The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency.Supplemental material is available for this article.Received January 25, 2014; revised version accepted April 16, 2014. Embryonic cells are characterized by a large degree of plasticity, which is the ability to generate different cell types upon differentiation and is necessary to start a full developmental program. After fertilization, the mouse embryo has the transient capacity to generate both embryonic and extraembryonic cell types, a feature that is referred to as totipotency (Tarkowski 1959;Ishiuchi and Torres-Padilla 2013). This is in contrast to pluripotent cells, which contribute to all three germ layers of the embryo proper, but not to extraembryonic lineages, and therefore have a more restricted potential than totipotent cells. In mice, only the zygote and two-cell stage blastomeres are, strictly speaking, totipotent, since they have the ability to develop into a full organism without the need of carrier cells (Tarkowski 1959;Tarkowski and Wroblewska 1967;Kelly et al. 1978;Ishiuchi and Torres-Padilla 2013). As development progresses, pluripotent cells form in the inner cell mass (ICM) of the blastocyst, accompanied by the activation of pluripotency-associated transcription factors like Nanog and Pou5f1/Oct4 (Nichols et al. 1998;Chambers et al. 2003). The first differentiated embryonic tissue, the trophectoderm (TE), appears morphologically distinguishable and surrounds the ICM in the blastocyst. Thus, during the early stages of development, the mouse embryo undergoes dramatic changes in cellular plasticity.Upon fertilization, embryonic chromatin undergoes intense chromatin remodeling. Indeed, this epigenetic reprogramming of the gametes is thought to be essential to gain totipotency (Sado and Ferguson-Smith 2005;Surani et al. 2007;Hemberger et al. 2009). However, the precise conformation of embryonic chromatin and the way it...

Upon fertilization in mammals the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodeling of constitutive heterochromatin, although the functional relevance of this is unknown. Here we show that heterochromatin establishment relies on the stepwise expression and regulated activity of Suv39h enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, demonstrating that heterochromatin remodeling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation in the paternal pronucleus after fertilization is catalyzed by Suv39h2 and that pericentromeric RNAs inhibit Suv39h2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression but instead can bookmark promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.

Pluripotent stem cells are thought of as a surrogate of early developmental stages that sustain the capacity to generate all cell types in the body, thereby constituting an invaluable tool to address the mechanisms underlying cellular plasticity. In the mouse, cells resembling totipotent 2-cell-stage embryos (2-cell-like cells) arise at a very low frequency in embryonic stem cell (ESC) cultures. However, the extent to which these early-embryonic-like cells recapitulate the molecular features of the early embryo is unclear. Here, we have undertaken a characterization of some of the metabolic features of early-embryonic-like cells in culture. Our data indicate that early-embryonic-like cells exhibit decreased glycolytic and respiratory activity, lower levels of reactive oxygen species and increased glucose uptake, suggesting a shift of the metabolic programme during 2-cell-like cell reprogramming. Accordingly, we find that 2-cell-like cells can be induced by defined metabolites. Thus, in addition to their transcriptional and chromatin features, 2-cell-like cells recapitulate some of the metabolic features of their in vivo counterpart. Altogether, our work underscores a distinct metabolic state of early-embryonic-like cells and identifies compounds that can induce their emergence in vitro.

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Extensive chromatin remodeling after fertilization is thought to take place to allow a new developmental program to start. This includes dynamic changes in histone methylation and, in particular, the remodeling of constitutive heterochromatic marks such as histone H4 Lys20 trimethylation (H4K20me3). While the essential function of H4K20me1 in preimplantation mouse embryos is well established, the role of the additional H4K20 methylation states through the action of the SUV4-20 methyltransferases has not been addressed. Here we show that Suv4-20h1/h2 are mostly absent in mouse embryos before implantation, underscoring a rapid decrease of H4K20me3 from the two-cell stage onward. We addressed the functional significance of this remodeling by introducing Suv4-20h1 and Suv4-20h2 in early embryos. Ectopic expression of Suv4-20h2 leads to sustained levels of H4K20me3, developmental arrest, and defects in S-phase progression. The developmental phenotype can be partially overcome through inhibition of the ATR pathway, suggesting that the main function for the remodeling of H4K20me3 after fertilization is to allow the timely and coordinated progression of replication. This is in contrast to the replication program in somatic cells, where H4K20me3 has been shown to promote replication origin licensing, and anticipates a different regulation of replication during this early developmental time window.

An intense period of chromatin remodeling takes place after fertilization in mammals, which is thought necessary for epigenetic reprogramming to start a new developmental program. While much attention has been given to the role of Polycomb Repressive Complex 2 (PRC2) and to canonical PRC1 complexes during this process, little is known as to whether there is any contribution of non-canonical PRC1 in shaping the chromatin landscape after fertilization. Here, we first describe in detail the temporal dynamics and abundance of H2A ubiquitylation (H2AK119ub), a histone modification catalyzed by PRC1, during preimplantation mouse development. In addition, we have analyzed the presence of the 2 characteristic subunits of non-canonical PRC1 complexes, RYBP and its homolog YAF-2. Our results indicate that H2AK119ub is inherited from the sperm, rapidly removed from the paternal chromatin after fertilization, but detected again prior to the first mitosis, suggesting that PRC1 activity occurs as early as the zygotic stage. RYBP and YAF-2, together with the non-canonical subunit L3MBTL2, are all present during preimplantation development but show different temporal dynamics. While RYBP is absent in the zygote, it is strongly induced from the 4-cell stage onwards. YAF-2 is inherited maternally and localizes to the pericentromeric regions in the zygote, is strongly induced between the 2-and 4-cell stages but then remains weak to undetectable subsequently. All together, our data suggest that non-canonical PRC1 is active during pre-implantation development and should be regarded as an additional component during epigenetic reprogramming and in the establishment of cellular plasticity of the early embryo.

Organisation de Coopération et de Développement Économiques Organisation for Economic Co-operation and Development 25-Mar-2015 ___________________________________________________________________________________________ _____________ English -Or. English ECONOMICS DEPARTMENT RAISING THE POTENTIAL OF THE DOMESTICALLY ORIENTED SECTOR IN GERMANY ECONOMICS DEPARTMENT WORKING PAPERS No. 1194 By André Eid and Andrés Fuentes Hutfilter OECD Working Papers should not be reported as representing the official views of the OECD or of its member countries. The opinions expressed and arguments employed are those of the author(s).