scholarly journals Xist RNA Is Confined to the Nuclear Territory of the Silenced X Chromosome throughout the Cell Cycle

2008 ◽  
Vol 28 (18) ◽  
pp. 5583-5594 ◽  
Author(s):  
Iris Jonkers ◽  
Kim Monkhorst ◽  
Eveline Rentmeester ◽  
J. Anton Grootegoed ◽  
Frank Grosveld ◽  
...  
Keyword(s):  
Cell Cycle ◽  
X Chromosome ◽  
Es Cells ◽  
Embryonic Stem ◽  
Chromosome Analysis ◽  
Tetraploid Cell ◽  
Mouse Es Cells ◽  
Xist Rna ◽  
Mammalian Female ◽  
Dose Difference

ABSTRACT In mammalian female cells, one X chromosome is inactivated to prevent a dose difference in the expression of X-encoded proteins between males and females. Xist RNA, required for X chromosome inactivation, is transcribed from the future inactivated X chromosome (Xi), where it spreads in cis, to initiate silencing. We have analyzed Xist RNA transcription and localization throughout the cell cycle. It was found that Xist transcription is constant and that the mature RNA remains attached to the Xi throughout mitosis. Diploid and tetraploid cell lines with an MS2-tagged Xist gene were used to investigate spreading of Xist. Most XXXXMS2 tetraploid mouse embryonic stem (ES) cells inactivate the XMS2 chromosome and one other X chromosome. Analysis of cells with two Xi's indicates that Xist RNA is retained by the Xi of its origin and does not spread in trans. Also, in XXMS2 diploid mouse ES cells with an autosomal Xist transgene, there is no trans exchange of Xist RNA from the Xi to the autosome. We propose that Xist RNA does not dissociate from the Xi of its origin, which precludes a model of diffusion-mediated trans spreading of Xist RNA.

scholarly journals T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal

2012 ◽  
Vol 302 (3) ◽  
pp. C494-C504 ◽  
Author(s):  
José A. Rodríguez-Gómez ◽  
Konstantín L. Levitsky ◽  
José López-Barneo
Keyword(s):  
Cell Cycle ◽  
Alkaline Phosphatase ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Mrna Levels ◽  
Es Cell ◽  
External Application ◽  
Self Renewal ◽  
Mouse Es Cells

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na+ currents as well as transient Ca2+ currents abolished by the external application of Ni2+. Biophysical and pharmacological data indicated that the Ca2+ current is predominantly mediated by T-type (Cav3.2) channels. The number of cells expressing T-type channels and Cav3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca2+ currents with Ni2+ induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Cav3.2) Ca2+ channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Cav3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca2+ entry mediated by Cav3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.

scholarly journals Histone Macroh2a1.2 Relocates to the Inactive X Chromosome after Initiation and Propagation of X-Inactivation

1999 ◽  
Vol 147 (7) ◽  
pp. 1399-1408 ◽  
Author(s):  
Jacqueline E. Mermoud ◽  
Carl Costanzi ◽  
John R. Pehrson ◽  
Neil Brockdorff
Keyword(s):  
X Chromosome ◽  
Es Cells ◽  
Embryonic Stem ◽  
X Inactivation ◽  
Dense Region ◽  
Rich Domain ◽  
Inactive X Chromosome ◽  
Initiation And Propagation ◽  
Xist Rna

The histone macroH2A1.2 has been implicated in X chromosome inactivation on the basis of its accumulation on the inactive X chromosome (Xi) of adult female mammals. We have established the timing of macroH2A1.2 association with the Xi relative to the onset of X-inactivation in differentiating murine embryonic stem (ES) cells using immuno-RNA fluorescence in situ hybridization (FISH). Before X-inactivation we observe a single macroH2A1.2-dense region in both undifferentiated XX and XY ES cells that does not colocalize with X inactive specific transcript (Xist) RNA, and thus appears not to associate with the X chromosome(s). This pattern persists through early stages of differentiation, up to day 7. Then the frequency of XY cells containing a macroH2A1.2-rich domain declines. In contrast, in XX cells there is a striking relocalization of macroH2A1.2 to the Xi. Relocalization occurs in a highly synchronized wave over a 2-d period, indicating a precisely regulated association. The timing of macroH2A1.2 accumulation on the Xi suggests it is not necessary for the initiation or propagation of random X-inactivation.

