Methylation pseudotime analysis for label-free profiling of the temporal chromatin landscape with long-read and single-molecule resolution [ATAC-seq]. Methylation pseudotime analysis for label-free profiling of the temporal chromatin landscape with long-read and single-molecule resolution [ATAC-seq]

Faithful epigenetic inheritance across cell divisions is essential to maintaining cell identity and involves numerous epigenetic modifications, whose roles in coordinating chromatin architecture are less understood. Technological approaches to temporally order epigenetic modifications throughout the cell cycle often face limitations in sequence resolution and rely on potentially damaging mitotic labeling or conversion steps. Herein, we present Methylation Pseudotime Analysis Through read-level Heterogeneity (MPATH), a label- and conversion-free method to infer post-replication DNA strand maturity from methylation patterns across single molecules. We use MPATH to temporally order hydroxymethylation throughout mitotic inheritance revealing, for the first time, that CpGs within cis-regulatory elements undergo transitions between methylation states at sub-cell-cycle timescales. When applied to long reads generated by NOMe-seq, MPATH uncovered relationships between nucleosome occupancy and DNA maturity. Finally, extension of MPATH to phased reads reveals allele-specific trends in pseudotime distribution associated with X chromosome inactivation. Our findings suggest that when coupled with multimodal sequencing strategies, MPATH could provide valuable insights into chromatin restoration dynamics. Overall design: HUES64 hESCs were subjected to several different experimental conditions prior to DNA extraction for long-read sequencing. In the first experiment, hESCs were treated with 2µg/ml nocodazole for 16h. BrdU pulse-chase experiments were conducted on HUES64 hESCs to investigate DNA methylation patterns during chromatin maturation. For the pulse optimization, cells were subjected to 50 µM BrdU for durations of 10 minutes, 20 minutes, 30 minutes, and 1 hour, followed by genomic DNA collection at 0h (nascent chromatin) and 16h (mature chromatin). Mock controls were included for both 0h and 16h timepoints. Optimized labeling conditions included a 500 µM BrdU pulse (10 minute duration) with 0h and 16h chase points. Unlabeled HUES64 samples were also generated (sequenced to a total of 62x coverage across 2 technical replicates). All long-read data was acquired using ONT sequencing platforms. In a separate sequencing experiment, hESCs were subjected to Repli-ATAC sequencing in order to temporally profile chromatin accessibility across replication-associated timescales.

跨细胞分裂的表观遗传忠实遗传，对于维持细胞身份至关重要，且涉及众多表观遗传修饰；然而这类修饰在协调染色质架构中的作用仍未得到充分阐释。目前用于在整个细胞周期中对表观遗传修饰进行时间排序的技术方法，往往在序列分辨率上存在局限，且需依赖可能具有损伤性的有丝分裂标记或转化步骤。为此，我们提出了基于读段水平异质性的甲基化伪时间分析（Methylation Pseudotime Analysis Through read-level Heterogeneity，MPATH）——一种无需标记与转化的方法，可从单分子水平的甲基化模式中推断复制后DNA链的成熟度。我们利用MPATH对有丝分裂遗传过程中的羟甲基化进行时间排序，首次揭示了顺式调控元件内的CpG位点会在亚细胞周期时间尺度上发生甲基化状态的转变。当将MPATH应用于NOMe-seq产生的长读段数据时，其揭示了核小体占据与DNA成熟度之间的关联。最后，将MPATH拓展至分相读段后，我们发现了与X染色体失活相关的伪时间分布等位基因特异性趋势。我们的研究结果表明，若将MPATH与多模态测序策略相结合，可为染色质修复动力学研究提供极具价值的见解。实验整体设计：在进行DNA提取以开展长读段测序前，HUES64人胚胎干细胞（human embryonic stem cells，hESCs）接受了多种不同的实验处理。在第一项实验中，hESCs经2μg/ml诺考达唑处理16小时。我们针对HUES64 hESCs开展了BrdU脉冲追踪实验，以探究染色质成熟过程中的DNA甲基化模式。在脉冲标记优化实验中，细胞经50μM BrdU分别处理10分钟、20分钟、30分钟及1小时，随后分别在0小时（新生染色质）与16小时（成熟染色质）两个时间点收集基因组DNA。0小时与16小时两个时间点均设置了空白对照。优化后的标记条件为：以500μM BrdU进行10分钟脉冲标记，并设置0小时与16小时两个追踪时间点。我们还制备了未标记的HUES64样本（两个技术重复样本的总测序覆盖度达62×）。所有长读段数据均通过ONT测序平台获取。在另一项独立测序实验中，我们对hESCs开展了Repli-ATAC测序，以在与复制相关的时间尺度上对染色质可及性进行时间维度的表征。