• BRG1
• Glycolysis
• H3K9me3
• PI3K/AKT signaling pathway
• Porcine iPSCs
• Transcriptional activity

• Animals
• Glycolysis/drug effects
• Glycolysis/genetics
• Cellular Reprogramming/genetics
• Induced Pluripotent Stem Cells/metabolism
• Induced Pluripotent Stem Cells/cytology
• Swine
• Proto-Oncogene Proteins c-akt/metabolism
• Signal Transduction
• Phosphatidylinositol 3-Kinases/metabolism
• Transcription Factors/metabolism
• Transcription Factors/genetics
• DNA Helicases/metabolism
• DNA Helicases/genetics
• Morpholines/pharmacology
• Chromones/pharmacology
• Nuclear Proteins/metabolism
• Nuclear Proteins/genetics
• Cells, Cultured

• 32060754 National Natural Science Funding
• 32460833 National Natural Science Funding
• 2020GXNSFDA297009 Guangxi Natural Science Funding
• 20220030 Project of Bama County for Talents in Science and Technology
• 2024AFB787 Natural Science Foundation of Hubei Province

• embryonic stem cells
• stem-cell differentiation
• growth factor signalling
• differentiation

• Animals
• Mice
• Pluripotent Stem Cells/metabolism
• Gene Regulatory Networks
• Cell Differentiation/genetics
• Proto-Oncogene Proteins c-akt/metabolism
• Proto-Oncogene Proteins c-akt/genetics
• PTEN Phosphohydrolase/metabolism
• PTEN Phosphohydrolase/genetics
• Forkhead Transcription Factors/metabolism
• Forkhead Transcription Factors/genetics
• Forkhead Box Protein O1/metabolism
• Forkhead Box Protein O1/genetics
• Phosphorylation
• Mouse Embryonic Stem Cells/metabolism
• Embryonic Stem Cells/metabolism
• Gene Expression Regulation, Developmental

• I 5958 Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung)
• DOC 72 Austrian Science Fund FWF
• W 1261 Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung)
• VRG14-006 Vienna Science and Technology Fund (Wiener Wissenschafts-, Forschungs- und Technologiefonds)
• DOC/25622 Österreichischen Akademie der Wissenschaften (Austrian Academy of Sciences)

• EGCs
• gatekeeper
• migratory primordial germ cells
• p38 MAPK
• pluripotency
• reprogramming
• teratoma

• bovine fetuses
• bovine papillomavirus
• ddPCR
• ovine papillomavirus
• transplacental coinfection

• Animals
• Female
• Cattle
• Pregnancy
• Papillomavirus Infections/veterinary
• Papillomavirus Infections/virology
• Coinfection/veterinary
• Coinfection/virology
• Cattle Diseases/virology
• Papillomaviridae/genetics
• Infectious Disease Transmission, Vertical/veterinary
• Fetus/virology
• Pregnancy Complications, Infectious/veterinary
• Pregnancy Complications, Infectious/virology
• Placenta/virology
• Placenta/pathology
• Sheep Diseases/virology
• Urinary Bladder Neoplasms/virology
• Urinary Bladder Neoplasms/veterinary

• Cytoophidium
• Embryonic development
• Enzyme polymerisation
• GTP biosynthesis
• IMPDH
• Pluripotent stem cells

• Animals
• Female
• Mice
• Cell Proliferation
• DNA Damage
• Fetal Development/genetics
• Guanosine Triphosphate/metabolism
• IMP Dehydrogenase/metabolism
• IMP Dehydrogenase/genetics
• Mice, Inbred C57BL
• Pluripotent Stem Cells/metabolism
• Pluripotent Stem Cells/cytology
• Cellular Structures/metabolism

• MC_UU_12021/3; MC_U137788471 Medical Research Council
• National Science and Technology Council
• National Natural Science Foundation of China

• ATP synthesis
• Aging
• Alpha-ketoglutarate
• DNA methylation
• Healthspan
• Lifespan
• mTOR

• Cancer cell reprogramming
• Cancer-iPSCs
• Induced pluripotent cancer cells
• Induced pluripotent stem cells

