Department of Urology, Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
Genome Res. 2012 Dec;22(12):2497-506. doi: 10.1101/gr.143008.112. Epub 2012 Sep 7.
DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline, we show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions that is not present at promoters. We further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome-depleted. Importantly, NOMe-seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule, and thus, single cell level, that can be used to monitor disease progression and response to therapy.
DNA 甲基化和核小体定位共同作用，生成调节基因表达的染色质结构。核小体通常使用需要大量材料和不同酶浓度的核酸酶消化来进行作图。我们开发了一种方法（NOMe-seq），该方法使用 GpC 甲基转移酶（M.CviPI）和下一代测序，使用不到 100 万个细胞，在保留同一 DNA 链上内源性 DNA 甲基化信息的同时，生成全基因组核小体定位的高分辨率足迹。使用一种新的生物信息学管道，我们发现在 CTCF 区域，核小体占有率与 DNA 甲基化之间存在显著的反相关关系，而这种关系在启动子中不存在。我们进一步表明，启动子处核小体耗竭的程度与表达水平直接相关，可以容纳多个核小体，并提供全基因组证据表明表达的非 CpG 岛启动子是核小体耗竭的。重要的是，NOMe-seq 从同一 DNA 分子获得 DNA 甲基化和核小体定位信息，首次在单分子水平上进行全基因组 DNA 甲基化和核小体定位相关性分析，并且可以用于监测疾病进展和治疗反应。
