Abstract
Extrachromosomal circular DNA is an emerging regulatory element implicated in genomic stability and gene regulation, yet its role in preimplantation development remains elusive. Here, we report the widespread presence of extrachromosomal circular DNA in preimplantation embryos, characterized by homologous junction sequences and originating from genomic regions enriched for active histone marks and RNA Polymerase II occupancy. Functional perturbations demonstrate that RNA Polymerase II inhibition suppresses extrachromosomal circular DNA production, whereas disruption of the Fanconi anemia pathway elevates it, suggesting that transcription-replication conflicts affect its biogenesis. Notably, extrachromosomal circular DNA levels surge during major zygotic genome activation. Synthetic extrachromosomal circular DNAs carrying putative enhancers for the zygotic genome activation genes Mycn and Egfl7, and the developmental gene Emx1, significantly upregulate the expression of their respective genes upon transfection into fibroblasts and zygotes. Collectively, this study unveils the extrachromosomal circular DNA landscape in preimplantation embryos, elucidates a transcription-replication conflict mechanism underlying its generation, and establishes its regulatory potential during mammalian preimplantation development.
Similar content being viewed by others
Data availability
Raw sequencing datasets of MDA and scRNA-seq reported in this paper have been deposited in the Genome Sequence Archive (GSA) in the National Genomics Data Center, China National Center for Bioinformation, Chinese Academy of Sciences, under accession number CRA019281. Publicly available ATAC-seq and other datasets used in this study were obtained from the Gene Expression Omnibus (GEO) under the following accession codes: GSE66582, GSE44183, GSE124718, GSE71434, GSE66390, GSE72784, GSE135457. Source data are provided with this paper.
Code availability
All scripts used for eccDNA quantification in this study are publicly available at GitHub: https://github.com/duck-rong/eccDNA_quantification_scripts. No software was developed beyond these scripts; all analyses were performed using these scripts together with publicly available tools as described in the Methods section.
References
Yang, L. et al. Extrachromosomal circular DNA: biogenesis, structure, functions and diseases. Signal Transduct. Target Ther. 7, 342 (2022).
Zhao, X. et al. CircleBase: an integrated resource and analysis platform for human eccDNAs. Nucleic Acids Res. 50, D72–D82 (2022).
Wei, L. et al. CircleBase V2: an eccDNA annotation platform across cancers and species. Nucleic Acids Res. 54, D66–D77 (2026).
Wu, N. et al. Innovative insights into extrachromosomal circular DNAs in gynecologic tumors and reproduction. Protein Cell 15, 6–20 (2024).
Shibata, Y. et al. Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues. Science 336, 82–86 (2012).
Moller, H. D., Parsons, L., Jorgensen, T. S., Botstein, D. & Regenberg, B. Extrachromosomal circular DNA is common in yeast. Proc. Natl. Acad. Sci. USA 112, E3114–3122 (2015).
Dillon, L. W. et al. Production of extrachromosomal MicroDNAs IS Linked To Mismatch Repair Pathways And Transcriptional Activity. Cell Rep. 11, 1749–1759 (2015).
Moller, H. D. et al. Circular DNA elements of chromosomal origin are common in healthy human somatic tissue. Nat. Commun. 9, 1069 (2018).
Wang, Y. et al. eccDNAs are apoptotic products with high innate immunostimulatory activity. Nature 599, 308–314 (2021).
Moller, H. D., Ramos-Madrigal, J., Prada-Luengo, I., Gilbert, M. T. P. & Regenberg, B. Near-random distribution of chromosome-derived circular DNA in the Condensed genome of pigeons and the larger, more repeat-rich human genome. Genome Biol. Evol. 12, 3762–3777 (2020).
Henriksen, R. A. et al. Circular DNA in the human germline and its association with recombination. Mol. Cell 82, 209–217 e207 (2022).
Paulsen, T. et al. MicroDNA levels are dependent on MMEJ, repressed by c-NHEJ pathway, and stimulated by DNA damage. Nucleic Acids Res. 49, 11787–11799 (2021).