Exploiting the pluripotent properties of embryonic stem cells (ESCs) holds great promise for regenerative medicine. Nevertheless, directing ESC differentiation into specialized cell lineages requires intricate control governed by both intrinsic and extrinsic factors along with the actions of specific signaling networks. Here, we reveal the involvement of the p21-activated kinase 4 (Pak4), a serine/threonine kinase, in sustaining murine ESC (mESC) pluripotency. Pak4 is highly expressed in R1 ESC cells compared with embryonic fibroblast cells and its expression is progressively decreased during differentiation. Manipulations using knockdown and overexpression demonstrated a positive relationship between Pak4 expression and the clonogenic potential of mESCs. Moreover, ectopic Pak4 expression increases reprogramming efficiency of Oct4-Klf4-Sox2-Myc-induced pluripotent stem cells (iPSCs) whereas Pak4-knockdown iPSCs were largely incapable of generating teratomas containing mesodermal, ectodermal and endodermal tissues, indicative of a failure in differentiation. We further establish that Pak4 expression in mESCs is transcriptionally driven by the core pluripotency factor Nanog which recognizes specific binding motifs in the Pak4 proximal promoter region. In turn, the increased levels of Pak4 in mESCs fundamentally act as an upstream activator of the Akt pathway. Pak4 directly binds to and phosphorylates Akt at Ser473 with the resulting Akt activation shown to attenuate downstream GSK3β signaling. Thus, our findings indicate that the Nanog-Pak4-Akt signaling axis is essential for maintaining mESC self-renewal potential with further importance shown during somatic cell reprogramming where Pak4 appears indispensable for multi-lineage specification.

• Akt
• Nanog
• Pak4
• iPSCs
• mESCs
• pluripotency

ERas is a new gene of the Ras family found in murine embryonic stem (ES) cells. Its human ortholog is not expressed in human ES cells. So far ERas gene has only been found to be expressed in the tissues of adult cynomolgus monkeys and cattle; however, information about ERAS expression or its potential functions in equine tissues is lacking. This study was performed to investigate whether Eras is an equine functional gene and whether ERAS is expressed in the tissues of adult horses and determine its potential physiological role. Expression of the ERas gene was detected in all examined adult tissues, and the RT-PCR assay revealed ERAS transcripts. Protein expression was also detected by Western blot analysis. Quantitative real time RT-qPCR analysis revealed that different expression levels of ERAS transcripts were most highly expressed in the testis. Immunohistochemically, ERAS was found to be localized prevalently in the plasmatic membrane as well as cytoplasm of the cells. ERAS was a physical partner of activated PDGFβR leading to the AKT signaling. ERAS was found to interact with a network of proteins (BAG3, CHIP, Hsc70/Hsp70, HspB8, Synpo2, and p62) known to play a role in the chaperone-assisted selective autophagy (CASA), which is also known as BAG3-mediated selective macroautophagy, an adaptive mechanism to maintain cellular homeostasis. Furthermore, ERAS was found to interact with parkin. PINK1, BNIP3, laforin. All these proteins are known to play a role in parkin-dependent and -independent mitophagy. This is the first study demonstrating that Eras is a functional gene, and that ERAS is constitutively expressed in the tissues of adult horses. ERAS appears to play a physiological role in cellular proteostasis maintenance, thus mitigating the proteotoxicity of accumulated misfolded proteins and contributing to protection against disease. Finally, it is conceivable that activation of AKT pathway by PDGFRs promotes actin reorganization, directed cell movements, stimulation of cell growth.

• Endoplasmic reticulum
• Glycolysis
• Metabolism
• Mitochondria
• Pluripotent stem cells

The genes of the RAS family are among the group of genes most frequently mutated in human cancer. ERAS is a relatively unknown gene of this family. Although ERAS is overexpressed in some tumoral samples and in several cancer cell lines of human origin, it is not known if its expression drives tumor formation or if, alternatively, its expression is a secondary event in tumoral transformation. In this report, in order to clarify the role of ERAS in mammary tumorigenesis, we studied transgenic mice expressing ERAS in myoepithelial cells of mammary and other exocrine glands and in basal cells of stratified epithelia. These mice displayed an altered development and function of the mammary glands, and suffered high-frequency tumoral lesions in the mammary glands resembling a rare human breast tumor named malignant adenomyoepithelioma. Our results clearly demonstrate that ERAS is a true oncogene able to produce mammary tumors when inappropriately expressed.

