Mutations within Sox2/SOX2 are associated with abnormalities in the hypothalamo-pituitary-gonadal axis in mice and humans
J. Clin. Invest. Daniel Kelberman, et al. 116:2442 doi:10.1172/JCI28658 [Go to this article.]

Figure 5
Functional analysis of SOX2 mutants. (A) Loss of the HMG box results in impaired nuclear localization. Cells were transfected with 50 ng plasmid construct containing wild-type or mutant SOX2 fused to an N-terminal FLAG epitope, fixed, and stained with anti-FLAG antibody; nuclei were counterstained with DAPI. Wild-type SOX2 showed predominantly nuclear staining. Mutant proteins lacking the HMG box (c.60insG and c.70del20) showed a majority of staining in the cytoplasm and impaired nuclear localization. Mutant proteins retaining the HMG box showed staining similar to wild-type. (B) SOX2 mutants show variable DNA binding. Identical amounts of in vitro translated wild-type (lanes 3 and 10) or individual variant SOX2 proteins (lanes 4–8 and 11–13) were incubated with a radiolabeled DNA probe. Specific binding of SOX2 to the probe was demonstrated by duplicate experiments using in vitro translated empty vector (lane 2) or 100 times excess cold probe (lane 9). (C) SOX2 mutants show impaired transcriptional activation. Expression constructs containing wild-type or variant SOX2 (10–50 ng) were cotransfected with 20 ng luciferase reporter construct containing part of the proximal promoter of HESX1. Wild-type SOX2 led to dose-dependent activation of the reporter (up to 20-fold activation), whereas mutant truncated SOX2 proteins showed impaired activation correlating with the extent of the truncation (dashed arrow). c.60insG, c.70del20, and L97P showed similar levels of activation to empty expression vector. In all experiments described here, c.479delA showed identical results to Y160X (not shown). Results are mean ± SD of 3 independent experiments performed in triplicate.