The events that prime pluripotent cells for differentiation are not well understood. has been made in establishing the factors that maintain pluripotency (Chambers and Smith, 2004). In contrast, little is known about the transcription factors that guide the transition from pluripotency to somatic lineage commitment. Pluripotent cells are maintained by a network of pluripotency factors that include Oct4, Sox2, Nanog, Klf4, and Esrrb. In the early blastocyst, fibroblast growth factor (FGF) 4 drives a subpopulation of cells toward a primitive endoderm fate (Nichols et?al., 2009; Yamanaka et?al., 2010). Cells that escape FGF action and retain high levels of Nanog go on to become restricted to an epiblast fate by around embryonic day 4.25 (E4.25) (Nichols and Smith, 2009; Yamanaka et?al., 2010). Experiments MK-5108 using embryonic stem cells (ESCs) show that FGF signaling is required not only for primitive endoderm differentiation but also for competence to differentiate into somatic cell types (Kunath et?al., 2007). FGF is necessary but not sufficient to drive lineage commitment: further progression to overt differentiation is restrained by the combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP) signaling, both of which restrict cells from progressing to a postimplantation epiblast-like state (Ying et?al., 2003). The transcription factors that act downstream of FGF in order to drive epiblast cells toward this differentiation-primed state are not known. A clue to their identity comes from the finding that inhibitor of DNA binding/differentiation (Id) proteins are able Mouse monoclonal to SUZ12 to block the transition of ESCs MK-5108 to epiblast stem cells (EpiSC) (Zhang MK-5108 et?al., 2010). Id proteins classically function through the inhibition of active basic helix-loop-helix (bHLH) transcription factors. We thus hypothesized that epiblast priming is driven by specific bHLH factors that are expressed in pluripotent cells but held in an inactive state through the action of Id proteins. As soon as Id proteins are downregulated, the bHLH activity of these primed cells would be released from inhibition, allowing epiblast maturation to proceed. In other cell types, Id proteins act through either direct binding and inhibition of bHLH transcription factors or indirect inhibition of bHLH transcription factor function through binding and sequestration of their essential heterodimerization partners E proteins (including E47 and E12) (Norton, 2000). Thus, we set out to identify the targets of Id inhibition by determining the direct binding partners of both Id and E proteins in ESCs. To achieve this, we performed a series of yeast two-hybrid (Y2H) screens for binding partners of Id1, E47, and E12 within a library generated from the messenger RNA (mRNA) of pluripotent mouse ESCs. This revealed three Id-regulated bHLH factors that are expressed in ESCs, of which one, Tcf15, is also expressed in the inner cell mass of the E4.5 embryo. Despite a known function in controlling somite development (Burgess et?al., 1996), a role for Tcf15 at this earlier development stage has been unknown. Here, we MK-5108 demonstrate a distinct wave of Tcf15 expression in the late preimplantation embryo in?vivo and a transient spike MK-5108 of expression during the early stages of ESC differentiation in?vitro. We show that an Id-resistant form of Tcf15 rapidly downregulates and accelerates the transition of ESCs through the epiblast state while suppressing primitive endoderm differentiation. Efforts to understand the balance between pluripotency and lineage commitment have been hampered by the lack of a marker that can be used to monitor exit from the pluripotent state toward somatic lineages. Tcf15 acts as a marker of this transition state: it.