Selective impairment of methylation maintenance is the major cause of DNA methylation reprogramming in the early embryo

DNA methylomes are extensively reprogrammed during mouse pre-implantation and early germ cell development. The main feature of this reprogramming is a genome-wide decrease in 5-methylcytosine (5mC). Standard high-resolution single-stranded bisulfite sequencing techniques do not allow discrimination of the underlying passive (replication-dependent) or active enzymatic mechanisms of 5mC loss. We approached this problem by generating high-resolution deep hairpin bisulfite sequencing (DHBS) maps, allowing us to follow the patterns of symmetric DNA methylation at CpGs dyads on both DNA strands over single replications.We compared DHBS maps of repetitive elements in the developing zygote, the early embryo, and primordial germ cells (PGCs) at defined stages of development. In the zygote, we observed distinct effects in paternal and maternal chromosomes. A significant loss of paternal DNA methylation was linked to replication and to an increase in continuous and dispersed hemimethylated CpG dyad patterns. Overall methylation levels at maternal copies remained largely unchanged, but showed an increased level of dispersed hemi-methylated CpG dyads. After the first cell cycle, the combined DHBS patterns of paternal and maternal chromosomes remained unchanged over the next three cell divisions. By contrast, in PGCs the DNA demethylation process was continuous, as seen by a consistent decrease in fully methylated CpG dyads over consecutive cell divisions.The main driver of DNA demethylation in germ cells and in the zygote is partial impairment of maintenance of symmetric DNA methylation at CpG dyads. In the embryo, this passive demethylation is restricted to the first cell division, whereas it continues over several cell divisions in germ cells. The dispersed patterns of CpG dyads in the early-cleavage embryo suggest a continuous partial (and to a low extent active) loss of methylation apparently compensated for by selective de novo methylation. We conclude that a combination of passive and active demethylation events counteracted by de novo methylation are involved in the distinct reprogramming dynamics of DNA methylomes in the zygote, the early embryo, and PGCs

Selective impairment of methylation maintenance is the major cause of DNA methylation reprogramming in the early embryo

BACKGROUND: DNA methylomes are extensively reprogrammed during mouse pre-implantation and early germ cell development. The main feature of this reprogramming is a genome-wide decrease in 5-methylcytosine (5mC). Standard high-resolution single-stranded bisulfite sequencing techniques do not allow discrimination of the underlying passive (replication-dependent) or active enzymatic mechanisms of 5mC loss. We approached this problem by generating high-resolution deep hairpin bisulfite sequencing (DHBS) maps, allowing us to follow the patterns of symmetric DNA methylation at CpGs dyads on both DNA strands over single replications. RESULTS: We compared DHBS maps of repetitive elements in the developing zygote, the early embryo, and primordial germ cells (PGCs) at defined stages of development. In the zygote, we observed distinct effects in paternal and maternal chromosomes. A significant loss of paternal DNA methylation was linked to replication and to an increase in continuous and dispersed hemimethylated CpG dyad patterns. Overall methylation levels at maternal copies remained largely unchanged, but showed an increased level of dispersed hemi-methylated CpG dyads. After the first cell cycle, the combined DHBS patterns of paternal and maternal chromosomes remained unchanged over the next three cell divisions. By contrast, in PGCs the DNA demethylation process was continuous, as seen by a consistent decrease in fully methylated CpG dyads over consecutive cell divisions. CONCLUSIONS: The main driver of DNA demethylation in germ cells and in the zygote is partial impairment of maintenance of symmetric DNA methylation at CpG dyads. In the embryo, this passive demethylation is restricted to the first cell division, whereas it continues over several cell divisions in germ cells. The dispersed patterns of CpG dyads in the early-cleavage embryo suggest a continuous partial (and to a low extent active) loss of methylation apparently compensated for by selective de novo methylation. We conclude that a combination of passive and active demethylation events counteracted by de novo methylation are involved in the distinct reprogramming dynamics of DNA methylomes in the zygote, the early embryo, and PGCs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1756-8935-8-1) contains supplementary material, which is available to authorized users

