Fig. 4: Casilio visualization of RAD21-dependent and -independent interactions in the presence and absence of RAD21. | Nature Communications

Fig. 4: Casilio visualization of RAD21-dependent and -independent interactions in the presence and absence of RAD21.

From: CRISPR-mediated multiplexed live cell imaging of nonrepetitive genomic loci with one guide RNA per locus

Fig. 4

a Schematic of Casilio imaging of chromatin interactions between IER5L promoter (IER5L-P) and a super enhancer (IER5L-SE) in HCT116/RAD21-mAID cells. Hi-C map shows interactions in this region in HCT116/RAD21-mAID cells derived from a previous study29. Upper right off-diagonal and lower left off-diagonal plots pairwise interaction between genomic bins in untreated and auxin-treated cells, respectively. Dotted squares mark the area of interest. Chr, chromosome. Mb, megabase. b, c Representative images of IER5L-P and IER5L-SE labeled by Clover (green, stealth arrowhead), and iRFP670 (red, triangle arrowhead), respectively, in nuclei of HCT116/RAD21-mAID cells without (b, see Supplementary Video 2) or with auxin treatment (c, see Supplementary Video 3). Scale bars, 5 μm. Experiments repeated 7 times. d Boxplot of pairwise spot distances in untreated (n = 1325 measurements of 33 pairs from 18 nuclei; mean = 1.52 μm; median = 1.21 μm) and auxin-treated (n = 1435 measurements of 35 pairs from 20 nuclei; mean =  2.26 μm; median = 2.09 μm) cells. Boxplot displays a median line, mean x-marked, interquartile range boxes, and min to max whiskers. ***Mann-Whitney test two-sided p = 1.69 × 10−53 < 0.00001. See Supplementary Figs. 4, 5 for all distance plots of imaged nuclei. e Hi-C map showing the pairwise interaction between genomic bins encompassing a RAD21-independent loop in untreated (upper right off-diagonal) and auxin-treated (lower left off-diagonal) cells. Dotted squares mark the area of interest. f, g Representative images of the RAD21-independent interaction labeled by Clover (green, stealth arrowhead), and iRFP670 (red, triangle arrowhead), respectively, in nuclei of HCT116/RAD21-mAID cells without (f, see Supplementary Video 4) or with auxin treatment (g, see Supplementary Video 5). Scale bars, 5 μm. Experiment repeated 4 times. h Boxplot of pairwise spot distances in untreated (n = 1046 measurements of 27 pairs from 18 nuclei; mean = 1.44 μm; median = 1.24 μm) and auxin-treated (n = 921 measurements of 25 pairs from 20 nuclei; mean =  0.76 μm; median =  0.57 μm) cells. Boxplot displays a median line, mean x-marked, interquartile range boxes, and min to max whiskers. ***Mann-Whitney test two-sided p = 1.81 × 10−108 < 0.00001. Source data are provided as a Source Data file.

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