Extended Data Fig. 2: HiDRO screen validated hits are region non-specific regulators of TAD boundaries, Related to Fig. 2.
From: High-throughput Oligopaint screen identifies druggable 3D genome regulators
a) Protein classes of genes in the Druggable Genome library. Targeted genes encode proteins across diverse classes including kinases, membrane and extracellular matrix proteins, and proteases. n = 3083. b) Protein class designations of all primary hits from Druggable Genome HiDRO screen (n = 58). c) Protein class designations of primary hits that increase inter-TAD interactions (n = 33). d) Protein class designations of primary hits that decrease inter-TAD interactions (n = 25). e) Representative images of chr22:D1-D2 for hits altering inter-TAD interactions. Solid white line indicates nuclear edge. Scale bar nucleus, 5 µm; Scale bar spots, 1 µm. Each gene was tested in two biological replicates. f) Correlation heatmap of 21 image-based phenotypes with colour-coded squares outlining the five measurement categories used for the phenotypic tree in Fig. 2f. The five categories are overlap metrics, domain area, domain shape, nuclear area and nuclear shape. g) Histogram of nuclear area for non-targeting control wells from replicate 1 of Fig. 1b data. Black lines denote the 20th (142 µm2) and 30th percentiles (170 µm2) of nuclear area, representing G1 nuclei. Red lines denote the 70th (283 µm2) and 80th percentiles (354 µm2) of nuclear area, representing G2 nuclei. n = 128 wells. h) Violin plot of D1 spots detected per nucleus per well in G1 and G2 nuclei. Solid line is median, dotted lines are 25th and 75th percentiles. [Data from n = 128 wells for each bar.] i) Violin plot of mean CCD per well in G1 and G2 nuclei. Solid line is median, dotted lines are 25th and 75th percentiles. [Data from n = 128 wells for each bar.] ns P-value > 0.05, two-tailed t-test. j)Violin plot of mean D1 overlap per well in G1 and G2 nuclei. Solid line is median, dotted lines are 25th and 75th percentiles. [Data from n = 128 wells for each bar.] ns P-value > 0.05, two-tailed t-test. k) Validation HiDRO screen workflow tests each primary hit with four independent siRNA duplexes in separate wells, then applies DNA FISH to chr22 domains D1 and D2. l) Hi-C contact matrix and Oligopaint design for three adjacent TADs on chr3. Hi-C data from ref. 4. Fold change in spatial overlap between chr3 D1 and D2 for top hits. [Data from one well per condition of HiDRO experiment; Number of Alleles for Control (n = 1,075), WAPL (n = 426), GSK3A (n = 855), CALM1 (n = 1,321), FBXL14 (n = 1,184)]. *** P-value < 0.001, **** P-value < 0.0001, two-tailed Mann-Whitney U test. m) Heatmap displaying fold change in CCD at 13 boundaries across the human genome for WAPL, GSK3A, CALM1 and FBXL14 KD. Each boundary was tested with 3-4 biological replicates per gene KD. n) Fold change in CCD versus insulation score at boundary for top siRNA KD. For each graph, x-axis is insulation score of the boundary. Insulations scores from ref. 4. Y-axis is fold change in centre-centre distance between domains relative to control. Each point is the mean of 4 biological replicates except WAPL KD at insulation scores 91, 119, 142, 149, 195 (n = 3 biological replicates) error bars are +/− SEM. o) Number of significantly altered boundaries as measured by D1 overlap and D2 overlap for different gene KD.