scholarly journals BRCa1 participates in XIST RNa localization on inactive x chromosome

2019 ◽  
Vol 18 (2) ◽  
pp. 21-26
Author(s):  
E. A. Shestakova ◽  
T. A. Bogush
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Confocal Microscopy ◽  
X Chromosome ◽  
Fluorescent Microscopy ◽  
S Phase ◽  
Immunofluorescent Staining ◽  
Heterochromatin Protein ◽  
Inactive X Chromosome ◽  
Xist Rna

Introduction . Inactive X chromosome (Xi) is associated with noncoding XIST RNA, series of proteins and contains multiple epigenetic modifications that altogether determine a silence of the most of X-linked genes. Recently the data were obtained that tumor suppressor BRCA1 is also associated with Xi. The purpose of this study was to reveal the colocalization of BRCA1 and XIST RNA and precise spatial organization on Xi with the high resolution of confocal microscopy.Materials and methods . The object of the study is IMR90hTERT diploid immortalized fibroblast cell line. For BRCA1 and XIST RNA colocalization analysis on Xi the method of fluorescent hybridization in situ associated with immunofluorescent cell staining (immunoFISH) and confocal microscopy were used. For BRCA1 and heterochromatin protein-1 colocalization study the method of double immunofluorescent staining and common fluorescent microscopy were applied. Results . The study using confocal fluorescent microscopy with higher resolution has demonstrated at first the colocalization of BRCA1 with XIST RNA region of Xi revealed with XIST RNA probes and with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. Altogether, the data obtained suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the regulation of XIST RNA association with Xi. Moreover, according to the results of confocal microscopy, BRCA1 also colocalizes with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. This indicates a possible involvement of this protein in the replication of pericentromeric repeats in cellular chromosomes. Colocalization of BRCA1 with heterochromatin protein-1α presented in pericentromeric regions of all chromosomes supports this suggestion.Conclusions . Altogether, the data obtained in this study suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the association with noncoding inhibiting XIST RNA and in replication of heterochromatin regions. 

Human embryonic stem cells

2000 ◽  
Vol 113 (1) ◽  
pp. 5-10 ◽  
Author(s):  
M.F. Pera ◽  
B. Reubinoff ◽  
A. Trounson
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Embryo ◽  
Carcinoma Cells ◽  
Mouse Es Cells ◽  
Undifferentiated State ◽  
Human Es Cells

Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Candidate ES and EG cell lines from the human blastocyst and embryonic gonad can differentiate into multiple types of somatic cell. The phenotype of the blastocyst-derived cell lines is very similar to that of monkey ES cells and pluripotent human embryonal carcinoma cells, but differs from that of mouse ES cells or the human germ-cell-derived stem cells. Although our understanding of the control of growth and differentiation of human ES cells is quite limited, it is clear that the development of these cell lines will have a widespread impact on biomedical research.

X chromosome retains the memory of its parental origin in murine embryonic stem cells

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 813-821 ◽  
Author(s):  
T. Tada ◽  
M. Tada ◽  
N. Takagi
Keyword(s):  
X Chromosome ◽  
Polar Region ◽  
Biochemical Study ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Parental Origin ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Preferential Inactivation

A cytogenetic and biochemical study of balloon-like cystic embryoid bodies, formed by newly established embryonic stem (ES) cell lines having a cytogenetically or genetically marked X chromosome, revealed that the paternally derived X chromosome was inactivated in the majority of cells in the yolk sac-like mural region consisting of the visceral endoderm and mesoderm. The nonrandomness was less evident in the more solid polar region containing the ectodermal vesicle, mesoderm and visceral endoderm. Since the same was true in embryoid bodies derived from ES cells at the 30th subculture generation, it was concluded that the imprinting responsible for the preferential inactivation of the paternal X chromosome that was limited to non-epiblast cells of the female mouse embryos, was stably maintained in undifferentiated ES cells. Differentiating epiblast cells should be able to erase or avoid responding to the imprint.

