glucose transport proteins br Results br Discussion The plur

Results
Discussion The pluripotency status of ES glucose transport proteins or iPS cells is maintained by a tightly regulated transcription factor network (Boyer et al., 2005). OCT4 is at the center of this network (Pardo et al., 2010; van den Berg et al., 2010) and is associated with chromatin modifiers to maintain epigenetic identity of pluripotent cells (Liang et al., 2008; Zhao et al., 2007). Its expression level is strictly controlled in pluripotent cells in which changes in protein level, either higher or lower, can lead to lineage-specific differentiation (Niwa et al., 2000; Wang et al., 2012). Wnt signaling is well known as an inducer of primitive streak formation (Gadue et al., 2006) and an indispensable component for efficient endoderm differentiation (Han et al., 2011). Endogenous WNT3 expression in ES cells can predict endoderm differentiation efficiency, highlighting the importance of this pathway in endoderm induction (Jiang et al., 2013). While Wnt can directly activate primitive streak genes such as T (Gadue et al., 2006), we demonstrate a secondary effect that this pathway has on regulating OCT4 function. Activation of β-catenin was critical for increased OCT4 binding to target genes and presumably regulates the transcription status of these genes during activin A-induced differentiation. Without Wnt signaling, mesendoderm genes (T, EOMES, and GSC) and DE genes (FOXA2 and SOX17) maintain the repressive PRC2 complex and the H3K27me3 mark. These findings are supported by recent work in the mouse ES cell system where it was demonstrated that a composite Oct4-Tcf/Lef site was critical for Mesp1 induction and cardiomyocyte differentiation (Li et al., 2013). Based on our observations, we propose a preliminary working model of how OCT4 cooperates with β-catenin to evict the PRC2 complex and remove the repressive histone mark at differentiation initiation (Figure S3D). Two recent studies have reported opposite conclusions on the effect of OCT4 knockdown in hES cell differentiation. Teo et al. (2011) demonstrated that OCT4 knockdown led to precocious upregulation of endodermal genes, while Wang et al. (2012) reported that OCT4 knockdown inhibits mesendoderm differentiation. Our data support the findings from both sides and shed light on the mechanism of these seemingly contradictory results. Like Teo et al., we also find endodermal gene expression upon OCT4 knockdown. However, the level of endodermal and primitive streak gene induction, while increased over that seen in ES cells, was orders of magnitude less than expression of these genes during normal differentiation (Figure S2B, ii, iii). In both hES cells and iPS cells, when OCT4 knockdown cells were subjected to DE differentiation, expression of primitive streak and DE genes was drastically downregulated compared with scramble siRNA transfected cells, which supported the findings of Wang et al. (2012). These seemingly contradictory phenotypes of OCT4 knockdown may underlie multiple roles for OCT4 in ES cell maintenance and differentiation. OCT4 is well known to repress differentiation genes (Boyer et al., 2005) such that lack of its expression leads to de-repression of these genes but at low levels without other differentiation induction signals. The second role of OCT4 that we uncovered is its involvement in the removal of PRC2 complex from mesendodermal genes and its requirement for the physiologic expression of these genes during directed differentiation of hES cells. Recent studies examining the role of Oct4 showed that Oct4 is critical in lineage specification in the mouse, especially for the endodermal program and supports our results. Oct4 ES cells and mouse iPS cells expressing low levels of Oct4 can be maintained in the undifferentiated state and express increased levels of many pluripotency markers (Karwacki-Neisius et al., 2013; Radzisheuskaya et al., 2013). Both pluripotent stem cell lines also displayed an inhibition of differentiation into embryonic lineages. In addition, mouse blastocysts that lack Oct4 fail to upregulate primitive endoderm genes in a cell autonomous manner. Our results show that OCT4 is not only critical for mesendoderm gene activation, but also crucial for DE gene expression by pre-patterning epigenetic marks prior to gene expression.