Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, 17177, Sweden.
Science for Life Laboratory, Tomtebodavägen 23A, Solna, 17165, Sweden.
Nat Commun. 2024 Feb 27;15(1):1768. doi: 10.1038/s41467-024-45972-y.
Extrachromosomal circular DNAs (eccDNAs) have emerged as important intra-cellular mobile genetic elements that affect gene copy number and exert in trans regulatory roles within the cell nucleus. Here, we describe scCircle-seq, a method for profiling eccDNAs and unraveling their diversity and complexity in single cells. We implement and validate scCircle-seq in normal and cancer cell lines, demonstrating that most eccDNAs vary largely between cells and are stochastically inherited during cell division, although their genomic landscape is cell type-specific and can be used to accurately cluster cells of the same origin. eccDNAs are preferentially produced from chromatin regions enriched in H3K9me3 and H3K27me3 histone marks and are induced during replication stress conditions. Concomitant sequencing of eccDNAs and RNA from the same cell uncovers the absence of correlation between eccDNA copy number and gene expression levels, except for a few oncogenes, including MYC, contained within a large eccDNA in colorectal cancer cells. Lastly, we apply scCircle-seq to one prostate cancer and two breast cancer specimens, revealing cancer-specific eccDNA landscapes and a higher propensity of eccDNAs to form in amplified genomic regions. scCircle-seq is a scalable tool that can be used to dissect the complexity of eccDNAs across different cell and tissue types, and further expands the potential of eccDNAs for cancer diagnostics.
染色体外环状 DNA（eccDNA）已成为影响基因拷贝数的重要细胞内可移动遗传元件，并在细胞核内发挥跨基因调控作用。在这里，我们描述了 scCircle-seq 方法，用于分析 eccDNA 并揭示其在单细胞中的多样性和复杂性。我们在正常和癌细胞系中实施和验证了 scCircle-seq，证明大多数 eccDNA 在细胞间差异很大，并在细胞分裂过程中随机遗传，尽管它们的基因组景观是细胞类型特异性的，并可用于准确聚类相同来源的细胞。eccDNA 优先从富含 H3K9me3 和 H3K27me3 组蛋白标记的染色质区域产生，并在复制应激条件下诱导产生。从同一细胞中同时对 eccDNA 和 RNA 进行测序，揭示了 eccDNA 拷贝数与基因表达水平之间除少数癌基因（包括结直肠癌细胞中的 MYC）外不存在相关性，这些癌基因包含在一个大的 eccDNA 中。最后，我们将 scCircle-seq 应用于一个前列腺癌和两个乳腺癌标本，揭示了癌症特异性的 eccDNA 景观以及 eccDNA 在扩增基因组区域中形成的更高倾向。scCircle-seq 是一种可扩展的工具，可用于剖析不同细胞和组织类型中 eccDNA 的复杂性，并进一步扩展了 eccDNA 在癌症诊断中的潜力。
