Tbx3 directs cell fate decision towards mesendoderm, pancreas and liver in mouse embryonic stem cells

MicroRNAs (miRNAs) play important roles in cell fate decisions. However, the miRNAs and their targets involved in the regulation of cardiac lineage specification are largely unexplored. Here, we report novel functions of miR-142-3p in the regulation of cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). With a miRNA array screen, we identified a number of miRNAs significantly changed during mESC differentiation into the mesodermal and cardiac progenitor cells, and miR-142-3p was one among the markedly downregulated miRNAs. Ectopic expression and inhibition of miR-142-3p did not alter the characteristics of undifferentiated ESCs, whereas ectopic expression of miR-142-3p impaired cardiomyocyte formation. In addition, ectopic expression of miR-142-3p inhibited the expression of a cardiac mesodermal marker gene Mesp1 and downstream cardiac transcription factors Nkx2.5, Tbx5, and Mef2c but not the expression of three germ layer-specific genes. We further demonstrated that miR-142-3p targeted the 3′-untranslated region of Mef2c. These results reveal miR-142-3p as an important regulator of early cardiomyocyte differentiation. Our findings provide new knowledge for further understanding of roles and mechanisms of miRNAs as critical regulators of cardiomyocyte differentiation.

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Inhibition of β-catenin–TCF1 interaction delays differentiation of mouse embryonic stem cells

The Journal of Cell Biology ◽

10.1083/jcb.201503017 ◽

2015 ◽

Vol 211 (1) ◽

pp. 39-51 ◽

Cited By ~ 17

Author(s):

Sujash S. Chatterjee ◽

Abil Saj ◽

Tenzin Gocha ◽

Matthew Murphy ◽

Foster C. Gonsalves ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Protein Interactions ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Protein Protein Interactions ◽

Nanog Expression ◽

Context Specific

The ability of mouse embryonic stem cells (mESCs) to self-renew or differentiate into various cell lineages is regulated by signaling pathways and a core pluripotency transcriptional network (PTN) comprising Nanog, Oct4, and Sox2. The Wnt/β-catenin pathway promotes pluripotency by alleviating T cell factor TCF3-mediated repression of the PTN. However, it has remained unclear how β-catenin’s function as a transcriptional activator with TCF1 influences mESC fate. Here, we show that TCF1-mediated transcription is up-regulated in differentiating mESCs and that chemical inhibition of β-catenin/TCF1 interaction improves long-term self-renewal and enhances functional pluripotency. Genetic loss of TCF1 inhibited differentiation by delaying exit from pluripotency and conferred a transcriptional profile strikingly reminiscent of self-renewing mESCs with high Nanog expression. Together, our data suggest that β-catenin’s function in regulating mESCs is highly context specific and that its interaction with TCF1 promotes differentiation, further highlighting the need for understanding how its individual protein–protein interactions drive stem cell fate.

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Bdh2 Deficiency Promotes Endoderm-Biased Early Differentiation of Mouse Embryonic Stem Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.655145 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yuting Fu ◽

Fangyuan Liu ◽

Shuo Cao ◽

Jia Zhang ◽

Huizhi Wang ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Iron Homeostasis ◽

Embryonic Stem ◽

Cell Fate Decisions ◽

Rate Limiting Step ◽

Fate Decision

3-hydroxybutyrate dehydrogenase-2 (Bdh2), a short-chain dehydrogenase, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore, playing a key role in iron homeostasis, energy metabolism and apoptosis. However, the function of Bdh2 in embryonic stem cells (ESCs) remains unknown. To gain insights into the role of Bdh2 on pluripotency and cell fate decisions of mouse ESCs, we generated Bdh2 homozygous knockout lines for both mouse advanced embryonic stem cell (ASC) and ESC using CRISPR/Cas9 genome editing technology. Bdh2 deficiency in both ASCs and ESCs had no effect on expression of core pluripotent transcription factors and alkaline phosphatase activity, suggesting dispensability of Bdh2 for self-renewal and pluripotency of ESCs. Interestingly, cells with Bdh2 deficiency exhibited potency of endoderm differentiation in vitro; with upregulated endoderm associated genes revealed by RNA-seq and RT-qPCR. We further demonstrate that Bdh2 loss inhibited expression of multiple methyltransferases (DNMTs) at both RNA and protein level, suggesting that Bdh2 may be essentially required to maintain DNA methylation in ASCs and ESCs. Overall, this study provides valuable data and resources for understanding how Bdh2 regulate earliest cell fate decision and DNA methylation in ASCs/ESCs.

