human colorectal cancer cell lines colo320dm  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: The prometaphase spread technique untethers ecDNA and chromosomes by decompacting mitotic chromatin. A , schematic of an ecDNA-containing cell undergoing mitosis (from left to right : prophase, metaphase, anaphase, telophase). Acentric ecDNA ( red ) have been observed to tether to or “hitchhike” on chromosomes ( blue ) during mitosis to ensure their proper segregation and inheritance by daughter cells. B , prometaphase spreads performed on colcemid-arrested COLO320DM cells with the conventional hypotonic solution (75 mM KCl) and higher osmolarity solutions (100 mM and 125 mM KCl), producing varying amounts of chromosome (DAPI, blue ) individualization and ecDNA ( MYC FISH, red ) untethering. C , quantification of panel B . Left : boxplots quantifying chromosome individualization in prometaphase spreads performed with varying solution osmolarity; from left to right , n = 3, 3, 3 biological replicates and 95, 92, 105 cells; one-way ANOVA, F = 100.7, p < 0.001; ∗∗ p < 0.01 by Tukey’s honestly significant difference (HSD), ns = not significant. Right : quantification of ecDNA untethering; one-way ANOVA, F = 48.3, p < 0.001. Chromosome individualization is represented by the number of connected components identified as chromosomes by ecSeg. ecDNA untethering is represented by the number of ecDNA unattached to chromosomes divided by the total number of ecDNA not completely surrounded by chromosomes. D , prometaphase spreads performed with incubation in 1× PBS using COLO320DM cells pretreated for 8 h with vehicle (0.1% DMSO) or varying concentrations of Trichostatin A (TSA). E , quantification of panel D . Left : quantification of chromosome individualization in prometaphase spreads performed with 1× PBS on cells pretreated with TSA; n = 3, 3, 3, 3, 3, 3 biological replicates and 88, 113, 96, 80, 76, 84 cells; one-way ANOVA, F = 38.5, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant. Right : quantification of ecDNA untethering; one-way ANOVA, F = 166.5, p < 0.001. F , linear regression analysis of median chromosome individualization v er s us ecDNA untethering of prometaphase spread conditions in panels B – E (n = 9). G , schematic: electrostatic/hydrophobic mitotic compaction forces at the level of nucleosomes tether ecDNA ( red ) to mitotic chromosomes ( blue ). In panels B–E , scale bar = 10 μm and each data point in graphs represents one cell.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Hypotonic conditions and HDAC inhibition untether ecDNA. A , fixed metaphase COLO320DM cells cultured on glass coverslips treated for 15 min with 1× media, 0.75× media (not shown), 0.5× media, 1:1 mix of 1× PBS with 1× media (1× PBS-media, not shown), and 1:1 mix of 1.5× PBS with 1× media (1.25× PBS-media); dashed outlines indicate metaphase plate chromosomes (DAPI, blue ), arrowheads indicate untethered ecDNA (identified by MYC FISH signal, red ). B , boxplots quantifying untethered ecDNA per cell from panel A , represented by % of MYC FISH signal in a cell unattached to chromosomes aligned at the metaphase plate (non-overlapping pixels); from left to right, n = 4, 6, 6, 7, 4 biological replicates and 427, 672, 691, 532, 686 cells; one-way ANOVA, F = 882.3, p < 0.001; ∗ p < 0.05, ∗∗ p < 0.01 by Tukey’s HSD. C , live imaging of COLO320DM cells expressing H2B-emiRFP670 (chromatin, blue ) and tetR-mNeonGreen which binds to a tetO-96mer repeat sequence inserted near MYC loci ( MYC , red ). Cells were arrested at metaphase with 10 μM MG132 and placed in hypotonic 0.5× media at t = 0 min to decompact chromatin; arrows indicate untethered ecDNA; after image acquisition at t = 15 min, cells were placed in relatively normotonic 1× PBS-media to recompact chromatin; arrows indicate ecDNA-ecDNA tethering. Representative images of n = 5 biological replicates and >50 cells. D , fixed metaphase COLO320DM cells cultured on glass coverslips treated for 8 h with vehicle (0.1% DMSO) or indicated concentrations of TSA; dashed outlines indicate metaphase plate chromosomes (DAPI, blue ), arrowheads indicate untethered ecDNA (identified by MYC FISH signal, red ). E , boxplots quantifying untethered ecDNA per cell from panel D ; from left to right , n = 3, 3, 3, 3 biological replicates and 98, 144, 161, 246 cells; one-way ANOVA, F = 34.9, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD. F , modified volcano plot summarizing the