It is widely acknowledged that DNA is the fundamental genetic unit for most living organisms1. Although we are generally aware that DNA is arranged linearly on chromosomes, our inherent cognition was broken with the discovery of a type of circular DNA structure in 19652. In particular, extrachromosomal circular DNA (eccDNA) has gained increasing attention since it was discovered common in yeast in 20153 and later found to play a critical role in tumor pathogenesis and evolution in 20174 and 20195. The growing interest in eccDNA has highlighted its prevalence and significance across various organisms6-9. Although a considerable amount of research is currently being performed to understand its role in cancer, little is still known about the biology of eccDNA in healthy non-cancerous mammalian cells.
What We Want to Learn
Our study aims to investigate:
(1) How eccDNA is distributed across different somatic tissues in healthy mice at various ages?
(2) Does eccDNA accumulate in healthy tissues as mice age?
(3) The potential link between eccDNA formation and transcriptional activity.
How We Studied It
We created our atlas by collecting tissues from wild-type C57BL/6NRj male mice across four ages (E17.5, 3M, 12M, 22M). Using Circle-Seq and high-throughput sequencing, we identified 567,963 high-confidence eccDNAs through the Circle-Map pipeline. RNA-seq and DESeq2 were performed to analyze gene expression further. To explore the relationship between transcription levels and eccDNA formation, we applied quantile regression with cubic spline fitting and LOESS smoothing. This approach led to two key findings, offering crucial insights into how mammalian genes mutate and evolve to reduce mutational load.
Key Findings
1. EccDNA formation is affected by genes with high transcriptional activity.
Firstly, our analysis reveals a strong correlation between transcriptional activity and eccDNA formation, with the number of eccDNA increasing logarithmically as a function of transcript levels (Figure 1). This provides compelling evidence that eccDNA formation is directly linked to transcription. Notably, genes that play the most significant roles in shaping cellular phenotype, those with high RNA transcription, are also the most susceptible to eccDNA formation. Such genomic alterations may ultimately compromise their wild-type function.
If we draw an analogy between a cell and a busy library filled with thousands of books (genes), certain books are frequently read by visitors (genes with high transcriptional activity). Over time, repeated handing causes some pages to wear out or even fall out (just like DNA double-strand break-induced eccDNA formation). Similarly, highly transcribed genes are more prone to "losing pages", leading to the formation of eccDNA.
This finding is important because it suggests that the transcriptional activity in mammalian somatic tissues is inherently limited by the load of eccDNA arising from these genes.
2. Genes with more splice forms and higher intron density tend to be protected from eccDNA formation.
Our study further reveals that certain genes are partly protected from transcription-induced circularization. Just like some frequently borrowed books remain intact due to more blank pages and more extra copies, genes with high intron density and multiple splice variants seem to possess a "self-protection mechanism" against eccDNA formation (Figure 2).
Notably, these protected genes are tissue-specific, characterized by many splice forms and high intron-to-exon density (Figure 3). This finding is important because it addresses a longstanding question in genetics about evolutionary forces driving intron gain and splicing in eukaryotic genomes10.
In summary, our manuscript provides new insight into the mechanisms of gene evolution and opens new directions for future research on eccDNA (Figure 4).
Surprising Results
Our eccDNA atlas shows that eccDNA does not accumulate in healthy mouse tissue with aging, which is unexpected given studies in yeast have demonstrated eccDNA accumulation as a major driver of replicative aging in this eukaryote11. This finding also contrasts with the age-related accumulation of other mutation types, such as CNVs and Indels, in epithelial12 and stem cells13,14. One possible explanation is that eccDNA does not replicate and is gradually diluted as cells divide, similar to observations in yeast. Another possibility is that animal cells possess mechanisms to actively clear eccDNA, although no direct evidence for this has been found. Alternatively, cells accumulating eccDNA may enter senescence and disappear, causing the loss of eccDNA.
Why This Matters
Our findings reveal a complex relationship between transcriptional activity, gene structure, and eccDNA formation, providing new insights into genome stability and mutation regulation. We demonstrate that highly transcribed genes are more prone to eccDNA formation, whereas genes with high intron density and multiple splice forms appear to be protected, shedding light on a longstanding question in genetics regarding the evolutionary pressures shaping gene architecture. These features may serve as a natural defense mechanism, reducing the risk of deleterious circularization events in essential genes. Importantly, eccDNA plays a crucial role in cancer by amplifying oncogenes, promoting tumor progression, and driving therapy resistance4,9,15-17. Understanding which genes are most affected by eccDNA formation and how gene structure influences this process could provide new strategies for targeting cancer cells while preserving normal cellular function.
References
Genetic Alliance & The New York-Mid-Atlantic Consortium for Genetic and Newborn ScreeningServices. Understanding Genetics: A New York, Mid-Atlantic Guide for Patients and Health Professionals. (Genetic Alliance, Washington (DC), 2009).
Hotta, Y. & Bassel, A. MOLECULAR SIZE AND CIRCULARITY OF DNA IN CELLS OF MAMMALS AND HIGHER PLANTS. Proc. Natl. Acad. Sci. U.S.A. 53, 356–362 (1965).
Møller, H. D., Parsons, L., Jørgensen, T. S., Botstein, D. & Regenberg, B. Extrachromosomal circular DNA is common in yeast. Proc Natl Acad Sci U S A 112, E3114-3122 (2015).
Turner, K. M. et al. Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity. Nature 543, 122–125 (2017).
Wu, S. et al. Circular ecDNA promotes accessible chromatin and high oncogene expression. Nature 575, 699–703 (2019).
Møller, H. D. et al. Circular DNA elements of chromosomal origin are common in healthy human somatic tissue. Nat Commun 9, 1069 (2018).
Chamorro González, R. et al. Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells. Nat Genet 1–11 (2023).
Yang, F. et al. Retrotransposons hijack alt-EJ for DNA replication and eccDNA biogenesis. Nature 1–8 (2023).
Luebeck, J. et al. Extrachromosomal DNA in the cancerous transformation of Barrett’s oesophagus. Nature 1–8 (2023).
Gozashti, L. et al. Transposable elements drive intron gain in diverse eukaryotes. Proceedings of the National Academy of Sciences 119, e2209766119 (2022).
Sinclair, D. A. & Guarente, L. Extrachromosomal rDNA circles—a cause of aging in yeast. Cell 91, 1033–1042 (1997).
Huang, Z. et al. Single-cell analysis of somatic mutations in human bronchial epithelial cells in relation to aging and smoking. Nat Genet 54, 492–498(2022).
Blokzijl, F. et al. Tissue-specific mutation accumulation in human adult stem cells during life. Nature 538, 260–264 (2016)
Brunner, S. F. et al. Somatic mutations and clonal dynamics in healthy and cirrhotic human liver. Nature 574, 538–542 (2019).
deCarvalho, A. C. et al. Discordant inheritance of chromosomal and extrachromosomal DNA elements contributes to dynamic disease evolution in glioblastoma. Nat. Genet. 50, 708–717 (2018).
Lange, J. T. et al. The evolutionary dynamics of extrachromosomal DNA in human cancers. Nat. Genet. 54, 1527–1533 (2022).
Stöber, M. C. et al. Intercellular extrachromosomal DNA copy-number heterogeneity drives neuroblastoma cell state diversity. Cell Rep. 43, 114711 (2024).