Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells

Abstract

ES cells represent a valuable model for investigating early embryo development and hold promise for future regenerative medicine strategies. The self-renewal of pluripotent mouse ES cells has been shown to require extrinsic stimulation by the bone morphogenetic protein (BMP) and leukemia inhibitory factor signaling pathways and the expression of the transcription factors Oct4 and Nanog. However, the network of interactions among extrinsic and intrinsic determinants of ES cell pluripotency is currently poorly understood. Here, we show that Nanog expression is up-regulated in mouse ES cells by the binding of T (Brachyury) and STAT3 to an enhancer element in the mouse Nanog gene. We further show that Nanog blocks BMP-induced mesoderm differentiation of ES cells by physically interacting with Smad1 and interfering with the recruitment of coactivators to the active Smad transcriptional complexes. Taken together, our findings illustrate the existence of ES cellspecific regulatory networks that underlie the maintenance of ES cell pluripotency and provide mechanistic insights into the role of Nanog in this process.pluripotency ͉ T (Brachyury) ͉ self-renewal ͉ mesoderm differentiation ͉ leukemia inhibitory factor M ouse ES cells are self-renewing, pluripotent cell lines derived from preimplantation embryos (1, 2). Strict culture conditions must be followed to maintain the self-renewal of pluripotent mouse ES cells. Two extrinsic culture requirements, a feeder layer of fibroblasts and the addition of FBS, have been identified as necessary to sustain proliferation of undifferentiated mouse ES cells and their activities pinpointed to specific molecules (reviewed in ref. 3). Thus, self-renewal of mouse ES cells can be sustained in feeder-free conditions by supplementing the culture media with the cytokine leukemia inhibitory factor (LIF) (4, 5). In the absence of LIF, ES cell colonies flatten and form epithelium-like sheets (4, 5). More recently, the self-renewal promoting activity of animal serum has been identified as being mediated by ligands of specific families of the TGF-␤ superfamily, including the bone morphogenetic protein (BMP) family members BMP2 and BMP4 and the growth and differentiation factor (GDF) family member GDF6 (6). In the absence of BMP͞GDF signals, LIF is not sufficient to prevent the neural differentiation of ES cells, whereas the absence of both BMP͞GDF and LIF stimulation results in a flattened cell phenotype similar to that observed during LIF withdrawal (6).The intracellular signaling cascades initiated by both LIF and BMP͞GDF that sustain self-renewal of mouse ES cells have been worked out to a significant degree of detail (reviewed in ref.3). In summary, binding of LIF to its cognate LIF receptor results in the recruitment of gp130 and the formation of a ternary complex that catalyzes the tyrosine phosphorylation, dimerization, and nuclear translocation of the downstream signal transducer STAT3. BMP͞ GDF, in turn, promotes ES cell self-renewal by inducing the expression of members of the inhibit...

Hypoxia-inducible Factor-1α Inhibits Self-renewal of Mouse Embryonic Stem Cells in Vitro via Negative Regulation of the Leukemia Inhibitory Factor-STAT3 Pathway

“…However, there appears to be cross-talk between Nanog and the LIF-STAT3 pathway to promote maximal self-renewal efficiency of mESCs (32,33). In addition, STAT3 activated by LIF regenerates pluripotent mESCs from early mesoderm-specified progenitor cells via up-regulation of Nanog expression (34). These findings suggest that such intrinsic and extrinsic factors responsible for mESC pluripotency could act cooperatively in vivo.…”

The Pluripotency Factor NANOG Binds to GLI Proteins and Represses Hedgehog-mediated Transcription

“…We show that Gli1 is expressed at high levels in ES cells along with Nanog. Interestingly, previous studies have shown that NANOG also binds to and inhibits the transcriptional effectors of both the BMP and NF-B pathways in ES cells (18,19). Collectively, these results suggest that, by binding to multiple transcriptional effectors, NANOG may help to buffer ES cells from external signals.…”

“…In this study, NANOG was found to bind to SMAD1 and inhibit BMP-mediated responses that would normally drive ES cells to differentiate (19). NANOG has also been shown to prevent NF-B-induced differentiation by binding to NF-B family transcription factors (18).…”

Critical Roles of Coactivator p300 in Mouse Embryonic Stem Cell Differentiation and Nanog Expression

“…However, the inability of Nanog overexpression to rescue the aberrant expression of other lineage markers implies that factors other than Nanog may mediate p300 deficiency-dependent phenotypes, which needs further analysis. In addition, we discovered that Nanog expression is under direct regulation of p300, which adds another factor to the list of the known regulators of Nanog expression, including Oct4 and Sox2 (8,29), p53 (10), Tcf3 (9), and T and Stat3 (11,30).…”

“…In contrast, p53 and Tcf3 were reported to suppress Nanog expression in mouse ES cells, respectively (9,10), suggesting that the steady-state level of Nanog expression in undifferentiated ES cells is controlled by the balance between its activators and repressors. In addition, regulation of Nanog expression by T (Brachyury) and Stat3 was investigated during an early stage of ES cell specification toward mesoderm lineage (11). The finding suggests that strict control of Nanog expression exists in differentiating as well as undifferentiated ES cells.…”