The transcription factor Oct6 regulates the dissolution of the pluripotent state by inhibiting Nanog and inducing differentiation associated transcrip tion factors

ARIEL WAISMAN

Mendoza

Congreso; Congreso de la Sociedad Argentina de Investigaciones Bioquímicas y en Biología Molecular (SAIB); 2022

Animal development is controlled by gene regulatory networks (GRNs) that govern the nearly irreversible changes that occur when cells differentiate. However, the study of GRNs in mammalian development is hampered by technical and ethical constraints. Embryonic stem cells (ESCs) provide a good in vitro model to study this, as they can generate all the cell types in the organism and their differentiation recapitulates early embryonic development. In this work, we identified Oct6 as one of the earliest transcription factors (TFs) induced during the onset of mouse ESCs differentiation. By generating an Oct6 knock-out cell line using CRISPR, we show that differentiating cells failed to acquire the typical morphology associated with differentiation. RNA-seq analysis of differentiating cells identified 300 differentially expressed genes compared to wild-type cells, which included master pluripotency TFs that failed to correctly downregulate. Overexpression of Oct6 in pluripotent cells induced a rapid morphological change that resembled the one observed in differentiating cells while also inducing the expression of differentiation TFs such as Sox3, Zic2/3, Foxp1 as well as the endogenous Oct6. Strikingly, quantitative immunofluorescence showed that Oct6 also induced the repression of the core pluripotency TF Nanog. RT-qPCR and single molecule RNA-FISH analysis of Nanog confirmed that this regulation was at the transcriptional level. In summary, our results suggest that Oct6 is a key TF in the dissolution of the pluripotent state and that, together with Nanog, they form a double negative feedback loop which could act as a toggle switch important for this cell identity transition.