scholarly journals Integrated analysis of Xist upregulation and X-chromosome inactivation with single-cell and single-allele resolution

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guido Pacini ◽  
Ilona Dunkel ◽  
Norbert Mages ◽  
Verena Mutzel ◽  
Bernd Timmermann ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Single Cell ◽  
X Chromosome ◽  
Embryonic Stem ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Cellular Heterogeneity ◽  
Integrated Analysis ◽  
X Chromosomes ◽  
Multiple Levels

AbstractTo ensure dosage compensation between the sexes, one randomly chosen X chromosome is silenced in each female cell in the process of X-chromosome inactivation (XCI). XCI is initiated during early development through upregulation of the long non-coding RNA Xist, which mediates chromosome-wide gene silencing. Cell differentiation, Xist upregulation and gene silencing are thought to be coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes. Here we perform an integrated analysis of all three processes through allele-specific single-cell RNA-sequencing. Specifically, we assess the onset of random XCI in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploiting the inter-cellular heterogeneity of XCI onset, we identify putative Xist regulators. Moreover, we show that transient Xist upregulation from both X chromosomes results in biallelic gene silencing right before transitioning to the monoallelic state, confirming a prediction of the stochastic model of XCI. Finally, we show that genetic variation modulates the XCI process at multiple levels, providing a potential explanation for the long-known X-controlling element (Xce) effect, which leads to preferential inactivation of a specific X chromosome in inter-strain crosses. We thus draw a detailed picture of the different levels of regulation that govern the initiation of XCI. The experimental and computational strategies we have developed here will allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.

scholarly journals Integrated analysis of Xist upregulation and gene silencing at the onset of random X-chromosome inactivation at high temporal and allelic resolution

2020 ◽  
Author(s):  
Guido Pacini ◽  
Ilona Dunkel ◽  
Norbert Mages ◽  
Verena Mutzel ◽  
Bernd Timmermann ◽  
...  
Keyword(s):  
Gene Silencing ◽  
X Chromosome ◽  
Embryonic Stem ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Cellular Heterogeneity ◽  
Integrated Analysis ◽  
High Temporal Resolution ◽  
Negative Feedback Regulation ◽  
Multiple Levels

AbstractTo ensure dosage compensation between the sexes, one randomly chosen X chromosome is silenced in each female cell in the process of X-chromosome inactivation (XCI). XCI is initiated during early development through upregulation of the long non-coding RNA Xist, which mediates chromosome-wide gene silencing. Cell differentiation, Xist upregulation and silencing are thought to be coupled at multiple levels to ensure inactivation of exactly one out of two X chromosomes. Here we perform an integrated analysis of all three processes through allele-specific single-cell RNA-sequencing. Specifically, we assess the onset of random XCI with high temporal resolution in differentiating mouse embryonic stem cells, and develop dedicated analysis approaches. By exploiting the inter-cellular heterogeneity of XCI onset, we identify Nanog downregulation as its main trigger and discover additional putative Xist regulators. Moreover, we confirm several predictions of the stochastic model of XCI where monoallelic silencing is thought to be ensured through negative feedback regulation. Finally, we show that genetic variation modulates the XCI process at multiple levels, providing a potential explanation for the long-known Xce effect, which leads to preferential inactivation of a specific X chromosome in inter-strain crosses. We thus draw a detailed picture of the different levels of regulation that govern the initiation of XCI. The experimental and computational strategies we have developed here will allow us to profile random XCI in more physiological contexts, including primary human cells in vivo.

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.

scholarly journals Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ava C Carter ◽  
Jin Xu ◽  
Meagan Y Nakamoto ◽  
Yuning Wei ◽  
Brian J Zarnegar ◽  
...  
Keyword(s):  
Gene Silencing ◽  
X Chromosome ◽  
Protein Interactions ◽  
Sex Chromosome ◽  
Embryonic Stem ◽  
Structural Similarity ◽  
Chromatin Accessibility ◽  
Endogenous Retrovirus ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation

The Xist lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an Xist-binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.

scholarly journals Epigenetic-structural changes in X chromosomes promote Xic pairing during early differentiation from mouse embryonic stem cells

2021 ◽  
Author(s):  
Tetsushi Komoto ◽  
Masashi Fujii ◽  
Akinori Awazu
Keyword(s):  
X Chromosome ◽  
Structural Changes ◽  
Es Cells ◽  
Embryonic Stem ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Coarse Grained ◽  
Dynamics Model ◽  
X Chromosomes ◽  
Inactivation Center

