scholarly journals Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

10.3791/51628 ◽  
2014 ◽  
Author(s):  
Tahsin Stefan Barakat ◽  
Joost Gribnau
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
X Chromosome ◽  
Female Mouse ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation

scholarly journals SPEN is required for Xist upregulation during initiation of X chromosome inactivation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Teresa Robert-Finestra ◽  
Beatrice F. Tan ◽  
Hegias Mira-Bontenbal ◽  
Erika Timmers ◽  
Cristina Gontan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
X Chromosome ◽  
Female Mouse ◽  
Antisense Transcript ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Chromatin Remodelers

AbstractAt initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen−/− ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in initiation of the XCI process.

scholarly journals SPEN is Required for Xist Upregulation during Initiation of X Chromosome Inactivation

2020 ◽  
Author(s):  
Teresa Robert-Finestra ◽  
Beatrice F. Tan ◽  
Hegias Mira-Bontenbal ◽  
Erika Timmers ◽  
Cristina Gontan-Pardo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
X Chromosome ◽  
Female Mouse ◽  
Antisense Transcript ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Chromatin Remodelers

AbstractAt initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in the initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen−/− ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in the initiation of the XCI process.

scholarly journals Elements at the 5′ end of Xist harbor SPEN-independent transcriptional antiterminator activity

2020 ◽  
Author(s):  
Jackson B. Trotman ◽  
David M. Lee ◽  
Rachel E. Cherney ◽  
Sue O. Kim ◽  
Kaoru Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Gene Silencing ◽  
X Chromosome ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Sequence Elements ◽  
Cleavage And Polyadenylation

AbstractThe Xist lncRNA requires Repeat A, a conserved RNA element located in its 5′ end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for the production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of multiple downstream polyadenylation sequences. Readthrough required Repeat A and the ~750 nucleotides downstream but did not require SPEN. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a transgene comprising Xist’s first 5.5 kilobases robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5′ end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes.

Aberrant Patterns of X Chromosome Inactivation in a New Line of Human Embryonic Stem Cells Established in Physiological Oxygen Concentrations

2014 ◽  
Vol 10 (4) ◽  
pp. 472-479 ◽  
Author(s):  
Juliana Andrea de Oliveira Georges ◽  
Naja Vergani ◽  
Simone Aparecida Siqueira Fonseca ◽  
Ana Maria Fraga ◽  
Joana Carvalho Moreira de Mello ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
X Chromosome ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Oxygen Concentrations

scholarly journals Histone H3 Lysine 36 Trimethylation Is Established over theXistPromoter by AntisenseTsixTranscription and Contributes to RepressingXistExpression

2015 ◽  
Vol 35 (22) ◽  
pp. 3909-3920 ◽  
Author(s):  
Tatsuya Ohhata ◽  
Mika Matsumoto ◽  
Martin Leeb ◽  
Shinwa Shibata ◽  
Satoshi Sakai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
X Chromosome ◽  
Antisense Rna ◽  
Histone H3 ◽  
Embryonic Stem ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
X Chromosomes ◽  
Histone H3.3

One of the two X chromosomes in female mammals is inactivated by the noncodingXistRNA. In mice, X chromosome inactivation (XCI) is regulated by the antisense RNATsix, which repressesXiston the active X chromosome. In the absence ofTsix, PRC2-mediated histone H3 lysine 27 trimethylation (H3K27me3) is established over theXistpromoter. Simultaneous disruption ofTsixand PRC2 leads to derepression ofXistand in turn silencing of the single X chromosome in male embryonic stem cells. Here, we identified histone H3 lysine 36 trimethylation (H3K36me3) as a modification that is recruited byTsixcotranscriptionally and extends over theXistpromoter. Reduction of H3K36me3 by expression of a mutated histone H3.3 with a substitution of methionine for lysine at position 36 causes a significant derepression ofXist. Moreover, depletion of the H3K36 methylaseSetd2leads to upregulation ofXist, suggesting H3K36me3 as a modification that contributes to the mechanism ofTsixfunction in regulating XCI. Furthermore, we found that reduction of H3K36me3 does not facilitate an increase in H3K27me3 over theXistpromoter, indicating that additional mechanisms exist by whichTsixblocks PRC2 recruitment to theXistpromoter.

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