scholarly journals Xist spatially amplifies SHARP recruitment to balance chromosome-wide silencing and specificity to the X chromosome

2021 ◽  
Author(s):  
Joanna W Jachowicz ◽  
Mackenzie Strehle ◽  
Abhik K Banerjee ◽  
Mario R Blanco ◽  
Jasmine Thai ◽  
...  
Keyword(s):  
Gene Silencing ◽  
X Chromosome ◽  
Target Genes ◽  
General Mechanism ◽  
X Chromosomes ◽  
Xist Rna ◽  
Mammalian Genomes ◽  
Males And Females ◽  
Amplification Mechanism ◽  
Low Levels

Although thousands of lncRNAs are encoded in mammalian genomes, their mechanisms of action are largely uncharacterized because they are often expressed at significantly lower levels than their proposed targets. One such lncRNA is Xist, which mediates chromosome-wide gene silencing on one of the two X chromosomes to achieve gene expression balance between males and females. How a limited number of Xist molecules can mediate robust silencing of a significantly larger number of target genes (~1 Xist RNA: 10 gene targets) while maintaining specificity to genes on the X within each cell is unknown. Here, we show that Xist drives non-stoichiometric recruitment of the essential silencing protein SHARP (also called Spen) to amplify its abundance across the inactive X, including at regions not directly occupied by Xist. This amplification is achieved through concentration dependent homotypic assemblies of SHARP on the X and is required for chromosome-wide silencing. We find that expressing Xist at higher levels leads to increased localization at autosomal regions, demonstrating that low levels of Xist are critical for ensuring its specificity to the X chromosome. We show that Xist (through SHARP) acts to suppress production of its own RNA which may act to constrain overall RNA levels and restrict its ability to spread beyond the X. Together, our results demonstrate a spatial amplification mechanism that allows Xist to achieve two essential but countervailing regulatory objectives: chromosome-wide gene silencing and specificity to the X. Our results suggest that this spatial amplification mechanism may be a more general mechanism by which other low abundance lncRNAs can balance specificity to, and robust control of, their regulatory targets.

scholarly journals Localized accumulation of Xist RNA in X chromosome inactivation

Open Biology ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 190213 ◽  
Author(s):  
Neil Brockdorff
Keyword(s):  
X Chromosome ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
X Chromosomes ◽  
Non Coding Rna ◽  
Xist Rna ◽  
Analogous Manner ◽  
Mammalian Embryos ◽  
Non Coding Rnas ◽  
In Cis

The non-coding RNA Xist regulates the process of X chromosome inactivation, in which one of the two X chromosomes present in cells of early female mammalian embryos is selectively and coordinately shut down. Remarkably Xist RNA functions in cis , affecting only the chromosome from which it is transcribed. This feature is attributable to the unique propensity of Xist RNA to accumulate over the territory of the chromosome on which it is synthesized, contrasting with the majority of RNAs that are rapidly exported out of the cell nucleus. In this review I provide an overview of the progress that has been made towards understanding localized accumulation of Xist RNA, drawing attention to evidence that some other non-coding RNAs probably function in a highly analogous manner. I describe a simple model for localized accumulation of Xist RNA and discuss key unresolved questions that need to be addressed in future studies.

Review Lecture: Mechanisms and evolutionary origins of variable X-chromosome activity in mammals

1974 ◽  
Vol 187 (1088) ◽  
pp. 243-268 ◽  
Keyword(s):  
X Chromosome ◽  
Germ Cells ◽  
Gene Dosage ◽  
Control Mechanisms ◽  
Compensation Mechanism ◽  
X Chromosomes ◽  
Animal Group ◽  
Evolutionary Origins ◽  
Genetic Activity ◽  
Males And Females

The X-chromosome of mammals is remarkable for its variable genetic activity. In somatic cells only a single X-chromosome is active, no matter how many are present, thus providing a dosage compensation mechanism by which males and females effectively have the same gene dosage of X-linked genes. In germ cells, however, it appears that all X-chromosomes present are active. Female germ cells require the presence of two X-chromosomes for normal survival, whereas male germ cells die if they have more than one X-chromosome. This system is found in all eutherian mammals and in marsupials, but is not known in any other animal group. In marsupials the X-chromosome derived from the father seems to be preferentially inactivated, whereas in eutherian mammals that from either parent may be so in different cells of the same animal. The differentiation of a particular X-chromosome as active or inactive is initiated in early embryogeny, and thereafter maintained through all further cell divisions in that individual. The mechanisms by which this is achieved are of great interest in relation to genetic control mechanisms in general. Various recent hypotheses concerning these mechanisms are discussed.

