data are presented as means ± SD from three independent experiments. Statistical significance of differences is indicated (***, P < 0.001; Student's unpaired t test).