Yang, F. et al. Retrotransposons hijack alt-EJ for DNA replication and eccDNA biogenesis. Nature 620, 218–225 (2023).
Wu, S. et al. Circular ecDNA promotes accessible chromatin and high oncogene expression. Nature 575, 699–703 (2019).
Noer, J. B., Horsdal, O. K., Xiang, X., Luo, Y. & Regenberg, B. Extrachromosomal circular DNA in cancer: history, current knowledge, and methods. Trends Genet. 38, 766–781 (2022).
Lv, W. et al. Extrachromosomal circular DNA orchestrates genome heterogeneity in urothelial bladder carcinoma. Theranostics 14, 5102–5122 (2024).
Koche, R. P. et al. Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma. Nat. Genet. 52, 29–34 (2020).
Zhu, Y. et al. Oncogenic extrachromosomal DNA functions as mobile enhancers to globally amplify chromosomal transcription. Cancer Cell 39, 694–707 e697 (2021).
Nathanson, D. A. et al. Targeted therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA. Science 343, 72–76 (2014).
Lv. W, et al. Spatial-temporal diversity of extrachromosomal DNA shapes urothelial carcinoma evolution and tumor-immune microenvironment. Cancer Discov. https://doi.org/10.1158/2159-8290.CD-24-1532 (2025).
Ling, X. et al. Small extrachromosomal circular DNA (eccDNA): major functions in evolution and cancer. Mol. Cancer 20, 113 (2021).
Shi, B., Yang, P., Qiao, H., Yu, D. & Zhang, S. Extrachromosomal circular DNA drives dynamic genome plasticity: emerging roles in disease progression and clinical potential. Theranostics 15, 6387–6411 (2025).
Zhou, M. et al. Plasma extrachromosomal circular DNA as a potential diagnostic biomarker for nodular thyroid disease. Clin. Transl. Med. 14, e1740 (2024).
Hu, J. et al. Microhomology-mediated circular DNA formation from oligonucleosomal fragments during spermatogenesis. Elife 12, RP87115 (2023).
Schulz, K. N. & Harrison, M. M. Mechanisms regulating zygotic genome activation. Nat. Rev. Genet. 20, 221–234 (2019).
Vastenhouw, N.L., Cao, W.X., Lipshitz, H.D. The maternal-to-zygotic transition revisited. Development 146, dev161471 (2019).
Abe, K. I. et al. Minor zygotic gene activation is essential for mouse preimplantation development. Proc. Natl. Acad. Sci. USA 115, E6780–E6788 (2018).
Messerschmidt, D. M., Knowles, B. B. & Solter, D. DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos. Genes Dev. 28, 812–828 (2014).
Reik, W., Dean, W. & Walter, J. Epigenetic reprogramming in mammalian development. Science 293, 1089–1093 (2001).
Nakatani, T. et al. Emergence of replication timing during early mammalian development. Nature 625, 401–409 (2024).
Kumar, P. et al. ATAC-seq identifies thousands of extrachromosomal circular DNA in cancer and cell lines. Sci. Adv. 6, eaba2489 (2020).
Fan, X. et al. SMOOTH-seq: single-cell genome sequencing of human cells on a third-generation sequencing platform. Genome Biol. 22, 195 (2021).
Flach, G., Johnson, M. H., Braude, P. R., Taylor, R. A. & Bolton, V. N. The transition from maternal to embryonic control in the 2-cell mouse embryo. EMBO J. 1, 681–686 (1982).
Chen, Z. & Zhang, Y. Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development. Nat. Genet. 51, 947–951 (2019).
Kang, X. et al. Extrachromosomal DNA replication and maintenance couple with DNA damage pathway in tumors. Cell 188, 3405–3421 e3427 (2025).
Paulsen, T., Kumar, P., Koseoglu, M. M. & Dutta, A. Discoveries of extrachromosomal circles of DNA in normal and tumor cells. Trends Genet 34, 270–278 (2018).
Ji, S. et al. OBOX regulates mouse zygotic genome activation and early development. Nature 620, 1047–1053 (2023).