ERAS is a relatively uncharacterized gene of the Ras superfamily. It is expressed in ES cells and in the first stages of embryonic development; later on, it is silenced in the majority of cell types and tissues. Although there are several reports showing ERAS expression in tumoral cell lines and human tumor samples, it is unknown if ERAS deregulated expression is enough to drive tumor development. In this report, we have generated transgenic mice expressing ERAS in myoepithelial basal cells of the mammary gland and in basal cells of stratified epithelia. In spite of the low level of ERAS expression, these transgenic mice showed phenotypic alterations resembling overgrowth syndromes caused by the activation of the AKT-PI3K pathway. In addition, their mammary glands present developmental and functional disabilities accompanied by morphological and biochemical alterations in the myoepithelial cells. These mice suffer from tumoral transformation in the mammary glands with high incidence. These mammary tumors resemble, both histologically and by the expression of differentiation markers, malignant adenomyoepitheliomas. In sum, our results highlight the importance of ERAS silencing in adult tissues and define a truly oncogenic role for ERAS in mammary gland cells when inappropriately expressed.

• ERAS
• Ras pathway
• adenomyoepithelioma
• breast cancer
• transgenic mice

Phosphoinositide-3 kinase (PI3K)/AKT signaling participates in cellular proliferation, survival and tumorigenesis. The activation of AKT signaling promotes the cellular reprogramming including generation of induced pluripotent stem cells (iPSCs) and dedifferentiation of primordial germ cells (PGCs). Previous studies suggested that AKT promotes reprogramming by activating proliferation and glycolysis. Here we report a line of evidence that supports the notion that AKT signaling is involved in TET-mediated DNA demethylation during iPSC induction.

AKT signaling was activated in mouse embryonic fibroblasts (MEFs) that were transduced with OCT4, SOX2 and KLF4. Multiomics analyses were conducted in this system to examine the effects of AKT activation on cells undergoing reprogramming.

We revealed that cells undergoing reprogramming with artificially activated AKT exhibit enhanced anabolic glucose metabolism and accordingly increased level of cytosolic α-ketoglutarate (αKG), which is an essential cofactor for the enzymatic activity of the 5-methylcytosine (5mC) dioxygenase TET. Additionally, the level of TET is upregulated. Consistent with the upregulation of αKG production and TET, we observed a genome-wide increase in 5-hydroxymethylcytosine (5hmC), which is an intermediate in DNA demethylation. Moreover, the DNA methylation level of ES-cell super-enhancers of pluripotency-related genes is significantly decreased, leading to the upregulation of associated genes. Finally, the transduction of TET and the administration of cell-permeable αKG to somatic cells synergistically enhance cell reprogramming by Yamanaka factors.

These results suggest the possibility that the activation of AKT during somatic cell reprogramming promotes epigenetic reprogramming through the hyperactivation of TET at the transcriptional and catalytic levels.

The online version contains supplementary material available at 10.1186/s13287-021-02578-1.

• AKT signal
• DNA demethylation
• Reprogramming
• TET
• iPS cells
• αKG

• HDAC4
• PTHrP
• cyclin D1
• p-AKT
• preimplantation embryo

• cancer metabolism
• glycolysis
• lipid metabolism
• mTOR
• metabolic reprogramming

Cancer stem cells (CSC) may be the most relevant and elusive cancer cell population, as they have the exquisite ability to seed new tumors. It is plausible, that highly mutated cancer genes, such as KRAS, are functionally associated with processes contributing to the emergence of stemness traits. In this review, we will summarize the evidence for a stemness driving activity of oncogenic Ras. This activity appears to differ by Ras isoform, with the highly mutated KRAS having a particularly profound impact. Next to established stemness pathways such as Wnt and Hedgehog (Hh), the precise, cell cycle dependent orchestration of the MAPK-pathway appears to relay Ras activation in this context. We will examine how non-canonical activities of K-Ras4B (hereafter K-Ras) could be enabled by its trafficking chaperones calmodulin and PDE6D/PDEδ. Both dynamically localize to the cellular machinery that is intimately linked to cell fate decisions, such as the primary cilium and the centrosome. Thus, it can be speculated that oncogenic K-Ras disrupts fundamental polarized signaling and asymmetric apportioning processes that are necessary during cell differentiation.

