We present a method, named Mx-TOP, for profiling of three epigenetic regulatory layers—chromatin accessibility, general DNA modification, and DNA hydroxymethylation—from a single library. The approach is based on chemo-enzymatic covalent tagging of unmodified CG sites and hydroxymethylated cytosine (5hmC) along with GC sites in chromatin, which are then mapped using tag-selective base-resolution TOP-seq sequencing. Our in-depth validation of the approach revealed its sensitivity and informativity in evaluating chromatin accessibility and DNA modification interactions that drive transcriptional regulation. We employed the technology in a study of chromatin and DNA demethylation dynamics during neuronal differentiation. The study highlighted the involvement of gene body 5hmC in modulating an extensive decoupling between promoter accessibility and transcription. The importance of 5hmC in chromatin remodeling was further demonstrated by the observed resistance of the developmentally acquired open loci to the global 5hmC erasure in neuronal progenitors.

中文翻译：

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非甲基化、羟甲基化和开放染色质位点的一锅三峰图谱揭示了 5hmC 在动态染色质位点上的独特作用

我们提出了一种名为 Mx-TOP 的方法，用于从单个文库中分析三个表观遗传调控层——染色质可及性、一般 DNA 修饰和 DNA 羟甲基化。该方法基于未修饰的 CG 位点和羟甲基化胞嘧啶 (5hmC) 以及染色质中的 GC 位点的化学酶共价标记，然后使用标记选择性碱基分辨率 TOP-seq 测序进行定位。我们对该方法的深入验证揭示了其在评估驱动转录调控的染色质可及性和 DNA 修饰相互作用方面的敏感性和信息性。我们利用该技术研究神经元分化过程中的染色质和 DNA 去甲基化动态。该研究强调了基因体 5hmC 参与调节启动子可及性和转录之间的广泛解耦。观察到发育获得的开放基因座对神经元祖细胞中整体 5hmC 擦除的抵抗性，进一步证明了 5hmC 在染色质重塑中的重要性。