br Introduction In mammals cells from the blastocyst stage c

Introduction In mammals, cells from the abacavir stage can be used to form embryonic stem (ES) cell lines that can self-renew in culture while maintaining the ability to differentiate into cells of the three germ layers (Reubinoff et al., 2000). Directed in vitro differentiation protocols generally attempt to utilize our knowledge of the normal signaling pathways guiding embryogenesis and mimic this process in vitro, theoretically providing an unlimited supply of any desired cell type (Murry and Keller, 2008). The molecular mechanisms underlying pluripotent stem cell differentiation are of great interest for understanding development, disease, and regenerative medicine. A growing body of evidence underscores the importance of pluripotency factors during differentiation. Human and mouse ES cell studies have demonstrated that the core pluripotency transcription factors, SOX2, OCT4, and NANOG, play distinct roles in coordinating ES cell lineage commitment (Lu et al., 2009; Thomson et al., 2011; Wang et al., 2012). NANOG promotes definitive endoderm (DE) formation by coordinating with the activation of the TGF-β signaling pathway through the induction of EOMES (Teo et al., 2011). SOX2 is important in ectoderm formation by limiting mesendoderm formation (Wang et al., 2012). The role of OCT4 in lineage commitment is somewhat less clear. In zebrafish, the OCT4 homolog spg is required for endoderm induction (Lunde et al., 2004). The role of OCT4 during human ES (hES) cell differentiation and lineage commitment is controversial because knockdown studies performed by different labs have resulted in the induction of trophectoderm, primitive endoderm, or neuroectoderm cell fates (Niwa et al., 2000; Wang et al., 2012). Another study suggested that OCT4 knockdown induced DE formation, even though gene expression was low (Teo et al., 2011). These studies also demonstrated a critical role for OCT4 in maintaining the undifferentiated state. Several studies have demonstrated that OCT4 may play a role in regulating the epigenetic landscape in ES cells and during differentiation. Pull-down assays indicate that major Oct4 partners in mouse ES cells are related to chromatin remodeling (Pardo et al., 2010; van den Berg et al., 2010). Bernstein et al. (2006) defined a specific chromatin modification pattern called the “bivalent domain,” which harbors both the inhibitory, H3K27me3, and activating, H3K4me3, histone modifications at genes important in regulating early development in ES cells. Genes present in bivalent domains are typically silent in ES cells, but are poised for activation. Genome-wide studies have shown that OCT4 co-localizes with Polycomb2 (PRC2) components, which are responsible for laying down the inhibitory H3K27me3 mark and generating bivalent domains in hES cells (Boyer et al., 2005, 2006). Based on these findings, OCT4 may play an important role in chromatin remodeling during differentiation in response to external signals. The Wnt signaling pathway is important for both maintaining pluripotency (Sokol, 2011; Wang and Wynshaw-Boris, 2004) and inducing differentiation to primitive streak and mesendoderm (Cheng et al., 2008; Gadue et al., 2006; Lyashenko et al., 2011). In mouse ES cells, Oct4 has been shown to play a role in the Wnt signaling pathway by physically interacting with β-catenin to reinforce pluripotency (Kelly et al., 2011). In this study, we define a role for OCT4 and Wnt signaling in establishing an appropriate chromatin signature during hES cell specification into endoderm. By utilizing a directed differentiation approach coupled with siRNA knockdown of OCT4, we show that OCT4 and Wnt signaling play a role in the chromatin pre-patterning of endoderm genes such as SOX17 and FOXA2. During mesendoderm commitment, OCT4 physically associates with β-catenin while the knockdown of OCT4 eliminates the mesendoderm differentiation capacity of hES cells. OCT4 knockdown also led to a failure to remove the PRC2 complex from primitive streak and endodermal genes. In the absence of Wnt pathway activation during endoderm induction, hES cells maintain high levels of OCT4 protein but fail to evict the PRC2 complex and downregulate H3K27me3 on primitive streak and endodermal genes. In summary, OCT4 plays a key role in the pluripotency core network and is indispensable for lineage commitment by coordinating with WNT signaling to target bivalent genes for activation. These data underscore the importance of pluripotent transcription factors in differentiation as well as for maintenance of the pluripotent state.