He, A. et al. Dynamic GATA4 enhancers shape the chromatin landscape central to heart development and disease. Nat. Commun. 5, 4907 (2014).
Cirillo, L. A., Lin, F. R., Cuesta, I., Friedman, D., Jarnik, M. & Zaret, K. S. Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol. Cell 9, 279–289 (2002).
Yan, J., Xu, L., Crawford, G., Wang, Z. & Burgess, S. M. The forkhead transcription factor FoxI1 remains bound to condensed mitotic chromosomes and stably remodels chromatin structure. Mol. Cell Biol. 26, 155–168 (2006).
Lalmansingh, A. S., Karmakar, S., Jin, Y. & Nagaich, A. K. Multiple modes of chromatin remodeling by Forkhead box proteins. Biochim Biophys. Acta 1819, 707–715 (2012).
Zhang, B. et al. Allelic reprogramming of the histone modification H3K4me3 in early mammalian development. Nature 537, 553–557 (2016).
Wu, J. et al. The landscape of accessible chromatin in mammalian preimplantation embryos. Nature 534, 652–657 (2016).
Dahl, J. A. et al. Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition. Nature 537, 548–552 (2016).
Liu, B. et al. The landscape of RNA Pol II binding reveals a stepwise transition during ZGA. Nature 587, 139–144 (2020).
Xia, W. et al. Resetting histone modifications during human parental-to-zygotic transition. Science 365, 353–360 (2019).
Lorch, Y., Maier-Davis, B. & Kornberg, R. D. Role of DNA sequence in chromatin remodeling and the formation of nucleosome-free regions. Genes Dev. 28, 2492–2497 (2014).
Yang, Y. et al. Transcription-replication conflicts in primordial germ cells necessitate the Fanconi anemia pathway to safeguard genome stability. Proc. Natl. Acad. Sci. USA 119, e2203208119 (2022).
Xue, Z. et al. Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing. Nature 500, 593–597 (2013).
Futschik, M. E. & Carlisle, B. Noise-robust soft clustering of gene expression time-course data. J. Bioinform. Comput Biol. 3, 965–988 (2005).
Xu, R. et al. Stage-specific H3K9me3 occupancy ensures retrotransposon silencing in human pre-implantation embryos. Cell Stem Cell 29, 1051–1066 e1058 (2022).
Paulsen, T., Shibata, Y., Kumar, P., Dillon, L. & Dutta, A. Small extrachromosomal circular DNAs, microDNA, produce short regulatory RNAs that suppress gene expression independent of canonical promoters. Nucleic Acids Res. 47, 4586–4596 (2019).
Gan, W. et al. R-loop-mediated genomic instability is caused by impairment of replication fork progression. Genes Dev. 25, 2041–2056 (2011).
Huertas, P. & Aguilera, A. Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination. Mol. Cell 12, 711–721 (2003).
Palmerola, K. L. et al. Replication stress impairs chromosome segregation and preimplantation development in human embryos. Cell 185, 2988–3007 e2920 (2022).
Chen S, et al. The urinary eccDNA landscape in prostate cancer reveals associations with genome instability and vital roles in cancer progression. J. Adv. Res. 77, 637–652 (2025).
Tang, L. et al. Circular single-stranded DNA as switchable vector for gene expression in mammalian cells. Nat. Commun. 14, 6665 (2023).
Joubert, P. M. & Krasileva, K. V. The extrachromosomal circular DNAs of the rice blast pathogen Magnaporthe oryzae contain a wide variety of LTR retrotransposons, genes, and effectors. BMC Biol. 20, 260 (2022).
Tei, C. et al. Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging. Commun. Biol. 8, 749 (2025).
Sfeir, A., Tijsterman, M. & McVey, M. Microhomology-MEdiated End-joining Chronicles: Tracing The Evolutionary Footprints Of Genome Protection. Annu. Rev. Cell Dev. Biol. 40, 195–218 (2024).
Chazaud, C., Yamanaka, Y., Pawson, T. & Rossant, J. Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Dev. Cell 10, 615–624 (2006).
Werling, D. M. et al. An analytical framework for whole-genome sequence association studies and its implications for autism spectrum disorder. Nat. Genet. 50, 727–736 (2018).