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

2020 ◽  
Vol 48 (18) ◽  
pp. 10500-10517
Author(s):  
Jackson B Trotman ◽  
David M Lee ◽  
Rachel E Cherney ◽  
Susan O Kim ◽  
Kaoru Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Gene Silencing ◽  
X Chromosome ◽  
Embryonic Stem ◽  
X Chromosome Inactivation ◽  
Xist Rna ◽  
Sequence Elements ◽  
Cleavage And Polyadenylation ◽  
Xist Locus

Abstract The 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 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 downstream polyadenylation sequences. Readthrough required Repeat A and the ∼750 nucleotides downstream, did not require SPEN, and was attenuated by splicing. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a 5.5-kb Xist transgene 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 Signatures of replication timing, recombination, and sex in the spectrum of rare variants on the human X chromosome and autosomes

2019 ◽  
Vol 116 (36) ◽  
pp. 17916-17924 ◽  
Author(s):  
Ipsita Agarwal ◽  
Molly Przeworski
Keyword(s):  
X Chromosome ◽  
Rare Variants ◽  
Low Frequency ◽  
Replication Timing ◽  
Double Strand Breaks ◽  
X Chromosomes ◽  
Strand Breaks ◽  
Biochemical Processes ◽  
Males And Females ◽  
Mutational Processes

The sources of human germline mutations are poorly understood. Part of the difficulty is that mutations occur very rarely, and so direct pedigree-based approaches remain limited in the numbers that they can examine. To address this problem, we consider the spectrum of low-frequency variants in a dataset (Genome Aggregation Database, gnomAD) of 13,860 human X chromosomes and autosomes. X-autosome differences are reflective of germline sex differences and have been used extensively to learn about male versus female mutational processes; what is less appreciated is that they also reflect chromosome-level biochemical features that differ between the X and autosomes. We tease these components apart by comparing the mutation spectrum in multiple genomic compartments on the autosomes and between the X and autosomes. In so doing, we are able to ascribe specific mutation patterns to replication timing and recombination and to identify differences in the types of mutations that accrue in males and females. In particular, we identify C > G as a mutagenic signature of male meiotic double-strand breaks on the X, which may result from late repair. Our results show how biochemical processes of damage and repair in the germline interact with sex-specific life history traits to shape mutation patterns on both the X chromosome and autosomes.

scholarly journals A system for imaging the regulatory noncoding Xist RNA in living mouse embryonic stem cells

2011 ◽  
Vol 22 (14) ◽  
pp. 2634-2645 ◽  
Author(s):  
Karen Ng ◽  
Nathalie Daigle ◽  
Aurélien Bancaud ◽  
Tatsuya Ohhata ◽  
Peter Humphreys ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
X Chromosome ◽  
Single Point ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Chromatin Domain ◽  
Dynamic Binding ◽  
X Chromosomes ◽  
Xist Rna

In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2‑tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome‑bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement.

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 Xist-seeded nucleation sites form local concentration gradients of silencing proteins to inactivate the X-chromosome

2020 ◽  
Author(s):  
Yolanda Markaki ◽  
Johnny Gan Chong ◽  
Christy Luong ◽  
Shawn Y.X. Tan ◽  
Yuying Wang ◽  
...  
Keyword(s):  
Gene Silencing ◽  
X Chromosome ◽  
Effector Proteins ◽  
Local Concentration ◽  
Concentration Gradients ◽  
Rna Molecules ◽  
Intrinsically Disordered ◽  
Chromatin Interactions ◽  
Non Coding Rna ◽  
Xist Rna

AbstractThe long non-coding RNA Xist exploits numerous effector proteins to progressively induce gene silencing across the X chromosome and form the inactive X (Xi)-compartment. The mechanism underlying formation of the chromosome-wide Xi-compartment is poorly understood. Here, we find that formation of the Xi-compartment is induced by ∼50 locally confined granules, where two Xist RNA molecules nucleate supra-molecular complexes (SMCs) of interacting proteins. Xist-SMCs are transient structures that concentrate rapidly recycling proteins in the X by increasing protein binding affinity. We find that gene silencing originates at Xist-SMCs and propagates across the entire chromosome over time, achieved by Polycomb-mediated coalescence of chromatin regions and aggregation, via its intrinsically disordered domains, of the critical silencing factor SPEN. Our results suggest a new model for X chromosome inactivation, in which Xist RNA induces macromolecular crowding of heterochromatinizing proteins near distinct sites which ultimately increases their density throughout the chromosome. This mechanism enables deterministic gene silencing without the need for Xist ribonucleoprotein complex-chromatin interactions at each target gene.

scholarly journals FITNESS EFFECTS OF EMS-INDUCED MUTATIONS ON THE X CHROMOSOME OF DROSOPHILA MELANOGASTER. I. VIABILITY EFFECTS AND HETEROZYGOUS FITNESS EFFECTS

