Coordinated changes of DNA (de)methylation, nucleosome positioning, and chromatin binding of the architectural protein CTCF play an important role for establishing cell-type-specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes, we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the Tet1 and Tet2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC), or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation, and gene expression in DKO ESCs and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, whereas unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild-type (WT) cells but not from 5fC-enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, for example, at promoters, boundaries of chromatin loops, and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to down-regulation of neighboring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions, and DNA sequence that determines differential CTCF binding and regulates gene expression.

中文翻译：

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胚胎干细胞中的 DNA（去）甲基化控制 CTCF 依赖性染色质边界。

结构蛋白 CTCF 的 DNA（去）甲基化、核小体定位和染色质结合的协调变化对于在分化过程中建立细胞类型特异性染色质状态起着重要作用。为了阐明连接这些过程的分子机制，我们研究了携带 Tet1 和 Tet2 双加氧酶双敲除 (DKO) 的小鼠胚胎干细胞 (ESC) 中扰动的 DNA 修饰景观。这些酶负责将 5-甲基胞嘧啶 (5mC) 转化为其羟甲基化 (5hmC)、甲酰化 (5fC) 或羧化 (5caC) 形式。我们确定了 DKO ESC 中核小体定位、CTCF 结合、DNA 甲基化和基因表达的变化，并开发了生物物理模型来预测差异 CTCF 结合。甲基化敏感的核小体重新定位占 DKO ESC 中 CTCF 结合损失的重要部分，而未甲基化和核小体耗尽的 CpG 岛因仍被占用的 CTCF 位点而富集。许多 CTCF 位点也显示出与 CpG 修饰状态的直接相关性：CTCF 优先从野生型 (WT) 细胞中标记为 5hmC 的位点丢失，而不是从富含 5fC 的位点丢失。此外，我们发现一些 CTCF 位点可以作为定义差异甲基化景观的分叉点。这些位点的 CTCF 丢失，例如，在启动子、染色质环的边界和拓扑相关域 (TAD) 处，与 DNA 甲基化/去甲基化扩散相关，并且可能与邻近基因的下调有关。