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Profilin2 (PFN2) is a target of the embryonic stem cell (ESC)-enriched miR-290 family of microRNAs (miRNAs) and an actin/dynamin-binding protein implicated in endocytosis. Here we show that the miR-290-PFN2 pathway regulates many aspects of ESC biology. In the absence of miRNAs, PFN2 is up-regulated in ESCs, with a resulting decrease in endocytosis. Reintroduction of miR-290, knockout of Pfn2 , or disruption of the PFN2-dynamin interaction domain in miRNA-deficient cells reverses the endocytosis defect. The reduced endocytosis is associated with impaired extracellular signal-regulated kinase (ERK) signaling, delayed ESC cell cycle progression, and repressed ESC differentiation. Mutagenesis of the single canonical conserved 3' UTR miR-290-binding site of Pfn2 or overexpression of the Pfn2 open reading frame alone in otherwise wild-type cells largely recapitulates these phenotypes. Taken together, these findings define an axis of posttranscriptional control, endocytosis, and signal transduction that is important for ESC proliferation and differentiation., Competing Interests: The authors declare no competing interest.

Pluripotent stem cells give rise to all cells of the adult organism, making them an invaluable tool in regenerative medicine. In response to differentiation cues, they can activate markedly distinct lineage-specific gene networks while turning off or rewiring pluripotency networks. Recent innovations in chromatin and nuclear structure analyses combined with classical genetics have led to novel insights into the transcriptional and epigenetic mechanisms underlying these networks. Here, we review these findings in relation to their impact on the maintenance of and exit from pluripotency and highlight the many factors that drive these processes, including histone modifying enzymes, DNA methylation and demethylation, nucleosome remodeling complexes and transcription factor-mediated enhancer switching., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The enhancer landscape of pluripotent stem cells undergoes extensive reorganization during early mammalian development. The functions and mechanisms behind such reorganization, however, are unclear. Here, we show that the transcription factor GRHL2 is necessary and sufficient to activate an epithelial subset of enhancers as naive embryonic stem cells (ESCs) transition into formative epiblast-like cells (EpiLCs). Surprisingly, many GRHL2 target genes do not change in expression during the ESC-EpiLC transition. Instead, enhancers regulating these genes in ESCs diminish in activity in EpiLCs while GRHL2-dependent alternative enhancers become activated to maintain transcription. GRHL2 therefore assumes control over a subset of the naive network via enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency. These data evoke a model where the naive pluripotency network becomes partitioned into smaller, independent networks regulated by EpiLC-specific transcription factors, thereby priming cells for lineage specification., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Metabolites and cofactors are emerging as key regulators of cell plasticity and reprogramming, and their role in the control of pluripotency is just being discovered. Here we provide unprecedented evidence that embryonic stem cell (ESC) pluripotency relies on the relative levels of two physiological metabolites, namely ascorbic acid (vitamin C, VitC) and l-proline (l-Pro), which affect global DNA methylation, transcriptional profile, and energy metabolism. Specifically, while a high VitC/l-Pro ratio drives ESCs toward a naive state, the opposite condition (l-Pro excess) captures a fully reversible early primed pluripotent state, which depends on autocrine fibroblast growth factor and transforming growth factor β signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Early development is governed by the ability of pluripotent cells to retain the full range of developmental potential and respond accurately to developmental cues. This property is achieved in large part by the temporal and contextual regulation of gene expression by enhancers. Here, we evaluated regulation of enhancer activity during differentiation of embryonic stem to epiblast cells and uncovered the forkhead transcription factor FOXD3 as a major regulator of the developmental potential of both pluripotent states. FOXD3 bound to distinct sites in the two cell types priming enhancers through a dual-functional mechanism. It recruited the SWI/SNF chromatin remodeling complex ATPase BRG1 to promote nucleosome removal while concurrently inhibiting maximal activation of the same enhancers by recruiting histone deacetylases1/2. Thus, FOXD3 prepares cognate genes for future maximal expression by establishing and simultaneously repressing enhancer activity. Through switching of target sites, FOXD3 modulates the developmental potential of pluripotent cells as they differentiate., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Establishment, maintenance, and exit from pluripotency require precise coordination of a cell's molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Individual microRNAs (miRNAs) can target hundreds of mRNAs forming networks of presumably cooperating genes. To test this presumption, we functionally screened miRNAs and their targets in the context of dedifferentiation of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Along with the miR-302-miR-294 family, the miR-181 family arose as a previously unidentified enhancer of the initiation phase of reprogramming. Endogenous miR-181 miRNAs were transiently elevated with the introduction of Pou5f1 (also known as Oct4), Sox2 and Klf4 (referred to as OSK), and miR-181 inhibition diminished iPSC colony formation. We tested the functional contribution of 114 individual targets of the two families, revealing 25 genes that normally suppress initiation. Coinhibition of targets cooperatively promoted both the frequency and kinetics of OSK-induced reprogramming. These data establish two of the largest functionally defined networks of miRNA-mRNA interactions and reveal previously unidentified relationships among genes that act together to suppress early stages of reprogramming.

In the past decade, significant progress has been made in understanding both microRNA function and cellular pluripotency. Here we review the intersection of these two exciting fields. While microRNAs are not required for the establishment and maintenance of pluripotency in early development and cell culture, respectively, they are critically important in the regulation of the cell cycle structure of pluripotent stem cells as well as the silencing of the pluripotency program upon differentiation. Pluripotent cells, both in vivo and in vitro, dominantly express a single family of microRNAs, which can promote the reprogramming of a somatic cell back to a pluripotent state. Here, we review the known mechanisms by which these and other microRNAs regulate the different aspects of the pluripotent stem cell program in both mouse and human.

Stem cell differentiation requires a complex coordination of events to transition from a self-renewing to a differentiated cell fate. Stem cells can be pluripotent (capable of giving rise to all embryonic lineages), multipotent (possessing the potential to give rise to multiple lineages) and unipotent (capable of given rise to a single cell lineage). Regardless of their potency all stem cells must silence their self-renewal program during differentiation. The self-renewal program can be defined as the integration of external and internal stimuli that enables a cell to proliferate while maintaining its potency. Two hallmarks of the self-renewal program are a self-reinforcing transcriptional network and a specialized cell-cycle profile. In this chapter we discuss the impact of various microRNAs (miRNAs) to either reinforce or inhibit the self-renewal program of stem cells and how this added regulatory layer provides robustness to cell-fate decisions. We will focus on embryonic stem cells (ESCs) describing miRNA function in self-renewal, differentiation and de-differentiation. We will compare and contrast miRNA functions in ESCs with miRNA function in lineage specific somatic stem cells and in cancer.