Mouse embryonic stem (ES) cells can self-renew in the presence of leukemia inhibitory factor (LIF). Several essential transcription factors have been identified for the self-renewal of mouse ES cells, including STAT3, Oct-3/4, and Nanog. The molecular mechanism of ES cell self-renewal, however, is not fully understood. In the present study, we identified Eed, a core component of Polycomb repressive complex 2, as a downstream molecule of STAT3 and Oct-3/4. Artificial activation of STAT3 resulted in increased expression of Eed, whereas expression of a dominant negative mutant of STAT3 or suppression of Oct-3/4 expression led to down-regulation of Eed. Reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays revealed that STAT3 and Oct-3/4 directly bind to the promoter region of Eed, suggesting that Eed is a common target molecule of STAT3 and Oct-3/4. We also found that suppression of STAT3, Oct-3/4, or Eed causes induction of differentiation-associated genes as well as loss of Lys 27 -trimethylated histone H3 at the promoter regions of the differentiation-associated genes. Suppression of STAT3 and Oct-3/4 also resulted in the absence of Eed at the promoter regions. These results suggest that STAT3 and Oct-3/4 maintain silencing of differentiation-associated genes through up-regulation of Eed in self-renewing ES cells. Embryonic stem (ES)5 cells are derived from the inner cell mass of the mammalian blastocyst and have two major characteristics, pluripotency and self-renewal (1, 2). Previous studies have identified several essential transcription factors for the self-renewal of mouse ES cells, such as Oct-3/4, Nanog, and STAT3 (3). Oct-3/4 is a POU-family transcription factor involved in inner cell mass formation (4). A precise level of Oct-3/4 expression is required for maintenance of ES cells: repression of Oct-3/4 leads to trophectodermal differentiation, and overexpression of Oct-3/4 stimulates differentiation, mainly to extraembryonic endoderm (5). Nanog is a homeodomain transcription factor whose overexpression sustains ES cell self-renewal (6). Targeted disruption of the nanog gene results in ES cell differentiation, primarily along the primitive endoderm lineage, suggesting that Nanog prevents ES cells from endoderm differentiation (7).The pluripotency and self-renewal of mouse ES cells can be maintained by the presence of leukemia inhibitory factor (LIF). LIF stimulation leads to the activation of transcription factor STAT3. Previously, using a fusion protein consisting of STAT3 and the ligand-binding domain of estrogen receptor (STAT3ER), we demonstrated that the self-renewal of ES cells can be maintained by activation of STAT3ER with a synthetic estrogen receptor ligand, 4-hydroxytamoxifen (4HT), even in the absence of LIF (8). Another study showed that expression of a dominant negative mutant of STAT3 causes differentiation of ES cells (9). These observations indicate that the activation of STAT3 is essential and sufficient for the self-renewal of mouse ES cells.Despite having ...

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

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Self-renewal capacity and pluripotency, which are controlled by the Oct3/4-centered transcriptional regulatory network, are major characteristics of embryonic stem (ES) cells. Nuclear hormone receptor Dax1 is one of the crucial factors in the network. Here, we identified an orphan nuclear receptor, Esrrb (estrogen-related receptor beta), as a Dax1-interacting protein. Interaction of Dax1 and Esrrb was mediated through LXXLL motifs of Dax1 and the activation-and ligand-binding domains of Esrrb. Furthermore, Esrrb enhanced the promoter activity of the Dax1 gene via direct binding to Esrrb-binding site 1 (ERRE1, where "ERRE" represents "Esrrb-responsive element") of the promoter. Expression of Dax1 was suppressed followed by Oct3/4 repression; however, overexpression of Esrrb maintained expression of Dax1 even in the absence of Oct3/4, indicating that Dax1 is a direct downstream target of Esrrb and that Esrrb can regulate Dax1 expression in an Oct3/4-independent manner. We also found that the transcriptional activity of Esrrb was repressed by Dax1. Furthermore, we revealed that Oct3/4, Dax1, and Esrrb have a competitive inhibition capacity for each complex. These data, together with previous findings, suggest that Dax1 functions as a negative regulator of Esrrb and Oct3/4, and these molecules form a regulatory loop for controlling the pluripotency and self-renewal capacity of ES cells. P luripotency and self-renewal capacity are major characteristics of murine embryonic stem (ES) cells. Leukemia inhibitory factor (LIF) plays an important role for the self-renewal of ES cells, and depletion of LIF from ES cell culture medium leads to spontaneous differentiation of cells and results in a failure of self-renewal (1, 2). A large number of transcription factors function downstream of signaling by LIF, and several transcription factors, including STAT3, Oct3/4, Sox2, and Nanog, play crucial roles for pluripotency and self-renewal of ES cells (3-5). Artificial activation of STAT3, which is achieved by 4-hydroxytamoxifen stimulation of nuclear localization of the STAT3-estrogen receptor fusion protein (STAT3ER), as well as forced expression of Nanog, accelerates the self-renewal in a LIF-independent manner (6-8). Oct3/4-deficient embryos develop to the blastocyst stage, but their inner cell mass (ICM), from which ES cells are established, loses pluripotency, and the deletion of Oct3/4 expression in ES cells promotes differentiation into extraembryonic trophectodermal cells (9, 10). Sox2-deficient blastocysts form abnormal ICM and fail to achieve outgrowth (11). ES cells lacking the Sox2 gene differentiate into trophoblast cells (12).Actually, these transcription factors collaboratively regulate gene expression with other factors and contribute to maintenance of pluripotency and self-renewal of ES cells. For instance, Oct3/4 interacts with Sox2, and this complex enhances expression of ES cell-specific genes, including Fgf4, Lefty1, Nanog, UTF1, and Sox2 (13). ␤-Catenin is also a binding partner of Oct3/4, and the complex re...

Partially covered stent-grafts can be safely placed in relatively small animals and potentially used in research.