results of the targeted screen for drugs with the ability to untether ecDNA and chromosomes, quantified by the area of the convex hull of the prometaphase spread produced without hypotonic solution incubation. TSA (0.5 μM) was included as positive control. padj = adjusted two-tailed Student’s t test p value using Bonferroni multiple test correction (44 total comparisons were made). Each data point represents the average of all cells for each condition. G , metaphase COLO320DM cells cultured on glass coverslips treated for 24 h with vehicle (0.1% DMSO) or indicated concentrations of LMK235; dashed outlines indicate metaphase plate chromosomes, arrowheads indicate untethered ecDNA. H , quantification of untethered ecDNA per cell from panel G; n = 11, 3, 7, 4, 3, 3 biological replicates and 1278, 501, 875, 472, 571, 388 cells; one-way ANOVA, F = 735.1, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant. In all panels, scale bar = 10 μm. Except in panel F , each data point in graphs represents one cell.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Inhibition, Cell Culture, Imaging, Expressing, Sequencing, Modification, Produced, Incubation, Positive Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Hypotonic conditions and HDAC inhibition lead to mis-segregation of ecDNA into micronuclei. A , live cell imaging of COLO320DM cells undergoing mitosis (chromatin labeled by H2B-emiRFP670, blue ) and ( MYC loci labeled by tetR-mNeonGreen, red ). Mitotic cells were placed in 0.5× media at t = 0 min; dashed outlines indicate chromosomes/primary nuclei, arrowheads indicate untethered ecDNA, arrows indicate micronuclei. Representative image of n = 5 biological replicates and >50 cells. B , fixed newly-divided daughter COLO320DM cells, as identified by the presence of Aurora B staining, treated for 6 h with 1× media, 0.75× media (not shown), 0.5× media, 1× PBS-media (not shown), and 1.25× PBS-media; dashed outlines indicate primary nuclei, arrowheads indicate micronuclei. C , quantification of panel B . Left : quantification of the percentage of daughter cell pairs with micronuclei; chi-squared, ∗ p < 0.05, ∗∗ p < 0.01. Right : quantification of the percentage of all MYC FISH signal per daughter cell pair inside micronuclei as opposed to the primary nuclei (symlog scale, linear ≤2, log >2); one-way ANOVA, F = 151.4, p < 0.001, ∗ p < 0.05, ∗∗ p < 0.01; n = 3, 3, 3, 6, 4 biological replicates and 601, 233, 148, 881, 836 daughter cell pairs. D and E , fixed newly-divided daughter COLO320HSR cells, treated similarly as in panels B and C . E , left : chi-squared, ∗∗ p < 0.01, ns = not significant. Right : one-way ANOVA, F = 0.9, p = 0.49; n = 3, 3, 3, 5, 3 biological replicates and 680, 256, 179, 578, 487 daughter cell pairs. F , quantification of pan-Histone H3 acetylation (pan-H3ac) IF in cytospin preparations of COLO320DM prometaphase spreads with chromosome and ecDNA segmentation by ecSeg (see C ); cells were treated in vehicle or LMK235 for 24 h; n = 2, 2, 2, 2 biological replicates and 52, 44, 75, 84 cells; one-way ANOVA, chromosomes: F = 67.6, p < 0.001, ecDNA: F = 81.3, p < 0.001, ∗ p < 0.05, ∗∗ p < 0.01 by Tukey’s HSD. G , newly-divided daughter COLO320DM cells, as identified by the presence of Aurora B staining (not shown), treated for 24 h with vehicle (0.1% DMSO) or indicated concentrations of LMK235; dashed outlines indicate primary nuclei, arrowheads indicate micronuclei. H , quantification of panel G . Left : quantification of the percentage of daughter cell pairs with micronuclei; chi-squared, ∗∗ p < 0.01. Right : quantification of the percentage of all MYC FISH signal per daughter cell pair inside micronuclei (symlog scale, linear ≤2, log >2); one-way ANOVA, F = 151.4, p < 0.001, ∗ p < 0.05, ∗∗ p < 0.01; n = 5, 5, 4 biological replicates and 713, 683, 420 daughter cell pairs. I , same as panel F , for COLO320HSR cells (chromosomes only; see E ); n = 2, 2, 2, 2 biological replicates and 85, 85, 63, 99 cells; one-way ANOVA, F = 204.1, p < 0.001, ∗∗ p < 0.01 by Tukey’s HSD. J and K , newly-divided daughter COLO320HSR cells, treated similarly as in panels G and H. K , left : chi-squared. Right : one-way ANOVA, F = 0.1, p = 0.88; n = 4, 3, 3 biological replicates and 486, 383, 335 daughter cell pairs. In all panels, scale bar = 10 μm and each data point in graphs represents one cell or daughter cell pair.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Inhibition, Live Cell Imaging, Labeling, Staining

