Staševskij Zdislav, Gibas Povilas, Gordevičius Juozas, Kriukienė Edita, Klimašauskas Saulius
Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-10257, Lithuania.
Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius LT-10257, Lithuania; Department of Systems Analysis, Institute of Mathematics and Informatics, Vilnius University, Vilnius LT-08663, Lithuania.
Mol Cell. 2017 Feb 2;65(3):554-564.e6. doi: 10.1016/j.molcel.2016.12.012. Epub 2017 Jan 19.
Modification of CG dinucleotides in DNA is part of epigenetic regulation of gene function in vertebrates and is associated with complex human disease. Bisulfite sequencing permits high-resolution analysis of cytosine modification in mammalian genomes; however, its utility is often limited due to substantial cost. Here, we describe an alternative epigenome profiling approach, named TOP-seq, which is based on covalent tagging of individual unmodified CG sites followed by non-homologous priming of the DNA polymerase action at these sites to directly produce adjoining regions for their sequencing and precise genomic mapping. Pilot TOP-seq analyses of bacterial and human genomes showed a better agreement of TOP-seq with published bisulfite sequencing maps as compared to widely used MBD-seq and MRE-seq and permitted identification of long-range and gene-level differential methylation among human tissues and neuroblastoma cell types. Altogether, we propose an affordable single CG-resolution technique well suited for large-scale epigenome studies.
DNA中CG二核苷酸的修饰是脊椎动物基因功能表观遗传调控的一部分，并且与复杂的人类疾病相关。亚硫酸氢盐测序能够对哺乳动物基因组中的胞嘧啶修饰进行高分辨率分析；然而，由于成本高昂，其应用常常受到限制。在此，我们描述了一种名为TOP-seq的替代表观基因组分析方法，该方法基于对单个未修饰的CG位点进行共价标记，随后在这些位点对DNA聚合酶的作用进行非同源引发，以直接产生相邻区域用于测序和精确的基因组定位。对细菌和人类基因组的初步TOP-seq分析表明，与广泛使用的MBD-seq和MRE-seq相比，TOP-seq与已发表的亚硫酸氢盐测序图谱具有更好的一致性，并且能够识别出人类组织和神经母细胞瘤细胞类型之间的长程和基因水平的差异甲基化。总之，我们提出了一种经济实惠的单CG分辨率技术，非常适合大规模表观基因组研究。
