CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function

•The orientation of Pcdh CBSs determines the direction of topological DNA looping•Directional CTCF binding to CBSs is crucial for loop topology and gene expression•The CTCF binding orientation functions similarly in β-globin and the whole genome•CTCF/cohesin-mediated directional DNA-looping determines chromosome architecture

SummaryCTCF and the associated cohesin complex play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and β-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context, the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. These findings reveal how 3D chromosome architecture can be encoded by linear genome sequences.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (215 K)Download as PowerPoint slide