NANOG is a homeodomain bearing protein that is transcribed specifically in pluripotent cells in ESCs. Oct4 and Sox2 are the major transcription factors that bind to the NANOG promoter to promote its transcription. FoxD3 transcription factor also activates the NANOG promoter. NANOG is also a direct downstream effector of the LIF-STAT3 pathway in maintaining ESC pluripotency. Besides, the BMP 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 Brachyury and prevent neural-ectoderm differentiation. In the presence of LIF, STAT3 activated by JAK, which is recruited by LIFR-GP130 heterodimer on binding LIF, interacts with Brachyury and binds the NANOG promoter resulting in upregulation of NANOG expression. BMPs mediate signaling through their downstream effectors - SMAD1, SMAD5 and SMAD8. NANOG physically interacts with SMAD1 and interferes with further recruitment of the coactivators to the active SMAD1 complexes thus inhibiting the activity of BMP signaling and limiting mesoderm progression. Ctnn-β, a key regulator of the WNT pathway, also upregulates NANOG. Ctnn-β stability is negatively regulated through its phosphorylation by GSK3β, APC and AXIN complex. Extracellular WNT proteins activate the canonical WNT signaling pathway by binding the Fz receptor and stabilizing intracellular Ctnn-β, which then translocates to the nucleus where it interacts with TCF/LEF transcription factors to regulate expression of target genes. Ctnn-β is also stabilized by the LIF pathway. LIF stimulation leads to inactivation of GSK3β through the PI3K/Akt pathway, thus stabilizing Ctnn-β. Ctnn-β is involved in upregulation of NANOG through binding with Oct3/4 protein in the nucleus. Oct4 binds to PSBP leading to NANOG gene expression. SalL4 interacts with NANOG and exists as a complex with it. NANOG and SalL4 co-target many genomic sites. They also bind to and regulate the respective regulatory regions of their own genes.Once expressed NANOG blocks differentiation. p53 binds to the NANOG promoter and acts as negative regulator of NANOG. TCF3 also binds to a regulatory region on the NANOG promoter and repress promoter activity in ESCs. Signaling molecules involved in NANOG repression by primitive endoderm specification include GRB2/SOS/Ras and ERK. The canonical ERK cascade is stimulated upon the binding of extracellular growth factors to their respective transmembrane RTKs. The subsequent auto phosphorylation of the cytoplasmic tails of the receptor leads to the tyrosine phosphorylation of the adaptor protein SHC. SHC then recruits the GRB2-SOS complex to the membrane. SOS exchanges the GDP bound to Ras for GTP. Once Ras binds GTP, it activates ERK1/2 via Raf and MEK1/2. ERK1/2 then inhibits NANOG expression.
NANOG also regulates the expression of REX1/ZFP42 by binding to its promoter. Sox2 cooperates with NANOG in regulating REX1 activity. NANOG directly repress GATA6, which results in repression of GATA4, thereby inhibiting ESC differentiation. NANOG also regulates the expression of Oct4 and Sox2 genes. It 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 targets in mouse include ESRRB and RIF1. Other genes regulated by NANOG include Tbx3 and TCL1. Oct4, Sox2 and NANOG also repress Cdx2 expression which is necessary for trophectoderm development. In human ESCs, about 352 genes are bound by Oct4, NANOG and Sox2 simultaneously which may be expressed or repressed. Identification of NANOG is a crucial step in understanding early embryogenesis and in exploiting pluripotent cells for therapeutic goals.