scholarly journals H3K9 tri-methylation at Nanog times differentiation commitment and enables the acquisition of primitive endoderm fate

2021 ◽  
Author(s):  
Agnes Dubois ◽  
Loris Vincenti ◽  
Sandrine Vandormael-Pournin ◽  
Michel Cohen-Tannoudji ◽  
Pablo Navarro
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Endoderm ◽  
Cell Fates ◽  
Master Regulators ◽  
Nanog Expression ◽  
Impaired Ability ◽  
Gene Regulatory ◽  
Erk Activity ◽  
Pluripotency Genes

Mouse Embryonic Stem (ES) cells have an inherent propensity to explore distinct gene-regulatory states associated with either self-renewal or differentiation. This property is largely dependent on ERK activity, which promotes silencing of pluripotency genes, most notably of the transcription factor Nanog. Here, we aimed at identifying repressive histone modifications that would mark the Nanog locus for inactivation in response to ERK activity. We found histone H3 lysine 9 tri-methylation (H3K9me3) focally enriched between the Nanog promoter and its -5kb enhancer. While in undifferentiated ES cells H3K9me3 at Nanog depends on ERK activity, in somatic cells it becomes ERK-independent. Moreover, upon deletion of the region harbouring H3K9me3, ES cells display reduced heterogeneity of NANOG expression, delayed commitment into differentiation and impaired ability to acquire a primitive endoderm fate. We suggest that establishment of irreversible H3K9me3 at specific master regulators allows the acquisition of particular cell fates during differentiation.

scholarly journals The Grb2/Mek Pathway Represses Nanog in Murine Embryonic Stem Cells

2006 ◽  
Vol 26 (20) ◽  
pp. 7539-7549 ◽  
Author(s):  
Takashi Hamazaki ◽  
Sarah M. Kehoe ◽  
Toru Nakano ◽  
Naohiro Terada
Keyword(s):  
Es Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Cell Aggregation ◽  
Cell Aggregates ◽  
Es Cell ◽  
Sodium Vanadate ◽  
Primitive Endoderm ◽  
Endoderm Differentiation

ABSTRACT The homeobox gene Nanog is a key intrinsic determinant of self renewal in embryonic stem (ES) cells, and its repression leads ES cells to selectively differentiate into primitive endoderm. Although Nanog repression occurs at the outermost layer of ES cell aggregates independent of the leukemia inhibitory factor (LIF)/STAT3 pathway, it is largely undetermined what external cues and intracellular signals cause the event. Of interest, addition of the tyrosine phosphatase inhibitor, sodium vanadate, selectively repressed Nanog transcription without any detectable changes in upstream transcriptional regulators Oct3/4 and Sox2. Furthermore, sodium vanadate induced primitive endoderm differentiation, even in the inner cells of ES cell aggregates. Expression of Gata6 and Zfp42, two putative downstream Nanog effectors, was also increased and decreased by the addition of sodium vanadate, respectively, but these changes were eliminated by exogenous Nanog expression. The effects of sodium vanadate were abrogated by Grb2 deficiency or by the addition of the Mek inhibitor, PD98059. Indeed, PD98059 prevented Nanog repression induced by ES cell aggregation as well. Furthermore, transfection of a constitutive active Mek mutant into ES cells induced Nanog repression and primitive endoderm differentiation. These data indicate that the Grb2/Mek pathway primarily mediates Nanog gene repression upon ES cell differentiation into primitive endoderm.

