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Dissection of two routes to naive pluripotency using different kinase inhibitors.

ABSTRACT: Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors.

SUBMITTER: Martinez-Val A

PROVIDER: S-EPMC7994667 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Dissection of two routes to naïve pluripotency using different kinase inhibitors

Project description:Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors.

2021-02-10 | MTBLS301 | MetaboLights

YAP Induces Human Naive Pluripotency.

Project description:The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP(-/-) cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology.

| S-EPMC4807727 | biostudies-literature

Splenic immune response to Plasmodium infection initiated by two different routes

Project description:Plasmodium chabaudi parasite obtained from recently mosquito transmitted infections give rise to an attenuated blood-stage infection when compared to serially blood passaged parasites. We compared the immune response in the spleens induced by these two types of parasites in order to determine what may cause this attenuation. Spleens were taken at several timepoints during the blood-stage infection and RNA-seq analysis was performed.

2021-09-15 | GSE169752 | GEO

Inhibitors of AKT kinase increase LDL receptor mRNA expression by two different mechanisms.

Project description:Protein kinase B (AKT) is a serine/threonine kinase that functions as an important downstream effector of phosphoinositide 3-kinase. We have recently shown that MK-2206 and triciribine, two highly selective AKT inhibitors increase the level of low density lipoprotein receptor (LDLR) mRNA which leads to increased amount of cell-surface LDLRs. However, whereas MK-2206 induces transcription of the LDLR gene, triciribine stabilizes LDLR mRNA, raising the possibility that the two inhibitors may actually affect other kinases than AKT. In this study, we aimed to ascertain the role of AKT in regulation of LDLR mRNA expression by examining the effect of five additional AKT inhibitors on LDLR mRNA levels. Here we show that in cultured HepG2 cells, AKT inhibitors ARQ-092, AKT inhibitor VIII, perifosine, AT7867 and CCT128930 increase LDLR mRNA levels by inducing the activity of LDLR promoter. CCT128930 also increased the stability of LDLR mRNA. To study the role of AKT isoforms on LDLR mRNA levels, we examined the effect of siRNA-mediated knockdown of AKT1 or AKT2 on LDLR promoter activity and LDLR mRNA stability. Whereas knockdown of either AKT1 or AKT2 led to upregulation of LDLR promoter activity, only knockdown of AKT2 had a stabilizing effect on LDLR mRNA. Taken together, these results provide strong evidence for involvement of AKT in regulation of LDLR mRNA expression, and point towards the AKT isoform specificity for upregulation of LDLR mRNA expression.

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Sox2 modulation increases naive pluripotency plasticity.

Project description:Induced pluripotency provides a tool to explore mechanisms underlying establishment, maintenance, and differentiation of naive pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, upon the removal of self-renewing cues Sox2-low nPSCs differentiate into both embryonic and extraembryonic cell fates in vitro and in vivo. This differs from previous studies which only identified conditions that allowed cells to differentiate to one fate or the other. At the single-cell level self-renewing Sox2-low nPSCs exhibit a naive molecular signature. However, they display a nearer trophoblast identity than controls and decreased ability of Oct4 to bind naïve-associated regulatory sequences. In sum, this work defines wild-type levels of Sox2 as a restrictor of developmental potential and suggests perturbation of naive network as a mechanism to increase cell plasticity.

| S-EPMC7903329 | biostudies-literature

Acute coronary syndrome by two different spontaneous coronary artery dissection types in two different vessels.

Project description: Not available

| S-EPMC8926971 | biostudies-literature

Distinct Cell-Cycle Control in Two Different States of Mouse Pluripotency.

Project description:Mouse embryonic stem cells (ESCs) cultured in serum are characterized by hyper-phosphorylated RB protein, lack of G1 control, and rapid progression through the cell cycle. Here, we show that ESCs grown in the presence of two small-molecule inhibitors (2i ESCs) have a longer G1-phase with hypo-phosphorylated RB, implying that they have a functional G1 checkpoint. Deletion of RB, P107, and P130 in 2i ESCs results in a G1-phase similar to that of serum ESCs. Inhibition of the ERK signaling pathway in serum ESCs results in the appearance of hypo-phosphorylated RB and the reinstatement of a G1 checkpoint. In addition, induction of a dormant state by the inhibition of MYC, resembling diapause, requires the presence of the RB family proteins. Collectively, our data show that RB-dependent G1 restriction point signaling is active in mouse ESCs grown in 2i but abrogated in serum by ERK-dependent phosphorylation.

| S-EPMC5658514 | biostudies-literature

The Current State of Naive Human Pluripotency.

