Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping.
Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping.
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DOI：
10.1093/nar/gkac460
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发表时间：
2022-09-09
影响因子：
14.9
通讯作者：
Ebenstein, Yuval
中科院分区：
文献类型：
--
作者：
Gabrieli, Tslil;Michaeli, Yael;Avraham, Sigal;Torchinsky, Dmitry;Margalit, Sapir;Schutz, Leonie;Juhasz, Matyas;Coruh, Ceyda;Arbib, Nissim;Zhou, Zhaohui Sunny;Law, Julie A.;Weinhold, Elmar;Ebenstein, Yuval
DNA 甲基化，特别是 5 个碳位置 (5-mC) 的胞嘧啶 (C) 核苷酸的甲基化，是研究最多且最重要的表观遗传修饰。在这里，我们开发了一种化学酶程序，可以在 CpG 背景下荧光标记非甲基化胞嘧啶，从而对跨越数十万个碱基对的单个 DNA 分子进行表观遗传分析。我们使用 CpG 甲基转移酶和合成的 S-腺苷-L-甲硫氨酸辅因子类似物将叠氮化物转移到胞嘧啶而不是天然甲基。然后将荧光团点击到 DNA 上，报告非甲基化 CpG 的数量和位置。我们发现，通过添加核苷酶，标记效率提高了 2 倍，大概是通过在标记后降解辅因子的无活性副产物，防止其抑制作用。我们使用该方法确定了慢性淋巴细胞白血病患者的整体 DNA 甲基化下降情况，然后对模型植物拟南芥进行了全基因组甲基化作图。我们的基因组图谱与已发表的亚硫酸氢盐测序甲基化图谱高度一致。尽管光学检测将作图分辨率限制在 500-1000 bp，但通过这种方法产生的标记 DNA 分子有数十万个碱基对长，允许访问长重复且结构可变的基因组区域。
DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and significant epigenetic modification. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in CpG context, allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. We used a CpG methyltransferase with a synthetic S-adenosyl-l-methionine cofactor analog to transfer an azide to cytosines instead of the natural methyl group. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased up to 2-fold by the addition of a nucleosidase, presumably by degrading the inactive by-product of the cofactor after labeling, preventing its inhibitory effect. We used the method to determine the decline in global DNA methylation in a chronic lymphocytic leukemia patient and then performed whole-genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published bisulfite sequencing methylation maps. Although mapping resolution is limited by optical detection to 500–1000 bp, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.
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影响因子：
48
作者：
Flusberg, Benjamin A.;Webster, Dale R.;Lee, Jessica H.;Travers, Kevin J.;Olivares, Eric C.;Clark, Tyson A.;Korlach, Jonas;Turner, Stephen W.
通讯作者：
Turner, Stephen W.
影响因子：
64.5
作者：
Kawakatsu T;Huang SC;Jupe F;Sasaki E;Schmitz RJ;Urich MA;Castanon R;Nery JR;Barragan C;He Y;Chen H;Dubin M;Lee CR;Wang C;Bemm F;Becker C;O'Neil R;O'Malley RC;Quarless DX;1001 Genomes Consortium;Schork NJ;Weigel D;Nordborg M;Ecker JR
通讯作者：
Ecker JR
影响因子：
15
作者：
Lee, Bobby W. K.;Sun, He G.;Zang, Tianzhu;Kim, Byung Ju;Alfaro, Joshua F.;Zhou, Zhaohui Sunny
通讯作者：
Zhou, Zhaohui Sunny
影响因子：
16.6
作者：
Deschamps S;Zhang Y;Llaca V;Ye L;Sanyal A;King M;May G;Lin H
通讯作者：
Lin H
影响因子：
7
作者：
Jiao WB;Accinelli GG;Hartwig B;Kiefer C;Baker D;Severing E;Willing EM;Piednoel M;Woetzel S;Madrid-Herrero E;Huettel B;Hümann U;Reinhard R;Koch MA;Swan D;Clavijo B;Coupland G;Schneeberger K
通讯作者：
Schneeberger K