Developmental functions of the dynamic DNA methylome and hydroxymethylome in the mouse and zebrafish: similarities and differences

5-methylcytosine (5mC) is the best understood DNA modification and is generally believed to be associated with repression of gene expression. Over the last decade, sequentially oxidized forms of 5mC (oxi-mCs) have been discovered within the genomes of vertebrates. Their discovery was accompanied by that of the ten-eleven translocation (TET) methylcytosine dioxygenases, the enzymes that catalyse the formation of the oxi-mCs. Although a number of studies performed on different vertebrate models and embryonic stem cells demonstrated that both TET enzymes and oxi-mCs are likely to be important for several developmental processes it is currently unclear whether their developmental roles are conserved among vertebrates. Here, we summarise recent developments in this field suggesting that biological roles of TETs/oxi-mCs may significantly differ between mice and zebrafish. Thus, although the role of TET proteins in late organogenesis has been documented for both these systems; unlike in mice the enzymatic oxidation of 5mC does not seem to be involved in zygotic reprogramming or gastrulation in zebrafish. Our analysis may provide an insight into the general principles of epigenetic regulation of animal development and cellular differentiation

A lexicon of DNA modifications: their roles in embryo development and the germline

5-methylcytosine (5mC) on CpG dinucleotides has been viewed as the major epigenetic modification in eukaryotes for a long time. Apart from 5mC, additional DNA modifications have been discovered in eukaryotic genomes. Many of these modifications are thought to be solely associated with DNA damage. However, growing evidence indicates that some base modifications, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), and N6-methadenine (6mA), may be of biological relevance, particularly during early stages of embryo development. Although abundance of these DNA modifications in eukaryotic genomes can be low, there are suggestions that they cooperate with other epigenetic markers to affect DNA-protein interactions, gene expression, defense of genome stability and epigenetic inheritance. Little is still known about their distribution in different tissues and their functions during key stages of the animal lifecycle. This review discusses current knowledge and future perspectives of these novel DNA modifications in the mammalian genome with a focus on their dynamic distribution during early embryonic development and their potential function in epigenetic inheritance through the germ line

Mechanisms of demethylation in primordial germ cells and the importance of stage-specific demethylation in safeguarding against precocious differentiation

Primordial germ cells (PGCs) are the cellular precursors for mature gametes which are responsible for giving rise to embryonic development and the next generation of PGCs. During development, proper PGC differentiation results in high quality gametes, which are essential for normal development and future child health. Problems during PGC differentiation can lead to impaired fertility, poor quality germ cells, or developmental defects in the next generation. One of the essential events that occurs during PGC development is whole-genome reprogramming of DNA methylation. The reprogramming of DNA methylation in the context of PGC development is required for appropriate cell lineage differentiation. This process is essential in establishing the correct epigenetic landscape which will impact differentiation, and maturation of PGCs. My goal is to focus on two aspects of genome-wide reprogramming in Primordial Germ Cells (PGCs). First, the molecular mechanisms of DNA demethylation during the gonadal stage of development, as well as the mechanisms involved protecting specific loci from demethylation in order to allow for correct temporal expression of germ cell genesPrimordial germ cells (PGCs) undergo genome-wide demethylation in two distinct stages. Stage 1 consists of global demethylation before embryonic (e) day e9.5 of mouse development. Stage 2 The second phase occurs once PGCs colonize the genital ridge between e10.5-e13.5, and happens in a temporal and locus-specific manner. Results indicate that the second phase is regulated in part by Ten eleven translocation (Tet) protein Tet 1, and conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) at specific loci. The major working model for Tet-dependent DNA demethylation involves replication-coupled loss of methylated cytosines from the genome. However an alternate model would predict active removal of 5hmC at specific loci independent of cell division. In order to address this directly, we have established a new organ culture model involving the growth of dissected aorta/gonad/mesonephros (AGM) tissues isolated from the mouse embryo at e10.5. During three days of organ culture, we show that PGCs divide on average three times. We also show that in the background of global hypomethylation established in phase 1, PGCs isolated from the organ culture undergo locus-specific DNA demethylation, and 5hmC reorganization, and this occurs within three days. Using this model we have targeted the PGC cell cycle using a P-AKT inhibitor, and have determined that imprint erasure can happen in proliferation dependent and independent ways depending on the genomic locusAlternatively, during the removal of DNA methylation in stage 1, some loci are protected from demethylation and the mechanism for this process remains unknown. In the current study we tested the hypothesis that Dnmt1 is responsible for maintaining methylation by being recruited at specific genomic sites during whole genome demethylation. To address this, we created a conditional germline knockout of Dnmt1. Analysis of Dnmt1 conditional knockout (DCKO) PGCs revealed that Dnmt1 is the major methyltransferase that functions during whole genome demethylation to maintain DNA methylation at discreet genomic regions including intracisternal A particle (IAP) transposons, as well as maternal and paternal imprinting control centers. Furthermore, the absence of Dnmt1 results in precocious differentiation that leads to germ cell loss in both male and female embryos. Taken together, we propose a model in which maintenance of cytosine methylation by Dnmt1 is essential to maintain cytosine methylation at discreet regions of the genome during whole genome DNA methylation reprogramming

