Project description:DNA and Histone-3 Lysine 27 methylation typically function as repressive modifications and operate within distinct genomic compartments. In mammals, the majority of the genome is kept in a DNA methylated state, whereas the Polycomb Repressive Complexes regulate the unmethylated CpG-rich promoters of developmental genes. In contrast to this general framework, the extraembryonic lineages display non-canonical, globally intermediate DNA methylation levels that includes disruption of local Polycomb domains. To better understand this unusual landscape’s molecular properties, we genetically and chemically perturbed major epigenetic pathways in mouse Trophoblast Stem Cells (TSCs). We find that the extraembryonic epigenome reflects ongoing and dynamic de novo methyltransferase recruitment, which is continuously antagonized by Polycomb to maintain intermediate, locally disordered methylation. Despite its disorganized molecular appearance, our data point to a highly controlled equilibrium between counteracting repressors within extraembryonic cells, one that can seemingly persist indefinitely without bistable features typically seen for embryonic forms of epigenetic regulation.
Project description:DNA and Histone-3 Lysine 27 methylation typically function as repressive modifications and operate within distinct genomic compartments. In mammals, the majority of the genome is kept in a DNA methylated state, whereas the Polycomb Repressive Complexes regulate the unmethylated CpG-rich promoters of developmental genes. In contrast to this general framework, the extraembryonic lineages display non-canonical, globally intermediate DNA methylation levels that includes disruption of local Polycomb domains. To better understand this unusual landscape’s molecular properties, we genetically and chemically perturbed major epigenetic pathways in mouse Trophoblast Stem Cells (TSCs). We find that the extraembryonic epigenome reflects ongoing and dynamic de novo methyltransferase recruitment, which is continuously antagonized by Polycomb to maintain intermediate, locally disordered methylation. Despite its disorganized molecular appearance, our data point to a highly controlled equilibrium between counteracting repressors within extraembryonic cells, one that can seemingly persist indefinitely without bistable features typically seen for embryonic forms of epigenetic regulation. Dataset 1: EED co-immunoprecipitation of wild type mouse trophoblast stem cells (TSCs) and Eed knockout TSCs as control, with 3 biological replicates per condition.
Project description:One of the most important topic in mammalian embryogenesis is cell lineage segregation. Briefly, one totipotent zygote will develop into inner cell mass (ICM) and trophectoderm (TE) at blastocyst stage, then the ICM will finally develop into multiple somatic cell lineages and TE will majorly become the placenta tissue which supports and protects the development of the embryo proper. Multiple extrinsic and intrinsic regulatory pathways are involved in facilitating the appropriate development of the embryo. Epigenetic reprogramming is one of the most pervasive events during mouse embryo development(Li, 2002). Recent studies had implied that distinct features for the establishment of DNA methylation(Monk et al., 1987) and histone modifications especially H3K27me3(Liu et al., 2016) during mouse early embryo development. The re-establishment of DNA methylation in early mouse embryos starts at blastocyst stage (about embryonic day 3.5, E3.5) and peaks around the gastrulation stage, while the re-establishment of H3K27me3 exhibits a great level of dynamics and gradually increased CpG preference during pre-implantation embryo development(Liu et al., 2016). However, the underlying epigenetic mechanism concerning the lineage segregation and developmental competence restriction between the pluripotent embryo proper and the supporting extraembryonic tissues especially extraembryonic ectoderm (ExE) remains largely unknown. Surprisingly, no significant difference exists for the distribution of H3K27me3 and DNA methylation between ICM and TE in the preimplantation embryos. Therefore, it is of great importance for unveiling the interplays between H3K27me3 and DNA methylation involving in the restriction of developmental competence between embryonic cells and extraembryonic cells in post-implantation embryos.
Project description:Tet-mediated DNA oxidation is a new type of epigenetic modification in mammals and its role in the regulation of cell fate transition remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capability to be reprogrammed into iPS cells. We demonstrate that these Tet-deficient MEFs cannot be reprogrammed due to a blockage in the mesenchymal-to-epithelial transition (MET). Reprogramming of MEFs deficient in TDG is similarly blocked. The blockage is caused by impaired activation of crucial microRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either miR-200c or catalytically active Tet and TDG restores reprogramming to the respective knockout MEFs. Thus, oxidative demethylation is essential for somatic cell reprogramming. These findings provide mechanistic insights into the operation of epigenetic barriers in cell lineage conversion. Reduced Representation Bisulfite (RRBS, MspI,~75-400bp size fraction) and Tet-Assisted RRBS (TARRBS) of MEFs & reprogramming MEFs at Day 5