Nucleus-cytoskeleton communication impacts on OCT4-chromatin interactions in embryonic stem cells

Abstract Background The cytoskeleton is a key component of the system responsible for transmitting mechanical cues from the cellular environment to the nucleus, where they trigger downstream responses. This communication is particularly relevant in embryonic stem (ES) cells since forces can regulate cell fate and guide developmental processes. However, little is known regarding cytoskeleton organization in ES cells, and thus, relevant aspects of nuclear-cytoskeletal interactions remain elusive. Results We explored the three-dimensional distribution of the cytoskeleton in live ES cells and show that these filaments affect the shape of the nucleus. Next, we evaluated if cytoskeletal components indirectly modulate the binding of the pluripotency transcription factor OCT4 to chromatin targets. We show that actin depolymerization triggers OCT4 binding to chromatin sites whereas vimentin disruption produces the opposite effect. In contrast to actin, vimentin contributes to the preservation of OCT4-chromatin interactions and, consequently, may have a pro-stemness role. Conclusions Our results suggest roles of components of the cytoskeleton in shaping the nucleus of ES cells, influencing the interactions of the transcription factor OCT4 with the chromatin and potentially affecting pluripotency and cell fate.

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H4K20me3 methyltransferase SUV420H2 shapes the chromatin landscape of pluripotent embryonic stem cells

Development ◽

10.1242/dev.188516 ◽

2020 ◽

Vol 147 (23) ◽

pp. dev188516

Author(s):

Jiji T. Kurup ◽

Zhijun Han ◽

Wenfei Jin ◽

Benjamin L. Kidder

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Es Cells ◽

Three Dimensional ◽

Embryonic Stem ◽

Pericentric Heterochromatin ◽

Es Cell ◽

Chromatin Interactions ◽

Repressive Histone Modification ◽

Dna Elements

ABSTRACTHeterochromatin, a densely packed chromatin state that is transcriptionally silent, is a critical regulator of gene expression. However, it is unclear how the repressive histone modification H4K20me3 or the histone methyltransferase SUV420H2 regulates embryonic stem (ES) cell fate by patterning the epigenetic landscape. Here, we report that depletion of SUV420H2 leads to a near-complete loss of H4K20me3 genome wide, dysregulated gene expression and delayed ES cell differentiation. SUV420H2-bound regions are enriched with repetitive DNA elements, which are de-repressed in SUV420H2 knockout ES cells. Moreover, SUV420H2 regulation of H4K20me3-marked heterochromatin controls chromatin architecture, including fine-scale chromatin interactions in pluripotent ES cells. Our results indicate that SUV420H2 plays a crucial role in stabilizing the three-dimensional chromatin landscape of ES cells, as loss of SUV420H2 resulted in A/B compartment switching, perturbed chromatin insulation, and altered chromatin interactions of pericentric heterochromatin and surrounding regions, indicative of localized decondensation. In addition, depletion of SUV420H2 resulted in compromised interactions between H4K20me3 and gene-regulatory regions. Together, these findings describe a new role for SUV420H2 in regulating the chromatin landscape of ES cells.

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Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1642 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1642-1651 ◽

Cited By ~ 240

Author(s):

M J Weiss ◽

C Yu ◽

S H Orkin

Keyword(s):

Transcription Factor ◽

Cell Line ◽

Zinc Finger ◽

Es Cells ◽

Embryonic Stem ◽

Erythroid Cell ◽

Transactivation Domain ◽

Stage Of Development ◽

Erythroid Maturation

The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.

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Human embryonic stem cells: challenges and opportunities

Reproduction Fertility and Development ◽

10.1071/rd06113 ◽

2006 ◽

Vol 18 (8) ◽

pp. 839 ◽

Cited By ~ 3

Author(s):

Steven L. Stice ◽

Nolan L. Boyd ◽

Sujoy K. Dhara ◽

Brian A. Gerwe ◽

David W. Machacek ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Cell Line ◽

Cell Fate ◽

Neural Development ◽

Es Cells ◽

Embryonic Stem ◽

Normal Karyotype ◽

Es Cell ◽

Cell Therapies

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.

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Transcription Factor Lbx1 Expression in Mouse Embryonic Stem Cell-Derived Phenotypes

Stem Cells International ◽

10.4061/2011/130970 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 5

Author(s):

Stefanie Schmitteckert ◽

Cornelia Ziegler ◽

Liane Kartes ◽

Alexandra Rolletschek

Keyword(s):

Transcription Factor ◽

Developmental Stages ◽

Troponin T ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Cardiac Cells ◽

Cell Stage

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. Duringin vitrodifferentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation ofLbx1at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.