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Histone Acetyltransferase KAT2A Stabilises Pluripotency with Control of Transcriptional Heterogeneity

10.1101/347476 ◽

2018 ◽

Cited By ~ 1

Author(s):

Naomi Moris ◽

Shlomit Edri ◽

Denis Seyres ◽

Rashmi Kulkarni ◽

Ana Filipa Domingues ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Regulatory Networks ◽

Environmental Changes ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Histone Acetyl Transferase ◽

Unicellular Organisms

ABSTRACTCell fate transitions in mammalian stem cell systems have often been associated with transcriptional heterogeneity, however existing data have failed to establish a functional or mechanistic link between the two phenomena. Experiments in unicellular organisms support the notion that transcriptional heterogeneity can be used to facilitate adaptability to environmental changes and have identified conserved chromatin-associated factors that modulate levels of transcriptional noise. Herein, we show destabilisation of pluripotency-associated gene regulatory networks through increased transcriptional heterogeneity of mouse embryonic stem cells in which paradigmatic histone acetyl-transferase, and candidate noise modulator, Kat2a (yeast orthologue Gcn5) has been inhibited. Functionally, network destabilisation associates with reduced pluripotency and accelerated mesendodermal differentiation, with increased probability of transitions into lineage commitment. Thus, we functionally link transcriptional heterogeneity to cell fate transitions through manipulation of the histone acetylation landscape of mouse embryonic stem cells and establish a general paradigm that could be exploited in other normal and malignant stem cell fate transitions.

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Slc25a36 modulates pluripotency of mouse embryonic stem cells by regulating mitochondrial function and glutathione level

Biochemical Journal ◽

10.1042/bcj20190057 ◽

2019 ◽

Vol 476 (11) ◽

pp. 1585-1604 ◽

Cited By ~ 1

Author(s):

Yanli Xin ◽

Yanliang Wang ◽

Liang Zhong ◽

Bingbo Shi ◽

Hui Liang ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Mitochondrial Dysfunction ◽

Cell Fate ◽

Mitochondrial Function ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Glutathione Metabolism ◽

Pluripotency Maintenance

Abstract Mitochondria play a central role in the maintenance of the naive state of embryonic stem cells. Many details of the mechanism remain to be fully elucidated. Solute carrier family 25 member 36 (Slc25a36) might regulate mitochondrial function through transporting pyrimidine nucleotides for mtDNA/RNA synthesis. Its physical role in this process remains unknown; however, Slc25a36 was recently found to be highly expressed in naive mouse embryonic stem cells (mESCs). Here, the function of Slc25a36 was characterized as a maintenance factor of mESCs pluripotency. Slc25a36 deficiency (via knockdown) has been demonstrated to result in mitochondrial dysfunction, which induces the differentiation of mESCs. The expression of key pluripotency markers (Pou5f1, Sox2, Nanog, and Utf1) decreased, while that of key TE genes (Cdx2, Gata3, and Hand1) increased. Cdx2-positive cells emerged in Slc25a36-deficient colonies under trophoblast stem cell culture conditions. As a result of Slc25a36 deficiency, mtDNA of knockdown cells declined, leading to impaired mitochondria with swollen morphology, decreased mitochondrial membrane potential, and low numbers. The key transcription regulators of mitochondrial biogenesis also decreased. These results indicate that mitochondrial dysfunction leads to an inability to support the pluripotency maintenance. Moreover, down-regulated glutathione metabolism and up-regulated focal adhesion reinforced and stabilized the process of differentiation by separately enhancing OCT4 degradation and promoting cell spread. This study improves the understanding of the function of Slc25a36, as well as the relationship of mitochondrial function with naive pluripotency maintenance and stem cell fate decision.

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LncRNA Mrhl orchestrates differentiation programs in mouse embryonic stem cells through chromatin mediated regulation

10.1101/713404 ◽

2019 ◽

Cited By ~ 1

Author(s):

Debosree Pal ◽

C V Neha ◽

Utsa Bhaduri ◽

Zenia ◽

Subbulakshmi Chidambaram ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Loss Of Function ◽

Genome Wide ◽

Development And Differentiation ◽

Lineage Specific Genes

AbstractLong non-coding RNAs (lncRNAs) have been well-established to act as regulators and mediators of development and cell fate specification programs. LncRNA Mrhl (meiotic recombination hotspot locus) has been shown to act in a negative feedback loop with WNT signaling to regulate male germ cell meiotic commitment. In our current study, we have addressed the role of Mrhl in development and differentiation using mouse embryonic stem cells (mESCs) as our model system of study. We found Mrhl to be a nuclear-localized, chromatin-bound lncRNA with moderately stable expression in mESCs. Transcriptome analyses and loss-of-function phenotype studies revealed dysregulation of developmental processes and lineage-specific genes along with aberrance in specification of early lineages during differentiation of mESCs. Genome-wide chromatin occupancy studies suggest regulation of chromatin architecture at key target loci through triplex formation. Our studies thus reveal a role for lncRNA Mrhl in regulating differentiation programs in mESCs in the context of appropriate cues through chromatin-mediated responses.

