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Nanog in Mammalian ESC Pluripotency
ESCs (embryonic stem cells) are pluripotent cells derived from the ICM (inner cell mass) of blastocyst-stage embryos. These cells have two distinctive properties: an unlimited capacity for self-renewal and pluripotency. The capability for self-renewal and the pluripotency of ESCs seem to be under the control of multiple transcriptional factors, most common among them being Nanog (Nanog homeobox), Oct4 (Octamer Binding Transcription Factor-4), and SOX2 [SRY (Sex Determining Region-Y) Box-2]. Functions of these transcription factors depend on the stage of development of a pluripotent cell, indicating that these factors function in combination with other processes. The activity of these transcription factors also depends on the accessibility of their target genes, which are made accessible by the modification of their DNA, histones, or chromatin structure. These transcription factors activate or repress patterns of gene expression that mediate phenotypic changes during stem cell differentiation [1,2].
The transcription factor Nanog is a homeodomain-bearing protein that is transcribed specifically in pluripotent cells in mouse preimplantation embryos, ESCs, and EGCs (embryonic germ cells), and monkey and human ESCs. Nanog protein consists of three domains with the homeodomain separating the N-terminal serine-rich 96 amino acids from the C-terminal 150 amino acids. Both N- and C-terminal domains in mouse Nanog show trans-activator function, but the activity of the C-terminal domain is much higher than the N-terminal domain. In human Nanog, transactivation potential is retained in the C-terminal domain but not in the N-terminal domain. The prominent feature of the C-terminal domain is the presence of a 10 pentapeptide repeat (WR), each starting with a tryptophan (W). This repeat is well conserved between humans and mice, although one of the tryptophans is replaced by a glutamine (Q) in human Nanog. In addition to WR, the C-terminal domain also contains another trans-activator module, the 58 amino acids region C-terminal to the WR (CD2). Both WR and CD2 can separately transactivate the expression of a reporter gene [3,4].
Nanog expression is restricted to pluripotent cells and is downregulated upon differentiation, but little is known about how Nanog expression is regulated. Nanog gene is transcribed under the control of a regulatory region that lies within 332 bp upstream of the transcriptional start site. This region contains Octamer and SOX elements, which are highly conserved among the 5’-flanking regions of the mouse, monkey, and human Nanog genes. Oct4 and SOX2 are the major transcription factors that bind to the Nanog promoter in vitro and in vivo to promote Nanog transcription. However, high levels of Nanog are beneficial to ES cell self-renewal, but overexpressed Oct4 induces differentiation. Other pluripotent factors also contribute to the regulation of Nanog expression. FoxD3 (Forkhead Box Protein-D3), a Forkhead family transcription factor, is highly expressed in mouse ES cells and in pluripotent cells of the early embryo. FoxD3 activates the Nanog promoter through an ESC-specific enhancer localized at –270 upstream of the transcription start site. Nanog is also a direct downstream effector of the LIF (Leukemia Inhibitory Factor)-STAT3 (Signal Transducer and Activator of Transcription-3) pathway in maintaining ESC pluripotency. Besides, the BMP (Bone Morphogenetic Protein) pathway also regulates Nanog expression. In the absence of LIF, low concentrations of BMPs promote mesoderm differentiation of mouse ESCs with upregulation of the mesoderm marker-T (Brachyury) and prevent the neural-ectoderm differentiation. In the presence of LIF, STAT3 activated by JAK (Janus tyrosine Kinase), which is recruited by LIFR–GP130 (GP130 Transducer Chain) heterodimer on binding LIF, interacts with Brachyury and binds the Nanog promoter, resulting in upregulation of Nanog expression. BMPs belong to the TGF-Beta (Transforming Growth Factor-Beta) Superfamily and mediate signaling through their downstream effectors – SMAD1 [Sma and MAD (Mothers Against Decapentaplegic) Related Protein-1] or the closely related SMAD5 [Sma and MAD (Mothers Against Decapentaplegic) Related Protein-5] and SMAD8 (Sma and MAD (Mothers Against Decapentaplegic) Related Protein-8). Nanog can physically interact with SMAD1 and interfere with the further recruitment of the coactivators to the active SMAD1 complexes, thus inhibiting the activity of BMP signaling and limiting mesoderm progression and ultimately maintaining the undifferentiated state of Mouse ESCs (Ref.5 & 6). Ctnn-Beta (Catenin-Beta), a key regulator of WNT (Wingless-Type MMTV Integration Site Family Member) Pathway, also upregulates Nanog. In general, Ctnn-Beta stability is negatively regulated through its phosphorylation by GSK3Beta (Glycogen Synthase Kinase-3-Beta), APC (Adenomatous Polyposis Coli), and AXIN (Axis Inhibitor) complex. Extracellular WNT proteins activate the canonical WNT signaling pathway by binding the Fz (Frizzled) seven transmembrane span receptor and stabilizing intracellular Ctnn-Beta, which then translocates to the nucleus, where it interacts with TCF (T-cell Factor)/ LEF (Lymphocyte Enhancer Factor) transcription factors to regulate expression of target genes. Ctnn-Beta can also be stabilized by LIF Pathway. LIF stimulation leads to inactivation of GSK3Beta through the PI3K (Phosphoinositide 3-Kinase)/Akt pathway, thus stabilizing Ctnn-Beta. Ctnn-Beta is involved in upregulation of Nanog through binding with Oct3/4 protein in nucleus. Oct4 can also bind to a novel PSBP (pluripotential cell-specific SOX element-binding protein) leading to Nanog gene expression. Oct4 dominantly bound to the Octamer element, while PSBP preferentially bound to the SOX element. SalL4 [Sal-Like-4 (Drosophila)], a member of Spalt-like protein family, interacts with Nanog and exists as a complex with Nanog. The SalL4 and Nanog complex resembles the network configuration for the Oct4 and SOX2 complex. Nanog and SalL4 co-target many genomic sites. SalL4 and Nanog also bind to and regulate the respective regulatory regions of their own genes [1,7-9].
