Kaiso reads methylated CpGs at nucleosome entry/exit and displaces the H3 tail

Abstract The zinc finger transcription factor Kaiso recognizes methylated CpG dinucleotides at silenced promoters and imprinted loci, but how it engages methylated DNA within the nucleosome remains unclear. To address this, we developed a DNMT1-based strategy for preparing site-specifically methylated nucleosomes with defined position and methylation state of the Kaiso recognition motif. Electrophoretic mobility shift assays show that Kaiso binds methylated nucleosomes with strong positional preference, with high-affinity engagement at the entry/exit site (SHL 6.5; Kd ≈ 100 nM), reduced affinity at SHL 5.5 (Kd ≈ 170 nM), and no methylation-dependent enhancement at dyad-proximal positions. Hemi- and fully methylated substrates bind Kaiso comparably at SHL 6.5, and the E535A mutation, which disrupts a key methyl-CpG contact, reduces binding in a methylation- and position-dependent manner. Solution NMR titrations of 15N-labeled H3 nucleosomes reveal that Kaiso binding perturbs a discrete set of H3 N-terminal tail residues, with chemical shifts trending toward free-peptide values, indicating release of the tail from its nucleosomal DNA contacts. This pattern closely resembles that produced by the pioneer factor Sox2 at the same nucleosomal region, suggesting H3 tail displacement is a general consequence of factor engagement at the nucleosome edge, independent of DNA-recognition mode. These results establish Kaiso as an active reader of methylated nucleosomal DNA that may prime local chromatin by exposing the H3 tail. Competing Interest Statement The authors have declared no competing interest.