Project description:Naïve or ground state pluripotency is a cellular state in vitro which resembles cells of the preimplantation epiblast in vivo. This state was first observed in mouse embryonic stem cells and is characterized by high rates of proliferation, the ability to differentiate widely, and global hypomethylation. Human pluripotent stem cells (hPSCs) correspond to a later or "primed" stage of embryonic development. The conversion of hPSCs to a naïve state is desirable as their features should facilitate techniques such as gene editing and more efficient differentiation. Here we review protocols which now allow derivation of naïve human pluripotent stem cells by transgene expression or the use of media formulations containing inhibitors and growth factors and correlate this with pathways involved. Maintenance of these ground state cells is possible using a combination of basic fibroblast growth factor and human leukemia inhibitory factor together with dual inhibition of glycogen synthase kinase 3 beta, and mitogen-activated protein kinase kinase (MEK). Close similarity between the ground state hPSC and the in vivo preimplantation epiblast have been shown both by demonstrating similar upregulation of endogenous retroviruses and correlation of global RNA-seq data. This suggests that the human naïve state is not an in vitro artifact.

| S-EPMC4833179 | biostudies-literature

Suppression of YAP safeguards human naive pluripotency.

Project description:Propagation of human naïve pluripotent stem cells (nPSCs) relies on the inhibition of MEK/ERK signalling. However, MEK/ERK inhibition also promotes differentiation into trophectoderm (TE). Therefore, robust self-renewal requires suppression of TE fate. Tankyrase inhibition using XAV939 has been shown to stabilise human nPSCs and is implicated in TE suppression. Here, we dissect the mechanism of this effect. Tankyrase inhibition is known to block canonical Wnt/β-catenin signalling. However, we show that nPSCs depleted of β-catenin remain dependent on XAV939. Rather than inhibiting Wnt, we found that XAV939 prevents TE induction by reducing activation of YAP, a co-factor of TE-inducing TEAD transcription factors. Tankyrase inhibition stabilises angiomotin, which limits nuclear accumulation of YAP. Upon deletion of angiomotin-family members AMOT and AMOTL2, nuclear YAP increases and XAV939 fails to prevent TE induction. Expression of constitutively active YAP similarly precipitates TE differentiation. Conversely, nPSCs lacking YAP1 or its paralog TAZ (WWTR1) resist TE differentiation and self-renewal efficiently without XAV939. These findings explain the distinct requirement for tankyrase inhibition in human but not in mouse nPSCs and highlight the pivotal role of YAP activity in human naïve pluripotency and TE differentiation. This article has an associated 'The people behind the papers' interview.

| S-EPMC9845734 | biostudies-literature

microRNAs Regulating Human and Mouse Naive Pluripotency.

Project description:microRNAs are ~22bp nucleotide non-coding RNAs that play important roles in the post-transcriptional regulation of gene expression. Many studies have established that microRNAs are important for cell fate choices, including the naïve to primed pluripotency state transitions, and their intermediate state, the developmentally suspended diapause state in early development. However, the full extent of microRNAs associated with these stage transitions in human and mouse remain under-explored. By meta-analysis of microRNA-seq, RNA-seq, and metabolomics datasets from human and mouse, we found a set of microRNAs, and importantly, their experimentally validated target genes that show consistent changes in naïve to primed transitions (microRNA up, target genes down, or vice versa). The targets of these microRNAs regulate developmental pathways (e.g., the Hedgehog-pathway), primary cilium, and remodeling of metabolic processes (oxidative phosphorylation, fatty acid metabolism, and amino acid transport) during the transition. Importantly, we identified 115 microRNAs that significantly change in the same direction in naïve to primed transitions in both human and mouse, many of which are novel candidate regulators of pluripotency. Furthermore, we identified 38 microRNAs and 274 target genes that may be involved in diapause, where embryonic development is temporarily suspended prior to implantation to uterus. The upregulated target genes suggest that microRNAs activate stress response in the diapause stage. In conclusion, we provide a comprehensive resource of microRNAs and their target genes involved in naïve to primed transition and in the paused intermediate, the embryonic diapause stage.

| S-EPMC6929104 | biostudies-literature

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