Identification and analysis of extrachromosomal circular DNAs in pancreatic islets during the early and late stages of T2DM mice.

Penetrance and low concordance in monozygotic twins in disease: are they the results of alterations in somatic genomes?

Rationale: Extrachromosomal circular DNA is a hallmark of cancer, but its role in shaping the genome heterogeneity of urothelial bladder carcinoma (UBC) remains poorly understood. Here, we comprehensively analyzed the features of extrachromosomal circular DNA in 80 UBC patients.Methods: We performed whole-genome/exome sequencing (WGS/WES), Circle-Seq, single-molecule real-time (SMRT) long-read sequencing of circular DNA, and RNA sequencing (RNA-Seq) on 80 pairs of tumor and AT samples. We used our newly developed circular DNA analysis software, Circle-Map++ to detect small extrachromosomal circular DNA from Circle-Seq data.Results: We observed a high load and significant heterogeneity of extrachromosomal circular DNAs in UBC, including numerous single-locus and complex chimeric circular DNAs originating from different chromosomes. This includes highly chimeric circular DNAs carrying seven oncogenes and circles from nine chromosomes. We also found that large tumor-specific extrachromosomal circular DNAs could influence genome-wide gene expression, and are detectable in time-matched urinary sediments. Additionally, we found that the extrachromosomal circular DNA correlates with hypermutation, copy number variation, oncogene amplification, and clinical outcome.Conclusions: Overall, our study provides a comprehensive extrachromosomal circular DNA map of UBC, along with valuable data resources and bioinformatics tools for future cancer and extrachromosomal circular DNA research.

Extrachromosomal DNA circularization is a common event in cancer cells and frequently serves as a vehicle for cancer oncogene amplification. Random segregation of oncogene-containing extrachromosomal circular DNA promotes rapid intercellular heterogeneity, conferring tumors the ability to rapidly evolve and escape therapy. Smaller, copy-number neutral extrachromosomal circular DNAs are also abundantly identified in both healthy and malignant tissues, but their function in cancer is still unknown. Understanding how extrachromosomal circular DNAs contribute to intercellular heterogeneity in cancer cells remains crucial, however methods for an unbiased characterization of extrachromosomal circular DNAs in single cells are lacking. We introduce scEC&T-seq (single cell extrachromosomal circular DNA and transcriptomic sequencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cells. We demonstrate the ability of our method to isolate and detect extrachromosomal circular DNAs genome-wide from all range of sizes in single cells. We observed that whereas large oncogene-containing circular DNAs are clonally present in most cancer cells, only a very small fraction of small circular DNAs are recurrently identified in single cells, indicating yet unknown prerequisites for maintenance and propagation. Our method was able to capture and recapitulate the structural complexity of oncogene-containing extrachromosomal circular DNAs in single cells, and the matching transcriptomic data allowed us to identify fusion transcripts resulting from the rearranged extrachromosomal structures. In addition, we observed that whereas the main structure of extrachromosomal circular DNAs is mostly stable in single cells, intercellular differences in extrachromosomal circular DNAs’ content can drive differences in oncogene transcription levels in single cells. We envision that by integrating extrachromosomal circular DNA and mRNA sequencing, our method will not only be useful to investigate the impact of intercellular heterogeneity in extrachromosomal circular DNA in tumor evolution, but also to interrogate its function in other biological and pathological processes. Citation Format: Rocio Chamorro Gonzalez, Thomas Conrad, Robin Xu, Madalina Giurgiu, Maja Cwikla, Katharina Kasack, Lotte Brückner, Eric van Leen, Elias Rodriguez-Fos, Konstantin Helmsauer, Heathcliff Dorado Garcia, Yi Bei, Karin Schmelz, Sascha Sauer, Angelika Eggert, Johannes H. Schulte, Roland F. Schwarz, Kerstin Haase, Richard P. Koche, Anton G. Henssen. Dissecting intercellular extrachromosomal circular DNA heterogeneity in single cancer cells with scEC&T-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1693.

Still unresolved is the question of how a lifetime accumulation of somatic gene copy number alterations impact organ functionality and aging and age-related pathologies. Such an issue appears particularly relevant in the broadly post-mitotic central nervous system (CNS), where non-replicative neurons are restricted in DNA-repair choices and are prone to accumulate DNA damage, as they remain unreplaced over a lifetime. Both DNA injuries and consecutive DNA-repair strategies are processes that can evoke extrachromosomal circular DNA species, apparently from either part of the genome. Due to their capacity to amplify gene copies and related transcripts, the individual cellular load of extrachromosomal circular DNAs will contribute to a dynamic pool of additional coding and regulatory chromatin elements. Analogous to tumor tissues, where the mosaicism of circular DNAs plays a well-characterized role in oncogene plasticity and drug resistance, we suggest involvement of the “circulome” also in the CNS. Accordingly, we summarize current knowledge on the molecular biogenesis, homeostasis and gene regulatory impacts of circular extrachromosomal DNA and propose, in light of recent discoveries, a critical role in CNS aging and neurodegeneration. Future studies will elucidate the influence of individual extrachromosomal DNA species according to their sequence complexity and regional distribution or cell-type-specific abundance.

