Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

Abstract

DNA methylation (5mC) is central to cellular identity. The global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of gametes to the 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 5mC erasure remains unclear. Here, we report a single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, allowing us to systematically probe the dynamics of global demethylation. When applied to 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 suggesting loss of maintenance methylation. Next, in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse 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.

Single-cell bisulfite-free 5mC and 5hmC sequencing with high sensitivity and scalability

“…In this way, hemi-5mC and hemi-5hmC landscapes in mouse embryos were mapped (Fig. 3c), which reflects passive demethylation activity (41). The abundance of hemi-5mC sharply increased after fertilization, reflecting a surge in passive demethylation; hemi-5mC abundance gradually elevated after late 2-cell when 5hmC declined (Fig.…”

Omics Views of Mechanisms for Cell Fate Determination in Early Mammalian Development

“…DNA methylation, usually referred to as 5-methylcytosine (5mC) methylation, plays a vital role in early mammalian embryogenesis [73] , [74] , [75] , [76] . The acquisition of a whole-scale DNA methylome is frequently based on bisulfite treatment followed by sequencing (BS-seq) using a large amount of input material [77] .…”

“…The acquisition of a whole-scale DNA methylome is frequently based on bisulfite treatment followed by sequencing (BS-seq) using a large amount of input material [77] . Advances in some techniques, such as single-cell reduced representation bisulfite sequencing (scRRBS), single-cell post-bisulfite adaptor tagging DNA methylome sequencing (scPBAT-seq), single-cell BS-seq (scBS-seq), and single-cell MspJI-based strand-specific 5mC sequencing method (scMspJI-seq), have overcome the inherent difficulty of input cells, through which single-cell dynamic DNA methylation landscapes in early mouse and human embryogenesis have been successfully obtained [73] , [74] , [75] , [76] . In mouse 4-cell embryos, genetic lineage can be traced by DNA 5mC [75] .…”

Introduction to Single-Cell DNA Methylation Profiling Methods

“…Preimplantation plays an important role in transforming terminally differentiated gametes into pluripotent cells through a mechanism that removes methylation during cell development. As a result of examining strand-specific changes using single-cell DNA methylation, it was observed that the loss of methylation maintenance was strand-specific [104]. CpG methylation was established broadly at the immature germinal vesicle stage using single-cell methylation sequencing to study not only the process of embryonic development (Figure 7, left) but also the process of human germ cell maturation.…”