Much attention has focused on a small set of transcription factors that maintain human or mouse embryonic stem (ES) cells in a pluripotent state. To gain a more complete understanding of the regulatory network that maintains this state, we identified target promoters of nine transcription factors, including somatic cell reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) and others (Nanog, Dax1, Rex1, Zpf281, and Nac1), on a global scale in mouse ES cells. We found that target genes fall into two classes: promoters bound by few factors tend to be inactive or repressed, whereas promoters bound by more than four factors are largely active in the pluripotent state and become repressed upon differentiation. Furthermore, we propose a transcriptional hierarchy for reprogramming factors and broadly distinguish targets of c-Myc versus other factors. Our data provide a resource for exploration of the complex network maintaining pluripotency.

Embryonic stem (ES) cells are pluripotent and of therapeutic potential in regenerative medicine. Understanding pluripotency at the molecular level should illuminate fundamental properties of stem cells and the process of cellular reprogramming. Through cell fusion the embryonic cell phenotype can be imposed on somatic cells, a process promoted by the homeodomain protein Nanog, which is central to the maintenance of ES cell pluripotency. Nanog is thought to function in concert with other factors such as Oct4 (ref. 8) and Sox2 (ref. 9) to establish ES cell identity. Here we explore the protein network in which Nanog operates in mouse ES cells. Using affinity purification of Nanog under native conditions followed by mass spectrometry, we have identified physically associated proteins. In an iterative fashion we also identified partners of several Nanog-associated proteins (including Oct4), validated the functional relevance of selected newly identified components and constructed a protein interaction network. The network is highly enriched for nuclear factors that are individually critical for maintenance of the ES cell state and co-regulated on differentiation. The network is linked to multiple co-repressor pathways and is composed of numerous proteins whose encoding genes are putative direct transcriptional targets of its members. This tight protein network seems to function as a cellular module dedicated to pluripotency.