E80A and K88N mutations differently affect CRX HD DNA-binding cooperativity at P3 sequences. (A,B) Schematics showing the Coop-seq experimental pipelines. dsDNA oligo pools of P3 and/or P5 Coop-seq library are incubated with different HD peptides. The dimeric and monomeric binding complexes are separated from unbound DNAs by EMSA. DNAs are extracted from all three DNA bands and subjected to quantification by Illumina sequencing. (Bd) Dimeric band, (Bm) monomeric band, and (U) unbound band. (C) Diagram depicting the Coop-seq library design and strategy to match a P3 sequence with a P5 counterpart. Exact oligo sequences can be found in Supplemental Table S1. (D) Heatmap comparing the relative cooperativity of WT and variant CRX HDs on P3 and P5 libraries (ωp3/ωp5). Note the relative cooperativity is presented in the logarithmic scale and ordered by unsupervised hierarchical clustering. The ordered relative cooperativity matrix can be found in Supplemental Table S3.
