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 high-quality image (215 K)Download as PowerPoint slide