Hardwick Jack S, Dhir Somdutta, Kirchner Angie, Simeone Angela, Flynn Sean M, Edgerton James M, de Cesaris Araujo Tavares Rafael, Esain-Garcia Isabel, Tannahill David, Golder Paula, Monahan Jack M, Gosal Walraj S, Balasubramanian Shankar
Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom.
Cancer Research UK Cambridge institute, University of Cambridge, Cambridge CB2 0RE, United Kingdom.
Proc Natl Acad Sci U S A. 2025 Aug 5;122(31):e2512204122. doi: 10.1073/pnas.2512204122. Epub 2025 Jul 31.
In mammalian genomes, cytosine modifications form a layer of regulatory information alongside the genetic code. Decoding this information is crucial to our understanding of biology and disease. Established sequencing methods cannot simultaneously resolve cytosine's three most common forms-cytosine (C), 5-methylcytosine (mC), and 5-hydroxymethylcytosine (hmC)-across both strands of the DNA double helix. Thus, how epigenetic information is distributed in DNA remains unclear. Here, we present trand-upled andem ytosine ydroxymethylation and methylation sequencing (SCoTCH-seq): an accurate and quantitative, base-resolution approach to sequence genomes, together with mC and hmC, in both strands of the same DNA fragment. We show that different forms of cytosine combine across the double helix at CpG sites to form discrete information states in the mouse epigenome. These CpG states have distinct genomic distributions-including at promoters, enhancers, and gene bodies-and have different relationships with transcription. We show that while all possible forms of hydroxymethylation occur, hmC is predominantly asymmetric and that different forms of asymmetric hmC are not equivalent. Our findings demonstrate that 5-hydroxymethylcytosine combines with different cytosine variants across the DNA double helix to form distinct states of regulatory information.
在哺乳动物基因组中，胞嘧啶修饰与遗传密码一起构成了一层调控信息。解读这些信息对于我们理解生物学和疾病至关重要。现有的测序方法无法同时解析胞嘧啶的三种最常见形式——胞嘧啶（C）、5-甲基胞嘧啶（mC）和5-羟甲基胞嘧啶（hmC）——在DNA双螺旋的两条链上的情况。因此，表观遗传信息在DNA中是如何分布的仍不清楚。在这里，我们展示了双链和单链胞嘧啶羟甲基化与甲基化测序（SCoTCH-seq）：一种准确且定量的、碱基分辨率的方法，用于对同一DNA片段的两条链上的基因组以及mC和hmC进行测序。我们表明，不同形式的胞嘧啶在CpG位点跨双螺旋结合，在小鼠表观基因组中形成离散的信息状态。这些CpG状态具有不同的基因组分布——包括在启动子、增强子和基因体处——并且与转录有不同的关系。我们表明，虽然所有可能形式的羟甲基化都会发生，但hmC主要是不对称的，并且不同形式的不对称hmC并不等同。我们的研究结果表明，5-羟甲基胞嘧啶与DNA双螺旋上不同的胞嘧啶变体结合，形成不同的调控信息状态。