X chromosome inactivation center (Xic) pairing is robustly observed during the differentiation of embryonic stem (ES) cells from female mouse embryos, and this process is related to X chromosome inactivation, the circadian clock, intra-nucleus architecture, and metabolism. However, the mechanisms underlying the identification and approach of X chromosome pairs in the crowded nucleus are unclear. To elucidate the driving force of Xic pairing, we developed a coarse-grained molecular dynamics model of intranuclear chromosomes in ES cells and in cells 2 days after the onset of differentiation (2-days cells) by considering intrachromosome epigenetic-structural feature-dependent mechanics. The analysis of the experimental data showed X-chromosomes change to specifically softer than autosomes during the cell differentiation by the rearrangement of their distributions of open-close chromatin regions, and the simulations of these models exhibited such softening promoted the mutual approach of the Xic pair. These findings suggested that local intrachromosomal epigenetic features may contribute to the regulation of cell species-dependent differences in intranuclear architecture.

scholarly journals Species-specific differences in X chromosome inactivation in mammals

Reproduction ◽  
2013 ◽  
Vol 146 (4) ◽  
pp. R131-R139 ◽  
Author(s):  
Takashi Sado ◽  
Takehisa Sakaguchi
Keyword(s):  
X Chromosome ◽  
Molecular Basis ◽  
Embryonic Stem ◽  
X Chromosome Inactivation ◽  
X Inactivation ◽  
Chromosome Inactivation ◽  
X Chromosomes ◽  
Inactive X Chromosome ◽  
Species Specific ◽  
Genetically Manipulated

In female mammals, the dosage difference in X-linked genes between XX females and XY males is compensated for by inactivating one of the two X chromosomes during early development. Since the discovery of the X inactive-specific transcript (XIST) gene in humans and its subsequent isolation of the mouse homolog, Xist, in the early 1990s, the molecular basis of X chromosome inactivation (X-inactivation) has been more fully elucidated using genetically manipulated mouse embryos and embryonic stem cells. Studies on X-inactivation in other mammals, although limited when compared with those in the mice, have revealed that, while their inactive X chromosome shares many features with those in the mice, there are marked differences in not only some epigenetic modifications of the inactive X chromosome but also when and how X-inactivation is initiated during early embryonic development. Such differences raise the issue about what extent of the molecular basis of X-inactivation in the mice is commonly shared among others. Recognizing similarities and differences in X-inactivation among mammals may provide further insight into our understanding of not only the evolutionary but also the molecular aspects for the mechanism of X-inactivation. Here, we reviewed species-specific differences in X-inactivation and discussed what these differences may reveal.

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.

scholarly journals Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation

2019 ◽  
Author(s):  
Ava C. Carter ◽  
Jin Xu ◽  
Meagan Y. Nakamoto ◽  
Yuning Wei ◽  
Quanming Shi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
X Chromosome ◽  
Dosage Compensation ◽  
Sex Chromosome ◽  
Embryonic Stem ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Rna Domains ◽  
In Cis

Dosage compensation between the sexes has emerged independently multiple times during evolution, often harnessing long noncoding RNAs (lncRNAs) to alter gene expression on the sex chromosomes. In eutherian mammals, X chromosome inactivation (XCI) in females proceeds via the lncRNA Xist, which coats one of the two X chromosomes and recruits repressive proteins to epigenetically silence gene expression in cis1,2. How Xist evolved new functional RNA domains to recruit ancient, pleiotropic protein partners is of great interest. Here we show that Spen, an Xist-binding repressor protein essential for XCI3-7, binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen inactivation leads to de-repression of a subset of endogenous retroviral (ERV) elements in embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing8-9. ERV RNA and Xist A-repeat bind the RRM3 domain of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in local gene silencing in cis. These results suggest that insertion of an ERV element into proto-Xist may have been a critical evolutionary event, which allowed Xist to coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.

scholarly journals Escape from X Chromosome Inactivation and the Female Predominance in Autoimmune Diseases

2021 ◽  
Vol 22 (3) ◽  
pp. 1114
Author(s):  
Ali Youness ◽  
Charles-Henry Miquel ◽  
Jean-Charles Guéry
Keyword(s):  
Autoimmune Diseases ◽  
X Chromosome ◽  
Gene Dosage ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
X Chromosomes ◽  
Female Sex Hormones ◽  
Inactive X Chromosome ◽  
Immune Related Genes ◽  
Female Specific

