Simsir Özge, Walter Tobias, Sahin Hanife, Carell Thomas, Schneider Sabine
Department of Chemistry, Institute for Chemical Epigenetics, Ludwig-Maximilians Universität Munich Butenandtstr. 5-13 81377 Munich Germany
RSC Chem Biol. 2025 Mar 10;6(5):731-736. doi: 10.1039/d4cb00315b. eCollection 2025 May 8.
Epigenetic regulation of gene expression is essential for cellular development and differentiation processes in higher eukaryotes. Modifications of cytosine, in particular 5-methylcytosine (5mdC), in DNA play a central role through impacting chromatin structure, repressing transposons, and regulating transcription. DNA methylation is actively installed by DNA methyltransferases and reversed through Tet-dioxygenase-mediated oxidation of 5mdC to 5-hydroxylmethylcytosine (5hmdC), 5-formylcytosine (5fdC), and 5-carboxycytosine (5cadC). It is crucial to understand the role of these epigenetic DNA modifications in cellular differentiation and developmental processes, as well as in disease state mapping and tracing of 5mdC and its oxidized forms. In bisulfite sequencing, which has been the benchmark for mapping 5mdC for the last few decades, degradation of the majority of genetic material occurs through harsh chemical treatment. Alternative sequencing methods often utilize Tet-enzyme-mediated oxidation of 5mdC to locate 5mdC and 5hmdC in genomic DNA. Herein, we report the development of novel Tet3-variants for oxidation-based bisulfite-free 5mdC- sequencing.
基因表达的表观遗传调控对于高等真核生物的细胞发育和分化过程至关重要。DNA中胞嘧啶的修饰，特别是5-甲基胞嘧啶（5mdC），通过影响染色质结构、抑制转座子和调节转录发挥核心作用。DNA甲基化由DNA甲基转移酶主动安装，并通过Tet双加氧酶介导的5mdC氧化为5-羟甲基胞嘧啶（5hmdC）、5-甲酰基胞嘧啶（5fdC）和5-羧基胞嘧啶（5cadC）而逆转。了解这些表观遗传DNA修饰在细胞分化和发育过程中的作用，以及在疾病状态下5mdC及其氧化形式的定位和追踪至关重要。在过去几十年中一直是绘制5mdC基准的亚硫酸氢盐测序中，大多数遗传物质通过苛刻的化学处理发生降解。替代测序方法通常利用Tet酶介导的5mdC氧化来定位基因组DNA中的5mdC和5hmdC。在此，我们报告了用于基于氧化的无亚硫酸氢盐5mdC测序的新型Tet3变体的开发。
