Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117583, Singapore.
Institute for Health Innovation and Technology, National University of Singapore, Singapore 117599, Singapore.
Sci Adv. 2023 Sep 15;9(37):eadi0197. doi: 10.1126/sciadv.adi0197.
5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the most abundant DNA modifications that have important roles in gene regulation. Detailed studies of these different epigenetic marks aimed at understanding their combined effects and dynamic interconversion are, however, hampered by the inability of current methods to simultaneously measure both modifications, particularly in samples with limited quantities. We present DNA analysis by restriction enzyme for simultaneous detection of multiple epigenomic states (DARESOME), an assay based on modification-sensitive restriction digest and sequential tag ligation that can concurrently perform quantitative profiling of unmodified cytosine, 5mC, and 5hmC in CCGG sites genome-wide. DARESOME reveals the opposing roles of 5mC and 5hmC in gene expression regulation as well as their interconversion during aging in mouse brain. Implementation of DARESOME in single cells demonstrates pronounced 5hmC strand bias that reflects the semiconservative replication of DNA. Last, we showed that DARESOME enables integrative genomic, 5mC, and 5hmC profiling of cell-free DNA that uncovered multiomics cancer signatures in liquid biopsy.
5- 甲基胞嘧啶 (5mC) 和 5- 羟甲基胞嘧啶 (5hmC) 是最丰富的 DNA 修饰，它们在基因调控中起着重要作用。然而，对这些不同的表观遗传标记进行详细研究，旨在了解它们的联合作用和动态相互转化，受到当前方法无法同时测量这两种修饰的限制，特别是在数量有限的样本中。我们提出了通过限制酶分析 DNA 以同时检测多种表观基因组状态的方法 (DARESOME)，该方法基于修饰敏感的限制消化和顺序标签连接，可同时对 CCGG 位点全基因组中未修饰的胞嘧啶、5mC 和 5hmC 进行定量分析。DARESOME 揭示了 5mC 和 5hmC 在基因表达调控中的相反作用，以及它们在小鼠大脑衰老过程中的相互转化。在单细胞中实施 DARESOME 表明，5hmC 链偏向明显，反映了 DNA 的半保守复制。最后，我们表明，DARESOME 能够对无细胞游离 DNA 进行基因组、5mC 和 5hmC 的综合分析，在液体活检中揭示了多组学癌症特征。