• KRAS
• cancer stem cells
• centrosomes
• cilia
• trafficking

• ARF-suppression-induced senescence
• Alternative reading frame (ARF)
• Induced pluripotent stem (iPS) cells
• Pluripotency
• Reprogramming
• Xenograft
• Yamanaka factors

Induced pluripotent stem cells (iPSCs) were first established from differentiated somatic cells by gene introduction of key transcription factors, OCT4, SOX2, KLF4, and c-MYC, over a decade ago. Although iPSCs can be applicable for regenerative medicine, disease modeling and drug screening, several issues associated with the utilization of iPSCs such as low reprogramming efficiency and the risk of tumorigenesis, still need to be resolved. In addition, the molecular mechanisms involved in the somatic cell reprogramming to pluripotency are yet to be elucidated. Compared with their somatic counterparts, pluripotent stem cells, including embryonic stem cells and iPSCs, exhibit a high rate of glycolysis akin to aerobic glycolysis in cancer cells. This is known as the Warburg effect and is essential for maintaining stem cell properties. This unique glycolytic metabolism in iPSCs can provide energy and drive the pentose phosphate pathway, which is the preferred pathway for rapid cell proliferation. During reprogramming, somatic cells undergo a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis trigged by a transient OXPHOS burst, resulting in the initiation and progression of reprogramming to iPSCs. Metabolic intermediates and mitochondrial functions are also involved in the epigenetic modification necessary for the process of iPSC reprogramming. Among the key regulatory molecules that have been reported to be involved in metabolic shift so far, hypoxia-inducible factor 1 (HIF1) controls the transcription of many target genes to initiate metabolic changes in the early stage and maintains glycolytic metabolism in the later phase of reprogramming. This review summarizes the current understanding of the unique metabolism of pluripotent stem cells and the metabolic shift during reprogramming, and details the relevance of HIF1 in the metabolic shift.

• Glycolysis
• Hypoxia-inducible factor
• Induced pluripotent stem cells
• Metabolic shift
• Oxidative phosphorylation
• Regenerative medicine
• Reprogramming

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold a huge promise for regenerative medicine, drug development, and disease modeling. PSCs have unique metabolic features that are akin to those of cancer cells, in which glycolysis predominates to produce energy as well as building blocks for cellular components. Recent studies indicate that the unique metabolism in PSCs is not a mere consequence of their preference for a low oxygen environment, but is an active process for maintaining self-renewal and pluripotency, possibly in preparation for rapid response to the metabolic demands of differentiation. Understanding the regulatory mechanisms of this unique metabolism in PSCs is essential for proper derivation, generation, and maintenance of PSCs. In this review, we discuss the metabolic features of PSCs and describe the current understanding of the mechanisms of the metabolic shift during reprogramming from somatic cells to iPSCs, in which the metabolism switches from oxidative phosphorylation (OxPhos) to glycolysis.

• ESC
• OxPhos: glycolysis
• PSC
• hypoxia
• iPSC
• metabolism
• mitochondrion
• somatic cell reprogramming

• Animals
• Cellular Reprogramming
• Energy Metabolism
• Humans
• Mitochondria/metabolism
• Pluripotent Stem Cells/cytology
• Pluripotent Stem Cells/metabolism

Stem cells are undifferentiated cells that can give rise to several different cell types and can self-renew. Given their ability to differentiate into different lineages, stem cells retain huge therapeutic potential for regenerative medicine. Therefore, the understanding of the signaling pathways involved in stem cell pluripotency maintenance and differentiation has a paramount importance in order to understand these biological processes and to develop therapeutic strategies. In this review, we focus on phosphoinositide 3 kinase (PI3K) since its signaling pathway regulates many cellular processes, such as cell growth, proliferation, survival, and cellular transformation. Precisely, in human stem cells, the PI3K cascade is involved in different processes from pluripotency and induced pluripotent stem cell (iPSC) reprogramming to mesenchymal and oral mesenchymal differentiation, through different and interconnected mechanisms.