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SUMO conjugation susceptibility of Akt/protein kinase B affects the expression of the pluripotency transcription factor Nanog in embryonic stem cells

PLoS ONE ◽

10.1371/journal.pone.0254447 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0254447

Author(s):

Marcos Francia ◽

Martin Stortz ◽

Camila Vazquez Echegaray ◽

Camila Oses ◽

Paula Verneri ◽

...

Keyword(s):

Transcription Factor ◽

Es Cells ◽

Embryonic Stem ◽

Dependent Manner ◽

Cellular Functions ◽

Cellular Processes ◽

Sumo Conjugation ◽

Heterologous System ◽

Canonical Pathways ◽

The Impact

Akt/PKB is a kinase involved in the regulation of a wide variety of cell processes. Its activity is modulated by diverse post-translational modifications (PTMs). Particularly, conjugation of the small ubiquitin-related modifier (SUMO) to this kinase impacts on multiple cellular functions, such as proliferation and splicing. In embryonic stem (ES) cells, this kinase is key for pluripotency maintenance. Among other functions, Akt is known to promote the expression of Nanog, a central pluripotency transcription factor (TF). However, the relevance of this specific PTM of Akt has not been previously analyzed in this context. In this work, we study the effect of Akt1 variants with differential SUMOylation susceptibility on the expression of Nanog. Our results demonstrate that both, the Akt1 capability of being modified by SUMO conjugation and a functional SUMO conjugase activity are required to induce Nanog gene expression. Likewise, we found that the common oncogenic E17K Akt1 mutant affected Nanog expression in ES cells also in a SUMOylatability dependent manner. Interestingly, this outcome takes places in ES cells but not in a non-pluripotent heterologous system, suggesting the presence of a crucial factor for this induction in ES cells. Remarkably, the two major candidate factors to mediate this induction, GSK3-β and Tbx3, are non-essential players of this effect, suggesting a complex mechanism probably involving non-canonical pathways. Furthermore, we found that Akt1 subcellular distribution does not depend on its SUMOylatability, indicating that Akt localization has no influence on the effect on Nanog, and that besides the membrane localization of E17K Akt mutant, SUMOylation is also required for its hyperactivity. Our results highlight the impact of SUMO conjugation in the function of a kinase relevant for a plethora of cellular processes, including the control of a key pluripotency TF.

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169 CULTURE OF MURINE EMBRYONIC STEM CELLS ON NWPF DISCS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab169 ◽

2005 ◽

Vol 17 (2) ◽

pp. 235 ◽

Cited By ~ 1

Author(s):

G. Cetinkaya ◽

S. Arat ◽

H. Odaman Mercan ◽

M.A. Onur ◽

A. Tumer

Keyword(s):

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Es Cells ◽

Three Dimensional ◽

Embryonic Stem ◽

Signalling Pathways ◽

Feeder Layers ◽

The Matrix ◽

Matrix Bound

Murine embryonic stem cells derived from the inner cell mass of mouse blastocysts can be maintained in culture for extended periods by using feeder layers and leukemia inhibitory factor (LIF). Maintenance of undifferentiated status occurs via LIF-mediated signalling pathways. In this study we cultured embryonic stem (ES) cells in Knockout-DMEM with serum replacement on a three-dimensional matrix, non-woven polyester fabric (NWPF), which is formed from non-arrayed polyethylene teraphthalate fibers. The surface of the fibers was modified by immobilizing LIF. While stimulating the matrix-bound form of LIF in vitro, we also tried to induce LIF-mediated signalling pathways continually. Our goal was to constitute a synthetic microenvironment that would support the undifferentiated growth of murine ES cells. Experimental groups were examined according to colony morphology, alkaline phosphatase activity, SSEA-1 antibody immunoreactivity, and SEM analyses. It was shown that three dimensional macroporous fibrous matrix, NWPF could support growth of undifferentiated ES cells. However, the ratio of undifferentiated colonies was higher on feeder layers than an polymeric surfaces (93% on mouse embryonic fibroblasts; 63,7% on hydrolized polymeric surface, P < 0,05). Results showed that LIF-immobilized surfaces supported undifferentiated growth of ES cells better than hydrolyzed surfaces. Colonies cultured on LIF-immobilized surfaces, had higher alkaline phosphatase activity and undifferentiated phenotype ratio than those on hydrolyzed surfaces. When the soluble or the matrix-bound form of LIF was used, the number of undifferentiated colonies increased in the polymeric groups (77.8% soluble LIF; 81.6% matrix bound LIF P < 0,05). On NWPF discs, ES cells formed big cell aggregates which had high alkaline phosphatase activity but low SSEA-1 immunoreactivity . When they were passaged to feeder layers, SSEA-1 activity increased. We managed to obtain undifferentiated colonies on NWPF discs by using LIF but the skeletal structure of polymeric matrix would be more convenient for differentiation studies. This study was performed in TUBITAK-RIGEB and supported by a part of grant from Hacettepe University (0102601001).

