Abstract
Totipotency emerges in early embryogenesis, but its molecular underpinnings remain poorly characterized. In the present study, we employed DNA fiber analysis to investigate how pluripotent stem cells are reprogrammed into totipotent-like 2-cell-like cells (2CLCs). We show that totipotent cells of the early mouse embryo have slow DNA replication fork speed and that 2CLCs recapitulate this feature, suggesting that fork speed underlies the transition to a totipotent-like state. 2CLCs emerge concomitant with DNA replication and display changes in replication timing (RT), particularly during the early S-phase. RT changes occur prior to 2CLC emergence, suggesting that RT may predispose to gene expression changes and consequent reprogramming of cell fate. Slowing down replication fork speed experimentally induces 2CLCs. In vivo, slowing fork speed improves the reprogramming efficiency of somatic cell nuclear transfer. Our data suggest that fork speed regulates cellular plasticity and that remodeling of replication features leads to changes in cell fate and reprogramming.
Main
Cellular plasticity is an essential requirement for multicellular organisms. Cells in the early mammalian embryo are most plastic because they can generate every cell type in the body. In particular, the mouse zygote and each of the blastomeres in 2-cell-stage embryos are totipotent1,2, because they can generate a new organism on their own without the need for carrier cells. This contrasts with pluripotent cells, which can generate all the cells in the body, but not extraembryonic tissues3,4. Thus, totipotent cells have greater cellular plasticity. However, the mechanisms that sustain totipotency are poorly understood.
DNA replication is a fundamental process for genetic and epigenetic inheritance. However, how the early mammalian embryo replicates its DNA and whether the acquisition of totipotency is regulated through DNA-replication-dependent mechanisms is unknown. As the molecular properties of the replication fork are central to the regulation of replication5, we set out to investigate replication fork dynamics in totipotent cells in vivo and totipotent-like cells in culture.
Results
2CLCs and totipotent embryos have a slow replication fork speed
Totipotent-like cells resembling 2-cell-stage mouse embryos arise spontaneously in embryonic stem cell (ESC) cultures, but only in very low proportions of around 0.5%6. 2CLCs recapitulate several molecular features of the totipotent cells in mouse embryos and display expanded potency, including higher ability to be reprogrammed upon nuclear transfer6,7,8. Similar to 2-cell-stage embryos, 2CLCs express specific repeats such as MERVL6,9 and thus can be identified by a fluorescent reporter under the control of the MERVL long-terminal repeat6,10, enabling their characterization and isolation (Fig. 1a). We used DNA fiber analysis to study DNA replication and measure replication fork speed11,12. Analysis of replication fork speed in 2CLCs revealed a significantly slower fork speed compared with ESCs (Fig. 1b). Although ESCs displayed an expected rate of 1.34 kb min−1 (ref. 13), 2CLCs had approximately half this speed (0.56 kb min−1) (Fig. 1c). This suggested that totipotent-like cells in culture replicate DNA much more slowly than pluripotent stem cells. Importantly, the length of the S-phase did not change (see also below), suggesting that 2CLCs may use more origins than ESCs, to compensate for a slower fork progression. Indeed, analysis of the DNA fibers14 indicated an increase in DNA fibers in which replication stopped after the first label, implying more termination or blockage events (Fig. 1d), consistent with increased origin usage. In agreement, visualization of replication by 5-ethynyl-2′-deoxyuridine (EdU) incorporation revealed that 2CLCs displayed a more dispersed EdU pattern and higher number of replication clusters compared with ESCs (Extended Data Fig. 1a,b).