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Ki67 gain-of-function untethers ecDNA from chromosomes. A , fixed COLO320DM cells cultured on coverslip and incubated in 0.5× media for 15 min to visualize and colocalize individual ecDNA ( MYC FISH) with Ki67 IF ( arrowheads ). B , line profile of MYC FISH and Ki67 IF intensity along the line indicated in panel A; polynomial curves were fitted to the line profiles. C , metaphase COLO320DM cells cultured on glass coverslips 2 to 4 days post mCherry-only or Ki67-mCherry expression plasmid transfection; cells were categorized based on mCherry fluorescence: - indicates lack of mCherry expression, ++ indicates top 20% tile mCherry expression by fluorescence intensity, + indicates the remaining cells that express mCherry; dashed outlines indicate chromosomes aligned at the metaphase plate, arrowheads indicate untethered ecDNA. D , boxplots quantifying ecDNA untethering in plasmid transfected cells from panel C ; mCh = mCherry expression category, Plas = plasmid transfected; from left to right ; n = 4, 4, 4, 4, 4, 4 biological replicates and 20, 63, 22, 116, 221, 86 total cells; two-way ANOVA, mCherry expression category (- v er s us + v er s us ++): F = 123.9, p < 0.001; plasmid transfected (mCherry-only v er s us Ki67-mCherry): F = 40.5, p < 0.001; interaction: F = 33.8, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant. E , newly-divided daughter COLO320DM cells, as identified by the presence of Aurora B staining (not shown), 2 to 4 days post mCherry-only or Ki67-mCherry expression plasmid transfection (mCherry ++ cells are shown); dashed outlines indicate primary nuclei, arrowheads indicate micronuclei. F , quantification of panel E . Left : quantification of the percentage of daughter cell pairs with micronuclei; chi-squared, ∗∗ p < 0.01. Right: quantification of the percentage of all MYC FISH signal per daughter cell pair inside micronuclei (symlog scale, linear ≤2, log >2); two-way ANOVA, mCherry expression category (- v er s us + v er s us ++): F = 7.6, p < 0.001; plasmid transfected (mCherry-only v er s us Ki67-mCherry): F = 7.6, p = 0.0058; interaction: F = 4.9, p = 0.0073; ∗∗ p < 0.01; n = 7, 7, 7, 7, 7, 7 biological replicates and 93, 312, 104, 179, 400, 147 daughter cell pairs. G and H , newly-divided daughter COLO320HSR cells, transfected similarly as in panels E and F (mCherry ++ cells are shown). H , left : chi-squared, ∗ p < 0.05. Right : two-way ANOVA, mCherry expression category (- v er s us + v er s us ++): F = 0.7, p = 0.49; plasmid transfected (mCherry-only v er s us Ki67-mCherry): F = 0.1, p = 0.77; interaction: F = 0.9, p = 0.39; n = 4, 4, 4, 4, 4, 4 biological replicates and 168, 245, 107, 202, 227, 111 daughter cell pairs. In all panels, scale bar = 10 μm. Except in panel B , each data point in graphs represents one cell or daughter cell pair.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Cell Culture, Incubation, Expressing, Plasmid Preparation, Transfection, Fluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Ki67 loss-of-function decreases ecDNA untethering from chromosomes. A , metaphase COLO320DM wildtype (NT) and MKI67 knockout cells (gRNA #1 and #2), incubated for 15 min in 1× media (not shown), 0.75× media, and 0.5× media (not shown); dashed outlines indicate chromosomes aligned at the metaphase plate, arrowheads indicate untethered ecDNA. B , quantification of ecDNA untethering of COLO320DM wildtype and MKI67 knockout clones from panel A (each data point represents the weighted average of at least 95 cells from each clone) after 15 min incubation in 1× media ( left ; n = 4, 6, 5 clones; one-way ANOVA, F = 1.7, p = 0.23), 0.75× media ( middle ; n = 4, 6, 5 clones; one-way ANOVA, F = 12.4, p = 0.001), and 0.5× media ( right ; n = 4, 6, 5 clones; one-way ANOVA, F = 0.1, p = 