Fig. 5: CRE perturbation analyses.

a CREs selected for perturbation analysis. Variants derived from seq6846_R (*) were not sufficiently detected in the assays and were therefore excluded from subsequent analyses. b Two strategies were used for enhancer perturbation analyses. In the single-nucleotide substitution approach (left), each nucleotide in a 100-bp CRE was individually mutated to all three alternative nucleotides (300 variants per CRE). In the 6-bp window perturbation approach (right), consecutive 6-bp segments were randomly mutated across the enhancer sequence using a sliding window shifted by 1 bp increments, resulting in 95 variants per CRE replicate. This randomization was performed twice independently, generating a total of 190 variants per CRE. Analysis of single-nucleotide substitution effects on epigenetic activities (lentiMPRA, ATAC, and H3K27ac CUT&Tag) for seq68781_R (c) and POU5F1_DE_core (d). Annotated TF motifs⁴⁹ are shown above each plot. Bar colors represent statistical significance (black: significant at P < 0.01; gray: not significant). P values were obtained from multiple linear regression. Analysis of 6-bp window perturbation effects for seq68781_R (e) and POU5F1_DE_core (f). Positional effects of mutations were quantified as median absolute deviation (MAD) scores at each nucleotide position and smoothed using a Gaussian filter (line plot). Regions significantly affected by mutations, as identified by edge detection, are indicated by gray lines along the baseline. Source data are provided as a Source Data file.