SummaryPolycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.

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Sox2 is a transcription factor required for the maintenance of pluripotency. It also plays an essential role in different types of multipotent stem cells, raising the possibility that Sox2 governs the common stemness phenotype. Here we show that Sox2 is a critical downstream target of fibroblast growth factor (FGF) signaling, which mediates self-renewal of trophoblast stem cells (TSCs). Sustained expression of Sox2 together with Esrrb or Tfap2c can replace FGF dependency. By comparing genome-wide binding sites of Sox2 in embryonic stem cells (ESCs) and TSCs combined with inducible knockout systems, we found that, despite the common role in safeguarding the stem cell state, Sox2 regulates distinct sets of genes with unique functions in these two different yet developmentally related types of stem cells. Our findings provide insights into the functional versatility of transcription factors during embryogenesis, during which they can be recursively utilized in a variable manner within discrete network structures.

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