0.99); ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant; error bars = mean ± standard deviation. C , metaphase COLO320DM wildtype (NT) and MKI67 knockout cells (gRNA #1 and #2), treated for 24 h with vehicle (not shown), 0.2 μM LMK235, and 0.5 μM LMK235 (not shown); dashed outlines indicate chromosomes aligned at the metaphase plate, arrowheads indicate untethered ecDNA. D , quantification of ecDNA untethering of COLO320DM wildtype and MKI67 knockout clones from panel C (each data point represents the weighted average of at least 60 cells from each clone) after 24 h treatment with vehicle (0.1% DMSO, left ; n = 4, 3, 3 clones; one-way ANOVA, F = 3.9, p = 0.073), 0.2 μM LMK235 ( middle ; n = 4, 3, 3 clones; one-way ANOVA, F = 100.8, p < 0.001, ∗∗ p < 0.01 by Tukey’s HSD), and 0.5 μM LMK235 ( right ; n = 4, 3, 3 clones; one-way ANOVA, F = 0.8, p = 0.51); error bars = mean ± standard deviation; MKI67 knockout clones are gRNA #1 clones 1, 4, and 5, and gRNA #2 clones 1, 4, and 5 (see , A – C ). E , schematic: the biological surfactant Ki67 ( green ) coats the surface of mitotic ecDNA ( red ) and chromosomes ( blue ), helping to prevent tethering by electrostatic repulsion and steric hindrance. In all panels, scale bar = 10 μm.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Knock-Out, Incubation, Clone Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: HDAC inhibition leads to a loss of oncogene copy number in COLO320DM cells, but not in COLO320HSR cells. A and B , quantification of MYC DNA expression in COLO320DM ( A ) and COLO320HSR ( B ) cells treated with LMK235; qPCR, 2 -ΔΔCT analysis (normalized to LINE1 copy number and vehicle control); x-axis = symlog scale, linear ≤2, log >2. Error bars = mean ± 95% confidence interval. Drug-containing media was replaced every 2 to 3 days. A , treatment with vehicle (0.1% DMSO), 0.2 μM, 0.3 μM, or 1 μM LMK235 for 1 day (one-way ANOVA, F = 4.1, p = 0.036, n = 4, 4, 4, 3), 2 days (F = 31.3, p < 0.001, n = 4, 4, 4, 3), 10 days (F = 5.4, p = 0.017, n = 6, 6, 6, no 1 μM), 30 days (F = 19.8, p = 0.0023, n = 3, 3, 3, no 1 μM); 0 days: pretreatment (one-way ANOVA, F = 0.4, p = 0.77, n = 3, 3, 3, 3); # p < 0.05, ## p < 0.01 (0.2 μM LMK235 v er s us vehicle); ∗ p < 0.05, ∗∗ p < 0.01 (0.3 μM LMK235 v er s us vehicle); ˆˆ p < 0.01 (1 μM LMK235 v er s us vehicle) by Tukey’s HSD; B , treatment with vehicle, 0.3 μM LMK235, or 1 μM LMK235 for 2 days (one-way ANOVA, F = 2.2, p = 0.17, n = 4, 4, 4), 5 days (F = 0.5, p = 0.62, n = 4, 4, 4), 10 days (F = 10.1, p = 0.019, n = 4, 4), and 30 days (F = 2.7, p = 0.15, n = 4, 4); 0 days: pretreatment (one-way ANOVA, F = 0.8, p = 0.48); ∗ p < 0.05 (0.3 μM LMK235 v er s us vehicle). C , schematic, top : under normal mitotic conditions, ecDNA tether to chromosomes throughout mitosis to ensure their segregation into the primary nuclei of divided daughter cells; bottom : under conditions that decompact chromatin (hypotonic conditions and HDAC inhibition) or prevent ecDNA-chromosome interaction at their surfaces (Ki67 overexpression), ecDNA untether from chromosomes, leading some to be mis-segregated into micronuclei. D and E , schematics of a colloidal and surface chemistry-based framework for approaching ecDNA and chromosome compaction and tethering during mitosis. Arrow 1 represents biophysical changes to the colloidal particles (nucleosomes and chromatin molecules) or the solution/suspension medium (cytosol), such as alterations to the intracellular ionic strength or to the acetylation state of chromatin. Arrow 2 represents changes in the surfactant (Ki67) concentration of the system. Both sets of changes affect particle-particle and particle-solution interactions.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Inhibition, Expressing, Control, Over Expression, Suspension, Concentration Assay

