Fig. 5: ALX4 human disease variants impact DNA binding and/or cooperativity which affect transcriptional output on a P3 site.

A Five missense variants located within or near the DNA binding domain were characterized. B, D ALX4 and the corresponding variant protein (0, 37.5, 75, 150, and 300 nM) were combined with the P3 fluorescent probe. A single replicate is shown but all replicates are shown in Supplementary Fig. 10. Tau cooperativity factors were calculated for every lane in which protein was added. Bars depict the average Tau for each protein and each dot represents a Tau from an independent binding reaction (n = 12 binding reactions; biological replicates). Error bars denote standard deviation. Tau factors were compared with an unpaired, two-sided student t-test. Note, quantitation was derived from two gels from the same experiment that were processed in parallel. C ALX4 R216G (R3G) hindered protein’s ability to localize to the nucleus in HEK293T cells. H2B-mcherry was used as a transfection control. NLS Mapper³⁷ predicted the only ALX4 nuclear localization signal to be the N-terminal ARM. Scale bar represents 50 µM. E The Q225E (Q12E) variant is located near the hydrophobic core of ALX4. Protein schematic was created in PYMOL and residues are colored based on hydrophobicity. F ALX4 Q225E reduced protein stability. Melting temperatures derived from differential scanning fluorimetry were compared via a one-way ANOVA with a Tukey post-hoc correction. The p value of the variant compared to the wildtype ALX4 protein is shown. The melting curves are shown in Supplementary Fig. 13A. G ALX4 R218Q (R5Q) failed to bind the 14mer oligo duplex, CGCTAATTAGCTCG, in ITC whereas the wildtype ALX4 protein bound the sequence with an affinity of 15.5 nM. H Luciferase assays were performed in transfected HEK293T cells (n = 3 transfected wells; biological replicates). 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 one-way ANOVA with a Tukey post-hoc correction where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided on Figshare⁶⁹.