Fig. 4

RAD21 up-regulation affects genome contacts by stimulating cohesin activity. A Hi-C interaction frequency as a function of logarithmically increasing genomic distance bins for cells with and without RAD21 over-expression. B Hi-C contact matrices of chromosome 2 (0–100 Mb) in control and RAD21-OE HeLa cells. Direct minus between RAD21-OE and control matrices is on the right. C Normalized Hi-C interaction matrices for chromosome 5 (80–160 Mb) in control and RAD21-OE cells, and differential matrices of genomic regions between control and RAD21-OE cells (resolution: 150 kb). Below the heatmaps are PC1 values and gene density plots. Orange represents compartment A and blue represents compartment B. High gene density regions correlate with compartment A. D Ratios of inter-compartment interactions (AB) and intra-compartment interactions (AA + BB) for each chromosome (X chromosome excluded) in control and RAD21-OE cells (***P < 0.001, wilcoxon.test). E Average contact frequency enrichment showing the extent of compartmentalization in control and RAD21-OE cells. Direct minus between RAD21-OE and control matrices is on the right. F Genome-wide summary of genomic regions switching between A/B compartments in control and RAD21-OE cells. G Example immunofluorescence images of control and RAD21 over-expression HeLa cells using anti-H3K27me3 (Green) and H3K4me3 (Red) (scale bar is 5 μm). Transfected HeLa cells are used for the image without FACS sorting RAD21-GFP positive cells. 2 independent experiments. Deconvolution was applied to improve the imaging resolution and signal to noise ratio. H Example immunofluorescence images of control and RAD21 over-expression HeLa cells using anti-HP1α (Red) (scale bar is 5 μm). 2 independent experiments. Deconvolution was applied to improve the imaging resolution and signal to noise ratio. I Hi-C contact matrices for a zoomed in region on chromosome 2. Matrices are normalized to 160 million contacts, shown resolution is 40 kb. IS, insulation score, shows TAD pattern and insulation score distribution. Above and to the left of the contact matrices the union of CTCF sites identified in wild-type are shown. Red and blue triangles denote forward and reverse CTCF sites, respectively. Black histogram is CTCF ChIP-seq of wild-type HeLa. J Aggregate TAD analysis (ATA) calculates the average Hi-C signal across a selected set of TADs. The differential ATA signal between RAD21-OE and control is visualized for all TADs in the size range > 200 kb. Blue indicates a higher signal in the control, red indicates a higher signal in RAD21-OE cells. K Contact frequency ratio of intra-TAD and inter-TAD (TAD score) (***P < 0.001, paired t-test)