The pluripotency of embryonic stem (ES) cells is thought to be maintained by a few key transcription factors, including Oct3/4 and Sox2. The function of Oct3/4 in ES cells has been extensively characterized, but that of Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct-Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct-Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.

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SUMMARY To examine transcription factor (TF) network(s), we created mouse ES cell lines, in each of which one of 50 TFs tagged with a FLAG moiety is inserted into a ubiquitously controllable tetracycline-repressible locus. Of the 50 TFs, Cdx2 provoked the most extensive transcriptome perturbation in ES cells, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. ChIP-Seq revealed that CDX2 binds to promoters of up-regulated target genes. By contrast, genes down-regulated by CDX2 did not show CDX2 binding, but were enriched with binding sites for POU5F1, SOX2, and NANOG. Genes with binding sites for these core TFs were also down-regulated by the induction of at least 15 other TFs, suggesting a common initial step for ES cell differentiation mediated by interference with the binding of core TFs to their target genes. These ES cell lines provide a fundamental resource to study biological networks in ES cells and mice.

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Signaling molecules belonging to the Fibroblast growth factor (Fgf) family are necessary for directing bud outgrowth during tracheal development in Drosophila and lung development in mouse. A potential inhibitor of the Fgf signaling pathway, called Sprouty, has been identified in Drosophila. We have identified three potential mouse homologues of sprouty. One of them, called Sprouty4, exhibits a very restricted expression pattern. At 8.0 dpc (days post coitum) Sprouty4 is strongly expressed in the primitive streak region. At 9. 5 and 10.5 dpc, Sprouty4 is expressed in the nasal placode, the maxillary and mandibular processes, the otic vesicule, the second branchial arch, in the progress region of the limb buds and the presomitic mesoderm. Sprouty4 expression is also detected in the lateral region of the somites. In the developing lung, Sprouty4 is expressed broadly in the distal mesenchyme.

The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, the initial reprogramming mechanism is still poorly understood. Here we show that Zscan4, expressed transiently in 2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Interestingly, the forced expression of Zscan4 is required only for the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process and reactivates early embryonic genes.

Networks of transcription factors (TFs) are thought to determine and maintain the identity of cells. Here we systematically repressed each of 100 TFs with shRNA and carried out global gene expression profiling in mouse embryonic stem (ES) cells. Unexpectedly, only the repression of a handful of TFs significantly affected transcriptomes, which changed in two directions/trajectories: one trajectory by the repression of either Pou5f1 or Sox2; the other trajectory by the repression of either Esrrb, Sall4, Nanog, or Tcfap4. The data suggest that the trajectories of gene expression change are already preconfigured by the gene regulatory network and roughly correspond to extraembryonic and embryonic fates of cell differentiation, respectively. These data also indicate the robustness of the pluripotency gene network, as the transient repression of most TFs did not alter the transcriptomes.

Mouse embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, can self-renew in the presence of leukemia inhibitory factor (LIF) and maintain their pluripotency, the ability to differentiate into all types of somatic and germ cells (6, 11). In the self-renewal of mouse ES cells, STAT3, Oct3/4, Sox2, and Nanog play important roles (23). STAT3 is a well-known transcription factor downstream of LIF, and expression of its dominant-negative mutant induces differentiation of ES cells (19). Artificial activation of STAT3 using STAT3ER, a fusion protein consisting of STAT3 and the ligand-binding domain of estrogen receptor, can maintain ES cell self-renewal in the absence of LIF (14). These observations indicate that STAT3 activation is essential and sufficient for the maintenance of self-renewal. Nanog is a homeobox transcription factor whose overexpression can bypass the requirement of LIF for self-renewal (3, 15). Since a recent report has demonstrated that this homeobox transcription factor is dispensable for ES cell self-renewal (4), Nanog seems to be a self-renewal-promoting factor.Oct3/4 (encoded by pou5f1) belongs to the POU family of transcription factors and consists of three domains: the Nterminal, POU, and C-terminal domains (see Fig. 1D). The Nand C-terminal domains are transactivation domains with redundant functions (21), while the POU domain is a bipartite DNA-binding domain consisting of the POU-specific domain and the POU homeodomain. Although continuous expression of Oct3/4 fails to maintain the self-renewal of ES cells in the absence of LIF, targeted disruption of the pou5f1 gene results in loss of pluripotent inner cell mass, and conditional repression of this gene in ES cells leads to differentiation into trophectoderm, indicating that Oct3/4 is a central player in the self-renewal in ES cells (18,20). Furthermore, recent findings that Oct3/4 is one of the four factors required for the production of induced pluripotent stem cells suggest the importance of Oct3/4 for acquisition of pluripotency (30). Interestingly, not only suppression but also overexpression of Oct3/4 induces ES cell differentiation (20), suggesting that the proper expression/ activity level of Oct3/4 is required to maintain ES cell selfrenewal.In ES cells, the expression of Oct3/4 is regulated by several transcription factors (23). Previous studies have revealed that the upstream region of the pou5f1 gene contains two elements, proximal and distal enhancers, which regulate the stem cellspecific expression of Oct3/4 (36). An orphan nuclear receptor, liver receptor homolog 1 (LRH1, also known as Nr5a2), binds with the proximal enhancer (8). In LRH1-null ES cells, although Oct3/4 is still expressed, its downregulation during differentiation occurs more rapidly than in the wild-type cells, suggesting that LRH1 is involved in the maintenance of Oct3/4 expression (8). It is also documented that Oct3