• inositide signaling
• nucleus
• stem cells

• Cell Differentiation
• Cellular Reprogramming
• Human Embryonic Stem Cells/cytology
• Human Embryonic Stem Cells/metabolism
• Humans
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/metabolism
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/metabolism
• Phosphatidylinositol 3-Kinase/metabolism
• Signal Transduction

• Cytoskeleton
• EDG5
• GPCR
• Genome-wide RNAi screen
• Human somatic cell reprogramming
• hESCs/hiPSCs

Personalized medicine for cancer patients requires a deep understanding of the underlying genetics that drive cancer and the subsequent identification of predictive biomarkers. To discover new genes and pathways contributing to oncogenesis and therapy resistance in HER2+ breast cancer, we performed Mouse Mammary Tumor Virus (MMTV)-induced insertional mutagenesis screens in ErbB2/cNeu-transgenic mouse models. The screens revealed 34 common integration sites (CIS) in mammary tumors of MMTV-infected mice, highlighting loci with multiple independent MMTV integrations in which potential oncogenes are activated, most of which had never been reported as MMTV CIS. The CIS most strongly associated with the ErbB2-transgenic genotype was the locus containing Eras (ES cell-expressed Ras), a constitutively active RAS-family GTPase. We show that upon expression, Eras acts as a potent oncogenic driver through hyperactivation of the PI3K/AKT pathway, in contrast to other RAS proteins that signal primarily via the MAPK/ERK pathway and require upstream activation or activating mutations to induce signaling. We additionally show that ERAS synergistically enhances HER2-induced tumorigenesis and, in this role, can functionally replace ERBB3/HER3 by acting as a more powerful activator of PI3K/AKT signaling. Although previously reported as pseudogene in humans, we observed ERAS RNA and protein expression in a substantial subset of human primary breast carcinomas. Importantly, we show that ERAS induces primary resistance to the widely used HER2-targeting drugs Trastuzumab (Herceptin) and Lapatinib (Tykerb/Tyverb) in vivo, and is involved in acquired resistance via selective upregulation during treatment in vitro, indicating that ERAS may serve as a novel clinical biomarker for PI3K/AKT pathway hyperactivation and HER2-targeted therapy resistance.

• AKT
• Metabolism
• PI3K
• Pluripotency
• Proliferation and differentiation
• mTOR

• Cow
• ERas
• Immunohistochemistry
• PDGFβR
• Placenta
• Sequencing
• Western blot

Primordial germ cells (PGCs) are precursors of all gametes, and represent the founder cells of the germline. Although developmental potency is restricted to germ-lineage cells, PGCs can be reprogrammed into a pluripotent state. Specifically, PGCs give rise to germ cell tumors, such as testicular teratomas, in vivo, and to pluripotent stem cells known as embryonic germ cells in vitro. In this review, we highlight the current knowledge on signaling pathways, transcriptional controls, and post-transcriptional controls that govern germ cell differentiation and de-differentiation. These regulatory processes are common in the reprogramming of germ cells and somatic cells, and play a role in the pathogenesis of human germ cell tumors.

• Primordial germ cell
• Embryonic germ cell
• Germ cell tumor
• Reprogramming
• Induced pluripotent stem cell
• Small molecule compound
• Gene
• Signal
• Transcription factor

Reprogramming of somatic cells has become a versatile tool for biomedical research and for regenerative medicine. In the current study, we show that manipulating alternative splicing (AS) is a highly potent strategy to produce cells for therapeutic applications. We demonstrate that silencing of hTAF4-TAFH activity of TAF4 converts human facial dermal fibroblasts to melanocyte-like (iMel) cells. iMel cells produce melanin and express microphthalmia-associated transcription factor (MITF) and its target genes at levels comparable to normal melanocytes. Reprogramming of melanoma cells by manipulation with hTAF4-TAFH activity upon TAFH RNAi enforces cell differentiation towards chondrogenic pathway, whereas ectoptic expression of TAF4 results in enhanced multipotency and neural crest-like features in melanoma cells. In both cell states, iMels and cancer cells, hTAF4-TAFH activity controls migration by supporting E- to N-cadherin switches. From our data, we conclude that targeted splicing of hTAF4-TAFH coordinates AS of other TFIID subunits, underscoring the role of TAF4 in synchronised changes of Pol II complex composition essential for efficient cellular reprogramming. Taken together, targeted AS of TAF4 provides a unique strategy for generation of iMels and recapitulating stages of melanoma progression.

• HCC
• NASH
• NF-κB
• NRF2
• autophagy
• mTORC1
• p62/SQSTM1

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development.

