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. 2020 Sep 2:8:570107.
doi: 10.3389/fcell.2020.570107. eCollection 2020.

Characterization of DNA Methylation Patterns and Mining of Epigenetic Markers During Genomic Reprogramming in SCNT Embryos

Pengbo Cao  1 , Hanshuang Li  1 , Yongchun Zuo  1 , Buhe Nashun  1
Affiliations

Characterization of DNA Methylation Patterns and Mining of Epigenetic Markers During Genomic Reprogramming in SCNT Embryos

Pengbo Cao et al. Front Cell Dev Biol. .

Abstract

Somatic cell nuclear transfer (SCNT), also known as somatic cell cloning, is a commonly used technique to study epigenetic reprogramming. Although SCNT has the advantages of being safe and able to obtain pluripotent cells, early developmental arrest happens in most SCNT embryos. Overcoming epigenetic barriers is currently the primary strategy for improving reprogramming efficiency and improving developmental rate in SCNT embryos. In this study, we analyzed DNA methylation profiles of in vivo fertilized embryos and SCNT embryos with different developmental fates. Overall DNA methylation level was higher in SCNT embryos during global de-methylation process compared to in vivo fertilized embryos. In addition, promoter region, first intron and 3'UTR were found to be the major genomic regions that were hyper-methylated in SCNT embryos. Surprisingly, we found the length of re-methylated region was directly related to the change of methylation level. Furthermore, a number of genes including Dppa2 and Dppa4 which are important for early zygotic genome activation (ZGA) were not properly activated in SCNT embryos. This study comprehensively analyzed genome-wide DNA methylation patterns in SCNT embryos and provided candidate _target genes for improving efficiency of genomic reprogramming in SCNT embryos. Since SCNT technology has been widely used in agricultural and pastoral production, protection of endangered animals, and therapeutic cloning, the findings of this study have significant importance for all these fields.

Keywords: DNA methylation; SCNT; WGCNA; ZGA; epigenetic reprogramming.

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Figures

FIGURE 1
FIGURE 1
Aberrant DNA methylation occurs in SCNT embryos. (A) DNA methylation density distribution of different types of embryos. Higher probability density distribution of hyper-methylated regions was observed in nuclear transferred embryos. (B) PCA plot of DNA methylation from all samples used in this study. Different colors represent different samples indicated in the right. (C) Average DNA methylation levels were determined along the gene bodies, mm9 reference genome were split to 100 bins with equal proportion, the upstream and downstream 20 kb of the genes were split into 20 bins with the length of 1 kb. Methylation levels were average methylation levels on 1 bin. (D) Violinplot of DNA methylation levels of SCNT embryos at 2-cell and 4-cell stages.
FIGURE 2
FIGURE 2
Aberrant DNA methylation patterns revealed by differential DNA methylation region analysis in SCNT embryos. (A) Barplot of DMRs in the SCNT and in vivo fertilized samples at different developmental stages. The red and blue bars represent hyper-DMRs and hypo-DMRs, respectively. (B) Boxplot of hyper DMRs in SCNT and in vivo fertilized embryos. Each box represents a hyper DMR, and colors represent different types of embryos at different development stage. DMRs were classified into three categories: dDMRs (de-methylated differential methylated regions); rDMR (re-methylated differential methylated regions); and pDMR (persistent differential methylated regions). Differences are statistically significant. *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001, t-test. (C) Genomic distribution of rDMRs. Pie charts represent proportion of rDMRs in different genomic contexts. Inset graphs represent number of DMRs distributed in single or combined genomic regions. (D) Probability density distribution of rDMRs in 2-cell and 4-cell stage (left). Box plots show change in DNA methylation level within different length of rDMRs (right panel, less than 100 bp, between 100 and 500 bp, between 500 and 1000 bp, and greater than 1000 bp).
FIGURE 3
FIGURE 3
Functional analysis of rDMRs. (A,C) GO term analysis of rDMRs in 2-cell stage and 4-cell stage, respectively. (B,D) KEGG pathway enrichment of rDMRs in 2-cell stage and 4-cell stage, respectively. (E) Examples of genes with different DMR patterns. Different samples were represented by different bar colors. Ordinate represents DNA methylation level.
FIGURE 4
FIGURE 4
Identification of hub marker genes in SCNT embryos. (A) Network diagram showing interaction of genes in the two modules of WGCNA production. Hub node genes are highlighted by yellow. (B) Co-expression network of key genes. The genes in the three networks are highly connected in the networks calculated by module mining. (C) Promoter DNA methylation of certain transcription factors like Dapp2/Dapp4 leads to failed zygotic genome activation in SCNT embryos, while de-methylated promoter of these genes in fertilized embryo safeguard proper activation of zygote genome. Filled circle represents DNA methylation, open circle represents DNA de-methylation.
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