SRA

By CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CBSs, if located between enhancers and promoters in the clustered Pcdh and b-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CBS or not. Moreover, computational simulation in silico and genetic deletions in vivo revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem variable CBSs. Finally, gene expression levels are negatively correlated with CBS insulators located between enhancers and promoters on a genome-wide scale. Thus, single CBS insulators ensure proper enhancer insulation and promoter activation while tandem-arrayed CBS insulators determine balanced promoter choice. This finding has interesting implications on the role of topological insulators in 3D genome folding and developmental gene regulation. Overall design: To investigate the effects of CTCF sites on 3D chromatin conformation and insulation mechanisms, we inserted CTCF sites with different orientations into Pcdhalpha cluster. In addition, to pinpoint the effects to CTCF binding sequence, we mutated 5 bp of the binding site and inserted it into the same location as a control. After screening of the single-cell clones, we performed circular chromosome conformation capture experiments with enhancer or promoter as anchors in the CBS insertion and mutation single-cell clones.