henhaobio
Sign in

NatureCancer BiologyPeer-reviewed journal article

Genetic elements promote retention of extrachromosomal DNA in cancer cells

Venkat SankariD, King L. HungiD, Aditi GnanasekariD, Ivy Tsz-Lo WongiD, Quanming ShiiD, Katerina KraftiD, Matthew G. JonesiD, Britney Jiayu HeiD, Xiaowei YaniD, Julia A. BelkiD, Kevin J. LiuiD, Sangya AgarwaliD, Sean K. WangiD, Anton George HensseniD, Paul S. MischeliD, Howard Y. ChangiD

PDFDOIPreprintCodeData

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (12 Oct 2025) · CC BY 4.0

A genome-wide plasmid screen, Retain-seq, identifies thousands of CpG-rich promoter-like human elements that tether episomes to mitotic chromosomes, shape ecDNA structure, and lose function when methylated.

Circular, acentric ecDNAs segregate stochastically yet persist across generations, but the endogenous human DNA elements mediating their mitotic tethering were unknown. This work defines such retention elements and links them to ecDNA selection, amplicon structure and CpG methylation.

Key results

  1. ShownRetain-seq, a pooled episome screen using random human genomic inserts, identified a large set of retention elements; most appear specific to individual cell lines, and five of six individually cloned elements were retained above controls.

    14,353 combined retention elements across COLO320DM, GBM39 and K562 screens; five of six cloned elements were retained above empty vector and random-insert controls in K562 (qPCR); EBV oriP enriched only in EBNA1-positive GM12878. · Genetic elements drive episome retention; Figure legends

  2. ShownLive-cell imaging showed a plasmid carrying one retention element hitchhikes on mitotic chromosomes more reliably than an empty-vector control in COLO320DM cells.

    Failure of chromosome hitchhiking per mitotic event fell from 25% to 10.4% with one retention element (n = 51 control cells, n = 83 retention element cells; two-sided Wilcoxon rank-sums test). · Retention elements tether to chromosomes; Figure legends

  3. SuggestedHi-C aggregate peak analysis supports contacts between ecMYC retention elements and mitotically bookmarked chromosomal regions, mimicking promoter-enhancer contacts in trans.

    Contact enrichment persisted in mitotically arrested COLO320DM cells and vanished when regions were randomized; nearly half of bookmarked regions were retention elements; knockouts of three bookmarking factors did not widely untether ecDNA. · Episomal contact with mitotic bookmarks; Figure legends

  4. SuggestedRetention elements are nearly universal on patient oncogene ecDNAs and their local density correlates with amplicon size, consistent with selection shaping ecDNA structure; simulations show the need for high retention fidelity.

    98% of oncogene-containing ecDNAs (TCGA and PCAWG) contain retention elements; amplicon size decreases as local density rises; simulated ecDNA is lost when retention fidelity is below 0.9, near the ~10% failure rate seen by imaging. · Cancer ecDNAs contain retention elements; Analysis of ecDNA sequences in patient tumors; Figure legends

  5. ShownRetention element activity depends on CpG hypomethylation: native elements on GBM39 ecDNA are focally demethylated, and methylation abolishes plasmid retention and increases untethered ecDNA.

    Six of nine retention element intervals on GBM39 EGFR ecDNA are demethylated vs 1 kb intervals (n = 9 vs n = 1235); in vitro CpG methylation completely ablates plasmid retention; CRISPRoff raised untethered ecDNA foci (n = 60 non-targeting vs 50 targeting fields). · Methylation silences retention elements; Figure legends

Methods, data and reproducibility

Retain-seq transfects a pool of pUC19 plasmids with random GM12878 genomic inserts, passages cells, and compares recovered inserts with input in 1-kb windows. Findings are tested by live-cell imaging, Hi-C, patient amplicon analysis, simulation and CRISPRoff.

