Figure 6.

Consequences of Missense Mutations on SOX4 Protein Level and Activity

(A) Western blot of cytoplasmic (cy) and nuclear (nu) extracts from COS-1 cells transiently transfected with expression plasmids encoding wild-type (WT) or variant SOX4 proteins (C1 to C4) fused at the N terminus with a 3FLAG epitope. The proteins were identified using anti-FLAG antibody. The Mr of protein standards is indicated in k units. Note that each SOX4 protein exhibited the expected Mr of approximately 75 k. The bottom panel is a longer exposure of the same blot as in the top panel. It is limited to the SOX4 protein region and shows that SOX4 protein was present in all nuclear extracts, but at a lower level than in cytoplasmic extracts. Variant SOX4 proteins did not show significant differences in nuclear localization compared to wild-type SOX4 when several independent experiments were considered.

(B) EMSA comparing the binding efficiency of SOX4 wild-type and variants. The cell extracts were the same as in (A), but also included negative controls without SOX4 (−, no plasmid; E, empty plasmid). The arrow indicates the complex formed between wild-type SOX4 and the DNA probe.

(C) Assay of the ability of the four case subjects’ SOX4 variants to activate transcription. COS-1 cells were transiently transfected with a 6FXO-p89-Luc reporter, a pSV-beta-galactosidase plasmid, and expression plasmids for wild-type (WT) or variant SOX4 (C1 to C4), and POU3F2. Reporter activities are presented as the mean ± standard deviation obtained from triplicates for each condition. They were normalized for transfection efficiency and are reported as fold increase relative to the activity of the reporter in the absence of SOX4 and POU3F2. The presence (+) or absence (−) of SOX4 and POU3F2 plasmid is indicated beneath the bars. These data were reproduced in more than three independent experiments.

(D) Assay of the ability of the 12 gnomAD SOX4 HMG-domain missense variants (g1 to g12) to activate transcription. COS-1 cells were transfected as described in (C) and using SOX4 and POU3F2 expression plasmids as shown in the figure. Data were calculated and are presented as in (C). They were reproduced in four independent experiments. A western blot showing that similar amounts of SOX4 protein were made in all conditions is presented in Figure S3.