The transcription factor Oct6 promotes the dissolution of the naïve pluripotent state by repressing Nanog and activating a formative state gene regulatory network.

Animal development is controlled by large gene regulatory networks (GRNs) that govern the nearly irreversible changes that occur when a cell differentiates into another cell type. In this work we aimed to determine key transcription factors (TFs) associated with the dissolution of the naïve pluripotent state and the acquisition of a formative identity. We identified Oct6 as one of the earliest TFs induced during the onset of mouse embryonic stem cell (mESCs) differentiation. By generating an Oct6 knockout mESC cell line, we show that it failed to acquire the typical cell morphology associated with the formative state. Transcriptome analysis of these differentiating cells allowed for the identification of nearly 300 differentially expressed genes compared to wild-type cells. Among these were the pluripotency TFs Nanog, Klf2, Nr5a2, Prdm14, and Esrrb, that failed to correctly downregulate. Premature expression of Oct6 in naïve cells induced a rapid morphological transformation that resembled the one observed in differentiating cells. In these conditions, Oct6 induced its own expression as well as that of other TFs such as Sox3, Zic2/3, Foxp1, and formative genes like Dnmt3A and FGF5. Strikingly, Oct6 also repressed the expression of the key pluripotency TF Nanog. Gene expression and single molecule RNA-FISH analysis of Nanog confirmed that this regulation was at the transcriptional level. In summary, our results indicate that Oct6 is a key TF in the dissolution of the pluripotent state. Moreover, they support a model where Oct6 and Nanog form a double negative feedback loop which could act as a toggle switch important for the transition to the formative state. WT and Oct6 KO 46C mouse embryonic stem cells (mESCs) were differentiated to epiblast-like cells (EpiLCs) by culturing them for 48 h in N2B27 defined medium supplemented with 1% KSR, 12 µg/ml bFGF, and 20 µg/ml Activin A, in fibronectin-coated culture dishes. Three paired biological replicates were performed for both WT and KO EpiLCs. Total RNA was submitted to high-throughput sequencing.