Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9
Figure 5
Effect of Exp4 on Sox9 binding to its target gene.
(A) The effect of Exp4 depletion on the recruitment of Sox9 to its endogenous target gene. U2OS cells were treated with control siRNA (open bar) or siRNA for Exp4 (solid bar). After a 48 h incubation, fixed chromatin was prepared from both groups of treated cells and subjected to immunoprecipitation with anti-Sox9 antibody. The Col2a1 enhancer regions recovered in the two precipitated fractions were quantified by PCR. As a control, GAPDH genes recovered in the two precipitated fractions were quantified. The relative amount of enhancer DNA is shown: the amount of Col2a1 enhancer DNA obtained from the control siRNA cells is set at 100. (B) The DNA sequence of the probe used for DNA pull-down contains a biotinylated 18 base-pair Sox9 binding sequence of the Col2a1 enhancer region. (C) DNA pull-down assay with the indicated amounts (ng) of His-Sox9 and FLAG-Exp4. His-Sox9 and FLAG-Exp4 were incubated with a 5′ biotinylated double-stranded DNA probe. The complex was pulled down with streptavidin-agarose and the streptavidin-agarose bead bound fraction and flow-through fractions (FT) were analyzed by Western blotting.