Transcription factors that behave as master regulators during mammalian embryogenesis function as molecular rheostats

2008 ◽  
Vol 411 (2) ◽  
pp. e5-e7 ◽  
Author(s):  
Angie Rizzino
Keyword(s):  
Transcription Factors ◽  
Molecular Mechanisms ◽  
Es Cells ◽  
Embryonic Stem ◽  
Special Property ◽  
The Self ◽  
Leukaemia Inhibitory Factor ◽  
Self Renewal ◽  
Master Regulators ◽  
Mammalian Embryogenesis

Three transcription factors, Sox2, Oct-3/4 and Nanog, have been identified as master regulators that orchestrate mammalian embryogenesis as well as the self-renewal and pluripotency of ES (embryonic stem) cells. Efforts to understand how these transcription factors function have shown that they have a special property in common. Small changes in the expression of any one of these factors dramatically alter the self-renewal and pluripotency of ES cells. In this way, each functions as a molecular rheostat to control the behaviour of ES cells. Recent studies have begun to examine the molecular mechanisms that regulate the levels of these transcription factors. In this issue of the Biochemical Journal, Mullin and co-workers report that Nanog can self-associate to form dimers. Importantly, they also show that the domain responsible for dimerization is also needed for Nanog to sustain the self-renewal of ES cells in the absence of the cytokine LIF (leukaemia inhibitory factor). On the basis of their studies, they propose a novel mechanism for regulating the interactions between Nanog and other nuclear proteins.

scholarly journals Nanog fluctuations in ES cells highlight the problem of measurement in cell biology

2016 ◽  
Author(s):  
Rosanna C G Smith ◽  
Patrick S Stumpf ◽  
Sonya J Ridden ◽  
Aaron Sim ◽  
Sarah Filippi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Biology ◽  
Measurement Problem ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Genetically Engineered ◽  
Live Cells ◽  
Reporter Cell ◽  
Nanog Expression

A number of important pluripotency regulators, including the transcription factor Nanog, are observed to fluctuate stochastically in individual embryonic stem (ES) cells. By transiently priming cells for commitment to different lineages, these fluctuations are thought to be important to the maintenance of, and exit from, pluripotency. However, since temporal changes in intracellular protein abundances cannot be measured directly in live cells, these fluctuations are typically assessed using genetically engineered reporter cell lines that produce a fluorescent signal as a proxy for protein expression. Here, using a combination of mathematical modeling and experiment, we show that there are unforeseen ways in which widely used reporter strategies can systemically disturb the dynamics they are intended to monitor, sometimes giving profoundly misleading results. In the case of Nanog we show how genetic reporters can compromise the behavior of important pluripotency-sustaining positive feedback loops, and induce a bifurcation in the underlying dynamics that gives rise to heterogeneous Nanog expression patterns in reporter cell lines that are not representative of the wild-type. These findings help explain the range of published observations of Nanog variability and highlight a fundamental measurement problem in cell biology.

scholarly journals Networks of enhancers and microRNAs drive variation in cell states

2019 ◽  
Author(s):  
Meenakshi Chakraborty ◽  
Sofia Hu ◽  
Marco Del Giudice ◽  
Andrea De Martino ◽  
Carla Bosia ◽  
...  
Keyword(s):  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Feedback Loop ◽  
Embryonic Stem ◽  
Developmental Expression ◽  
Protein Coding ◽  
Pluripotency Factors ◽  
Gene Regulatory ◽  
Cell Variation ◽  
Pluripotency Genes

AbstractCell-to-cell variation in gene expression is a common feature of developmental processes. Yet, it remains unclear whether molecular mediators can generate variation and how this process is coordinated across loci to allow the emergence of new cell states. Using embryonic stem cells (ESCs) as a model of development, we found interconverting cell states that resemble developmental expression programs and vary in activity at specific enhancers, such as those regulating pluripotency genes Nanog and Sox2 but not Pou5f1 (Oct4). Variable enhancers drive expression of variable genes, including those encoding microRNAs (miRNAs). Notably, variable miRNAs increase cell-to-cell variation by acting on neighborhoods of pluripotency genes. The encoded, variable pluripotency factors bind variable enhancers, forming a feedback loop that amplifies variation and allows the emergence of new cell states. These findings suggest gene regulatory networks composed of enhancers, protein-coding genes, and miRNAs harness inherent variation into developmental outcomes.

scholarly journals Npac Is a Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse ES Cells

