Fig. 1: ALX4 genomic binding to the P3 dimer site is TWIST1 independent.

A Schematics of experiments performed in Kim et al.²⁸. Created in BioRender. Cain, B. (2025) https://BioRender.com/p80e992 (B) ALX4 bound 3250 regions in wildtype conditions. 2867 (88%) regions were no longer bound in dTAG^V-1 treated samples in which TWIST1 was depleted (TWIST1-dependent ALX4 regions). 383 (12%) regions were maintained after TWIST1 depletion (TWIST1-independent ALX4 regions). The color bar denotes the log2 ratio of the immunoprecipitated sample versus the IgG control. C A Homer de novo motif analysis revealed four distinct site types that were enriched in the ALX4 C&R: the Coordinator²⁸, the P3 dimer site, an independent E-box, and a monomer HD site. MNAse digestion patterns were used to discriminate between bound and unbound sites. TWIST1 dependence was assessed by footprinting the ALX4 bound sites with TWIST1 C&R and ALX4 C&R in a TWIST1 depleted background. D ALX4 binding signal decreases significantly after TWIST1 depletion for all peak sets except for peaks containing only the P3 dimer site. The 3250 ALX4 bound regions were categorized based on the presence of a coordinator site, a coordinator and dimer site, a dimer site, or only a monomer site or E-box or both independent sites. The average reads per million (RPM) after E. coli spike in normalization of the 200 bp window at the peak center were compared with a two-sided Wilcoxon rank sum test. Each datapoint represents the RPM for a peak center. E Chromatin accessibility decreases significantly after TWIST1 depletion for all peak sets except for peaks containing only the P3 dimer site. The average RPM of a 200 bp window at the peak centers was compared with a two-sided Wilcoxon rank sum test. Each datapoint represents the RPM for a peak center. Note, accessibility signals between TWIST1+ and TWIST- datasets were comparable at housekeeping genes: GAPDH and ACTB (Supplementary Fig. 5). For boxplots, the center line defines the median, the lower and upper box limits correspond with the 25^th and 75^th percentiles respectively, the lower and upper whiskers define either 1.5 times the interquartile range or the most extreme value. Data outside of the whisker limits are plotted as individual points. Source Data are provided on Figshare⁶⁹.