Journal: Nature

Article Title: Genetic elements promote retention of extrachromosomal DNA in cancer cells

doi: 10.1038/s41586-025-09764-8

Figure Lengend Snippet: a , Proposed mechanism of mitotic retention of ecDNAs in cancer cells through chromosome hitchhiking. b , Representative image of tethered (bottom arrowhead) and untethered (top arrowhead) ecDNA foci in mitotic PC3 cells ( n = 92 daughter cell pairs). Scale bar, 10 µm. c , Representative live-cell images ( n = 10 fields of view) showing ecDNA (labelled with TetR-mNeonGreen) colocalization with chromosomes during cancer cell division. Scale bar, 10 µm. d , Fractions of ecDNA with various oncogenes colocalizing with mitotic chromosomes in the following cancer cell lines: GBM39 glioblastoma cells, EGFR ecDNA from chromosome 7; PC3 prostate cancer cells, ec MYC from chromosome 8; SNU16 gastric cancer cells, ec MYC and FGFR2 ecDNA from chromosome 8 and chromosome 10, respectively; COLO320DM colorectal cancer cells, ec MYC. Raw images were obtained from a previous publication of IF–DNA-FISH of anaphase cells. e , Schematic of Retain-seq. f , Retain-seq enrichment of a known EBV sequence that promotes viral retention. EBNA1 ChIP–seq data in EBV-transformed GM12878 cells are shown at the bottom. g , Retain-seq signals at three representative enriched genomic loci. Red tracks represent loci that were significantly enriched in Retain-seq screens in the corresponding cell line, thus marking these loci as retention elements in that line; black tracks indicate that the sequence was not identified as a retention element in the corresponding experiment. h , Principal component analysis of Retain-seq in various cell lines at different time points. i , Individual validation by quantitative PCR (qPCR) of six episomally retained elements (RE-A–RE-F) identified by Retain-seq experiments in the K562 cell line and amplified on COLO320DM (RE-C) and GBM39 (others) ecDNAs. Each line in the plot for a given retention element represents a single replicate. The empty vector control is the pUC19 plasmid alone, whereas the random insert control comprises the pUC19 plasmid with random insert sequences from the genome of the human GM12878 cell line. P values were calculated using one-sided t -tests.

Article Snippet: The live-cell imaging cell line was engineered from COLO320DM cells obtained from the ATCC, as described in a previous publication .

Techniques: Sequencing, ChIP-sequencing, Transformation Assay, Biomarker Discovery, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Control

Journal: Nature

Article Title: Genetic elements promote retention of extrachromosomal DNA in cancer cells

doi: 10.1038/s41586-025-09764-8

Figure Lengend Snippet: a , Analyses of sequence features of retention elements. b , Input-normalized Retain-seq signals across annotated gene sequences. TTS, transcription termination site. c , Sequence annotations that overlap with retention elements identified in K562 cells. Percentages represent the proportion of retention elements that overlap with a given annotation class. d , ENCODE candidate cis -regulatory elements (cCREs) that overlap with retention elements identified in K562 cells. Fractions represent the proportion of retention elements that overlap with a given cCRE class. e , ENCODE ChIP–seq signals of the indicated histone marks and RNA polymerases II and III in K562 cells that surround retention elements identified in the same cell line. f , CpG density surrounding the combined set of retention elements. g , Number of CpG sites in genomic bins that overlap with retention elements ( n = 18,494) compared with those that do not ( n = 2,543,727). Box centre, line median; limits, upper and lower quartiles; whiskers, 1.5× the interquartile range. h , Fraction of origins of replication (identified by SNS-seq in K562 cells) that overlap with retention elements identified in K562 cells and random genomic intervals. i , Retention of plasmids that contain one, two or three copies of a retention element (RE-C; red segments in schematic) in COLO320DM cells, analysed by qPCR. Fold changes were computed using plasmid levels at day 14 after transfection, normalizing to levels at day 2 to adjust for different transfection efficiencies across conditions (three biological replicates). j , Left, schematic of transfection of plasmids with a CMV promoter and/or a retention element (RE-C) into COLO320DM cells. Right, retention of plasmids that contain a CMV promoter and/or a retention element in COLO320DM cells, assessed by qPCR (three biological replicates). Data for two different plasmid backbones, pUC19 and pGL4, are shown. P values were computed using two-sided Wilcoxon rank-sum tests ( g ), one-sided hypergeometric tests ( h ) or one-sided t -tests ( i , j ). NS, not significant.

