Analysis of the Foxo1 and VEGFA-regulated transcriptomes in mouse embryonic stem (ES) cells

Abstract Proper levels of gene expression are important for normal mammalian development. Typically, altered gene dosage caused by karyotypic abnormalities results in embryonic lethality or birth defects. Segmental aneuploidy can be compatible with life but often results in contiguous gene syndromes. The ability to manipulate the mouse genome allows the systematic exploration of regions that are affected by alterations in gene dosage. To explore the effects of segmental haploidy in the mouse t complex on chromosome 17, radiation-induced deletion complexes centered at the Sod2 and D17Leh94 loci were generated in embryonic stem (ES) cells. A small interval was identified that, when hemizygous, caused specific embryonic lethal phenotypes (exencephaly and edema) in most fetuses. The penetrance of these phenotypes was background dependent. Additionally, evidence for parent-of-origin effects was observed. This genetic approach should be useful for identifying genes that are imprinted or whose dosage is critical for normal embryonic development.

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The chromosomal protein SMCHD1 regulates DNA methylation and the 2c-like state of embryonic stem cells by antagonizing TET proteins

Science Advances ◽

10.1126/sciadv.abb9149 ◽

2021 ◽

Vol 7 (4) ◽

pp. eabb9149

Author(s):

Zhijun Huang ◽

Jiyoung Yu ◽

Wei Cui ◽

Benjamin K. Johnson ◽

Kyunggon Kim ◽

...

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Homeobox Gene ◽

Dna Demethylation ◽

Chromosomal Protein ◽

Dna Hypomethylation ◽

Tet Proteins ◽

Target Sites ◽

Structural Maintenance Of Chromosomes ◽

Hinge Domain

5-Methylcytosine (5mC) oxidases, the ten-eleven translocation (TET) proteins, initiate DNA demethylation, but it is unclear how 5mC oxidation is regulated. We show that the protein SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) is found in complexes with TET proteins and negatively regulates TET activities. Removal of SMCHD1 from mouse embryonic stem (ES) cells induces DNA hypomethylation, preferentially at SMCHD1 target sites and accumulation of 5-hydroxymethylcytosine (5hmC), along with promoter demethylation and activation of the Dux double-homeobox gene. In the absence of SMCHD1, ES cells acquire a two-cell (2c) embryo–like state characterized by activation of an early embryonic transcriptome that is substantially imposed by Dux. Using Smchd1/Tet1/Tet2/Tet3 quadruple-knockout cells, we show that DNA demethylation, activation of Dux, and other genes upon SMCHD1 loss depend on TET proteins. These data identify SMCHD1 as an antagonist of the 2c-like state of ES cells and of TET-mediated DNA demethylation.

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Retinoic acid signaling is critical during the totipotency window in early mammalian development

Nature Structural & Molecular Biology ◽

10.1038/s41594-021-00590-w ◽

2021 ◽

Author(s):

Ane Iturbide ◽

Mayra L. Ruiz Tejeda Segura ◽

Camille Noll ◽

Kenji Schorpp ◽

Ina Rothenaigner ◽

...

Keyword(s):

Retinoic Acid ◽

Regulatory Networks ◽

Es Cells ◽

Embryonic Stem ◽

Mammalian Development ◽

Cell Stage ◽

Cell Potential ◽

Cellular Models ◽

Early Embryos ◽

Genome Activation

AbstractTotipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

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The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽

10.7554/elife.03573 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 21

Author(s):

Yick W Fong ◽

Jaclyn J Ho ◽

Carla Inouye ◽

Robert Tjian

Keyword(s):

Stem Cell ◽

Es Cells ◽

Embryonic Stem ◽

In Vitro Transcription ◽

Specific Gene ◽

Transcriptional Level ◽

Ips Cell ◽

Ribonucleoprotein Complex ◽

Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

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Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients

AJP Cell Physiology ◽

10.1152/ajpcell.00188.2019 ◽

2019 ◽

Vol 317 (4) ◽

pp. C725-C736

Author(s):

Gurbind Singh ◽

Divya Sridharan ◽

Mahmood Khan ◽

Polani B. Seshagiri

Keyword(s):

Cell Biology ◽

Fluorescent Protein ◽

Es Cells ◽

Monochromatic Light ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Medical Diagnostics ◽

Calcium Transients ◽

Egfp Expression ◽

Mitochondrial Activity

We earlier established the mouse embryonic stem (ES) cell “GS-2” line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420–495), green (~510–575), and red (~600–700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

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Epigenetic Regulation of Mesenchymal Stem Cells: A Focus on Osteogenic and Adipogenic Differentiation

Stem Cells International ◽

10.4061/2011/201371 ◽

2011 ◽

Vol 2011 ◽

pp. 1-18 ◽

Cited By ~ 57

Author(s):

Chad M. Teven ◽

Xing Liu ◽

Ning Hu ◽

Ni Tang ◽

Stephanie H. Kim ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Epigenetic Regulation ◽

Adult Stem Cells ◽

Es Cells ◽

Adipogenic Differentiation ◽

Embryonic Stem ◽

Cell Types ◽

Differentiation Potential ◽

Msc Differentiation

Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES) cells. Mesenchymal stem cells (MSCs) are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.

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Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽

10.1210/en.2011-0219 ◽

2011 ◽

Vol 152 (7) ◽

pp. 2870-2882 ◽

Cited By ~ 24

Author(s):

Unmesh Jadhav ◽

J. Larry Jameson

Keyword(s):

Target Genes ◽

De Novo ◽

Embryoid Body ◽

Es Cells ◽

Embryonic Stem ◽

Cell Lineage ◽

Culture Conditions ◽

Steroidogenic Factor 1 ◽

Lineage Differentiation ◽

And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

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