  • Retain-seq screens in COLO320DM, GBM39, K562 and GM12878 cells (two biological replicates for calling elements) · human (cell lines) · n = two biological replicates · PRJNA1333946
  • Live-cell imaging of plasmid and ecMYC colocalization with mitotic chromosomes in COLO320DM · human (cell line) · n = n = 51 control, 83 retention element cells
  • Hi-C of mitotically arrested COLO320DM and asynchronous COLO320DM and GBM39 cells · human (cell lines) · GSM8523315, GSM8523316 (asynchronous COLO320DM)
  • Focal amplicons from patient tumors (TCGA and PCAWG) called by AmpliconArchitect · human (patient tumors) · n = n = 759 ecDNA, 364 BFB, 1295 linear amplicons
  • Nanopore methylation data of GBM39 EGFR ecDNA · human (cell line) · n = n = 9 retention element segments vs 1235 matched segments · PRJNA1110283
  • CRISPRoff targeting of five hypomethylated retention elements on GBM39 EGFR ecDNA, imaged by FISH · human (cell line) · n = n = 60 non-targeting, 50 targeting visual fields
  • Previously published circular microDNA sequences from five human cell lines (LNCaP, C4-2, PC-3, OVCAR8, ES-2) · human (cell lines)
  • COLO320DM GRO-seq (nascent transcription at retention elements) · human (cell line) · n = two replicates · GSM7956899, GSM7956900
Methods & tools
Retain-seq, BWA MEM 0.7.17-r1188, Trimmomatic 0.39, Picard MarkDuplicates 2.25.3, bedtools v.2.30.0, HiC-Pro, Juicer APA v.1.22.01, AmpliconArchitect, Cassiopeia forward-time simulation, CRISPRoff-v2.1, Megalodon 2.3.3, Sniffles 2.2
Software & environment
Versions stated for Trimmomatic 0.39, BWA MEM 0.7.17-r1188, Picard 2.25.3, bedtools v.2.30.0, Sniffles 2.2, Megalodon 2.3.3, Juicer v.1.22.01, deepTools 3.5.1, Fiji 2.1.0/1.53c and R v.3.6.1. No container or lockfile is stated.
Reproducibility gaps
Retention element coordinates are only said to be at figshare, with no URL. Retain-seq analysis scripts are not linked; only the simulation framework has a code link.
Limitations (5)
  • The Hi-C aggregate peak analysis in GBM39 was inconclusive, attributed to the small number of retention elements on its ecDNA. · stated by the authors
  • CRISPRoff targeting caused acute loss of viability, so cells were collected only at early time points and no reduction in total ecDNA copy number was seen at 5 days. · stated by the authors
  • Knockout of three individual bookmarking factors did not widely untether ecDNA, so the mediating proteins remain unidentified; the authors suggest redundancy among multiple factors. · stated by the authors
  • Most retention elements appear unique to each cell type, which the authors attribute to cell type specificity or technical variation across cell lines. · stated by the authors
  • The screen and validations use ectopic plasmids in a pUC19 backbone, with screen inserts size-selected to 400 bp to 1.5 kb, which may not reflect native ecDNA chromatin; the patient tumor analysis is correlative. · noted in this summary

Read this if you study ecDNA inheritance, mitotic bookmarking or episome maintenance and want a genome-scale functional screen linked to patient amplicon structure.

Citations
25
FWCI
14.62
Field percentile
top 1%

Abstract

Extrachromosomal DNA (ecDNA) is a prevalent and devastating form of oncogene amplification in cancer1,2. Circular megabase-sized ecDNAs lack centromeres and segregate stochastically during cell division3-6 yet persist over many generations. EcDNAs were first observed to hitchhike on mitotic chromosomes into daughter cell nuclei over 40 years ago with unknown mechanism3,7. Here we identify a family of human genomic elements, termed retention elements, that tether episomes to mitotic chromosomes to increase ecDNA transmission to daughter cells. We develop Retain-seq, a genome-scale assay that reveals thousands of human retention elements conferring generational persistence to heterologous episomes. Retention elements comprise a select set of CpG-rich gene promoters and act additively. Live-cell imaging and chromatin conformation capture show that retention elements physically interact with mitotic chromosomes at regions which are mitotically bookmarked by transcription factors and chromatin proteins, intermolecularly recapitulating promoter-enhancer interactions. Multiple retention elements are co-amplified with oncogenes on individual ecDNAs in human cancers and shape their sizes and structures. CpG-rich retention elements are focally hypomethylated; targeted cytosine methylation abrogates retention activity and leads to ecDNA loss, suggesting that methylation-sensitive interactions modulate episomal DNA retention. These results highlight the DNA elements and regulatory logic of mitotic ecDNA retention. Amplifications of retention elements promote the maintenance of oncogenic ecDNA across generations of cancer cells, revealing the principles of episome immortality intrinsic to the human genome.

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISHung '23Marom '25Yang '25Nichols '25Yan '26Kraft '25
68% semantic matchCancer Biology
K. L. Hung, M. G. Jones, I. T.-L. Wong+16 more·bioRxiv·Jul 18, 2023