The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline

Removal of cytosine methylation from the genome is critical for reprogramming and transdifferentiation and plays a central role in our understanding of the fundamental principles of embryo lineage development. One of the major models for studying cytosine demethylation is the mammalian germ line during the primordial germ cell (PGC) stage of embryo development. It is now understood that oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is required to remove cytosine methylation in a locus-specific manner in PGCs; however, the mechanisms downstream of 5hmC are controversial and hypothesized to involve either active demethylation or replication-coupled loss. In the current study, we used the aorta-gonad-mesonephros (AGM) organ culture model to show that this model recapitulates germ line reprogramming, including 5hmC reorganization and loss of cytosine methylation from Snrpn and H19 imprinting control centers (ICCs). To directly address the hypothesis that cell proliferation is required for cytosine demethylation, we blocked PI3-kinase-dependent PGC proliferation and show that this leads to a G1 and G2/M cell cycle arrest in PGCs, together with retained levels of cytosine methylation at the Snrpn ICC, but not at the H19 ICC. Taken together, the AGM organ culture model is an important tool to evaluate mechanisms of locus-specific demethylation and the role of PI3-kinase-dependent PGC proliferation in the locus-specific removal of cytosine methylation from the genome

The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline

Removal of cytosine methylation from the genome is critical for reprogramming and transdifferentiation and plays a central role in our understanding of the fundamental principles of embryo lineage development. One of the major models for studying cytosine demethylation is the mammalian germ line during the primordial germ cell (PGC) stage of embryo development. It is now understood that oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is required to remove cytosine methylation in a locus-specific manner in PGCs; however, the mechanisms downstream of 5hmC are controversial and hypothesized to involve either active demethylation or replication-coupled loss. In the current study, we used the aorta-gonad-mesonephros (AGM) organ culture model to show that this model recapitulates germ line reprogramming, including 5hmC reorganization and loss of cytosine methylation from Snrpn and H19 imprinting control centers (ICCs). To directly address the hypothesis that cell proliferation is required for cytosine demethylation, we blocked PI3-kinase-dependent PGC proliferation and show that this leads to a G1 and G2/M cell cycle arrest in PGCs, together with retained levels of cytosine methylation at the Snrpn ICC, but not at the H19 ICC. Taken together, the AGM organ culture model is an important tool to evaluate mechanisms of locus-specific demethylation and the role of PI3-kinase-dependent PGC proliferation in the locus-specific removal of cytosine methylation from the genome

PGC Reversion to Pluripotency Involves Erasure of DNA Methylation from Imprinting Control Centers followed by Locus-Specific Re-methylation

Primordial germ cells (PGCs) are fate restricted to differentiate into gametes in vivo. However, when removed from their embryonic niche, PGCs undergo reversion to pluripotent embryonic germ cells (EGCs) in vitro. One of the major differences between EGCs and embryonic stem cells (ESCs) is variable methylation at imprinting control centers (ICCs), a phenomenon that is poorly understood. Here we show that reverting PGCs to EGCs involved stable ICC methylation erasure at Snrpn, Igf2r, and Kcnqot1. In contrast, the H19/Igf2 ICC undergoes erasure followed by de novo re-methylation. PGCs differentiated in vitro from ESCs completed Snrpn ICC erasure. However, the hypomethylated state is highly unstable. We also discovered that when the H19/Igf2 ICC was abnormally hypermethylated in ESCs, this is not erased in PGCs differentiated from ESCs. Therefore, launching PGC differentiation from ESC lines with appropriately methylated ICCs is critical to the generation of germline cells that recapitulate endogenous ICC erasure