Cross-lineage induction of murine totipotency via epigenetically reprogramming of GC-rich retrotransposons

Abstract

Totipotent cells can give rise to all the differentiated cells in both embryonic and extraembryonic tissues. In mice, the zygote and blastomeres of the early 2-cell embryo are authentic totipotent cells. Despite the central importance of totipotency in establishing life, how does a totipotent embryo arise from two terminally differentiated gametes and the key factors and molecular mechanism by which the embryonic chromatin is naturally reprogrammed to a totipotent state to trigger zygotic genome activation (ZGA) are largely unknown in mammals. Establishment of totipotent-like stem cells in vitro constitutes an invaluable model for studying the molecular features of totipotent cells in normal development and for a number of downstream translational applications. Here, from pluripotent stem cells, we have identified a single molecule that enables establishment of totipotent zygotic genome activation-like cells (Z-cells) with high potency of generating both embryonic and extraembryonic cell lineages in in vivo chimeras and in vitro differentiation and blastoid generation. At this moment, our one molecule induction of totipotent-like cell system is the most efficient in vitro approach to generate totipotent-like cells. Importantly, Z-cells express the 2-cell blastomere enriched genes and retrotransposons and are more similar with 2-cell embryo than all other published mouse totipotent-like cells to date, indicating that Z-cells may represent a new cell source with enhanced totipotency. Mechanically, scATAC-seq analysis shows that the single molecule remodels the chromatin accessibility in pluripotent stem cells to open totipotent specific retrotransposons with high GC density, and thus these open DNA regions act as cis-regulatory elements to active zygotic gene expression. CRISPRi-based repression or CRISPRa-based activation of these retrotransposons abolishes or enhances the totipotency induction, respectively, further strengthening our conclusion. Interestingly, this novel protocol can also be used to successfully induce blastocysts and embryonic fibroblasts to regain 2C-like molecular features, suggesting that re-establishment of 2C-specific chromatin-accessible pattern is conserved for gaining totipotent properties in mouse different lineage cells. In summary, our one chemical approach for totipotency induction not only provides a defined in vitro system to generate high quality totipotent cells, but also identify the key regulatory genomic regions corelated with totipotent gene regulation, providing novel molecular insights of totipotency regulation and zygotic genome activation.