Epigenetic reprogramming of DNA methylation in the early embryogenesis of mouse

In der frühen Embryogenese von Säugetieren kommt es zu dramatischen epigenetischen Veränderungen. Kurz nach Befruchtung der Eizelle erfolgt die epigenetische Reprogrammierung früher Embryonen, die zur Entstehung eines totipotenten Entwicklungspotentials führt. Dabei kommt es über bisher unbekannte Mechanismen zu einer genomweiten Demethylierung des paternalen Genoms der Zygote. In dieser Arbeit wurden die molekularen Mechanismen der epigenetischen Reprogrammierung der DNA Methylierung in der frühen Embryogenese der Maus analysiert. Dabei wurde gezeigt, dass neben dem paternalen Genom auch das maternale Genom, wenn auch im geringeren Maße, von dieser Reprogrammierung betroffen ist. Der wohl wichtigste Befund dieser Arbeit ist die Entdeckung, dass die aktive Demethylierung in der Zygote maßgeblich über die enzymatische Oxidation von 5mC zu 5hmC erfolgt. Hier konnte die Dioxygenase Tet3 als verantwortliches Enzym identifiziert werden. Zudem konnte gezeigt werden, dass in Eizellen von Säugern die Hydroxylierung von 5mC ein konservierter Mechanismus ist, der zur Reprogrammierung exogener Genome von Spermien und somatischer Zellkerne führt. Neben der massiven Hydroxylierung des 5mC konnten auch Indizien auf eine indirekte aktive Demethylierung des paternalen Genoms über konstitutive DNA Reparaturwege gefunden werden. Diese Ergebnisse erfordern ein Umdenken des Begriffes "aktive DNA Demethylierung" und eröffnen neue Aspekte der epigenetischen Reprogrammierung höherer Säugertiere.In early mammalian embryos shortly after fertilization of the oocyte dramatic epigenetic changes occur. The epigenomes of early preimplantation embryos are substantially repro-grammed to ensure a totipotent developmental potential. Here, the paternal genome of the zygote undergoes genome-wide DNA demethylation by so far unknown mechanisms. In this study the molecular mechanisms of the epigenetic reprogramming of DNA methylation in the early murine preimplantation development are analyzed. This analysis has shown that not only the paternal genome but also the maternal genome is affected by this reprogramming process to some extent. Most striking, it was discovered that the active DNA demethylation in the zygote is primarily accomplished by the enzymatic oxidation of 5mC to 5hmC. In this process the dioxygenase Tet3 could be identified as responsible enzyme. Studying mouse, rabbit and bovine zygotes and also cloned mouse 1-cell embryos derived by somatic nuclear transfer, it was shown that the conversion of 5mC to 5hmC is driven by maternal factors and appears to be an evolutionary conserved mechanism of the mammalian oocyte. In addition to the massive enzymatic oxidation of 5mC, experimental evidences also suggest the involve-ment of ubiquitous DNA repair pathways in active DNA demethylation. Results of my thesis prompt the careful revision of the term "active DNA demethylation'; and open up new facets to epigenetic reprogramming in mammalian development

DNA methylation reprogramming in preimplantation development

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Functional topography of the fully grown human oocyte

In vivo maturation (IVM) of human oocytes is a technique used to increase the number of usable oocytes for in vitro fertilization (IVF) and represents a necessity for women with different ovarian pathologies. During IVM the oocytes progress from the germinal vesicle stage (GV) through the metaphase II and during this journey both nuclear and cytoplasmic rearrangements must be obtained to increase the probability to get viable and healthy zygotes/embryos after IVF. As the successful clinical outcomes of this technique are a reality, we wanted to investigate the causes behind oocytes maturation arrest. For obvious ethical reasons, we were able to analyze only few human immature oocytes discarded and donated to research by transmission electron microscopy showing that, as in the mouse, they have different chromatin and cytoplasmic organizations both essential for further embryo development.</p

Single cell expression analysis of primate-specific retroviruses-derived HPAT lincRNAs in viable human blastocysts identifies embryonic cells co-expressing genetic markers of multiple lineages

Chromosome instability and aneuploidies occur very frequently in human embryos, impairing proper embryogenesis and leading to cell cycle arrest, loss of cell viability, and developmental failures in 50–80% of cleavage-stage embryos. This high frequency of cellular extinction events represents a significant experimental obstacle challenging analyses of individual cells isolated from human preimplantation embryos. We carried out single cell expression profiling of 241 individual cells recovered from 32 human embryos during the early and late stages of viable human blastocyst (VHB) differentiation. Classification of embryonic cells was performed solely based on expression patterns of human pluripotency-associated transcripts (HPAT), which represent a family of primate-specific transposable element-derived lincRNAs highly expressed in human embryonic stem cells and regulating nuclear reprogramming and pluripotency induction. We then validated our findings by analyzing transcriptomes of 1,708 individual cells recovered from more than 100 human embryos and 259 mouse cells from more than 40 mouse embryos at different stages of preimplantation embryogenesis. HPAT's expression-guided spatiotemporal reconstruction of human embryonic development inferred from single-cell expression analysis of VHB differentiation enabled identification of telomerase-positive embryonic cells co-expressing key pluripotency regulatory genes and genetic markers of three major lineages. Follow-up validation analyses confirmed the emergence in human embryos prior to lineage segregation of telomerase-positive cells co-expressing genetic markers of multiple lineages. Observations reported in this contribution support the hypothesis of a developmental pathway of creation embryonic lineages and extraembryonic tissues from telomerase-positive pre-lineage cells manifesting multi-lineage precursor phenotype

YAP Induces Human Naive Pluripotency

The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP−/− cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology