Fig. 3: Retention elements promote extrachromosomal interactions with chromosomes during mitosis.

a, Schematic of the live-cell imaging experiment. b, Representative live-cell time-lapse images of dividing COLO320DM cells with labelled ecMYC after transfection with a plasmid containing a retention element or an empty vector control. Scale bar, 10 µm. c, Fraction of DNA signals not colocalizing with mitotic chromosomes during anaphase. n = 51 (control), n = 83 cells (retention element). Box plot parameters are as described in Fig. 2. d, Individual (left) and mean (right) cell trajectories of DNA signal colocalization with chromosomes throughout mitosis. n = 42 (control), n = 45 (retention element) cells. Mean cell trajectories include all time points with >3 cells. Error bars show the s.e.m. Vertical dashed lines indicate anaphase. e, Hi-C interaction maps in asynchronous or mitotically arrested COLO320DM cells. Numbers at bottom right below far right plots indicate maximum count values in corresponding color scales. Density plots show flow cytometry analyses of DNA content. f,g, APA of Hi-C data of asynchronous (f) and mitotically arrested (g) COLO320DM cells. Heatmaps are summed percentile matrices of pairwise interactions between chromosome bookmarked regions and a combined set of ecMYC retention elements with 5-kb resolution. h, Hi-C heatmap of pairwise interactions in mitotically arrested COLO320DM cells between ecMYC retention elements and chromosome bookmarked regions with ENCODE cCRE annotations. i, Mitotically bookmarked regions that overlap with retention elements or matched-size random genomic intervals. j, Cumulative distribution of retention elements that contain binding sites of bookmarking factors, ordered by factor enrichment relative to random genomic intervals. k, ecDNA–chromosome interactions recapitulate enhancer–promoter interactions. Gene expression in interphase cells is activated by an interaction between enhancer (blue) and promoter (red) sequences on the same chromosome. We propose that ecDNA retention in mitotic cells is mediated by an analogous intermolecular contact between promoter-like retention elements (red) on ecDNA and enhancer-like, or less commonly, promoter-like bookmarked sites (blue) on the chromosome. P values were calculated using two-sided Wilcoxon rank-sum tests (c), two-sided paired t-tests (d) or two-sided Fisher’s exact tests (i).