Final Report Summary - EPIPLUS (A novel epigenetic modification in pluripotent stem cells)
DNA methylation is critical during mammalian development. When present at gene promoters and enhancers, DNA methylation represses transcription and regulates cell lineage-specific gene expression. Conversely, during cellular reprogramming, efficient erasure of DNA methylation is essential for the reactivation of previously silenced genes and for attaining pluripotency. The Ten-Eleven-Translocation (TET) family of dioxygenases convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA, a novel epigenetic modification that can facilitate DNA demethylation and is associated with pluripotency. This discovery in 2009 has reshaped central dogma in epigenetics, by demonstrating the physiological presence of oxidized forms of 5mC in the mammalian genome. Indeed, other studies soon validated that TET enzymes can perform reiterative oxidation of 5mC into 5hmC, 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). At present, TET oxygenases are the only known mammalian enzymes capable of initiating a complete pathway of active DNA demethylation, with potentially vital roles in early mammalian embryogenesis.
As co-discoverer of the TET family, Dr Koh has previously demonstrated that Tet1 and Tet2 are highly expressed in mouse embryonic stem cells (ESCs) and regulate cell fate specification (Koh et al, 2011). However, the precise mechanisms by which TET proteins regulate gene expression in ESCs and in the mouse embryo remain unclear. This proposal aims to address: (1) The detailed mechanisms by which Tet proteins regulate the transition between ESC self-renewal and differentiation into distinct lineages; (2) How Tet1 regulates development of the early mouse embryo; (3) How Tet expression affects the differentiation potential of induced pluripotent stem cells (iPSCs).
First, EpiPluS researchers led by Dr Koh have characterized the promoter and enhancer domains regulating Tet1 and Tet2 genes in mouse ESCs and observed dynamic changes in promoter-enhancer coupling during early cell state transitions when cells exit pluripotency.Their results (Sohni et al, 2015) account for the developmental stage- and tissue-specific expression patterns of Tet1 and Tet2. They have further examined the kinetics of Tet1 and Tet2 expression during development of the early post-implantation stage mouse embryo. In two independent strains of Tet1 mutant mice, they have observed embryonic lethality in homozygous-null embryos, which resulted in Tet1-deficient mice born at below the expected Mendelian ratio. To identify Tet1 target genes in the primed epiblast, they have performed RNA-sequencing in distinct lineage compartments of the pre-streak mouse embryo in vivo as well as in primed epiblast-like cells in vitro. Together with base-resolution methylation analyses at target genes and on a genome-scale, they are discovering both direct and indirect effects of Tet1-mediated 5mC oxidation marks on gene regulation in the epiblast. Collectively, their results support an important role of Tet1 in primed epiblast cells, where hydroxymethylation regulates de novo DNA methylation and is accompanied by complex fine-tuning of lineage-specific gene expression prior to gastrulation.
Finally, EpiPluS researchers have generated transgenic mouse strains harbouring reporters for cell state-specific expression of Tet1. By crossing these new strains to existing strains harbouring a different fluorescent reporter for the master pluripotency factor Oct4, they have studied reprogramming kinetics using double-reporter transgenic mouse embryonic fibroblasts. Their studies have revealed that successful reprogramming towards iPSCs involves an ordered sequential activation of enhancers regulating both Oct4 and Tet1. By sorting stage-specific reprogramming intermediates based on reporter patterns, they are now clarifying the critical epigenetic changes occurring during the acquisition of fully naive pluripotency in iPSCs.
This work will contribute to a detailed epigenetic blueprint of an early stage of mammalian development. Dysregulation of TET activity is now widely recognized to be associated with developmental disorders and cancer. These studies will provide important new information of how abnormal DNA methylation marks in early development can result in disease later in life, and suggest new ways to treat diseases by finding out where the defective DNA marks are.
