Spec-seq data for human and macaque ZNF626 with replicates
Systematic analysis of available ChIP-exo data of human zinc finger proteins identified ZNF626 as a prominent binder to the protein-coding region of the HERVK9 gag gene, with a pronounced enrichment peak within a ~950-1000 bp window within HERVK9-int consensus site. Since conventional motif prediction algorithms failed to pinpoint the exact binding site with high confidence, we employed Spec-seq—a high-resolution, sequencing-based biophysical assay—to comprehensively map ZNF626's binding specificity. Alongside with human ZNF626, we ran Spec-seq on macaque ortholog of ZNF626, and compared it with human ZNF626. The resulting motifs differences can be directly attributed to their contact residues differences in fingers 1, 5, 9, and 10. Overall design: We designed a DNA binding sites library containing 18 triply randomized sequences spanning the entire 54 bp region of interest (positions 942-995), alongside 64 completely dissimilar sequences as a Non-specific background control. The resulting energy profiles and sequence logos for human ZNF626 unambiguously identified a 30 bp specific binding site (positions 967-995), which aligns perfectly with the expected motif length for a ten-finger protein.