Network plasticity of pluripotency transcription factors in embryonic stem cells

Abstract

Transcription factor (TF) networks are thought to regulate embryonic stem cell (ESC) pluripotency. However, TF expression dynamics and regulatory mechanisms are poorly understood. We use reporter mouse ESC lines allowing non-invasive quantification of Nanog or Oct4 protein levels and continuous long-term single-cell tracking and quantification over many generations to reveal diverse TF protein expression dynamics. For cells with low Nanog expression, we identified two distinct colony types: one re-expressed Nanog in a mosaic pattern, and the other did not re-express Nanog over many generations. Although both expressed pluripotency markers, they exhibited differences in their TF protein correlation networks and differentiation propensities. Sister cell analysis revealed that differences in Nanog levels are not necessarily accompanied by differences in the expression of other pluripotency factors. Thus, regulatory interactions of pluripotency TFs are less stringently implemented in individual self-renewing ESCs than assumed at present.

Dissecting mechanisms of mouse embryonic stem cells heterogeneity through a model-based analysis of transcription factor dynamics

“…In accordance with fluorescence measurements, quantitative measurements of Nanog mRNA molecules in single mESCs also show a broad and bimodal distribution of Nanog transcripts [15,16]. Most remarkable, mESCs possess the dynamic capacity to change between different Nanog expression states as demonstrated first by cell sorting experiments [5,13], and very recently also by live cell image analyses [15,17]. Although mESCs with low Nanog expression can sustain pluripotency and re-express Nanog, they possess a high propensity for differentiation [5,12,16,17].…”

Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation

“…In contrast to reduced NANOG expression, which is a reversible state (Chambers et al , 2007; Abranches et al , 2014; Filipczyk et al , 2015), loss of ESRRB commits cells to exit the naïve state. This can be clearly seen in clonal self‐renewal assays where Esrrb negative cells are essentially unable to form colonies in LIF.…”

“…Therefore, the NANOG low compartment is composed of two subpopulations, which differ in ESRRB expression and self‐renewal ability. Cell‐tracking experiments have also identified two distinct subpopulations of ESCs that have downregulated NANOG but differ in cell fate and differentiation potential (Filipczyk et al , 2015; Hastreiter et al , 2018). Our results extend these findings by showing that ESRRB can distinguish these subpopulations.…”

Nanog dynamics in single embryonic stem cells

“…4 Manipulations of Nanog levels showed its importance as a pluripotency factor, 6 but a tight deterministic regulation within the PGRN -at least in self-renewal conditions -and its use as a cell fate predictor are questionable. In contrast, our data agree with the notion that pluripotency is represented by an attractor basin in which ESCs are moving while cells that stay long-term low for Nanog are in a different attractor state with reduced developmental potential.…”

“…3 We therefore quantified Nanog protein expression dynamics in individual mouse ESCs over many generations. 4 Transitions from negative to high Nanog expression typically take longer than one cell generation, do not happen as part of oscillations and occur independently of the cell cycle phase. Nanog fluctuations are very heterogeneous between individual cells.…”