Iossifov, I. et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature 515, 216–221 (2014).
Zhao, G. et al. Gene4Denovo: an integrated database and analytic platform for de novo mutations in humans. Nucleic Acids Res. 48, D913–D926 (2020).
Layer, R. M., Chiang, C., Quinlan, A. R. & Hall, I. M. LUMPY: a probabilistic framework for structural variant discovery. Genome Biol. 15, R84 (2014).
Garvin, T. et al. Interactive analysis and assessment of single-cell copy-number variations. Nat. Methods 12, 1058–1060 (2015).
Deshpande, V. et al. Exploring the landscape of focal amplifications in cancer using AmpliconArchitect. Nat. Commun. 10, 392 (2019).
Picelli, S., Faridani, O. R., Bjorklund, A. K., Winberg, G., Sagasser, S. & Sandberg, R. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
Chen, L. et al. NAT10-mediated mRNA N(4)-acetylation is essential for the translational regulation during oocyte meiotic maturation in mice. Sci. Adv. 11, eadp5163 (2025).
Oka, Y., Bekker-Jensen, S. & Mailand, N. Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway. EMBO J. 34, 1385–1398 (2015).
Dexheimer, T.S. et al. Discovery of ML323 as a Novel Inhibitor of the USP1/UAF1 Deubiquitinase Complex. In: Probe Reports from the NIH Molecular Libraries Program) (2010).
Liang, Q. et al. A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses. Nat. Chem. Biol. 10, 298–304 (2014).
Yu, Z. et al. USP1-UAF1 deubiquitinase complex stabilizes TBK1 and enhances antiviral responses. J. Exp. Med. 214, 3553–3563 (2017).
Wu, J. et al. Chromatin analysis in human early development reveals epigenetic transition during ZGA. Nature 557, 256–260 (2018).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Faust, G. G. & Hall, I. M. SAMBLASTER: fast duplicate marking and structural variant read extraction. Bioinformatics 30, 2503–2505 (2014).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Pertea, M., Kim, D., Pertea, G. M., Leek, J. T. & Salzberg, S. L. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat. Protoc. 11, 1650–1667 (2016).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Danecek, P, et al. Twelve years of SAMtools and BCFtools. Gigascience 10, giab008 (2021).
Ramirez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–165 (2016).
Yu, G., Wang, L. G. & He, Q. Y. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 31, 2382–2383 (2015).
Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284–287 (2012).
Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).
Kumar, L. M EF. Mfuzz: a software package for soft clustering of microarray data. Bioinformation 2, 5–7 (2007).
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant Nos. 82288102 to JQ, 32470894 to QL, 32170493 to XLZ, 32470835 to FBM, 32400703 to LC), the National Key Research and Development Program of China (Grant No. 2025YFC2708100 to QL), the Beijing Natural Science Foundation (Grant Nos. L248056 to FBM, 7242169 to XLZ, 7244435 to LC), the fellowship of China National Postdoctoral Program for Innovative Talents (Grant No. BX20230031 to LC), the Key Clinical Projects of Peking University Third Hospital (Grant No. BYSYZD2024025 to XLZ), and the State Key Laboratory of Female Fertility Promotion, Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital (Grant Nos. BYSYSZKF2024002 to XLZ, BYSYSZKF2025008 to FBM).
Author information
Authors and Affiliations
Contributions
F.B.M., X.L.Z., Q.L. and J.Q. conceived and designed the study. L.W. collected the data, did the analysis, and wrote the paper. N.W. and L.C. performed the experiments. TW did the analysis of eccDNA overlapping with de novo mutations. LSS tested the AA pipeline. ZPZ tested the Ginkgo pipeline. F.B.M., X.L.Z., Q.L., J.Q. and X.X. performed a critical reading of the manuscript and modified it. All authors improved the manuscript and approved the submission.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks Joan Barau and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Wei, L., Wu, N., Chen, L. et al. The landscape and regulatory potential of eccDNAs in mammalian preimplantation embryos. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71227-z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-71227-z