Genetics ◽  
1977 ◽  
Vol 87 (4) ◽  
pp. 763-774
Author(s):  
Joyce A Mitchell
Keyword(s):  
Drosophila Melanogaster ◽  
X Chromosome ◽  
Strongly Correlated ◽  
Induced Mutations ◽  
X Chromosomes ◽  
Heterozygous Condition ◽  
Fitness Effects ◽  
Sucrose Solutions ◽  
Discrete Generation ◽  
Males And Females

ABSTRACT Drosophila melanogaster X chromosomes were mutagenized by feeding males sucrose solutions containing ethyl methanesulfonate (EMS); the concentrations of EMS in the food were 2.5 mm, 5.0 mm, and 10.0 mm. Chromosomes were exposed to the mutagen up to three times by treating males in succeeding generations. After treatment, the effective exposures were 2.5, 5.0, 7.5, 10.0, 15.0, and 30.0 mm EMS. X chromosomes treated in this manner were tested for effects on fitness in both hemizygous and heterozygous conditions, and for effects on viability in hemizygous and homozygous conditions. In addition, untreated X chromosomes were available for study. The viability and heterozygous fitness effects are presented in this paper, and the hemizygous fitness effects are discussed in the accompanying one (Mitchell and Simmons 1977). Hemizygous and homozygous viability effects were measured by segregation tests in vial cultures. For hemizygous males, viability was reduced 0.5 percent per mm EMS treatment; for homozygous females, it was reduced 0.7 percent per mm treatment. The decline in viability appeared to be a linear function of EMS dose. The viabilities of males and females were strongly correlated. Heterozygous fitness effects were measured by monitoring changes in the frequencies of treated and untreated X chromosomes in discrete generation populations which, through the use of an X-Y translocation, maintained them only in heterozygous condition. Flies that were heterozygous for a treated chromosome were found to be 0.4 percent less fit per m m EMS than flies heterozygous for an untreated one.

scholarly journals Ectopic Splicing Disturbs the Function of Xist RNA to Establish the Stable Heterochromatin State

2021 ◽  
Vol 9 ◽  
Author(s):  
Ruka Matsuura ◽  
Tatsuro Nakajima ◽  
Saya Ichihara ◽  
Takashi Sado
Keyword(s):  
Gene Silencing ◽  
X Chromosome ◽  
Molecular Basis ◽  
Essential Role ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Inactive State ◽  
Xist Rna ◽  
In Cis ◽  
Insight Into

Non-coding Xist RNA plays an essential role in X chromosome inactivation (XCI) in female mammals. It coats the X chromosome in cis and mediates the recruitment of many proteins involved in gene silencing and heterochromatinization. The molecular basis of how Xist RNA initiates chromosomal silencing and what proteins participate in this process has been extensively studied and elucidated. Its involvement in the establishment and maintenance of the X-inactivated state is, however, less understood. The XistIVS allele we previously reported is peculiar in that it can initiate XCI but fails to establish the inactive state that is stably maintained and, therefore, may provide an opportunity to explore how Xist RNA contributes to establish a robust heterochromatin state. Here we demonstrate that ectopic splicing taking place to produce XistIVS RNA disturbs its function to properly establish stable XCI state. This finding warrants the potential of XistIVS RNA to provide further insight into our understanding of how Xist RNA contributes to establish sustainable heterochromatin.

scholarly journals Advances in understanding chromosome silencing by the long non-coding RNA Xist

2013 ◽  
Vol 368 (1609) ◽  
pp. 20110325 ◽  
Author(s):  
Takashi Sado ◽  
Neil Brockdorff
Keyword(s):  
X Chromosome ◽  
Classical Model ◽  
X Inactivation ◽  
X Chromosomes ◽  
Current State ◽  
Non Coding Rna ◽  
Xist Rna ◽  
To Come ◽  
In Cis ◽  
Long Non Coding Rna

In female mammals, one of the two X chromosomes becomes genetically silenced to compensate for dosage imbalance of X-linked genes between XX females and XY males. X chromosome inactivation (X-inactivation) is a classical model for epigenetic gene regulation in mammals and has been studied for half a century. In the last two decades, efforts have been focused on the X inactive-specific transcript ( Xist ) locus, discovered to be the master regulator of X-inactivation. The Xist gene produces a non-coding RNA that functions as the primary switch for X-inactivation, coating the X chromosome from which it is transcribed in cis . Significant progress has been made towards understanding how Xist is regulated at the onset of X-inactivation, but our understanding of the molecular basis of silencing mediated by Xist RNA has progressed more slowly. A picture has, however, begun to emerge, and new tools and resources hold out the promise of further advances to come. Here, we provide an overview of the current state of our knowledge, what is known about Xist RNA and how it may trigger chromosome silencing.

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