A negative feedback loop of transcription factors that controls stem cell pluripotency and self‐renewal

Abstract

Embryonic stem (ES) cells possess the ability to renew themselves while maintaining the capacity to differentiate into virtually all cell types of the body. Current evidence suggests that ES cells maintain their pluripotent state by expressing a battery of transcription factors including Oct4 and Nanog. However, little is known about how ES cells maintain the expression of these pluripotent factors in ES cells. Here we present evidence that Oct4, Nanog, and FoxD3 form a negative feedback loop to maintain their expression in pluripotent ES cells. First, Oct4 maintains Nanog activity by directly activating its promoter at sub-steady-state concentration but repressing it at or above steady-state levels. On the other hand, FoxD3 behaves as a positive activator of Nanog to counter the repressive effect of Oct4. The expression of Oct4 is activated by FoxD3 and Nanog but repressed by Oct4 itself, thus, exerting an important negative feedback loop to limit its own activity. Indeed, overexpression of either FoxD3 or Nanog in ES cells failed to increase the concentration of Oct4 beyond the steady-state concentration, whereas knocking down either FoxD3 or Nanog reduces the expression of Oct4 in ES cells. Finally, overexpression of Oct4 or Nanog failed to compensate the loss of Nanog or Oct4, respectively, suggesting that both are required for ES self-renewal and pluripotency. Our results suggest the FoxD3-Nanog-Oct4 loop anchors an interdependent network of transcription factors that regulate stem cell pluripotency.

Aromatic Residues in the C-terminal Domain 2 Are Required for Nanog to Mediate LIF-independent Self-renewal of Mouse Embryonic Stem Cells

“…P19 cells and F9 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 15% fetal bovine serum (Hyclone, UT) and antibiotics (penicillin and streptomycin, 100 g/ml) (3,10). Mouse ES cells (CGR8 ES) were cultured on 0.1% gelatincoated substrates and cultured in Glasgow minimum essential medium (Sigma) supplemented with 20% fetal bovine serum (Invitrogen), 100 mM nonessential amino acids (Invitrogen), 0.55 mM mercaptoethanol (Sigma), 2 mM L-glutamine (Invitrogen), and 1,000 units/ml human recombinant LIF (Chemicon) as described (3,5,6,10).…”

“…The pluripotency of mouse ES cells appears to be governed by a network of transcription factors including Oct4, Sox2, FoxD3, and Nanog (1,(3)(4)(5)(6). Although volumes of data have been generated through large scale biological tools such as microarrays and proteomics about these core regulators of stem cell pluripotency (4,(7)(8)(9), little is known about the molecular mechanisms that govern their mechanism of action.…”

Regulation of the Pluripotency Marker Rex-1 by Nanog and Sox2

“…As shown in Fig. 3C, the luciferase activities driven by 286-mut-1, 286-mut-2, 286-mut-3, and 286-mut-4 mutant promoter sequences were no longer responsive to Nanog (lower portion, lanes [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], whereas Rexp286 was responsive to Nanog in a dose-dependent fashion (lanes 1-4). , lanes 1, 5, 9, 13, and 17; 0.25 g, lanes 2, 6, 10, 14, and 18; 0.5 g, lanes 3, 7, 11, 15, and 19; and 0.75 g, lanes 4, 8, 12, 16, and 20) into P19 cells.…”

“…Interestingly, Nanog is regulated by an adjacent pair of highly conserved Octamer-and Sox-binding sites through an interaction between Oct-3/4 and Sox2 (13). Our recent work has demonstrated that transcription factors Oct-3/4, Nanog, Sox2, and FoxD3 anchor a negative feedback loop to maintain the expression of pluripotent factors at a steady state (14). In this report, we present evidence that Rex-1 is regulated by Nanog, Oct-3/4, and Sox2 at the transcription level.…”

Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

“…Youth) is a key transcription factor regulating pluripotency in mammalian early embryos and pluripotent stem cells. Cooperating with other master regulators of pluripotency, NANOG plays a central role in pluripotency (1)(2)(3) and forms autoregulatory loops to maintain ES cell (ESC) identity (4)(5)(6)(7). NANOG was initially identified from its ability to confer mouse (m)ESC selfrenewal without dependence on leukemia inhibitory factor (LIF) when overexpressed in mESCs (8,9).…”