FoxO transcription factors actuate the formative pluripotency specific gene expression programme

Background:  Mouse embryo at the blastocyst stage consists of an outer layer of trophectoderm and an inner mass of pluripotent cells. These pluripotent cells have two core properties – the ability to self-renew and the potential to give rise to all the cell types in the body. What makes these cells even more unique is that as they self-renew, they also undergo many changes in their gene expression with time.  Therefore, rather than a fixed cell state, pluripotency is actually a continuum of states[1]. The stringent profiling of transcriptome, epigenome, and developmental potential has segregated these states into naïve, formative, and primed states of pluripotency [2, 3]. From a developmental perspective, it is important to understand the mechanism behind how a cell transitions from one state to the next over time. Santini et al. focused on the transition from a naïve to a formative pluripotent state wherein cells become competent for lineage specification. This is the time window for extensive remodeling of gene regulatory networks (GRNs); however, what exactly drives this remodeling has not been addressed before.

 

Key findings of the study:

 

AKT regulates cytoplasm to nuclear shuttling of FOXO transcription factors.

The PI3/AKT signaling cascade takes center stage in this preprint. Overexpression of constitutively active AKT arrests mouse embryonic stem cells in an undifferentiated state[4]. This study builds on the established finding that PTEN, a negative regulator of AKT, facilitates the exit from naïve pluripotency. A genetic screen identified three AKT targets necessary to exit the naïve state: TSC2, GSK3, and FOXO, to be necessary for exit from the naïve state [5]. In this study, the authors established that the AKT/FOXO axis indeed facilitates the exit of mouse pluripotent stem cells from the naïve state. They could show that FOXO1 localizes to the nucleus at E4.75-E5.5 in vivo and upon withdrawal of naïve culture conditions in vitro. Mechanistically, AKT-mediated phosphorylation retains FOXO transcription factors (TFs) in the cytoplasm. During the transition to the formative state, PTEN-mediated negative regulation of AKT abolishes cytoplasmic retention of FOXO transcription factors, facilitating their nuclear translocation. Additionally, the knockdown of FoxO transcription factors delayed the exit from naïve pluripotency, whereas complete deletion of FoxO compromised the self-renewal of mESCs

 

FOXO transcription factors enforce formative-state gene regulatory networks.

The authors delved deep into the function of FOXO transcription factors in embryonic stem cells. CHIP-sequencing analysis for FOXO1 and FOXO3 in naïve and formative state culture conditions showed an increase in genome occupancy by FOXO TFs 24 hours into the formative transition. The approach they adopted to analyze FOXO TF chromatin binding in various contexts led to several findings.

• Interestingly, FOXO TF peaks were located outside the promoter region and chromatin accessibility did not differ between wild-type and Pten-null cell lines, eliminating the possibility of FOXO transcription factors influencing the opening of chromatin.
• The FOXO peaks could be segregated into three categories- “naïve-only,” “formative-only” and “shared” peaks. The FOXO peaks overlapped with the bona fide “ESC-enhancer” and “EpiLCs-enhancer” peaks. The intersection of these peaks showed that FOXO1 “formative-only” peaks overlapped more with “EpiLCs-enhancers” than with the “ESC-enhancers,” suggesting that FOXO TFs play a role in activating formative-specific transcriptional programs via a set of EpiLC-specific enhancers.
• To assess the functional overlap between FOXO TFs and other state-specific pluripotency factors, the authors overlapped FOXO peaks with (i) OCT4, (ii) OTX2, (iii) ESSRB, and (iv) β-catenin peaks, which showed that FOXO TFs suppress naïve GRN and concomitantly enforce formative GRN by cooperating with major pluripotency TFs during the naïve to formative state transition.

 

What I like about the preprint: The mechanism-centric papers for the role of PI3/AKT signaling in pluripotent stem cells  have been sparse. What I find particularly impressive is how this study uncovers the previously underappreciated role of PI3/AKT in regulating the localization of FOXO transcription factors. The regulation of nuclear and cytoplasmic pools of FOXO transcription factors during pluripotent state transitions is an efficient and robust way to regulate developmental progression. This work is refreshing in terms of both concept and molecular mechanism. The experimental and analytical approaches used in this study led to balanced conclusions and this is an elegant model of state transitions in the mouse epiblast.

 References:

 

1. Pera, M.F. and J. Rossant, The exploration of pluripotency space: Charting cell state transitions in peri-implantation development. Cell Stem Cell, 2021. 28(11): p. 1896-1906.
2. Nichols, J. and A. Smith, Naive and primed pluripotent states. Cell stem cell, 2009. 4(6): p. 487-492.
3. Smith, A., Formative pluripotency: the executive phase in a developmental continuum. Development, 2017. 144(3): p. 365-373.
4. Watanabe, S., et al., Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells. Oncogene, 2006. 25(19): p. 2697-2707.
5. Lackner, A., et al., Cooperative genetic networks drive embryonic stem cell transition from naive to formative pluripotency. The EMBO journal, 2021. 40(8): p. e105776.

 

 

doi: https://doi.org/10.1242/prelights.36654

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