Fig. 6: Select ALX4 pathogenic variants can bind DNA and transcriptionally synergize with wildtype ALX4.
From: The ALX4 dimer structure provides insight into how disease alleles impact function
A Assessing pathogenic variants’ heterodimerization capacity and ability to compete with wildtype ALX4 for a P3 DNA site via EMSA. Wildtype ALX4209-274 and ALX4 R218Q209-274, and ALX4169-303 were tested at 200 and 400 nM individually to determine complex size (lane 2-7). Wildtype ALX4209-274 and ALX4 R218Q209-274 were then titrated (100, 200, 400, and 800 nM) with 400 nM of ALX4169-303 (lane 8−15). Experiment was repeated twice with similar results. B Pathogenic variants have distinct capacities to disrupt the ALX4169-303 dimer and form ALX4209-274 dimers. Percentage of probe bound by the ALX4169-303 dimer and ALX4209-274 dimer across increasing concentrations of ALX4. All quantified gels are shown in Supplementary Fig. 14. C Luciferase assays were performed in transfected HEK293T cells (n = 3 transfected wells; biological replicates) to assess the ability of ALX4 disease variants to synergize with wildtype ALX4 to induce transcriptional changes. Bars denote average luciferase fold change of the sample compared to reporter alone, while each dot represents an independent transfected well. Error bars denote standard deviation and luciferase fold changes between proteins were compared via a two-way ANOVA with a Tukey post-hoc correction where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for comparisons to 187.5 pg of ALX4 wildtype alone and +p < 0.05, ++p < 0.01, +++p < 0.001, ++++p < 0.0001 for comparisons to the concentration matched wildtype gradient. Source Data are provided on Figshare69.