Women represent 80% of people affected by autoimmune diseases. Although, many studies have demonstrated a role for sex hormone receptor signaling, particularly estrogens, in the direct regulation of innate and adaptive components of the immune system, recent data suggest that female sex hormones are not the only cause of the female predisposition to autoimmunity. Besides sex steroid hormones, growing evidence points towards the role of X-linked genetic factors. In female mammals, one of the two X chromosomes is randomly inactivated during embryonic development, resulting in a cellular mosaicism, where about one-half of the cells in a given tissue express either the maternal X chromosome or the paternal one. X chromosome inactivation (XCI) is however not complete and 15 to 23% of genes from the inactive X chromosome (Xi) escape XCI, thereby contributing to the emergence of a female-specific heterogeneous population of cells with bi-allelic expression of some X-linked genes. Although the direct contribution of this genetic mechanism in the female susceptibility to autoimmunity still remains to be established, the cellular mosaicism resulting from XCI escape is likely to create a unique functional plasticity within female immune cells. Here, we review recent findings identifying key immune related genes that escape XCI and the relationship between gene dosage imbalance and functional responsiveness in female cells.

Random X-chromosome inactivation in female primordial germ cells in the mouse

Development ◽  
1981 ◽  
Vol 64 (1) ◽  
pp. 251-258
Author(s):  
Andy McMahon ◽  
Mandy Fosten ◽  
Marilyn Monk
Keyword(s):  
X Chromosome ◽  
Germ Cells ◽  
Germ Line ◽  
Primordial Germ Cells ◽  
Phosphoglycerate Kinase ◽  
X Chromosome Inactivation ◽  
Yolk Sac ◽  
Chromosome Inactivation ◽  
X Chromosomes

The pattern of expression of the two X chromosomes was investigated in pre-meiotic germ cells from 12½-day-old female embryos heterozygous for the variant electrophoretic forms of the X-linked enzyme phosphoglycerate kinase (PGK-1). If such germ cells carry the preferentially active Searle's translocated X chromosome (Lyon, Searle, Ford & Ohno, 1964), then only the Pgk-1 allele on this chromosome is expressed. This confirms Johnston's evidence (1979,1981) that Pgk-1 expression reflects a single active X chromosome at this time. Extracts of 12½-day germ cells from heterozygous females carrying two normal X chromosomes show both the A and the B forms of PGK; since only one X chromosome in each cell is active, different alleles must be expressed in different cells, suggesting that X-chromosome inactivation is normally random in the germ line. This result makes it unlikely that germ cells are derived from the yolk-sac endoderm where the paternally derived X chromosome is preferentially inactivated. In their pattern of X-chromosome inactivation, germ cells evidently resemble other tissues derived from the epiblast.

Regulation of imprinted X-chromosome inactivation in mice by Tsix

Development ◽  
2001 ◽  
Vol 128 (8) ◽  
pp. 1275-1286 ◽  
Author(s):  
T. Sado ◽  
Z. Wang ◽  
H. Sasaki ◽  
E. Li
Keyword(s):  
X Chromosome ◽  
X Chromosome Inactivation ◽  
X Inactivation ◽  
Chromosome Inactivation ◽  
Maternal Allele ◽  
Xist Expression ◽  
Developmental Defects ◽  
X Chromosomes ◽  
Embryonic Tissues ◽  
Early Embryonic Lethality

In mammals, X-chromosome inactivation is imprinted in the extra-embryonic lineages with paternal X chromosome being preferentially inactivated. In this study, we investigate the role of Tsix, the antisense transcript from the Xist locus, in regulation of Xist expression and X-inactivation. We show that Tsix is transcribed from two putative promoters and its transcripts are processed. Expression of Tsix is first detected in blastocysts and is imprinted with only the maternal allele transcribed. The imprinted expression of Tsix persists in the extra-embryonic tissues after implantation, but is erased in embryonic tissues. To investigate the function of Tsix in X-inactivation, we disrupted Tsix by insertion of an IRES(β)geo cassette in the second exon, which blocked transcripts from both promoters. While disruption of the paternal Tsix allele has no adverse effects on embryonic development, inheritance of a disrupted maternal allele results in ectopic Xist expression and early embryonic lethality, owing to inactivation of both X chromosomes in females and single X chromosome in males. Further, early developmental defects of female embryos with maternal transmission of Tsix mutation can be rescued by paternal inheritance of the Xist deletion. These results provide genetic evidence that Tsix plays a crucial role in maintaining Xist silencing in cis and in regulation of imprinted X-inactivation in the extra-embryonic tissues.

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