The Transcription Factor Network in Embryonic Stem Cells: The virtue of promiscuity?

Embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and when cultured in vitro can self-renew indefinitely while retaining the capacity to differentiate into derivatives of the three germ layers. These key properties are regulated by a core transcriptional network that revolves around three transcription factors, Oct4, Sox2 and Nanog. Chapter 1 introduces basic aspects of eukaryotic transcription regulation and describes the role of transcription factors in mouse preimplantation development and in maintenance and reinstatement of pluripotency in vitro. Whereas for most ES cell transcription factors genomic binding sites and regulated genes have been reported, the scope of their interaction partners remains underinvestigated. Exploring the interactome of ES cell transcription factors, however, can help to elucidate the molecular mechanisms by which they regulate gene expression and potentially leads to identification of novel factors involved in ES cell maintenance. Chapter 3 describes an improved FLAG affinity based protein purification methodology that was used to purify complexes of transcription factors Oct4, Sall4, Dax1, Tcfcp2l1 and Esrrb from relatively small amounts of ES cell nuclear extract. Identification of associated proteins by mass spectrometry analysis resulted in an interactome comprised of 166 proteins, including transcription factors and chromatin modifying complexes with documented roles in pluripotency or self-renewal, but also factors that are novel to the ES cell network. It furthermore demonstrates association of Esrrb with the basal transcription machinery (i.e. Mediator complex, RNA pol2 and TFIID). Chapter 2 reports on the functional implications of the newly identified interaction between Oct4 and Esrrb in regulation of Nanog gene expression. Chapter 4 concerns the characterization of Mediator complexes in ES cells and reports reproducible identification of Esrrb in purifications of Mediator complex. Chapter 5 provides a general discussion of the studies presented in this thesis.