Nanog regulates Pou3f1 expression at the exit from pluripotency during gastrulation

Pluripotency is regulated by a network of transcription factors that maintains early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development its expression is restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog. Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation. Mouse embryonic stem cells were maintained in 2i+Lif conditions and at time 0h serum was add to the medium and 2i and Lif were removed, allowing the cells to differentiate. Three timepoints were obtain by triplicate: 0h, 12h and 24 starting at the adding of serum. RNA from NanogKO ES cells and their parental line was extracted using the RNeasy Mini Kit (Qiagen). Library preparation (New England Biolabs Nest Ultra RNA library prep Kit) and single read Next generation sequencing (Illumina HiSeq 2500) were performed at the Genomics Unit at Centro Nacional de Investigaciones Cardiovasculares (CNIC).