2020 ◽  
Author(s):  
Sue Yu ◽  
Jia Li ◽  
Guanxu Ji ◽  
Zhen Long Ng ◽  
Jiamin Siew ◽  
...  
Keyword(s):  
Es Cells ◽  
Chromatin Modification ◽  
Embryonic Stem ◽  
Transcriptional Elongation ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Mouse Es Cells ◽  
Reprogramming Efficiency ◽  
Pluripotency Maintenance ◽  
Pluripotency Genes

AbstractChromatin modification contributes to pluripotency maintenance in embryonic stem cells (ESCs). However, the related mechanisms remain obscure. Here, we show that Npac, a “reader” of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC pluripotency since knockdown of Npac causes mouse ESC differentiation. Depletion of Npac in mouse embryonic fibroblasts (MEFs) inhibits reprogramming efficiency. Furthermore, our Npac ChIP-seq results reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs. Interestingly, we find that Npac interacts with p-TEFb, RNA Pol II Ser2 and Ser5. Depletion of Npac disrupts transcriptional elongation of pluripotency genes Nanog and Rif1. Taken together, we propose that Npac is essential for transcriptional elongation of pluripotency genes by recruiting of p-TEFb and interacting with RNA Pol II Ser2 and Ser5.

Stem cells and lineage development in the mammalian blastocyst

2007 ◽  
Vol 19 (1) ◽  
pp. 111 ◽  
Author(s):  
Janet Rossant
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Es Cells ◽  
Mammalian Species ◽  
Embryonic Stem ◽  
Cell Types ◽  
Mouse Cell ◽  
Embryonic Cell ◽  
Primitive Endoderm ◽  
Key Factors

The mammalian blastocyst is the source of the most pluripotent stem cells known: embryonic stem (ES) cells. However, ES cells are not totipotent; in mouse chimeras, they do not contribute to extra-embryonic cell types of the trophectoderm (TE) and primitive endoderm (PrE) lineages. Understanding the genetic pathways that control pluripotency v. extra-embryonic lineage restriction is key to understanding not only normal embryonic development, but also how to reprogramme adult cells to pluripotency. The trophectoderm and primitive endoderm lineages also provide the first signals that drive patterned differentiation of the pluripotent epiblast cells of the embryo. My laboratory has produced permanent mouse cell lines from both the TE and the PrE, termed trophoblast stem (TS) and eXtra-embryonic ENdoderm (XEN) cells. We have used these cells to explore the genetic and molecular hierarchy of lineage restriction and identify the key factors that distinguish the ES cell v. the TS or XEN cell fate. The major molecular pathways of lineage commitment defined in mouse embryos and stem cells are probably conserved across mammalian species, but more comparative studies of lineage development in embryos of non-rodent mammals will likely yield interesting differences in terms of timing and details.

scholarly journals Characterisation of interactions between the pluripotency transcription factors Nanog, Oct4 and Sox2

2020 ◽  
Author(s):  
Tapan Kumar Mistri ◽  
David Kelly ◽  
John Mak ◽  
Douglas Colby ◽  
Nicholas Mullin ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Gene Regulatory Network ◽  
Single Molecule ◽  
Regulatory Network ◽  
Es Cells ◽  
Control Cell ◽  
Embryonic Stem ◽  
Correlation Spectroscopy ◽  
Dependent Manner ◽  
Gene Regulatory

ABSTRACTThe pluripotency transcription factors (TFs) Nanog, Sox2, and Oct4 are at the centre of the gene regulatory network that controls cell identity in embryonic stem (ES) cells. However, the mechanisms by which these factors control cell fate, and their interactions with one another are not fully understood. Here we combine biophysical and novel biochemical assays to assess how these factors interact with each other quantitatively. A new confocal microscopy method to detect binding of a target protein to a fluorescently labelled partner (coimmunoprecipitation bead imaging microscopy [CBIM]) is presented and used to demonstrate homotypic binding of Nanog and heterotypic binding between Nanog and Sox2 and between Nanog and Oct4. Using fluorescence correlation spectroscopy we show that in solution, Nanog but not Oct4 or Sox2 can form homodimers. Fluorescence Cross Correlation Spectroscopy shows that the affinity of Nanog for dimer formation is in the order Sox2 > Nanog > Oct4. Importantly, live cell analysis demonstrate the existence of Nanog homodimers in vivo. While Sox2 and Oct4 bind one another in a DNA-dependent manner, Nanog appears not to bridge Sox2 and Oct4, even though Nanog binds both Sox2 and Oct4 individually. Together these findings extend understanding of the molecular interactions occurring between these central mediators of the pluripotency gene regulatory network at the single molecule level.