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Fgf and Esrrb integrate epigenetic and transcriptional networks that regulate self-renewal of trophoblast stem cells

Nature Communications ◽

10.1038/ncomms8776 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 54

Author(s):

Paulina A. Latos ◽

Angela Goncalves ◽

David Oxley ◽

Hisham Mohammed ◽

Ernest Turro ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Rna Polymerase Ii ◽

Es Cells ◽

Embryonic Stem ◽

Transcriptional Networks ◽

Self Renewal ◽

Downstream Target ◽

Trophoblast Stem

Abstract Esrrb (oestrogen-related receptor beta) is a transcription factor implicated in embryonic stem (ES) cell self-renewal, yet its knockout causes intrauterine lethality due to defects in trophoblast development. Here we show that in trophoblast stem (TS) cells, Esrrb is a downstream target of fibroblast growth factor (Fgf) signalling and is critical to drive TS cell self-renewal. In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes. To elucidate the mechanisms whereby Esrrb controls the expression of its targets, we characterized its TS cell-specific interactome using mass spectrometry. Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex. Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

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Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides

Journal of Polymer Engineering ◽

10.1515/polyeng-2016-0193 ◽

2017 ◽

Vol 37 (7) ◽

pp. 647-660 ◽

Cited By ~ 5

Author(s):

Saradaprasan Muduli ◽

Li-Hua Chen ◽

Meng-Pei Li ◽

Zhao-wen Heish ◽

Cheng-Hui Liu ◽

...

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Extracellular Matrix ◽

Stem Cell ◽

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Poly Vinyl Alcohol ◽

Hematopoietic Stem ◽

Stem Cell Culture

Abstract The physical characteristics of cell culture materials, such as their elasticity, affect stem cell fate with respect to cell proliferation and differentiation. We systematically investigated the morphologies and characteristics of several stem cell types, including human amniotic-derived stem cells, human hematopoietic stem cells, human induced pluripotent stem (iPS) cells, and embryonic stem (ES) cells on poly(vinyl alcohol) (PVA) hydrogels immobilized with and without extracellular matrix-derived oligopeptide. Human ES cells did not adhere well to soft PVA hydrogels immobilized with oligovitronectin, whereas they did adhere well to PVA hydrogel dishes with elasticities greater than 15 kPa. These results indicate that biomaterials such as PVA hydrogels should be designed to possess minimum elasticity to facilitate human ES cell attachment. PVA hydrogels immobilized with and without extracellular matrix-derived oligopeptides are excellent candidates of cell culture biomaterials for investigations into how cell culture biomaterial elasticity affects stem cell culture and differentiation.

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Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells

10.1101/251611 ◽

2018 ◽

Cited By ~ 1

Author(s):

Constantine Mylonas ◽

Peter Tessarz

Keyword(s):

Rna Polymerase Ii ◽

Cell Fate ◽

Transcriptional Repression ◽

Mammalian Cells ◽

Es Cells ◽

Embryonic Stem ◽

Antisense Transcription ◽

Chromatin Accessibility ◽

Promoter Architecture ◽

Neuronal Lineage

The conserved and essential histone chaperone FACT (Facilitates Chromatin Transcription) reorganizes nucleosomes during DNA transcription, replication and repair and ensures both, efficient elongation of RNA Pol II and nucleosome integrity. In mammalian cells, FACT is a heterodimer, consisting of SSRP1 and SUPT16. Here, we show that in contrast to yeast, FACT accumulates at the transcription start site of genes reminiscent of RNA Polymerase II profile. Depletion of FACT in mouse embryonic stem cells leads to up-regulation of pro-proliferative genes and key pluripotency factors concomitant with hyper-proliferation of mES cells. Using MNase-, ATAC-, and Nascent Elongating Transcript Sequencing (NET-seq) we show that up-regulation of genes coincides with loss of nucleosomes upstream of the TSS and concomitant increase in antisense transcription, indicating that FACT impacts the promoter architecture to regulate expression of these genes. Finally, we demonstrate a role for FACT in cell fate determination and show that FACT depletion primes ES cells for the neuronal lineage.

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Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions

10.1101/299073 ◽

2018 ◽

Cited By ~ 4

Author(s):

Daniel Strebinger ◽

Cédric Deluz ◽

Elias T. Friman ◽

Subashika Govindan ◽

Andrea B. Alber ◽

...

Keyword(s):

Transcription Factors ◽

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Directed Differentiation ◽

Es Cell ◽

Endogenous Fluctuations ◽

Cell Fate Decisions ◽

Oct4 Protein

AbstractSOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. However, how temporal fluctuations in their expression levels bias lineage commitment is unknown. Here we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cell towards both neuroectodermal and mesendodermal fates. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

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