The Dynamic And Regulation Mechanism Of DNA Methylation And H3K9me2 In Mouse Zygotes

Epigenetic alterations are heritable changes in a cell not encoded by the DNA sequence. That meaning the genotype is same while the phenotype is different. And the changes can be delivered stably in development and cell proliferation. Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns in mammals. Epigenetic modifications of genomes involve two major mechanisms such as post-translational modification of histone proteins in chromatin and DNA methylation, which are regulated by distinct, but coupled, pathways. Thus, a comprehensive understanding of epigenetic mechanisms, their interactions and alterations, has become a priority in recent research. There are many kinds of epigenetic alterations in mouse zygote, including asymmetry of DNA methylation and some histone modification in male and female pronucleus. It’s a good model to research the epigenetic mechanisms.　In this paper we choose DNA methylation and H3K9me2 to research their alterations and mechanisms in zygote, as the two modifications have been well understood before.1. The dynamic and regulation mechanism of DNA methylation in mouse zygotesThe developmental dynamics of DNA methylation events have been well studied. Active demethylation of the paternal genome occurs in the zygote, followed by passive demethylation during cleavage stages, and remethylation occurs by the blastocyst stage. The paternal genome is less methylated during early development. However, in the present experiments, we provide direct and indirect evidence that genome-wide de novo DNA methylation of the paternal genome occurs during the first cell cycle in mouse embryos. Further investigations by testing the DNA methylation level of different stages with two hours interval before early two-cell embryos, we found that very low level de novo DNA methylation occurred on the male pronucleus in late zygote stage, but genome-wide de novo DNA methylation of the paternal genome occurred during the first mitosis. When aphidicolin or cycloheximide was added to the culture medium, the pronuclei didn’t disappear and the DNA methylation level of the male pronucleus was still very low at IVF 24h. This suggests the disappearance of pronucleus membrane during the first mitosis may promote the de novo DNA methylation. The localisation of the DNA methyltransferases (Dnmt3A and Dnmt3L) was detected, and we found Dnmt3A and Dnmt3L locate more obviously in the male pronucleus at IVF 14 h. In addition, on the male pronucleus in late zygote stage the methylation signal was more visible at the paternal heterochromatin regions. Comparing the distribution of trimethylated H4K20 on the pronucleus, it indicated that the epigenetic marks of heterochromatin were different between the paternal and maternal genome in zygotes.2. The dynamic and regulation mechanism of H3K9me2 in mouse zygotesPrevious research results showed that the asymmetric H3K9 methylation pattern between parental genomes is generated soon after fertilization, and methylated H3K9 showed a very weak or absent methylation signal in the male pronucleus, whereas a distinct methylation signal was detected in the female pronucleus. The mechanism that maintains the paternal genome in the undermethylated state is an active process depends on transcription and protein synthesis in fertilized egg, as inhibition of protein synthesis or gene expression increased methylation in the male pronucleus to a level that was similar to that of the female pronucleus at IVF12h. We checked H3K9me2 level in different stages of zygote, and found that low level H3K9me2 occurred in the male pronucleus after IVF 10h. This suggested that there were two distinct mechanisms to regulate the male pronucleus H3K9me2. It dosen’t depend on the novel protein synthesis, but inhibits the localization of histone methyltransferase into the male pronucleus before IVF8h. After IVF8h it depends on the novel protein synthesis. The two mechanisms transfer between IVF 8-10h, and the novel protein synthesis can’t inhibit the methyltransferase activity totally in time. This results in the low level H3K9me2 occurrence. Aphidicolin and Roscovitin don’t influence H3K9me2 level in the male pronucleus of zygote. The results indicate that male pronucleus H3K9me2 won’t change when DNA replication and cyclin/cyclin-depended kinase are inhibited. That meaning DNA replication and cell cycle regulation may not involve in the regulation of male pronucleus H3K9me2.Knock out and overexpression of G9A both can influence the H3K9me2, by zygote microinjection of G9A antibody and mRNA. The results indicate that G9A involves in the regulation of asymmetric H3K9me2, and G9A is the methyltransferase which induce the male pronucleus H3K9me2 occurrence in zygote after cycloheximide treated. We also checked the localization of GLP and the influence of GLP antibody to mouse zygote H3K9me2 after zygote microinjection of GLP antibody. GLP locates into the male and female pronucleus at early zygote stage, and cycloheximide may benefit the process. But GLP may not involve in the regulation of the zygote male pronucleus H3K9me2, as the localization of GLP dosen’t show the asymmetry between the male and female pronucleus.In this paper we detected the dynamic of DNA methylation and H3K9me2 in mouse zygote, and the regulation mechanisms were preliminarily investigated. We found that there may have specific epigenetic regulation mechanisms in mouse zygote, and zygotic genome activation (ZGA) may involve in the regulation progress. The results indicated that the alterations of DNA methylation and H3K9me2 may participate in the chromatin remodeling and cell reprogramming. The clarification of epigenetic regulation mechanisms in mouse zygote would benefit the research of the chromatin remodeling and cell reprogramming, and disclosure the mechanism of gene expression patterns regulation and cell differentiation in embryo development.