High-throughput gene screen reveals modulators of nuclear shape

Abstract

Irregular nuclear shapes characterized by blebs, lobules, micronuclei, or invaginations are hallmarks of many cancers and human pathologies. Despite the correlation between abnormal nuclear shape and human pathologies, the mechanism by which the cancer nucleus becomes misshapen is not fully understood. Motivated by recent evidence that modifying chromatin condensation can change nuclear morphology, we conducted a high-throughput RNAi screen to identify epigenetic regulators that are required to maintain normal nuclear shape in human breast epithelial MCF-10A cells. We silenced 608 genes in parallel using an epigenetics siRNA library and used an unbiased Fourier analysis approach to quantify nuclear contour irregularity from fluorescent images captured on a high-content microscope. Using this quantitative approach, which we validated with confocal microscopy, we significantly expand the list of epigenetic regulators that impact nuclear morphology.

FIGURE 1:

Quantification of nuclear contour irregularities with elliptical Fourier analysis. (A) Example images of single nuclei from nontransformed human MCF-10A mammary epithelial cells. Images of nuclei are arranged in descending order of regularity, from left to right. Scale bar is 5 µm. (B) Representative images of the detected nuclear contour in A overlaid onto the original raw image; nuclear contours were detected using MATLAB image processing algorithms. (C) Nuclear contours shown in B without the overlay of the original image (blue). (D) Elliptic Fourier approximations of the nuclear contours from C, calculated using 15 elliptic harmonics to fit each nuclear shape (Diaz et al., 1989; Lammerding et al., 2006). (E) Overlays of the detected nuclear contour and the elliptic Fourier approximation. (F) Representative EFC ratios (computed in MATLAB) and solidity measurements (where solidity = area/convex area; computed in ImageJ) for each nuclear shape depicted in A. (G) Quantification of the RMS error between the elliptic Fourier approximation and the nuclear boundary; each data point corresponds to a single nucleus (N = 3, n = 20 where N represents the number of biological replicates and n represents the total number of cells analyzed). (H) EFC ratio plotted against RMS error for each nucleus in (G; N = 3, n = 20). Error bars denote SEM. (I) Representative image after segmentation and quantification of EFC ratio. White outlines represent the detected nuclear boundary; green numbers overlaid onto nuclear centroids represent the EFC ratio for each respective nucleus. Scale bar is 20 µm. (J) Representative images of MCF-10A human breast epithelial cells and MDA-MB-231 human breast adenocarcinoma cells taken at 40× on a high-content microscope. Scale bar is 25 µm. (K) Mean EFC ratio and adjusted solidity (defined as [(1-solidity)*100]) for MCF-10A and MDA-MB-231 cells (N ≥ 15 and 3, n = 71,191 and 6952 for MCF-10A and MDA-MB-231, respectively). Error bars represent SEM. *p < 0.05 by the Bonferroni-corrected nonparametric Dunn’s test.

FIGURE 2:

High-throughput nuclear imaging screen identifies epigenetic regulators required for maintenance of normal nuclear shape. (A) Schematic workflow of the siRNA screen for nuclear irregularities. (B) The efficacy of siRNA-mediated knockdown of expression in a subset of genes from the screen identified to significantly decrease EFC ratio, selected randomly, assayed with probe-based RT-qPCR. (C) The efficacy of siRNA-mediated knockdown of expression in a subset of genes from the screen which had no significant effect on EFC ratio, selected randomly, and assayed with probe-based RT-qPCR. Data for both B and C was first internally normalized to GAPDH expression, then externally normalized against a scrambled siRNA negative control, and subsequently analyzed using the 2^-∆∆Ct method. Data represent mean of each condition from at least three biological replicates. Error bars represent SEM. (D) Results of the high-throughput screen for nuclear shape showing mean EFC ratio for each gene depletion condition plotted against arbitrary gene number (N = 3, n ≥ 216 for each condition). For D–F, gray data points represent gene depletions determined to have a statistically insignificant effect on the plotted parameter by the two-tailed Bonferroni-corrected nonparametric Dunn’s test; orange data points represent gene depletions which produce significant changes to nuclear shape (p < 0.05; all comparisons relative to the scrambled siRNA negative control). (E) Mean EFC ratio plotted against mean 2-D nuclear X-Y aspect ratio (length of major axis/length of minor axis) for each gene condition in the high-throughput screen (N = 3, n ≥ 216 for each condition). (F) Mean EFC ratio plotted against mean nuclear cross-sectional area (N = 3, n ≥ 216). Error bars represent SEM.

FIGURE 3:

Confocal screen of selected genes from the high-throughput screen. (A) Mean EFC ratio plotted against mean 3-D nuclear volume. Nuclear volume was calculated from 3-D reconstructions created in ImageJ from x-y confocal scans at discrete z-focal planes, as has previously been described (Tocco et al., 2018). (B) Mean EFC ratio plotted against mean 2-D nuclear X-Y aspect ratio. (C) Mean EFC ratio plotted against mean nuclear cross-sectional area for a subset of genes screened by confocal microscopy. Gray data points represent gene depletions which were determined to have a statistically insignificant effect on EFC ratio by the two-tailed Bonferroni-corrected nonparametric Dunn’s test; orange data points represent gene depletions which had a statistically significant effect on EFC ratio (p < 0.05; all comparisons relative to the scrambled siRNA negative control). Blue data point denotes the scrambled siRNA negative control. Error bars are SEM. N = 3, n ≥ 127 for all data shown. (D) Representative images of the nucleus for chosen gene depletions. MTA2, TAF7, FOXP1, SUZ12, DIDO1, T53, and ARID4A were chosen based on statistically significant low EFC ratios caused by the corresponding siRNA transfection in the confocal screen. Scale bar is 25 µm. The EFC ratio in the upper left hand corner of each image panel represents the mean EFC ratio for that gene depletion condition, plus or minus the SEM (N = 3 for all gene depletion conditions; n = 478, 187, 268, 159, 178, 146, 241, and 176 for Scrambled, MTA2, TAF7, FOXP1, SUZ12, DIDO1, T53, and ARID4A, respectively).