Effects of DNA Methylation on Nucleosome Stability

Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted high-throughput nucleosome reconstitution experiments on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The DNA loci from both species contained the genes that encode the serum albumin family, the MYC protein, and the tumor suppressor p53. The results demonstrated that a small subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. We propose that these features serve to enhance the affinity of methylated DNA for the histone octamer and that this effect could be involved in gene regulatory mechanisms such as silencing. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin. For the human data, there were 3 unmethylated nucleosome replicates, 2 methylated nucleosome replicates, an unmethylated MNase control, and an unmethylated sonicated control. For the mouse data, there were 2 unmethylated nucleosome replicates, 2 methylated nucleosome replicates, an unmethylated MNase control, a methylated MNase control, and an unmethylated sonicated control.

脊椎动物细胞中，CpG二核苷酸（CpG dinucleotides）位点的DNA甲基化，是调控基因表达的核心表观遗传机制之一。本研究针对572 KB的未甲基化或甲基化人类DNA，以及668 KB的未甲基化或甲基化小鼠DNA，开展了高通量核小体（nucleosome）重构实验，以探究该表观遗传修饰对核小体定位与稳定性的影响。两个物种的DNA位点均包含编码血清白蛋白家族、MYC蛋白以及肿瘤抑制因子p53的基因。实验结果显示，一小部分由核苷酸序列定位的核小体对甲基化敏感：该修饰可提升这些序列与组蛋白八聚体（histone octamer）的结合亲和力。相较于绝大多数对甲基化不敏感的核小体，这类敏感核小体具有两项特征：一是CpG二核苷酸的出现频率更高，二是CpG位点具有独特的旋转取向——其小沟（minor groove）更倾向于朝向核小体内的组蛋白，而非背离组蛋白。我们推测，上述特征可提升甲基化DNA与组蛋白八聚体的结合亲和力，且该效应可能参与基因沉默等基因调控机制。相较于内含子区域，这类甲基化敏感核小体更倾向于与外显子区域结合；而转录起始位点（transcription start sites）附近的未甲基化CpG岛（CpG islands）在发生甲基化后，核小体的富集程度会有所提升。本研究结果表明，体外实验中DNA甲基化对核小体稳定性的影响，可重现细胞内已观测到的现象，并为DNA甲基化与染色质（chromatin）的结构及功能之间搭建了直接关联。人类样本数据组中，包含3组未甲基化核小体重复实验、2组甲基化核小体重复实验、1组未甲基化MNase对照以及1组未甲基化超声裂解对照。小鼠样本数据组中，包含2组未甲基化核小体重复实验、2组甲基化核小体重复实验、1组未甲基化MNase对照、1组甲基化MNase对照以及1组未甲基化超声裂解对照。