Chromatin insulator mechanisms ensure accurate gene expression by controlling overall 3D genome organization

Chromatin insulators are DNA–protein complexes that promote specificity of enhancer–promoter interactions and maintain distinct transcriptional states through control of 3D genome organization. In this review, we highlight recent work visualizing how mammalian CCCTC-binding factor acts as a boundary to dynamic DNA loop extrusion mediated by cohesin. We also discuss new studies in both mammals and Drosophila that elucidate biological redundancy of chromatin insulator function and interplay with transcription with respect to topologically associating domain formation. Finally, we present novel concepts in spatiotemporal regulation of chromatin insulator function during differentiation and development and possible consequences of disrupted insulator activity on cellular proliferation. •Collision of cohesin with CTCF during loop extrusion can result in backtracking.•Drosophila chromatin insulator proteins act redundantly to form TAD borders.•Chromatin insulators and transcription together contribute to Drosophila TADs.•Delayed loop extrusion due to CTCF collision may serve as a developmental timer.