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Distinct SoxB1 networks are required for naïve and primed pluripotency

eLife ◽

10.7554/elife.27746 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 6

Author(s):

Andrea Corsinotti ◽

Frederick CK Wong ◽

Tülin Tatar ◽

Iwona Szczerbinska ◽

Florian Halbritter ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Neural Differentiation ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Self Renewal ◽

Cell Fate Decisions ◽

Pluripotent State

Deletion of Sox2 from mouse embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here, we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naïve pluripotency.

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STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells

Genome Biology ◽

10.1186/s13059-020-02156-3 ◽

2020 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Tianran Peng ◽

Yanan Zhai ◽

Yaser Atlasi ◽

Menno ter Huurne ◽

Hendrik Marks ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Control Cell ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Mouse Embryonic Stem Cells ◽

Chromatin Signature ◽

Genome Wide ◽

A Genome

Abstract Background Enhancers are distal regulators of gene expression that shape cell identity and control cell fate transitions. In mouse embryonic stem cells (mESCs), the pluripotency network is maintained by the function of a complex network of enhancers, that are drastically altered upon differentiation. Genome-wide chromatin accessibility and histone modification assays are commonly used as a proxy for identifying putative enhancers and for describing their activity levels and dynamics. Results Here, we applied STARR-seq, a genome-wide plasmid-based assay, as a read-out for the enhancer landscape in “ground-state” (2i+LIF; 2iL) and “metastable” (serum+LIF; SL) mESCs. This analysis reveals that active STARR-seq loci show modest overlap with enhancer locations derived from peak calling of ChIP-seq libraries for common enhancer marks. We unveil ZIC3-bound loci with significant STARR-seq activity in SL-ESCs. Knock-out of Zic3 removes STARR-seq activity only in SL-ESCs and increases their propensity to differentiate towards the endodermal fate. STARR-seq also reveals enhancers that are not accessible, masked by a repressive chromatin signature. We describe a class of dormant, p53 bound enhancers that gain H3K27ac under specific conditions, such as after treatment with Nocodazol, or transiently during reprogramming from fibroblasts to pluripotency. Conclusions In conclusion, loci identified as active by STARR-seq often overlap with those identified by chromatin accessibility and active epigenetic marking, yet a significant fraction is epigenetically repressed or display condition-specific enhancer activity.

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Mouse embryonic stem cells switch migratory behaviour during early differentiation

10.1101/2020.12.07.415307 ◽

2020 ◽

Author(s):

Irene M. Aspalter ◽

Wolfram Pönisch ◽

Kevin J. Chalut ◽

Ewa K. Paluch

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Migratory Behaviour ◽

Es Cell ◽

Early Differentiation ◽

Cell State

AbstractDevelopment relies on a series of precisely orchestrated cell fate changes. While studies of fate transitions often focus on changes in gene regulatory networks, most transitions are also associated with changes in cell shape and cell behaviour. Here, we investigate changes in migratory behaviour in mouse embryonic stem (ES) cells during their first developmental fate transition, exit from ES cell state. We show that naïve pluripotent ES cells cannot efficiently migrate on 2-dimensional substrates but are able to migrate in an amoeboid fashion when placed in confinement. Exit from ES cell state, typically characterised by enhanced cell spreading, is associated with decreased migration in confinement and acquisition of mesenchymal-like migration on 2D substrates. Interestingly, confined, amoeboid-like migration of ES cells strongly depends on Myosin IIA, but not Myosin IIB. In contrast mesenchymal-like migration of cells exiting the ES cell state does not depend on Myosin motor activity but relies on the activity of the Arp2/3 complex. Together, our data suggest that during early differentiation, cells undergo a switch in the regulation of the actin cytoskeleton, leading to a transition from amoeboid-to mesenchymal-like migration.Summary statementNaïve mouse embryonic stem cells display amoeboid-like migration in confinement, but switch to mesenchymal-like migration as they exit the ES cell state.

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