Tet enzymes are essential for early embryogenesis and completion of embryonic genome activation.

Mammalian development begins in transcriptional silence followed by a period of widespread activation of thousands of genes. DNA methylation reprogramming is integral to embryogenesis and linked to Tet enzymes, but their function in early development is not well understood. Here, we generate combined deficiencies of all three Tet enzymes in mouse oocytes using a morpholino-guided knockdown approach and study the impact of acute Tet enzyme deficiencies on preimplantation development. Tet1-3 deficient embryos arrest at the 2-cell stage with the most severe phenotype linked to Tet2. Individual Tet enzymes display non-redundant roles in the consecutive oxidation of 5-methylcytosine to 5-carboxylcytosine. Gene expression analysis uncovers that Tet enzymes are required for completion of embryonic genome activation (EGA) and fine-tuned expression of transposable elements and chimeric transcripts. Whole-genome bisulfite sequencing reveals minor changes of global DNA methylation in Tet-deficient 2-cell embryos, suggesting an important role of non-catalytic functions of Tet enzymes in early embryogenesis. Our results demonstrate that Tet enzymes are key components of the clock that regulates the timing and extent of EGA in mammalian embryos.

Reprogramming of DNA methylation is linked to successful human preimplantation development.

Human preimplantation development is characterized by low developmental rates that are poorly understood. Early mammalian embryogenesis is characterized by a major phase of epigenetic reprogramming, which involves global DNA methylation changes and activity of TET enzymes; the importance of DNA methylation reprogramming for successful human preimplantation development has not been investigated. Here, we analyzed early human embryos for dynamic changes in 5-methylcytosine and its oxidized derivatives generated by TET enzymes. We observed that 5-methylcytosine and 5-hydroxymethylcytosine show similar, albeit less pronounced, asymmetry between the parental pronuclei of human zygotes relative to mouse zygotes. Notably, we detected low levels of 5-formylcytosine and 5-carboxylcytosine, with no apparent difference in maternal or paternal pronuclei of human zygotes. Analysis of later human preimplantation stages revealed a mosaic pattern of DNA 5C modifications similar to those of the mouse and other mammals. Strikingly, using noninvasive time-lapse imaging and well-defined cell cycle parameters, we analyzed normally and abnormally developing human four-cell embryos for global reprogramming of DNA methylation and detected lower 5-methylcytosine and 5-hydroxymethylcytosine levels in normal embryos compared to abnormal embryos. In conclusion, our results suggest that DNA methylation reprogramming is conserved in humans, with human-specific dynamics and extent. Furthermore, abnormalities in the four-cell-specific DNA methylome in early human embryogenesis are associated with abnormal development, highlighting an essential role of epigenetic reprogramming for successful human embryogenesis. Further research should identify the underlying genomic regions and cause of abnormal DNA methylation reprogramming in early human embryos.

Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells.

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development.

Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes.

In mammalian zygotes, the 5-methyl-cytosine (5mC) content of paternal chromosomes is rapidly changed by a yet unknown but presumably active enzymatic mechanism. Here, we describe the developmental dynamics and parental asymmetries of DNA methylation in relation to the presence of DNA strand breaks, DNA repair markers and a precise timing of zygotic DNA replication. The analysis shows that distinct pre-replicative (active) and replicative (active and passive) phases of DNA demethylation can be observed. These phases of DNA demethylation are concomitant with the appearance of DNA strand breaks and DNA repair markers such as gammaH2A.X and PARP-1, respectively. The same correlations are found in cloned embryos obtained after somatic cell nuclear transfer. Together, the data suggest that (1) DNA-methylation reprogramming is more complex and extended as anticipated earlier and (2) the DNA demethylation, particularly the rapid loss of 5mC in paternal DNA, is likely to be linked to DNA repair mechanisms.

A transition phase in late mouse oogenesis impacts DNA methylation of the early embryo.

A well-orchestrated program of oocyte growth and differentiation results in a developmentally competent oocyte. In late oogenesis, germinal vesicle oocytes (GVOs) undergo chromatin remodeling accompanied by transcriptional silencing from an NSN (non-surrounded nucleolus) to an SN (surrounded nucleolus) chromatin state. By analyzing different cytoplasmic and nuclear characteristics, our results indicate that murine NSN-GVOs transition via an intermediate stage into SN-GVOs in vivo. Interestingly, this transition can also be observed ex vivo, including most characteristics seen in vivo, which allows to analyze this transition process in more detail. The nuclear rearrangements during the transition are accompanied by changes in DNA methylation and Tet enzyme-catalyzed DNA modifications. Early parthenogenetic embryos, derived from NSN-GVOs, show lower DNA methylation levels than SN-derived embryos. Together, our data suggest that a successful NSN-SN transition in oogenesis including proper DNA methylation remodeling is important for the establishment of a developmentally competent oocyte for the beginning of life.

Zscan4 binds nucleosomal microsatellite DNA and protects mouse two-cell embryos from DNA damage.

