Nanog, Oct4 and Tet1 interplay in establishing pluripotency

Abstract

A few central transcription factors inside mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are believed to control the cells' pluripotency. Characterizations of pluripotent state were put forward on both transcription factor and epigenetic levels. Whereas core players have been identified, it is desirable to map out gene regulatory networks which govern the reprogramming of somatic cells as well as the early developmental decisions. Here we propose a multiple level model where the regulatory network of Oct4, Nanog and Tet1 includes positive feedback loops involving DNA-demethylation around the promoters of Oct4 and Tet1. We put forward a mechanistic understanding of the regulatory dynamics which account for i) Oct4 overexpression is sufficient to induce pluripotency in somatic cell types expressing the other Yamanaka reprogramming factors endogenously; ii) Tet1 can replace Oct4 in reprogramming cocktail; iii) Nanog is not necessary for reprogramming however its over-expression leads to enhanced self-renewal; iv) DNA methylation is the key to the regulation of pluripotency genes; v) Lif withdrawal leads to loss of pluripotency. Overall, our paper proposes a novel framework combining transcription regulation with DNA methylation modifications which, takes into account the multi-layer nature of regulatory mechanisms governing pluripotency acquisition through reprogramming.

Figure 1. The transcription factors circuit.

The gene network for the mutual and self-regulatory interactions between Nanog, Oct4 and Tet1. The curly lines indicate that Nanog and Tet1 interact at protein level forming a complex. The arrows indicate positive DNA regulation. The dotted arrow depicts assumed Tet1 self-interaction. The model shown in Methods is based on: Nanog and Tet1 complex regulates Oct4 and Tet1, Oct4 regulates Nanog, Oct4 and Tet1. All the gene interactions in the network are positive.

Figure 2. Time series of Nanog, Oct4 and Tet1 for the dynamics of the gene regulatory networks under reprogramming.

Time series of Oct4 - green, Nanog -blue and Tet1 - black for the three reprogramming scenarios: i- exogenous Oct4 present and then removed, followed by ii- exogenous Nanog expressed and then removed and iii- Tet1 over expressed and then the exogenous Tet1 is turned off. When cells are in a non-pluripotent state we use - white background, when Oct4 is over-expressed - green background, when cells are pluripotent - red background, when Nanog is over-expressed - blue background, and when Tet1 is over-expressed-grey background.

Figure 3. Time series of Nanog, Oct4 and Tet1 for various reprogramming scenarios and differentiation.

(a) Time series of the three factors when levels of factors over expression are varied: N_over = O_over = T_over = 0.1 -bottom plot, over-expression prameters values are 0.2 for middle plot and 0.3 for top plot. The yellow background corresponds to pre-iPS cells state. (b) Time series showing that addition of Nanog helps reprogramming when too low levels of exogenous Oct4 are induced. Nanog and Oct4 over-expression is depicted by cyan background. (c) Time series showing the differentiation process occurring under Lif withdrawal.

Figure 4. Methylation of promoter CpG sites.

(a) Methylation status of Oct4 CpG sites. Data from bisulfite genomic sequencing by Takahashi and Yamanaka for MEF and iPS cells (closed circles are methylated and open circles unmethylated). (b) Schematic of the methylation model. Methylated CpG sites m are recruiting methylases with a rate σ and unmethylated u are recruiting demethylases with a rate κ, here assumed to correspond to the level of the Nanog-Tet1 complex. μ and β are noise on CpG conversions and affects the simple conversions between m and u. Simulated CpG sites of the Oct4 promoter (16 CpG sites) for low (MEF) and high (iPS) expression of Nanog-Tet1.

Figure 5. Simulations of the pluripotency network including the methylation status of the promoter CpG sites.

Upper panel: Time series of Oct4 - green, Nanog -blue and Tet1 - black under the same conditions as for Fig. 2 including simulations of the promoter CpG sites of Oct4 and Tet1. Lower panel: The methylation status of the promoter CpG sites of Oct4 - green and Tet1 - black for each time step in the simulation of the network of the three reprogramming scenarios. As for Fig. 2, when cells are in the somatic state we use - white background, when Oct4 is over-expressed - green background, when cells are pluripotent - red background, when Nanog is over-expressed - blue background, and when Tet1 is over-expressed-grey background.