2. Embryonic stem cells

2021 ◽  
pp. 21-37
Author(s):  
Jonathan Slack
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Practical Importance ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Human Embryos ◽  
Mouse Es Cells ◽  
Stage Embryo

‘Embryonic stem cells’ focuses on embryonic stem (ES) cells, which are grown in tissue culture from the inner cell mass of a mammalian blastocyst-stage embryo. Human ES cells offer a potential route to making the kinds of cells needed for cell therapy. ES cells were originally prepared from mouse embryos. Although somewhat different, cells grown from inner cell masses of human embryos share many properties with mouse ES cells, such as being able to grow without limit and to generate differentiated cell types. Mouse ES cells have so far been of greater practical importance than those of humans because they have enabled a substantial research industry based on the creation of genetically modified mice.

scholarly journals DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands

2020 ◽  
Vol 6 (35) ◽  
pp. eabb5820 ◽  
Author(s):  
Zhiming Li ◽  
Xu Hua ◽  
Albert Serra-Cardona ◽  
Xiaowei Xu ◽  
Songlin Gan ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Es Cells ◽  
Embryonic Stem ◽  
Endogenous Retroviruses ◽  
Histone Binding ◽  
Dna Polymerase Α ◽  
Mouse Es Cells ◽  
Dna Strands ◽  
Histone Deposition

How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication–linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.

Caveolin-1 plays important role in EGF-induced migration and proliferation of mouse embryonic stem cells: involvement of PI3K/Akt and ERK

2009 ◽  
Vol 297 (4) ◽  
pp. C935-C944 ◽  
Author(s):  
Jae Hong Park ◽  
Ho Jae Han
Keyword(s):  
Cell Cycle ◽  
Cell Migration ◽  
Kinase Inhibitor ◽  
Thymidine Incorporation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Akt Inhibitor ◽  
Expression Levels ◽  
Caveolin 1

The involvement of caveolin-1 in the regulation of embryonic stem (ES) cell growth by epidermal growth factor (EGF) is by no means clear cut. Thus we examined the relationship between EGF and caveolin-1 in mouse ES cell migration and proliferation. The results revealed that EGF increased Src, caveolin-1, focal adhesion kinase (FAK), Akt, and extracellular signal-regulated kinase-1/2 (ERK) phosphorylation levels. Especially, phosphorylation of caveolin-1 is attenuated by AG1478, herbimycin A (tyrosine kinase inhibitors), and pyrazolopyrimidine 2 (PP2, Src inhibitor) and EGF-induced ERK activation was blocked by PP2, methyl-β-cyclodextrin (MβCD), caveolin-1 small interfering RNA (siRNA), LY-294002 [phosphoinositol-3 kinase inhibitor (PI3K)], and Akt inhibitor. In addition, EGF promoted the cell migration, which was attenuated by PP2, caveolin-1 siRNA, FAK siRNA, LY-294002, Akt inhibitor, and PD-98059. EGF also increased matrix metalloproteinase (MMP-2) expression levels and EGF-induced MMP2 expression was inhibited by caveolin-1 siRNA, FAK siRNA, LY-294002, Akt inhibitor, and PD-98059. Furthermore, EGF-induced increase of cell cycle proteins expression level and [3H]thymidine incorporation was blocked by MMP inhibitor. EGF also significantly increases [3H]thymidine incorporation and cell number, which were significantly blocked by AG 1478, PP2, MβCD, caveolin-1 siRNA, FAK siRNA, LY-294002, and PD-98059 (ERK inhibitor). EGF-induced increase of protooncogenes (c- fos, c- myc, and c- Jun) and cell cycle regulatory proteins (cyclin D1, CDK4, cyclin E, and CDK2) expression levels were also attenuated by caveolin-1 siRNA and FAK siRNA. In conclusion, these results demonstrated that EGF-induced DNA synthesis and cell migration are mediated by caveolin-1, which is activated by Src, FAK, PI3K/Akt, ERK, and MMP-2 signals in mouse ES cells.

scholarly journals Recql5 and Blm RecQ DNA Helicases Have Nonredundant Roles in Suppressing Crossovers

