The transcription factor OCT6 promotes the dissolution of the naïve pluripotent state by repressing Nanog and activating a formative state gene regulatory network.

In the mouse embryo, the transition from the preimplantation to the postimplantation epiblast is governed by changes in the gene regulatory network (GRN) that lead to transcriptional, epigenetic, and functional changes. This transition can be faithfully recapitulated in vitro by the differentiation of mouse embryonic stem cells (mESCs) to epiblast-like cells (EpiLCs), that reside in naïve and formative states of pluripotency, respectively. However, the GRN that drives this conversion is not fully elucidated. Here we demonstrate that the transcription factor OCT6 is a key driver of this process. Firstly, we show that Oct6 is not expressed in mESCs but is rapidly induced as cells exit the naïve pluripotent state. By deleting Oct6 in mESCs, we find that knockout cells fail to acquire the typical morphological changes associated with the formative state when induced to differentiate. Additionally, the key naïve pluripotency TFs Nanog, Klf2, Nr5a2, Prdm14, and Esrrb were expressed at higher levels than in wild-type cells, indicating an incomplete dismantling of the naïve pluripotency GRN. Conversely, premature expression of Oct6 in naïve cells triggered a rapid morphological transformation mirroring differentiation, that was accompanied by the upregulation of the endogenous Oct6 as well as the formative genes Sox3, Zic2/3, Foxp1, Dnmt3A and FGF5. Strikingly, we found that OCT6 represses Nanog in a bistable manner and that this regulation is at the transcriptional level. Moreover, our findings also reveal that Oct6 is repressed by NANOG. Collectively, our results establish OCT6 as a key TF in the dissolution of the naïve pluripotent state and support a model where Oct6 and Nanog form a double negative feedback loop which could act as an important toggle mediating the transition to the formative state.

Discriminating between negative cooperativity and ligand binding to independent sites using pre-equilibrium properties of binding curves.

Negative cooperativity is a phenomenon in which the binding of a first ligand or substrate molecule decreases the rate of subsequent binding. This definition is not exclusive to ligand-receptor binding, it holds whenever two or more molecules undergo two successive binding events. Negative cooperativity turns the binding curve more graded and cannot be distinguished from two independent and different binding events based on equilibrium measurements only. The need of kinetic data for this purpose was already reported. Here, we study the binding response as a function of the amount of ligand, at different times, from very early times since ligand is added and until equilibrium is reached. Over those binding curves measured at different times, we compute the dynamic range: the fold change required in input to elicit a change from 10 to 90% of maximum output, finding that it evolves in time differently and controlled by different parameters in the two situations that are identical in equilibrium. Deciphering which is the microscopic model that leads to a given binding curve adds understanding on the molecular mechanisms at play, and thus, is a valuable tool. The methods developed in this article were tested both with simulated and experimental data, showing to be robust to noise and experimental constraints.

Cell cycle dynamics of mouse embryonic stem cells in the ground state and during transition to formative pluripotency.

Mouse embryonic stem cells (mESCs) can be maintained as homogeneous populations in the ground state of pluripotency. Release from this state in minimal conditions allows to obtain cells that resemble those of the early post-implantation epiblast, providing an important developmental model to study cell identity transitions. However, the cell cycle dynamics of mESCs in the ground state and during its dissolution have not been extensively studied. By performing live imaging experiments of mESCs bearing cell cycle reporters, we show here that cells in the pluripotent ground state display a cell cycle structure comparable to the reported for mESCs in serum-based media. Upon release from self-renewal, the cell cycle is rapidly accelerated by a reduction in the length of the G1 phase and of the S/G2/M phases, causing an increased proliferation rate. Analysis of cell lineages indicates that cell cycle variables of sister cells are highly correlated, suggesting the existence of inherited cell cycle regulators from the parental cell. Together with a major morphological reconfiguration upon differentiation, our findings support a correlation between this in vitro model and early embryonic events.