Extrachromosomal circular DNA molecules are prevalent in cancer cells and harbor amplified genes, such as oncogenes and drug resistance genes, that can provide a selective growth advantage to cancer cells. These circular DNA structures include double minute chromosomes (dmin), which can be detected with light microscopy following Giemsa staining, and submicroscopic circular DNA structures referred to as episomes. In this study, we investigated the fate of dmin and episomes in multidrug-resistant human epidermoid KB-VI cells undergoing cisplatin-induced apoptosis-a mode of cell death initially characterized by the fragmentation of chromosomal DNA, while the nuclear membrane remains intact. The circular DNA structures carry amplified copies of the multidrug resistance gene (MDR1). During cisplatin-induced apoptotic cell death, episomes and dmin, as well as native chromosomes, were degraded into high molecular weight DNA fragments of approximately 50 kb in length. DNA fragments in this size range appear to result from the preferential cleavage of matrix-associated regions in chromatin with the subsequent release of 20-30 nm loop domains of chromatin from the nuclear scaffold. Scanning electron microscopy studies were performed and confirmed the presence of 30 nm filaments in a higher-order DNA packing of MDR1-containing dmin and episomes. These combined data provide strong evidence that the higher-order DNA packing of episomes, as well as dmin, is similar to that of native chromosomes and underscore the potential for extrachromosomal DNA amplicons to study the structural and functional organization of chromatin. We discuss the implications of extra-chromosomal DNA matrix associated regions competing with native chromosomal DNA for binding to the nuclear matrix in tumor cells.

Spiroplasmavirus SVTS2, isolated from Spiroplasma melliferum TS2, produces plaques when inoculated onto lawns of Spiroplasma citri M200H, a derivative of the type strain Maroc R8A2. S. citri strains MR2 and MR3, originally selected as colonies growing within plaques on a lawn of M200H inoculated with SVTS2, were resistant to SVTS2. Genomic DNA fingerprints and electrophoretic protein profiles of M200H, MR2, and MR3 were similar, but three proteins present in M200H were missing or significantly reduced in both resistant lines. None of these three polypeptides reacted with antiserum against S. citri membrane proteins, indicating that they probably are not surface-located virus receptors. Electroporation with SVTS2 DNA produced 1.5 x 10(sup5) transfectants per (mu)g of DNA in M200H but none in MR2 or MR3, suggesting that resistance may result from inhibition of viral replication. The digestion patterns of the extrachromosomal double-stranded (ds) DNA of these lines were similar. Three TaqI fragments of MR2 extrachromosomal DNA that were not present in M200H extrachromosomal DNA hybridized strongly to an SVTS2 probe, and two of these fragments plus an additional one hybridized with the MR3 extrachromosomal DNA, indicating that a fragment of SVTS2 DNA was present in the extrachromosomal ds DNA of MR2 and MR3 but not of M200H. When the restricted genomes of all three lines were probed with SVTS2 DNA, strong hybridization to two EcoRI fragments of chromosomal MR2 and MR3 DNA but not M200H DNA indicated that SVTS2 DNA had integrated into the genomes of MR2 and MR3 but not of M200H. When MR3 extrachromosomal ds DNA containing a 2.1-kb SVTS2 DNA fragment was transfected into M200H, the transformed spiroplasmas were resistant to SVTS2. These results suggest that SVTS2 DNA fragments, possibly integrated into the chromosomal or extrachromosomal DNA of a previously susceptible spiroplasma, may function as viral incompatibility elements, providing resistance to superinfection by SVTS2.

Current methods for characterizing extrachromosomal nuclear DNA in mammalian cells do not permit single-cell analysis, are often semi-quantitative and frequently biased toward the detection of circular species. To overcome these limitations, we developed Halo-FISH to visualize and quantitatively analyze extrachromosomal DNA in single cells. We demonstrate Halo-FISH by using it to analyze extrachromosomal telomere-repeat (ECTR) in human cells that use the Alternative Lengthening of Telomeres (ALT) pathway(s) to maintain telomere lengths. We find that GM847 and VA13 ALT cells average ∼80 detectable G/C-strand ECTR DNA molecules/nucleus, while U2OS ALT cells average ∼18 molecules/nucleus. In comparison, human primary and telomerase-positive cells contain <5 ECTR DNA molecules/nucleus. ECTR DNA in ALT cells exhibit striking cell-to-cell variations in number (<20 to >300), range widely in length (<1 to >200 kb) and are composed of primarily G- or C-strand telomere-repeat DNA. Halo-FISH enables, for the first time, the simultaneous analysis of ECTR DNA and chromosomal telomeres in a single cell. We find that ECTR DNA comprises ∼15% of telomere-repeat DNA in GM847 and VA13 cells, but <4% in U2OS cells. In addition to its use in ALT cell analysis, Halo-FISH can facilitate the study of a wide variety of extrachromosomal DNA in mammalian cells.

