Fig. 5

Insulators for enhancers with no CTCF site. a Schematic of the HS5-1 CBS elements as an insulator of the HS7 enhancer for the Pcdhβ genes. b 5C interaction profiles of the Pcdh α and β clusters in the HS5-1 deletion (del) or inversion (inv) mice in vivo. The log2 ratios of chromatin interactions of HS5-1 with Pcdhα and of HS7 with 5′ isoforms of the Pcdhβ cluster are highlighted by blue or black rectangles, respectively. Note the significant increase of chromatin interactions between HS7 with 5′ isoforms of the Pcdhβ cluster upon HS5-1 deletion as indicated by the enlargement of insets C and D. c QHR-4C with HS7 or β3 as a viewpoint confirms the increased interactions between HS7 and 5′ isoforms of the Pcdhβ cluster in homozygous HS5-1 deletion mice. d QHR-4C with HS7 or β3 as a viewpoint confirms no significant alteration of interactions between HS7 and 5′ isoforms of the Pcdhβ cluster in homozygous HS5-1 inversion mice. e RNA-seq of cortical tissues of the WT and HS5-1 deletion mice. f RNA-seq of cortical tissues of the WT and HS5-1 inversion mice. g CTCF and Rad21 ChIP-seq of human single-cell β-globin CRISPR clones with insertion of a pair of reverse-forward CBS elements (“RF2”). h QHR-4C profiles with the human β-globin HBG2 promoter as a viewpoint. i RNA-seq reveal decreased expression levels (normalized to WT) of the human β-globin repertoire. The actual expression levels are shown in the inset. Data as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-tailed Student’s t test. j The pairwise contingency tables showing interrelationships of each pair of variables among genome-wide insulator strength, promoter activity, and enhancer and looping strength. k The optimal structure of relationships among genome-wide insulator strength, promoter activity, enhancer and looping strength during human epidermal differentiation learned by Bayesian networks. l Directional CTCF looping underlies stochastic monoallelic Pcdh α and βγ gene expression and balanced promoter usage. In particular, stochastic and monoallelic CTCF-mediated directional chromatin looping underlies activation of one and only one variable promoter in each chromosome in the Pcdhα cluster, while up to 4 promoters are activated in each chromosome in the Pcdhβ/γ clusters. m A polymer simulated 3D Hulu (gourd) model of tandem CTCF sites topologically balancing spatial chromatin contacts and enhancer-promoter selection. n Mechanistic interpretation of the 3D Hulu model in the context of bidirectional cohesin “loop extrusion” through tandem CTCF sites