Zinc finger protein Zscan4 is selectively expressed in mouse two-cell (2C) embryos undergoing zygotic genome activation (ZGA) and in a rare subpopulation of embryonic stem cells with 2C-like features. Here, we show that Zscan4 specifically recognizes a subset of (CA)n microsatellites, repeat sequences prone to genomic instability. Zscan4-associated microsatellite regions are characterized by low nuclease sensitivity and high histone occupancy. In vitro, Zscan4 binds nucleosomes and protects them from disassembly upon torsional strain. Furthermore, Zscan4 depletion leads to elevated DNA damage in 2C mouse embryos in a transcription-dependent manner. Together, our results identify Zscan4 as a DNA sequence-dependent microsatellite binding factor and suggest a developmentally regulated mechanism, which protects fragile genomic regions from DNA damage at a time of embryogenesis associated with high transcriptional burden and genomic stress.

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.

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.

Spatiotemporal Reconstruction of the Human Blastocyst by Single-Cell Gene-Expression Analysis Informs Induction of Naive Pluripotency.

Human preimplantation embryo development involves complex cellular and molecular events that lead to the establishment of three cell lineages in the blastocyst: trophectoderm, primitive endoderm, and epiblast. Owing to limited resources of biological specimens, our understanding of how the earliest lineage commitments are regulated remains narrow. Here, we examined gene expression in 241 individual cells from early and late human blastocysts to delineate dynamic gene-expression changes. We distinguished all three lineages and further developed a 3D model of the inner cell mass and trophectoderm in which individual cells were mapped into distinct expression domains. We identified in silico precursors of the epiblast and primitive endoderm lineages and revealed a role for MCRS1, TET1, and THAP11 in epiblast formation and their ability to induce naive pluripotency in vitro. Our results highlight the potential of single-cell gene-expression analysis in human preimplantation development to instruct human stem cell biology.

The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming.

Long intergenic noncoding RNAs (lincRNAs) are derived from thousands of loci in mammalian genomes and are frequently enriched in transposable elements (TEs). Although families of TE-derived lincRNAs have recently been implicated in the regulation of pluripotency, little is known of the specific functions of individual family members. Here we characterize three new individual TE-derived human lincRNAs, human pluripotency-associated transcripts 2, 3 and 5 (HPAT2, HPAT3 and HPAT5). Loss-of-function experiments indicate that HPAT2, HPAT3 and HPAT5 function in preimplantation embryo development to modulate the acquisition of pluripotency and the formation of the inner cell mass. CRISPR-mediated disruption of the genes for these lincRNAs in pluripotent stem cells, followed by whole-transcriptome analysis, identifies HPAT5 as a key component of the pluripotency network. Protein binding and reporter-based assays further demonstrate that HPAT5 interacts with the let-7 microRNA family. Our results indicate that unique individual members of large primate-specific lincRNA families modulate gene expression during development and differentiation to reinforce cell fate.

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.

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.

Dissecting the role of H3K64me3 in mouse pericentromeric heterochromatin.

To ensure genome stability, pericentromeric regions are compacted in a dense heterochromatic structure through a combination of specific 'epigenetic' factors and modifications. A cascadal pathway is responsible for establishing pericentromeric chromatin involving chromatin modifiers and 'readers', such as H3K9 histone methyltransferases (Suv)39h and heterochromatin protein 1. Here we define how H3K64me3 on the lateral surface of the histone octamer integrates within the heterochromatinization cascade. Our data suggest that enrichment of H3K64me3 at pericentromeric chromatin foci is dependent on H3K9me3 but independent of a number of central factors such as heterochromatin protein 1, DNA methyltransferases and Suv4-20h histone methyltransferases. Our results support a model in which pericentromeric heterochromatin foci are formed along distinct pathways upon H3K9 trimethylation, involving H3K64me3 to potentially stabilize DNA-histone interactions, as well as sequential recruitment of repressive histone tail and DNA modifications. We hence suggest that multiple mechanisms ensure heterochromatin integrity at pericentromeres, with H3K64me3 as an important factor.

5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming.

The epigenomes of early mammalian embryos are extensively reprogrammed to acquire a totipotent developmental potential. A major initial event in this reprogramming is the active loss/demethylation of 5-methylcytosine (5mC) in the zygote. Here, we report on findings that link this active demethylation to molecular mechanisms. We detect 5-hydroxymethylcytosine (5hmC) as a novel modification in mouse, bovine and rabbit zygotes. On zygotic development 5hmC accumulates in the paternal pronucleus along with a reduction of 5mC. A knockdown of the 5hmC generating dioxygenase Tet3 simultaneously affects the patterns of 5hmC and 5mC in the paternal pronucleus. This finding links the loss of 5mC to its conversion into 5hmC. The maternal pronucleus seems to be largely protected against this mechanism by PGC7/Dppa3/Stella, as in PGC7 knockout zygotes 5mC also becomes accessible to oxidation into 5hmC. In summary, our data suggest an important role of 5hmC and Tet3 for DNA methylation reprogramming processes in the mammalian zygote.

DNA methylation reprogramming and DNA repair in the mouse zygote.

Here, we summarize current knowledge about epigenetic reprogramming during mammalian preimplantation development, as well as the potential mechanisms driving these processes. We will particularly focus on changes taking place in the zygote, where the paternally derived DNA and chromatin undergo the most striking alterations, such as replacement of protamines by histones, histone modifications and active DNA demethylation. The putative mechanisms of active paternal DNA demethylation have been studied for over a decade, accumulating a lot of circumstantial evidence for enzymatic activities provided by the oocyte, protection of the maternal genome against such activities and possible involvement of DNA repair. We will discuss the various facets of dynamic epigenetic changes related to DNA methylation with an emphasis on the putative involvement of DNA repair in DNA demethylation.