Once expressed, Nanog blocks differentiation. Thus, negative regulation of Nanog is required to promote differentiation during embryonic development. The tumor suppressor protein p53 binds to the Nanog promoter and acts as a negative regulator of Nanog during ESC differentiation. TCF3 (Transcription Factor-3), a transcription factor that functions downstream of the WNT pathway, binds to a regulatory region on the Nanog promoter and represses the promoter activity in ESCs. Several signaling molecules are also involved in Nanog repression by primitive endoderm specification. Major signaling pathway involved in primitive endoderm differentiation is the GRB2 (Growth Factor Receptor-Bound Protein-2)/SOS (Son of Sevenless)/Ras/ERK (Extracellular Signal-Regulated Kinase) pathway. The canonical ERK cascade is stimulated upon the binding of extracellular growth factors to their respective transmembrane RTKs (receptor tyrosine kinases). The subsequent auto-phosphorylation of the cytoplasmic tails of the receptor on tyrosine leads to the tyrosine phosphorylation of the adapter protein SHC. SHC can then recruit the GRB2-SOS complex to the membrane via the SH2 domain of GRB2, binding to the phosphotyrosine on SHC. SOS, a GEF for Ras, can then exchange the GDP bound to Ras to GTP. Once Ras binds GTP, it can then recruit the serine/threonine kinase Raf to the membrane. When Raf translocates to the membrane, it becomes activated and then phosphorylates the dual-specificity kinases MEK1 (MAPK/ERK Kinase-1) and MEK2 (MAPK/ERK Kinase-1). MEKs (MAPK/ERK Kinases) then phosphorylate ERK1/2, which then inhibit Nanog expression [10-12].
The mechanism through which Nanog regulates stem cell pluripotency is not known properly. Nanog may assume a bifunctional role to repress those genes important for differentiation and activate the ones necessary for self-renewal. Nanog can regulate the expression of REX1/ZFP42 (Zinc Finger Protein-42) by binding to its promoter. This regulation is likely mediated by the C-terminal transactivator of Nanog. SOX2, not Oct3/4, cooperates with Nanog in regulating REX1 activity. GATA4 (GATA Binding Protein-4) and GATA6 (GATA Binding Protein-6) are endoderm transcription factors are also regulated by Nanog. Nanog directly represses GATA6, which results in repression of GATA4, thereby inhibiting ESC differentiation. Nanog also regulates the expression of Oct4 and SOX2 genes, which play important roles in pluripotency. Nanog also activates functions in concert with other factors, such as Oct4 and SOX2, to establish ESC identity. These factors regulate a core transcriptional circuit for ESC self-renewal. The major downstream target in mouse includes ESRRB (Estrogen Related Receptor-Beta) and RIF1 (Rap1 Interacting Factor-1 homolog (yeast)). ESRRB belongs to the superfamily of nuclear hormone receptors and is necessary to block the differentiation into mesoderm, ectoderm, and neural crest cells but is not required to repress trophectoderm differentiation. RIF1 is an ortholog of a yeast telomeric protein and is upregulated in mouse ES and germ cells. Other genes regulated by Nanog include Tbx3 (T-box 3) and TCL1 (T-Cell Lymphoma breakpoint-1). The function of TCL1 is to repress only a subset of neural crest genes, whereas Tbx3 blocks the mesoderm and ectoderm differentiation. Downregulation of Nanog, SOX2, ESRRB, Tbx3 or TCL1 leads to the immediate induction of Otx2 [Orthodenticle homolog 2 (Drosophila)], Pitx2 (Paired-like homeodomain Transcription factor-2), Sox18 [SRY (Sex determining region Y)-box 18] and probably additional genes. All three genes are expressed in the epiblast. Oct4, SOX2 and Nanog also repress Caudal-type homeodomain transcription factor Cdx2 (Caudal type homeobox-2) expression. Cdx2 is necessary for trophectoderm development. In undifferentiated human ES cells, about 352 genes are bound by Oct4, Nanog and SOX2 simultaneously, which may be expressed or repressed [13-15]. The ability of Nanog to robustly maintain ESC identity when overexpressed, without the normally obligatory LIF and BMP/GDF growth differentiation factor) signals, places Nanog firmly at the hub of the circuitry responsible for the properties of self-renewal and pluripotency. The mechanisms of Nanog function and regulation within these circuits are at present poorly perceived. However, as genes acting both upstream and downstream of Nanog are uncovered, and protein partners of Nanog are identified, a clearer vision is emerging of how Nanog functions. Identification of Nanog is a crucial step in understanding early embryogenesis and in exploiting pluripotent cells for therapeutic goals [1,16].
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