Article Snippet: The live-cell imaging cell line was engineered from COLO320DM cells obtained from the ATCC, as described in a previous publication .

Techniques: Sequencing, ChIP-sequencing, Plasmid Preparation, Transfection

Journal: Nature

Article Title: Genetic elements promote retention of extrachromosomal DNA in cancer cells

doi: 10.1038/s41586-025-09764-8

Figure Lengend Snippet: ( a ) Histograms and heatmaps of COLO320DM GRO-seq signal from biological replicate 1, computed over 50 bp bins within 3 kb of the midpoints of retention elements located within the genomic coordinates of the COLO320DM ecDNA. Retention elements were divided into 3 categories based on overlap with genomic annotations: those that overlap with coding gene promoters, other portions of coding genes, or noncoding regions. X-axis directionality is consistent for both strands. ( b ) Heatmap of COLO320DM GRO-seq signal from biological replicate 2 within 3 kb of the midpoints of retention elements located within the genomic coordinates of the COLO320DM ecDNA.

Article Snippet: The live-cell imaging cell line was engineered from COLO320DM cells obtained from the ATCC, as described in a previous publication .

Techniques:

Journal: Nature

Article Title: Genetic elements promote retention of extrachromosomal DNA in cancer cells

doi: 10.1038/s41586-025-09764-8

Figure Lengend Snippet: ( a ) ENCODE ChIP-seq signals of the indicated proteins in K562 cells surrounding retention elements identified in the same cell line. ( b ) ENCODE ChIP-seq signals of components of the replication licensing complex in K562 cells surrounding retention elements identified in the same cell line. ( c ) Motif enrichment (log2 fold change) of transcription factor motifs in retention element intervals identified in COLO320DM, GBM39, and K562 cells relative to random genomic intervals. ( d ) Episomal retention of plasmids containing 8 overlapping 500-bp tiles of a retention element (RE-C) in COLO320DM cells measured by quantitative PCR (six biological replicates for empty vector and retention element conditions, three for others). P -values computed by one-sided t-test.

Article Snippet: The live-cell imaging cell line was engineered from COLO320DM cells obtained from the ATCC, as described in a previous publication .

Techniques: ChIP-sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Nature

Article Title: Genetic elements promote retention of extrachromosomal DNA in cancer cells

doi: 10.1038/s41586-025-09764-8

Figure Lengend Snippet: a , Schematic of the live-cell imaging experiment. b , Representative live-cell time-lapse images of dividing COLO320DM cells with labelled ec MYC 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. . 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 ec MYC retention elements with 5-kb resolution. h , Hi-C heatmap of pairwise interactions in mitotically arrested COLO320DM cells between ec MYC 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 ).

Article Snippet: The live-cell imaging cell line was engineered from COLO320DM cells obtained from the ATCC, as described in a previous publication .

Techniques: Live Cell Imaging, Transfection, Plasmid Preparation, Control, Hi-C, Flow Cytometry, Binding Assay, Gene Expression

Journal: Nature

Article Title: Genetic elements promote retention of extrachromosomal DNA in cancer cells

