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Figure 7. Predicting causal mutations of evolutionary changes in MEL enhancers. (A,B) Example region upstream of APPL2 that is accessible (A) and active (B) in the MEL dog line Dog-OralMel-18249 but not in human MEL lines. (C) DeepMEL prediction score of each of the 24 topics for the dog and human APPL2 enhancer. (D) Effect on topic 4 DeepMEL score on the dog sequence when in silico simulating each of the single detected point mutations between the dog and human APPL2 enhancer. (E) DeepExplainer plots of the middle 120 bp of the dog and human APPL2 enhancer. In the middle, the effect of each possible point mutation between the dog and human sequence on the MEL DeepMEL score was in silico calculated and is represented by colored dots depending on the nucleotide to which the original dog nucleotide was in silico mutated. Truly existing point mutations between the dog and human sequence are highlighted by color-coded vertical dashed lines. Four mutations that decrease the motif score of the SOX10, MITF, and TFAP2A motifs are highlighted by a gray box and are encircled. (F) Bar plot showing the mean effect on the log2 delta ATAC-seq signal of a non-human region compared to the human homolog depending on the number of SOX10 motif hits lost or gained. Only regions having no change in the number of significant TFAP2A, MITF, and RUNX motifs hits were used. The y-axis is normalized to the category with no changes in the number of significant SOX10 motif hits. The number of regions in each of the categories is mentioned (#). (G) Luciferase assay on six human or dog enhancers. Significant motif hits per enhancer are shown with colored crosses. For the luciferase assays: luciferase activity in MM001 is shown relative to Renilla signal and is log10 transformed. P-values were determined using Student's t-test, and the error bars represent the standard deviation over three biological replicates.