Spatial organization of centromeres is cell-type specific in human cell lines.

a, Representative images of CENP-C (green) and DAPI (gray) stained nuclei in indicated human cell lines. Scale bar: 10 µm. b, c, Spatial organization of centromere quantified using Ripley K’s clustering score (b), CENP-C spot count (c). d, Nuclear area and e, mean radial distance in human cell lines. Statistical significance of difference between cell lines for clustering score, spot count, mean radial distance and nuclear area was tested using ANOVA (p-value or ‘Pr(>F)’ < 2e-16) following Tukey’s HSD test to compare means of all pairs of cell lines. Box plots represent the inter-quantile range (IQR) between first and third quantile (box), the median (horizontal bar), and the whiskers extend to the highest or lowest data point up to 1.5 times of IQR. Values are from one representative experiment with at least 7 technical replicates. At least 1000 cells were analyzed in each category per experiment.

Identification of the molecular determinants of spatial centromere distribution across cell types.

a, Schematics showing three stages of high-throughput imaging based arrayed CRISPR knockout screen employed to identify molecular determinants of spatial centromere distribution. b, Changes in spot count (mean Z-score of two replicates, y-axis) and clustering score (mean Z-score of two replicates, x-axis) for each of the 1064 sgRNAs. The most prominent hits were labelled and color coded as in Fig. 2d. Non-hits are colored in gray. c, Changes in clustering score in HCT116 (mean Z-score of two replicates, x-axis) and in RPE1 (mean Z-score of two replicates, y-axis) cells for each of the 1064 sgRNAs. Hits and non-hits are color coded and labelled as in b. A linear trend line (gray) was fitted to the data and Pearson’s correlation coefficient calculated is indicated at the top left corner of the plot. d, Network diagram with lines between 52 common hits drawn based on known physical and/or genetic interactions generated by the STRING database. Thickness of the lines indicates higher strength of data supporting the interaction. Broad categories are color coded as indicated. e, Changes in clustering (clustered or unclustered), count (higher or lower) and direction between two cell lines (same or opposite) for each of the common genes color coded based on their category as in Fig. 2d. Counts of genes in each subcategory are indicated. Values represent two biological replicates. Typically, 200-500 cells were analyzed for each target gene per experiment.

Changes in spatial organization of centromeres require progression through the cell cycle.

a, HCT116 and RPE1 cells at G1, S and G2/M phases stained with CENP-C (green), DAPI (gray) and EdU (red). Scale bar: 10 µm. b, EdU intensity (y-axis) and DAPI intensity (x-axis) showing separation between G1 (brown), S (gray), and G2/M (green) sub populations in cycling HCT116 cells. Comparison of cells at G1, S or G2/M stages (x-axis) for their clustering score (c), spot count (d), mean radial distance (e), or nuclear area (f). Statistical significance of differences was tested by pairwise t-test with Bonferroni correction. Asterisks indicate level of significance between a given pair reflecting the corresponding p-value of that comparison. g, A linear regression line (red) fitted through the single cell data for nuclear area (y-axis) and clustering score (x-axis) in cells at different cell cycle phases in HCT116 and RPE1 cells. Pearson correlation coefficient and respective adjusted p-values are indicated at the top of each panel. h, Experimental outline to test cell cycle stage-specific effect of knocking down select hits. i, Effect of siRNA knockdown for a panel of genes (x-axis) using three individual siRNAs per gene in HCT116 cells that are either arrested at G/S and G2 or cycling. Two control siRNAs for siNCAPH2 are in blue and siScrambled in yellow. Mean value for siScrambled is depicted by a horizontal yellow line. Statistical significance of differences was tested by performing pairwise t-tests with Bonferroni correction using siScrambled as control group. Box plots represent the inter-quantile range (IQR) between first and third quantile (box), the median (horizontal bar), and the whiskers that extend till the highest or lowest value up to 1.5 times of IQR. Values are from one representative experiment. Typically, 200 to 500 cells were analyzed in each category.

Progression through S-phase in the absence of select clustering factors does not alter interphase genome organization.

a, Experimental outline to compare centromere distribution during progression through S-phase in the presence or absence of clustering factors. b, c, Clustering score in all cells (b) or G2/M cells (c) in the presence (blue) or absence (red) of indicated clustering factors before and after S-phase release from G1/S arrest. Pair-wise comparisons were tested with t-test with Bonferroni correction and the level of significance is indicated by asterisks if any. Pairs without significant difference are not labelled. Box plots represent the inter-quantile range (IQR) between first and third quantile (box), the median (horizontal bar), and the whiskers that extend till the highest or lowest value up to 1.5 times of IQR. Values are from one representative experiment with three technical replicates. Typically, 200 to 500 cells were analyzed in each category.

Orderly progression through mitosis is required to re-establish centromere distribution.

a, Experimental outline to compare centromere distribution during mitotic progression in the presence or absence of clustering factors. b, c, Clustering score in all cells (b) or G1 cells (c) in the presence (blue) and absence (red) of indicated clustering factors before (0 h) and after (6 hrs) mitotic release from G2 arrest. Pair-wise comparisons were tested with t-test with Bonferroni correction and the level of significance is indicated by asterisks. Pairs without significant differences are not labelled. d, Representative images showing G1 nuclei stained with DAPI (gray) and CENP-C (green) in the presence or absence of indicated factors. Scale bar: 10 µm. e, Schematics for co-depletion of indicated factors. f, Clustering score (y-axis) in G1 cells after siRNA knockdown of indicated factors (x-axis) in presence (blue) or absence (red) of SPC24, KI67 or NCAPH2 as indicated. g, Clustering score (y-axis) in cells that were depleted (red) or depleted of indicated factors and then re-expressed (green) or remained unperturbed (blue). Box plots represent the inter-quantile range (IQR) between first and third quantile (box), the median (horizontal bar), and the whiskers that extend till the highest or lowest value up to 1.5 times of IQR. Values are from one representative experiment containing three technical replicates. Typically, 200 to 500 cells were analyzed per experiment.

Mitotic events shape interphase genome organization.

A model comparing events of normal and abnormal mitosis where defective loading of outer kinetochore (inset; green and red box) in late G2 leads to uncoordinated metaphase alignment and aberrant migration towards the spindle poles during anaphase that lowers chances of interactions between centromeres in the newly forming daughter nuclei during telophase and the lack of homotypic adhesion among centromeres contribute to further dispersion.