Cellular plasticity is essential for early embryonic cells. Unlike pluripotent cells, which form embryonic tissues, totipotent cells can generate a complete organism including embryonic and extraembryonic tissues. Cells resembling 2-cell-stage embryos (2C-like cells) arise at very low frequency in embryonic stem (ES) cell cultures. Although induced reprogramming to pluripotency is well established, totipotent cells remain poorly characterized, and whether reprogramming to totipotency is possible is unknown. We show that mouse 2C-like cells can be induced in vitro through downregulation of the chromatin-assembly activity of CAF-1. Endogenous retroviruses and genes specific to 2-cell embryos are the highest-upregulated genes upon CAF-1 knockdown. Emerging 2C-like cells exhibit molecular characteristics of 2-cell embryos and higher reprogrammability than ES cells upon nuclear transfer. Our results suggest that early embryonic-like cells can be induced by modulating chromatin assembly and that atypical histone deposition may trigger the emergence of totipotent cells.

Unlike pluripotent cells, which generate only embryonic tissues, totipotent cells can generate a full organism, including extraembryonic annexes. A rare population of cells resembling 2-cell stage embryos arises in pluripotent embryonic stem (ES) cell cultures. These 2-cell-like-cells display molecular features of totipotency and broader developmental plasticity. However, their specific nature and the process through which they arise remain outstanding questions. Here, we identify intermediate cellular states and molecular determinants during the emergence of 2-cell-like-cells. By deploying a quantitative single cell expression approach, we identified an intermediate population characterised by the expression of the transcription factor ZSCAN4 as precursor of 2-cell-like-cells. Using an siRNA screening, we uncovered novel epigenetic regulators of 2-cell-like-cell emergence, including the non-canonical PRC1 complex PRC1.6 and Ep400/Tip60. Our data shed light on the mechanisms underlying the exit from the ES cell state towards the formation of early-embryonic-like cells in culture and identify key epigenetic pathways that promote this transition.

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.