scholarly journals Simplified MethylRAD Sequencing to Detect Changes in DNA Methylation at Enhancer Elements in Differentiating Embryonic Stem Cells

Epigenomes ◽  
2020 ◽  
Vol 4 (4) ◽  
pp. 24
Author(s):  
Debapriya Saha ◽  
Allison B. Norvil ◽  
Nadia A. Lanman ◽  
Humaira Gowher
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Bioinformatic Analysis ◽  
Analysis Tool ◽  
Regulatory Regions ◽  
Genome Wide ◽  
Gene Regulatory ◽  
Pluripotency Genes

Differential DNA methylation is characteristic of gene regulatory regions, such as enhancers, which mostly constitute low or intermediate CpG content in their DNA sequence. Consequently, quantification of changes in DNA methylation at these sites is challenging. Given that DNA methylation across most of the mammalian genome is maintained, the use of genome-wide bisulfite sequencing to measure fractional changes in DNA methylation at specific sites is an overexertion which is both expensive and cumbersome. Here, we developed a MethylRAD technique with an improved experimental plan and bioinformatic analysis tool to examine regional DNA methylation changes in embryonic stem cells (ESCs) during differentiation. The transcriptional silencing of pluripotency genes (PpGs) during ESC differentiation is accompanied by PpG enhancer (PpGe) silencing mediated by the demethylation of H3K4me1 by LSD1. Our MethylRAD data show that in the presence of LSD1 inhibitor, a significant fraction of LSD1-bound PpGe fails to gain DNA methylation. We further show that this effect is mostly observed in PpGes with low/intermediate CpG content. Underscoring the sensitivity and accuracy of MethylRAD sequencing, our study demonstrates that this method can detect small changes in DNA methylation in regulatory regions, including those with low/intermediate CpG content, thus asserting its use as a method of choice for diagnostic purposes.

scholarly journals Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells

2010 ◽  
Vol 2010 ◽  
pp. 1-15 ◽  
Author(s):  
Callinice D. Capo-chichi ◽  
Jennifer L. Smedberg ◽  
Malgorzata Rula ◽  
Emmanuelle Nicolas ◽  
Anthony T. Yeung ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Gene Expression Pattern ◽  
Cell Types ◽  
Primitive Endoderm ◽  
Gata Factors ◽  
Endoderm Differentiation ◽  
Collagen Iii ◽  
Hepatocyte Nuclear Factor 4

Background. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonic primitive endoderm. Dab2, Laminin, GATA4, GATA5, and GATA6 are expressed in embryonic primitive endoderm and play critical roles in its lineage commitment.Results. We found that in the absence of GATA4 or GATA5, RA-induced primitive endoderm differentiation of ES cells was reduced. GATA4 (−/−) ES cells express higher level of GATA5, GATA6, and hepatocyte nuclear factor 4 alpha marker of visceral endoderm lineage. GATA5 (−/−) ES cells express higher level of alpha fetoprotein marker of early liver development. GATA6 (−/−) ES cells express higher level of GATA5 as well as mesoderm and cardiomyocyte markers which are collagen III alpha-1 and tropomyosin1 alpha. Thus, deletion of GATA6 precluded endoderm differentiation but promoted mesoderm lineages.Conclusions. GATA4, GATA5, and GATA6 each convey a unique gene expression pattern and influences ES cell differentiation. We showed that ES cells can be directed to avoid differentiating into primitive endoderm and to adopt unique lineages in vitro by modulating GATA factors. The finding offers a potential approach to produce desirable cell types from ES cells, useful for regenerative cell therapy.

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