Transitions in cell states are controlled by combinatorial actions of transcription factors. BLIMP1, the key regulator of primordial germ cell (PGC) specification, apparently acts together with PRDM14 and AP2γ. To investigate their individual and combinatorial functions, we first sought an in vitro system for transcriptional readouts and chromatin immunoprecipitation sequencing analysis. We then integrated this data with information from single-cell transcriptome analysis of normal and mutant PGCs. Here we show that BLIMP1 binds directly to repress somatic and cell proliferation genes. It also directly induces AP2γ, which together with PRDM14 initiates the PGC-specific fate. We determined the occupancy of critical genes by AP2γ-which, when computed altogether with those of BLIMP1 and PRDM14 (both individually and cooperatively), reveals a tripartite mutually interdependent transcriptional network for PGCs. We also demonstrate that, in principle, BLIMP1, AP2γ and PRDM14 are sufficient for PGC specification, and the unprecedented resetting of the epigenome towards a basal state.

Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice, where its precise role is yet unclear. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.