2005 ◽  
Vol 25 (9) ◽  
pp. 3431-3442 ◽  
Author(s):  
Yiduo Hu ◽  
Xincheng Lu ◽  
Ellen Barnes ◽  
Min Yan ◽  
Hua Lou ◽  
...  
Keyword(s):  
Cancer Susceptibility ◽  
Es Cells ◽  
Embryonic Stem ◽  
Bloom Syndrome ◽  
Embryonic Fibroblasts ◽  
Dna Helicases ◽  
Mouse Es Cells ◽  
Elevated Rate ◽  
Wild Type Control ◽  
Mitotic Cells

ABSTRACT In eukaryotes, crossovers in mitotic cells can have deleterious consequences and therefore must be suppressed. Mutations in BLM give rise to Bloom syndrome, a disease that is characterized by an elevated rate of crossovers and increased cancer susceptibility. However, simple eukaryotes such as Saccharomyces cerevisiae have multiple pathways for suppressing crossovers, suggesting that mammals also have multiple pathways for controlling crossovers in their mitotic cells. We show here that in mouse embryonic stem (ES) cells, mutations in either the Bloom syndrome homologue (Blm) or the Recql5 genes result in a significant increase in the frequency of sister chromatid exchange (SCE), whereas deleting both Blm and Recql5 lead to an even higher frequency of SCE. These data indicate that Blm and Recql5 have nonredundant roles in suppressing crossovers in mouse ES cells. Furthermore, we show that mouse embryonic fibroblasts derived from Recql5 knockout mice also exhibit a significantly increased frequency of SCE compared with the corresponding wild-type control. Thus, this study identifies a previously unknown Recql5-dependent, Blm-independent pathway for suppressing crossovers during mitosis in mice.

182. NOVEL SIGNALLING IN MOUSE EMBRYONIC STEM CELLS GENERATES PRIMITIVE ECTODERM-LIKE CELLS

2009 ◽  
Vol 21 (9) ◽  
pp. 100
Author(s):  
M. B. Morris ◽  
N. Hamra ◽  
A. C. Lonic ◽  
F. Felquer
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Leukaemia Inhibitory Factor ◽  
Signalling Pathways ◽  
Specific Cell ◽  
Self Renewal ◽  
Mouse Es Cells ◽  
Homogeneous Production ◽  
Extracellular Molecules

The phenotypic status of embryonic stem (ES) cells is controlled in part by signalling pathways which translate inputs mediated by extracellular molecules. An important extracellular protagonist in mouse ES cells is LIF (leukaemia inhibitory factor) which interacts with the gp130–LIFR receptor complex to activate a number of downstream signalling pathways, including the STAT3, MEK/ERK and PI3K/Akt. These pathways, together with others, interact in complex and sometimes competing ways to generate the well-known characteristics of mouse ES cells of self-renewal, high rates of proliferation, and pluripotence. The addition of a second molecule, L-proline, to the extracellular environment alters the pluripotent status of mouse ES cells, converting them to a second pluripotent population equivalent to the primitive ectoderm of the pre-gastrulating embryo. This conversion, from ES cells to primitive ectoderm-like cells, primes the latter for directed differentiation to specific cell types (1). Here we show, using inhibitor studies and kinome array analysis, that this small molecule appears to work by (i) changing the balance in activity of signalling pathways already stimulated by LIF and (ii) activating additional signalling pathways. Specifically, L-proline rapidly further activates the LIF-stimulated MEK/ERK pathway, tipping the balance in favour of primitive-ectoderm formation and away from ES-cell self-renewal sustained by LIF-mediated activation of the STAT3 pathway. In addition, L-proline rapidly stimulates other pathways including p38, mTOR and PI3K/Akt each of which contributes, to a greater or lesser extent, to the conversion to primitive ectoderm-like cells. These results indicate that (i) L-proline acts in novel ways to stimulate embryo-like developmental progression in ES cells and (ii) through the addition of small, nontoxic activators and inhibitors of signalling pathways, the differentiation of pluripotent ES cells might be controlled sufficiently well for the homogeneous production of specific cell types suitable for use in animal models of human disease.

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