PO.MCB06.01 · 分子与细胞生物学
通过长读长多组学测序和高内涵显微成像揭示 ecDNA 的空间组织和表观遗传景观
Unveiling ecDNA spatial organization and epigenetic landscapes through long-read multi-omic sequencing and high-content microscopy
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
癌基因扩增是癌症中最常见的遗传改变之一，在肿瘤发生中起核心作用。癌基因扩增的一个主要机制是染色体外 DNA（ecDNA），它存在于近 20% 的癌症患者中，并见于半数所有癌症类型。ecDNA 阳性肿瘤患者的生存期明显短于无 ecDNA 的患者。尽管 ecDNA 普遍存在且与不良治疗结局密切相关，但目前尚无获批的针对 ecDNA 阳性癌症的治疗方法。开发此类治疗需要识别 ecDNA 独特且可干预的特征。
ecDNA 有两个看似矛盾的独特特征值得特别关注。一方面，癌细胞的增殖依赖于 ecDNA 高水平的癌基因表达。另一方面，ecDNA 分子优先聚集在核周边区，而该区域通常与转录抑制相关。为什么癌细胞会将其大部分 ecDNA 定位于核周边？什么分子机制控制这种定位，其功能后果又是什么？这些是该领域长期存在的问题，之所以一直未能解答，很大程度上是由于缺乏能够解析 ecDNA 分子异质性并将其空间组织与癌基因表达联系起来的实验方法。
为填补这一技术空白并回答这些基本的 ecDNA 生物学问题，我们开发了 DINO-seq，一种长读长单分子测序技术，能够在单个 ecDNA 分子上同时分析蛋白质结合、DNA 可及性和 CpG 甲基化。使用 DINO-seq，我们发现 ecDNA 携带一种二价表观遗传景观——即活性和抑制性表观遗传标记共存——这与染色体 DNA 不同，使 ecDNA 即使在核周边也能保持转录活性。将 CRISPR 筛选与高内涵显微成像相结合，我们进一步鉴定出将 ecDNA 系留至核周边的分子锚点。在功能上，我们表明外周 ecDNA 定位改变复制时序、促进 ecDNA 枢纽（hub）形成，并增强 RNA 转录和输出。总之，这些发现揭示 ecDNA 具有独特的空间和表观遗传特性，使其区别于染色体 DNA，并代表可用于靶向 ecDNA 阳性癌症的潜在脆弱性。
查看英文原文 English abstract
Oncogene amplification is one of the most common genetic alterations in cancer, playing a central role in tumorigenesis. A major mechanism of oncogene amplification is extrachromosomal DNA (ecDNA), which is present in nearly 20% of cancer patients and found across half of all cancer types. Patients with ecDNA-positive tumors have significantly shorter survival compared to those without ecDNA. Despite its prevalence and strong association with poor treatment outcomes, there is no approved treatment targeting ecDNA-positive cancers. Developing such treatments requires identifying unique and actionable features of ecDNA.
Two unique features of ecDNA warrant special attention because they are seemingly paradoxical. On one hand, proliferation of cancer cells depends on high-level oncogene expression from ecDNA. On the other hand, ecDNA molecules preferentially cluster at the nuclear periphery, a region typically associated with transcriptional repression. Why would cancer cells position most of their ecDNA at the nuclear periphery? What molecular mechanisms control this localization, and what are its functional consequences? These are longstanding questions in the field that have remained unanswered largely due to the lack of experimental approaches capable of deciphering the molecular heterogeneity of ecDNA and linking its spatial organization to oncogene expression.
To fill the technical gap and answer these fundamental ecDNA biology questions, we developed DINO-seq, a long-read single-molecule sequencing technology that enables simultaneous profiling protein binding, DNA accessibility and CpG methylation on individual ecDNA molecules. Using DINO-seq, we discovered that ecDNA carries a bivalent epigenetic landscape-coexisting active and repressive epigenetic marks-that is distinct from chromosomal DNA and enables ecDNA to remain transcriptionally active even at the nuclear periphery. Combining a CRISPR screen with high-content microscopy, we further identified molecular anchors that tether ecDNA to the nuclear periphery. Functionally, we show that peripheral ecDNA localization alters replication timing, promotes ecDNA hub formation, and enhances RNA transcription and export. Together, these findings reveal that ecDNA possesses unique spatial and epigenetic properties that distinguish it from chromosomal DNA and represent potential vulnerabilities that could be exploited to target ecDNA-positive cancers.
利益披露 Disclosure
Y. Wang, None..
X. Yan, None..
N. E. Weiser, None..
S. Zhang, None..
I. Wong, None..
Y. Huang, None..
R. Li, None..
N. Altemose, None.