Methylation profiling by high throughput sequencing
Summary
DNA methylation (5mC) is central to cellular identity and the global erasure of 5mC from the parental genomes during preimplantation mammalian development. This process is critical to reset the methylome of terminally differentiated gametes to the become pluripotent cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to genome-wide 5mC erasure remains unclear. Here, we report a new high-throughput single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, thereby allowing us to systematically probe the dynamics of global demethylation. First, when applied to hybrid mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome and suggesting loss of maintenance methylation. Next, using scMspJI-seq in preimplantation mouse development, we discovered that maintenance methylation is active till the 16-cell stage followed by passive demethylation in a fraction of cells within 2 the early blastocyst at the 32-cell stage of development. Finally, we found that human preimplantation embryos qualitatively show slower yet similar demethylation dynamics as mouse preimplantation embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost effective method to map the strand-specific genome- wide patterns of 5mC in single cells, thereby enabling quantitative investigation of methylation dynamics in developmental systems.
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