Furthermore, understanding the fundamental roles of DNA methylation and demethylation dynamics in development will open new avenues to manipulate cell fate in vitro. The ability to re-acquire pluripotency in somatic cells through the generation of iPSCs already has considerable impact in the fields of stem cell and regenerative medicine, but clinical therapies are currently hindered by observations of aberrant DNA methylation marks gained during in vitro reprogramming. EpiPluS studies in the murine system is clarifying the critical epigenetic events for optimal reprogramming of iPSCs. In the near future, researchers will translate these findings to the research use of human somatic cells to derive iPSCs free of aberrant DNA methylation marks. Obtaining iPSCs of the highest quality will enhance derivation of diverse human cell lineages for broad applications in disease modelling, toxicology studies and cellular replacement therapies.
As co-discoverer of the TET family, Dr Koh has previously demonstrated that Tet1 and Tet2 are highly expressed in mouse embryonic stem cells (ESCs) and regulate cell fate specification (Koh et al, 2011). However, the precise mechanisms by which TET proteins regulate gene expression in ESCs and in the mouse embryo remain unclear. This proposal aims to address: (1) The detailed mechanisms by which Tet proteins regulate the transition between ESC self-renewal and differentiation into distinct lineages; (2) How Tet1 regulates development of the early mouse embryo; (3) How Tet expression affects the differentiation potential of induced pluripotent stem cells (iPSCs).
First, EpiPluS researchers led by Dr Koh have characterized the promoter and enhancer domains regulating Tet1 and Tet2 genes in mouse ESCs and observed dynamic changes in promoter-enhancer coupling during early cell state transitions when cells exit pluripotency.Their results (Sohni et al, 2015) account for the developmental stage- and tissue-specific expression patterns of Tet1 and Tet2. They have further examined the kinetics of Tet1 and Tet2 expression during development of the early post-implantation stage mouse embryo. In two independent strains of Tet1 mutant mice, they have observed embryonic lethality in homozygous-null embryos, which resulted in Tet1-deficient mice born at below the expected Mendelian ratio. To identify Tet1 target genes in the primed epiblast, they have performed RNA-sequencing in distinct lineage compartments of the pre-streak mouse embryo in vivo as well as in primed epiblast-like cells in vitro. Together with base-resolution methylation analyses at target genes and on a genome-scale, they are discovering both direct and indirect effects of Tet1-mediated 5mC oxidation marks on gene regulation in the epiblast. Collectively, their results support an important role of Tet1 in primed epiblast cells, where hydroxymethylation regulates de novo DNA methylation and is accompanied by complex fine-tuning of lineage-specific gene expression prior to gastrulation.
Finally, EpiPluS researchers have generated transgenic mouse strains harbouring reporters for cell state-specific expression of Tet1. By crossing these new strains to existing strains harbouring a different fluorescent reporter for the master pluripotency factor Oct4, they have studied reprogramming kinetics using double-reporter transgenic mouse embryonic fibroblasts. Their studies have revealed that successful reprogramming towards iPSCs involves an ordered sequential activation of enhancers regulating both Oct4 and Tet1. By sorting stage-specific reprogramming intermediates based on reporter patterns, they are now clarifying the critical epigenetic changes occurring during the acquisition of fully naive pluripotency in iPSCs.
This work will contribute to a detailed epigenetic blueprint of an early stage of mammalian development. Dysregulation of TET activity is now widely recognized to be associated with developmental disorders and cancer. These studies will provide important new information of how abnormal DNA methylation marks in early development can result in disease later in life, and suggest new ways to treat diseases by finding out where the defective DNA marks are.
Furthermore, understanding the fundamental roles of DNA methylation and demethylation dynamics in development will open new avenues to manipulate cell fate in vitro. The ability to re-acquire pluripotency in somatic cells through the generation of iPSCs already has considerable impact in the fields of stem cell and regenerative medicine, but clinical therapies are currently hindered by observations of aberrant DNA methylation marks gained during in vitro reprogramming. EpiPluS studies in the murine system is clarifying the critical epigenetic events for optimal reprogramming of iPSCs. In the near future, researchers will translate these findings to the research use of human somatic cells to derive iPSCs free of aberrant DNA methylation marks. Obtaining iPSCs of the highest quality will enhance derivation of diverse human cell lineages for broad applications in disease modelling, toxicology studies and cellular replacement therapies.