• Eras
• cell lineage
• embryonic stem cells
• mesoderm
• primitive streak

• Hippo pathway
• Raf kinase
• embryonic stem cell
• embryonic stem cell-expressed RAS
• embryonic stem cell-expressed RAS, ERAS
• embryonic stem cell-expressed RAS, ERAS, AKT
• hepatic stellate cell (HSC)
• mammalian target of rapamycin (mTOR)
• phosphatidylinositide 3-kinase (PI 3-kinase)
• signal transduction

• Akt
• Dual Leucine zipper-bearing Kinase (DLK)
• Nanog
• mouse embryonic stem cells
• self-renewal

• Cell cycle
• Cell proliferation
• E-Ras
• ES cell
• Reprogramming

• E-RAS
• H-RAS
• RAS protein
• Raf kinase
• effector selection
• embryonic stem cell-expressed RAS
• phosphatidylinositide 3-kinase (PI 3-kinase)
• phosphatidylinositol kinase (PI kinase)
• small GTPase
• specificity determining residues

The ultimate goal of regenerative medicine is to replace damaged tissues with new functioning ones. This can potentially be accomplished by stem cell transplantation. While stem cell transplantation for blood diseases has been increasingly successful, widespread application of stem cell therapy in the clinic has shown limited results. Despite successful efforts to refine existing methodologies and to develop better ones for reprogramming, clinical application of stem cell therapy suffers from issues related to the safety of the transplanted cells, as well as the low efficiency of reprogramming technology. Better understanding of the underlying mechanism(s) involved in pluripotency should accelerate the clinical application of stem cell transplantation for regenerative purposes. This review outlines the main decision-making factors involved in pluripotency, focusing on the role of microRNAs, epigenetic modification, signaling pathways, and toll-like receptors. Of special interest is the role of toll-like receptors in pluripotency, where emerging data indicate that the innate immune system plays a vital role in reprogramming. Based on these data, we propose that nongenetic mechanisms for reprogramming provide a novel and perhaps an essential strategy to accelerate application of regenerative medicine in the clinic.

• dedifferentiation
• epigenetic modifications
• microRNAs
• pluripotency
• reprogramming
• signaling pathways
• toll-like receptors
• transdifferentiation

• Cell Differentiation
• Cell Line
• Cellular Reprogramming
• Fibroblasts/cytology
• Fibroblasts/metabolism
• Gene Expression Regulation
• Genetic Vectors
• Humans
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/metabolism
• Kruppel-Like Factor 4
• Kruppel-Like Transcription Factors/genetics
• Kruppel-Like Transcription Factors/metabolism
• MicroRNAs/genetics
• MicroRNAs/metabolism
• Octamer Transcription Factor-3/genetics
• Octamer Transcription Factor-3/metabolism
• PTEN Phosphohydrolase/genetics
• PTEN Phosphohydrolase/metabolism
• Proto-Oncogene Proteins c-myc/genetics
• Proto-Oncogene Proteins c-myc/metabolism
• RNA, Long Noncoding
• Retroviridae/genetics
• SOXB1 Transcription Factors/genetics
• SOXB1 Transcription Factors/metabolism
• Signal Transduction

The self-renewal and pluripotency of embryonic stem cells (ESC) is regulated by a highly integrated network of essential transcription factors, which includes Sox2. Previous studies have shown that elevating Sox2 on its own in mouse ESC induces differentiation and inhibits the expression of endogenous Sox2 at the protein and mRNA level. These findings led us to hypothesize that increases in Sox2 activate a negative feedback loop that inhibits the transcription of the endogenous Sox2 gene. To test this hypothesis, we used i-OSKM-ESC, which elevate Sox2 in conjunction with Oct4, Klf4, and c-Myc when treated with doxycycline (Dox). Elevating the expression of these four transcription factors in i-OSKM-ESC does not induce differentiation, but it represses expression of endogenous Sox2. We determined that increases of Sox2 in i-OSKM-ESC lead to increases in activated AKT and inactivation of FoxO1 (an activator of Sox2), as well as decreases in binding of FoxO1 to the 5'flanking region of Sox2. Importantly, we determined that inhibition of AKT in Dox-treated i-OSKM-ESC leads to re-expression of endogenous Sox2 at the mRNA and protein level and reactivation of FoxO1. These findings argue that AKT signaling is part of the negative feedback loop that helps carefully control the transcription of Sox2 in ESC by modulating the binding of FoxO1 to the Sox2 gene. Collectively, our findings provide new insights into the mechanisms that enable ESC to carefully regulate the levels of Sox2 and retain their stem cell properties.

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