CTCF Mediates Dosage and Sequence-context-dependent Transcriptional Insulation through Formation of Local Chromatin Domains [Hi-C and PLAC-seq]

Insulators play a critical role in spatiotemporal gene expression in metazoans by separating active and repressive chromatin domains and preventing inappropriate enhancer-promoter contacts. The evolutionarily conserved CCCTC-binding factor (CTCF) is required for insulator function in mammals, but not all of its binding sites act as insulators. Here, we explore the sequence requirements of CTCF-mediated transcriptional insulation with the use of a sensitive insulator reporter assay in mouse embryonic stem cells. We find that potent insulation is provided by multiple CTCF binding sites. Furthermore, CTCF-mediated insulation is dependent on DNA sequences adjacent to its core binding motifs, and CTCF binding sites from topologically associating domains (TAD) boundaries are more likely to function as insulators than those outside TAD boundaries independent of binding strength. Using chromosomal conformation capture assays and high resolution chromatin imaging techniques, we demonstrate that insulators reduce enhancer-promoter contacts by forming local chromatin domains. Taken together, our results provide strong genetic, molecular and imaging evidence connecting chromatin topology to action of insulators in the mammalian genome. Overall design: Examination of insulator activity of CTCF binding seqences and their effects on chromatin topology in mouse embryonic stem cells. Chromosome coordinates of variants: '4CBS' was inserted between chr3:34,726,762-34,727,258; mm10 '4CBS downstream' was inserted between chr3:34,794,087-34,794,154; mm10.