During differentiation, human embryonic stem cells (hESCs) shut down the regulatory network conferring pluripotency in a process we designated pluripotent state dissolution (PSD). In a high-throughput RNAi screen using an inclusive set of differentiation conditions, we identify centrally important and context-dependent processes regulating PSD in hESCs, including histone acetylation, chromatin remodeling, RNA splicing, and signaling pathways. Strikingly, we detected a strong and specific enrichment of cell-cycle genes involved in DNA replication and G2 phase progression. Genetic and chemical perturbation studies demonstrate that the S and G2 phases attenuate PSD because they possess an intrinsic propensity toward the pluripotent state that is independent of G1 phase. Our data therefore functionally establish that pluripotency control is hardwired to the cell-cycle machinery, where S and G2 phase-specific pathways deterministically restrict PSD, whereas the absence of such pathways in G1 phase potentially permits the initiation of differentiation.

The limited number of in vivo germ cells poses an impediment to genome-wide studies. Here, we applied a small-scale chromatin immunoprecipitation sequencing (ChIP-seq) method on purified mouse fetal germ cells to generate genome-wide maps of four histone modifications (H3K4me3, H3K27me3, H3K27ac, and H2BK20ac). Comparison of active chromatin state between somatic, embryonic stem, and germ cells revealed promoters and enhancers needed for stem cell maintenance and germ cell development. We found the nuclear receptor Nr5a2 motif to be enriched at a subset of germ cell cis-regulatory regions, and our results implicate Nr5a2 in germ cell biology. Interestingly, in germ cells, the H3K27me3 histone modification occurs more frequently at regions that are enriched for retrotransposons and MHC genes, indicating that these loci are specifically silenced in germ cells. Together, our study provides genome-wide histone modification maps of in vivo germ cells and reveals the molecular chromatin signatures of germ cells.

The maintenance of mouse embryonic stem cells (mESCs) requires LIF and serum. However, a pluripotent "ground state," bearing resemblance to preimplantation mouse epiblasts, can be established through dual inhibition (2i) of both prodifferentiation Mek/Erk and Gsk3/Tcf3 pathways. While Gsk3 inhibition has been attributed to the transcriptional derepression of Esrrb, the molecular mechanism mediated by Mek inhibition remains unclear. In this study, we show that Krüppel-like factor 2 (Klf2) is phosphorylated by Erk2 and that phospho-Klf2 is proteosomally degraded. Mek inhibition hence prevents Klf2 protein phosphodegradation to sustain pluripotency. Indeed, while Klf2-null mESCs can survive under LIF/Serum, they are not viable under 2i, demonstrating that Klf2 is essential for ground state pluripotency. Importantly, we also show that ectopic Klf2 expression can replace Mek inhibition in mESCs, allowing the culture of Klf2-null mESCs under Gsk3 inhibition alone. Collectively, our study defines the Mek/Erk/Klf2 axis that cooperates with the Gsk3/Tcf3/Esrrb pathway in mediating ground state pluripotency.