Defining the DNA Binding Specificity of GRHL2

Grainyhead-like 2 (GRHL2) is an epithelial transcription factor with context-dependent regulatory roles, yet the sequence rules governing its DNA recognition remain incompletely defined. In this study, a high-density genomic Specificity and Affinity for Protein (SNAP) DNA-binding array containing 772,732 tiled probes derived from GRHL2 ChIP-seq regions was used to resolve GRHL2 binding specificity at 6 base pair resolution across genomic sequences. From high-affinity probes, de novo motif analysis recovered the canonical 5′-AACCGGTT-3′ motif. Sequence specificity landscapes revealed a stepwise reduction in binding as mismatches were introduced, with the strongest effects at the C (position 3) and G (position 6) within the motif, greater tolerance at the central CG dinucleotide, and intermediate tolerance at the A/T bases at the motif edges. This analysis also demonstrated the influence of nearby flanking sequences. Extended motif and spacing analyses indicated dimeric binding at paired motifs, with periodic helical spacing consistent with interactions on the same face of the DNA helix. Integration of SNAP array binding with ChIP-seq data distinguished direct, motif-encoded GRHL2 occupancy from indirect, cofactor-mediated recruitment at genomic sites. These results define the sequence specificity of GRHL2 interactions with variations in the DNA consensus motif and flanking sequences within an endogenous genomic context.