SummaryTranscription factors, such as Oct4, are critical for establishing and maintaining pluripotent cell identity. Whereas the genomic locations of several pluripotency transcription factors have been reported, the spectrum of their interaction partners is underexplored. Here, we use an improved affinity protocol to purify Oct4-interacting proteins from mouse embryonic stem cells (ESCs). Subsequent purification of Oct4 partners Sall4, Tcfcp2l1, Dax1, and Esrrb resulted in an Oct4 interactome of 166 proteins, including transcription factors and chromatin-modifying complexes with documented roles in self-renewal, but also many factors not previously associated with the ESC network. We find that Esrrb associated with the basal transcription machinery and also detect interactions between transcription factors and components of the TGF-β, Notch, and Wnt signaling pathways. Acute depletion of Oct4 reduced binding of Tcfcp2l1, Dax1, and Esrrb to several target genes. In conclusion, our purification protocol allowed us to bring greater definition to the circuitry controlling pluripotent cell identity.

SummaryEmbryonic stem cell (ESC) self-renewal efficiency is determined by the level of Nanog expression. However, the mechanisms by which Nanog functions remain unclear, and in particular, direct Nanog target genes are uncharacterized. Here we investigate ESCs expressing different Nanog levels and Nanog−/− cells with distinct functionally inducible Nanog proteins to identify Nanog-responsive genes. Surprisingly, these constitute a minor fraction of genes that Nanog binds. Prominent among Nanog-reponsive genes is Estrogen-related receptor b (Esrrb). Nanog binds directly to Esrrb, enhances binding of RNAPolII, and stimulates Esrrb transcription. Overexpression of Esrrb in ESCs maintains cytokine-independent self-renewal and pluripotency. Remarkably, this activity is retained in Nanog−/− ESCs. Moreover, Esrrb can reprogram Nanog−/− EpiSCs and can rescue stalled reprogramming in Nanog−/− pre-iPSCs. Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent ESC self-renewal. These findings are consistent with the functional placement of Esrrb downstream of Nanog.

Embryonic stem (ES) cell self-renewal is regulated by transcription factors, including Oct4, Sox2, and Nanog. A number of additional transcriptional regulators of ES cell self-renewal have recently been identified, including the orphan nuclear receptor estrogen-related receptor beta (Esrrb). However, the mode of action of Esrrb in ES cells is unknown. Here, using an Oct4 affinity screen, we identify Esrrb as an Oct4 partner protein. Esrrb can interact with Oct4 independently of DNA. Esrrb is recruited near the Oct-Sox element in the Nanog proximal promoter, where it positively regulates Nanog expression. Esrrb recruitment to the Nanog promoter requires both the presence of Oct4 and a degenerate estrogen-related receptor DNA element. Consistent with its role in Nanog regulation, expression of the Esrrb protein within the Oct4-positive ES cell population is mosaic and correlates with the mosaic expression of the Nanog protein. Together with previous reports that Nanog may regulate Esrrb gene expression, our results suggest that Esrrb and Nanog act as part of a feedback regulatory circuit that modulates the fluctuating self-renewal capacity of ES cell populations.The self-renewal of mouse ES cells is regulated by a network of transcription factors that includes Oct4, Nanog, and Sox2 (22). The expression level of Oct4 protein needs to be kept within a tight range in order to maintain ES cell self-renewal (23). Decreasing Oct4 levels below 50% induces differentiation into the trophectoderm, whereas a twofold increase causes differentiation into cells expressing markers of the endoderm and mesoderm (23). In contrast, overexpression of Nanog allows mouse embryonic stem (ES) cells to remain undifferentiated in the absence of the otherwise requisite stimulation by leukemia inhibitory factor and bone morphogenetic protein (5,19,34). Oct4 is thought to act together with Sox2 by binding to adjacent cognate DNA sequences in many genes (1), including Nanog (14, 26). Genome-wide chromatin immunoprecipitation (ChIP) studies have suggested that composite Oct-Sox motifs regulate the expression of many genes in mouse and human ES cells (2, 16). Recent evidence has shown that the critical role of Sox2 in maintaining ES cell self-renewal is regulating Oct4 expression, suggesting that the secondary role of gene regulation via Oct-Sox motifs is performed redundantly with Sox4, Sox11, and Sox15 (18).Recent reports have expanded the list of factors that contribute to ES cell self-renewal. Wang et al. (30) reported a proteomic analysis of interactors of Oct4 and Nanog and suggested that some Nanog interactors may assist in Nanogmediated gene regulation. A separate study using an RNA interference (RNAi) screen found that depletion of estrogenrelated receptor beta (Esrrb), Tbx3, or Tcl1 resulted in ES cell differentiation but that this differentiation could be attenuated by overexpression of Nanog (13). However, it is unclear how any of these novel regulatory factors mediate their function. Here we use an unbiased analysis of Oct4 binding...