Single-molecule imaging reveals DNA shape read-out by the INO80 chromatin remodeler.

ATP-dependent chromatin remodeling enzymes act together to construct complex chromatin architectures around functionally important regulatory regions like promoters and origins of replication (ORI). The INO80 complex accurately positions nucleosomes bordering these regions, which is crucial for proper transcription start site selection and efficient replication. How INO80 localizes these nucleosomes and regulates its remodeling activity to produce specific positions mechanistically is unclear, although recent findings suggest a role for DNA shape features and barrier factors. Here, we use single-molecule DNA curtains to directly observe interactions of INO80 with DNA, nucleosomes and barrier factor Reb1. We show that DNA shape features that are enriched in promoters and ORIs strongly regulate INO80 DNA binding and remodeling activity. Moreover, we find that INO80 performs 1D searches and intersegmental transfers to dynamically interact with nucleosomes and Reb1, but cannot bypass them, indicating that they could confine INO80 within promoters and thereby increase engagement with flanking nucleosomes. Our findings reveal how INO80's target-site search and remodeling activity are influenced by DNA shape recognition and molecular architecture and elucidate how remodelers can integrate different kinds of information to transform the chromatin landscape.