Methylation pseudotime analysis for label-free profiling of the temporal chromatin landscape with long-read sequencing. Methylation pseudotime analysis for label-free profiling of the temporal chromatin landscape with long-read sequencing

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 showed that MPATH is generally applicable across human embryonic stem cell (hESC) lines in which CpG methylation heterogeneity is coupled to replication, but not applicable to breast cancer cells, in which CpG methylation heterogeneity may also be associated with other factors. We used MPATH to track hydroxymethylated regions throughout mitotic inheritance in hESCs and observed that in the absence of ten-eleven translocation (TET) enzymes, these regions appeared to progress more quickly across replication-associated timescales, suggesting that enzymatic competition may drive temporal heterogeneity in CpG methylation patterns. When applied to long reads generated by NOMe-seq, MPATH revealed that nucleosome compaction and CpG methylation uniformity are coupled across replication-associated timescales. 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, H9 hESCs, HUES8 TKO hESCs and MDA-MB-231 breast cancer cells were subjected to several different experimental conditions prior to DNA extraction for long-read sequencing. In the first experiment, HUES64 hESCs were treated with 1µg/ml nocodazole for 16h. BrdU pulse-chase experiments were conducted on all cell lines 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. >>>Raw data in POD5 or FAST5 will be available upon request by the submitter.<<<