doi: 10.1038/s41586-025-09764-8

Figure Lengend Snippet: ( a-b ) Aggregated peak analysis (APA) of Hi-C data of asynchronous (a) and mitotically arrested (b) COLO320DM cells. Heatmaps are summed percentile matrices of pairwise interactions between previously reported chromosome bookmarked regions and a combined set of retention elements identified on the MYC ecDNA with 5-kb resolution, in which the chromosome bookmarked regions and/or the ecMYC retention elements are randomized. ( c ) Chromosome bookmarked regions or ecMYC retention elements with the indicated ENCODE cCRE annotations. ( d ) Hi-C heatmap of pairwise interactions between the MYC ecDNA retention elements and chromosome bookmarked regions with the indicated ENCODE cCRE annotations in asynchronous cells. Hi-C counts are normalized to number of interactions as well as bin sizes. ( e ) APA of Hi-C data of asynchronous GBM39 cells. ( f ) Importance scores (error bars show s.e.m.) indicating the relative contribution of each bookmarking factor to the cumulative distribution of retention elements. Scores represent the mean incremental number of retention elements containing binding sites for each factor over 1000 randomized cumulative distributions of the 20 bookmarking factors shown. Bookmarking factors are displayed in order of ChIP-seq peak enrichment within retention elements relative to random genomic intervals. ( g ) Fraction of tethered ecDNAs following CRISPR/Cas9 knockouts of selected bookmarking factors in mitotic COLO320DM cells. Box plot parameters as in Fig. . n = 55 (SMARCE1 NTC1), n = 42 (SMARCE1 KO1), n = 39 (SMARCE1 KO2), n = 34 (HEY1 NTC2), n = 33 (HEY1 KO1), n = 8 (CHD1 NTC1), n = 36 (CHD1 KO1) cells. ( h ) Mean immunofluorescence intensity of selected bookmarking factors in cells receiving targeting guide RNAs or non-targeting control (NTC) guides. n = 1874 (SMARCE1 NTC1), n = 2217 (SMARCE1 KO1), n = 1371 (SMARCE1 KO2), n = 1459 (HEY1 NTC2), n = 1976 (HEY1 KO1), n = 316 (CHD1 NTC1), n = 2730 (CHD1 KO1) cells. Box plot parameters as in Fig. .

Article Snippet: The live-cell imaging cell line was engineered from COLO320DM cells obtained from the ATCC, as described in a previous publication .

Techniques: Hi-C, Binding Assay, ChIP-sequencing, CRISPR, Immunofluorescence, Control

Journal: Nature

Article Title: Genetic elements promote retention of extrachromosomal DNA in cancer cells

doi: 10.1038/s41586-025-09764-8

Figure Lengend Snippet: a , Mean frequency (>10 independent replicates) of cells with ≥1 ecDNA in simulations. Shaded area, s.e.m. b , Analysis of retention element co-amplification with oncogenes on ecDNA in patient tumours. c , ecDNA amplicons that contain retention elements and/or oncogenes. d , Top, schematic of an ecDNA segment without retention elements co-amplified with a retention element. Bottom, frequency of co-amplification with retention elements in BFB, ecDNA or linear amplicons for genomic segments without retention elements. e , Top to bottom, oncogene sizes on ecDNA, frequency of genomic segments that contain retention elements sorted by size, and total ecDNA amplicon sizes. f , Schematic of experiment to analyse the distribution of retention element numbers among ecDNAs. g , Correlation (Pearson’s R with 95% confidence intervals) between local density of retention elements and amplicon size. The plot shows the linear fit using ordinary least squares with 95% confidence intervals. h , Circular microDNAs in five human cell lines that overlap with retention elements or matched-sized random genomic intervals detected using circle-seq. i , Increased WGS coverage of EGFR ecDNA in GBM39 cells and retention element positions. j , 5mC CpG methylation of retention elements ( n = 9 segments) compared with matched-sized sequence intervals ( n = 1,235 segments) in GBM39 ecDNA. k , 5mC methylation (Me + or Me – ) and density of CpG sites surrounding a retention element on GBM39 ecDNA. l , Site-specific methylation of retention elements by CRISPRoff. m , Frequency of GBM39 cells that contain untethered ecDNA foci 5 days after transfection. n = 60 (nontargeting) and n = 50 (targeting) visual fields. Box plot parameters are as described in Fig. . n , Plasmid retention after methylation in COLO320DM cells, as assessed by qPCR (three biological replicates). o , Retention elements and oncogenes on ecDNA (left) confer retention and selection, respectively, two processes that shape the evolution of cancer cell lineages (right). P values were calculated using one-sided tests of equal proportions ( d ), two-sided Fisher’s z -tests ( g ), two-sided Fisher’s exact tests ( h ), two-sided Wilcoxon rank-sum tests ( j ), two-sided Mann–Whitney–Wilcoxon tests ( m ) or one-sided t -tests ( n ).

Article Snippet: The live-cell imaging cell line was engineered from COLO320DM cells obtained from the ATCC, as described in a previous publication .

Techniques: Amplification, CpG Methylation Assay, Sequencing, Methylation, Transfection, Plasmid Preparation, Selection, MANN-WHITNEY