Global dynamic cohesin binding at active promoters correlates with H3K27 acetylation, but not gene expression, during erythroid differentiation. (A) Experimental system to determine the role of cohesin during normal erythroid differentiation. (B) Left: average profile for Rad21 binding at active promoters in the HPC (blue) and erythroid (red) cellular states. Right: representation of the erythroid differentiation tiers; density heat maps of Rad21-binding sites from a representative experiment (of 2) at active promoters that were split into 4 equal tiers, based on the incremental change of Rad21 binding during erythroid differentiation. (C) Box plots and 10th through 90th percentiles of the differences of Rad21 binding in erythroid cells and HPC (Rad21 Ery − Rad21 HPC) in the indicated erythroid differentiation tiers. Shown are means from 2 replicates. Box plots to the left of 0 indicate increased binding in HPC and to the right indicate increased binding in erythroid differentiating cells. (D) Box plots and 10th through 90th percentiles of the differences of H3K27ac binding for the same regions as in panel C. Shown are means from 2 replicates. (E) Box plots and 10th through 90th percentiles of the differences of significant interaction frequencies of the baits located at the promoters of the same regions as in panel C. Interactions were determined with the CHICAGO (Capture HiC Analysis of Genomic Organisation) pipeline. The threshold for significance was set at 5. Shown are results from 2 replicates. (F) Volcano plot showing gene expression changes during HPC to erythroid differentiation (3 biological replicates, Luc1, Luc2, and Luc3, were used per group for consistency with later experiments). The blue shaded area consists of genes enriched in HPC (immature genes), whereas the red shaded area shows genes whose expression significantly increased in the erythroid cell state (erythroid genes). (G) Venn diagrams of overlaps between annotated promoters from the erythroid differentiation tiers (center circle) and differentially expressed genes from panel F (outer circles). χ² analysis was performed for the trend of overlapping events across all tiers. (H) Examples of Rad21 and H3K27ac binding dynamics (upper), as well as interaction frequencies (lower arcs) between HPC and erythroid cellular states for a representative erythroid gene (Epor) and an immature gene (Etv5).