Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing

TLDR: The technique is highly sensitive and can detect the methylation status of up to 1.5 million CpG sites within the genome of an individual mouse embryonic stem cell (mESC) and shows that the demethylation dynamics of maternal and paternal genomes after fertilization can be traced within the individual pronuclei of mouse zygotes.

Abstract: Gene transcription is crucial for a cell to maintain its identity and physiological function and is regulated within individual cells. Epigenetic status is important in transcriptional regulation and is potentially heterogeneous even within a relatively homogeneous cell type due to the different cell subpopulations present (Jaenisch and Bird 2003; Toyooka et al. 2008). This is especially prominent in tumors in which both the genomes and epigenomes of the individual cells are heterogeneous (Rodriguez-Paredes and Esteller 2011; Marusyk et al. 2012). Moreover, it is very difficult to obtain large numbers of cells for epigenome analysis in some circumstances, such as for mammalian early embryos (Smallwood et al. 2011; Tang et al. 2011a; Smith et al. 2012). It is highly desirable to develop a single-cell epigenome analysis technique, ideally one that provides single-base resolution. As one of the most important epigenetic modifications, DNA methylation is critical for a wide variety of biological processes, including the regulation of genomic imprinting and X-chromosome inactivation, as well as the repression of transposable elements within the genome (Bird 2002; Lister et al. 2009; Hackett et al. 2012). DNA is methylated at the carbon atom occupying the fifth position of the cytosine ring (5mC) and is catalyzed by the DNA cytosine methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b (Reik 2007). DNA methylation is functionally important for mammalian development because both Dnmt1 and Dnmt3b knockout mice are embryonic lethal, whereas Dnmt3a mutant mice die within 1 mo after birth (Okano et al. 1999; Li 2002). The reduced representation bisulfite sequencing (RRBS) technique has been developed to dissect the methylomes of mammalian cells (Meissner et al. 2005). RRBS is based on the lack of even distribution for CpG sites within the mammalian genome; these sites tend to cluster together as CpG islands (CGIs) that are usually located near the promoter regions of annotated genes (Deaton and Bird 2011). Thus, after cutting the genome into short fragments via a restriction enzyme that recognizes CpG and its flanking sequences, a majority of the CGIs will be recovered and sequenced with high coverage even with relatively low numbers of total sequencing reads. RRBS has been shown to be effective for as few as 60 mouse early embryonic cells (Chan et al. 2012; Smallwood and Kelsey 2012) and has led to significant findings regarding global demethylation and remethylation processes during the early cleavage and post-implantation stages of mouse embryonic development, respectively (Smith et al. 2012). Recently, a method for the epigenetic analysis of histone modifications for an individual locus at single-cell resolution has been developed (Gomez et al. 2013). However, single-cell epigenome analysis at whole-genome scale has never been achieved. We report the development of a single-cell methylome analysis technique based on RRBS (scRRBS) and demonstrate its effective use for mouse embryonic stem cells (mESCs), sperm, and oocytes, as well as for male and female pronuclei of the zygotes. We were able to recover 0.5 to 1.5 million CpG sites from a single mESC, and the methylation levels for all analyzed genomic regions were comparable to those obtained from bulk mESCs (Table 1; Supplemental Table 1). Furthermore, we show for the first time that the methylome of the first polar body is comparable with that of the metaphase II oocyte within the same gamete. Finally, we used our method to prove that the demethylation process of the male pronucleus occurs more quickly than that of the female pronucleus in the same zygote. Table 1. Summary of the unique covered CpG sites and their mean coverage depths at 1×, 5×, and 10× in each single mESC cell and in bulk mESCs