Motif grammar and transcriptional programs decouple CTCF binding from nucleosome phasing to control cell-type-specific chromatin insulation.

CTCF organizes the genome via cohesin-mediated loop extrusion, insulating topologically associating domains (TADs) and constraining enhancer-promoter communication. Yet, CTCF binding varies across cell types and genomic contexts. In mouse, typically accessible sites combine strong motifs with higher CTCF/cohesin occupancy, while inaccessible sites use weaker motifs stabilized by flanking upstream (U) and downstream (D) sequences that engage peripheral zinc fingers to respectively enhance or dampen binding. Notably, TF motifs within ±35 bp bind cooperatively or competitively depending on expression and positional overlap, a finding supported by AlphaFold predictions and allele-specific perturbations. Favorable motif/TF environments can increase CTCF signal yet disrupt nucleosome phasing and weaken insulation, showing that binding strength and insulation can be uncoupled. Single-molecule maps show that nucleosome phasing regularity predicts insulation strength more reliably than CTCF peak intensity. Thus, differences in motif architecture and TF abundance between species and cell states provide a mechanism for cell-type-specific regulation of CTCF-mediated chromatin insulation.