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond.

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.

Extrachromosomal circular DNA was recently found to be particularly abundant in multiple human cancer cells, although its frequency varies among different tumor types. Elevated levels of extrachromosomal circular DNA have been considered an effective biomarker of cancer pathogenesis. Multiple reports have demonstrated that the amplification of oncogenes and therapeutic resistance genes located on extrachromosomal DNA is a frequent event that drives intratumoral genetic heterogeneity and provides a potential evolutionary advantage. This review highlights the current understanding of the extrachromosomal circular DNA present in the tissues and circulation of patients with advanced cancers and provides a detailed discussion of their substantial roles in tumor regulation. Confirming the presence of cancer-related extrachromosomal circular DNA would provide a putative testing strategy for the precision diagnosis and treatment of human malignancies in clinical practice.

Extrachromosomal circularization of DNA is an important genomic feature in cancer. However, the structure, composition and genome-wide frequency of extrachromosomal circular DNA have not yet been profiled extensively. Here, we combine genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multihit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer.

Several clones of reconstituted cells were isolated by fusion of karyoplasts of mouse melanoma B16 cells with cytoplasts of rat myoblastic L6 cells and processed by the mica-press-adsorption method for electron microscopy. Extrachromosomal small circular DNAs of less than 1 micron in contour length (smaller circular DNA) were present in both parental cells, and circles larger than 1 micron (larger circular DNA) were found more frequently in reconstituted cells at an early stage after the clonal isolation. Mitochondrial DNA was not released from mitochondria by this method. The new phenotypes of reconstituted cells were stable, but larger circles were lost after prolonged cultivation of the cells. The possibility that the larger circular DNAs result from intrachromosomal DNA rearrangement induced by rat cytoplasts is discussed.

COVALENTLY closed circular (CCC) duplex extrachromosomal DNAs are widespread in biological systems1. Because of their physical properties and small size, extra chromosomal circular DNAs are useful for studying the biology of genetic material. Their mechanism of replication is of particular interest and has been investigated extensively. Recent studies have shown that the replication of different types of circular DNA, such as plasmids of Escherichia coli, the DNA of polyoma and SV40 viruses, and mitochondrial DNA is basically similar2,3. Non-replicating circular DNA is isolated from cells largely as covalently closed supercoiled molecules (form I DNA). Because of its topological constraints, CCC DNA has a restricted affinity for intercalating dyes such as ethidium bromide and propidium diiodide; its buoyant density in gradients of CsCl containing such dyes is consequently much greater than that of non-circular or nicked circular (form II) DNA species, which can unwind to accommodate maximal amounts of dye4. DNA molecules in the process of replication have been shown to have buoyant densities intermediate to form I and form II DNA (refs 5–12 and L. Katz, P. H. Williams, S. Sato, R. W. Leavitt, and D. R. Helinski, unpublished). In a previous study of replicative forms of a constructed recombinant plasmid (pSC134) of E. coli13, we observed material pulse labelled with radioactive thymidine that had a buoyant density in CsCl–ethidium bromide gradients even greater than that of form I DNA5. Here we report results which suggest that this material, termed “heavy replicative DNA” or hrDNA, is a covalently closed circular DNA species that has a reduced number of supercoils, and is an intermediate in the replicative process.

The majority of cellular DNAs in eukaryotes are organized into linear chromosomes. In addition to chromosome DNAs, genes also reside on extrachromosomal elements. The extrachromosomal DNAs are commonly found to be circular, and they are referred to as extrachromosomal circular DNAs (eccDNAs). Recent technological advances have enriched our knowledge of eccDNA biology. There is currently increasing concern about the connection between eccDNA and cancer. Gene amplification on eccDNAs is prevalent in cancer. Moreover, eccDNAs commonly harbor oncogenes or drug resistance genes, hence providing a growth or survival advantage to cancer cells. eccDNAs play an important role in tumor heterogeneity and evolution, facilitating tumor adaptation to challenging circumstances. In addition, eccDNAs have recently been identified as cell-free DNAs in circulating system. The altered level of eccDNAs is observed in cancer patients relative to healthy controls. Particularly, eccDNAs are associated with cancer progression and poor outcomes. Thus, eccDNAs could be useful as novel biomarkers for the diagnosis and prognosis of cancer. In this review, we summarize current knowledge regarding the formation, characteristics and biological importance of eccDNAs, with a focus on the molecular mechanisms associated with their roles in cancer progression. We also discuss their potential applications in the detection and treatment of cancer. A better understanding of the functional role of eccDNAs in cancer would facilitate the comprehensive analysis of molecular mechanisms involved in cancer pathogenesis.

Analysis of recombination junctions in extrachromosomal circular DNA obtained by in-gel competitive reassociation