Onset Of X-chromosome Inactivation Research Articles

Initiation of X Chromosome Inactivation during Bovine Embryo Development.

X-chromosome inactivation (XCI) is a developmental process that aims to equalize the dosage of X-linked gene products between XY males and XX females in eutherian mammals. In female mouse embryos, paternal XCI is initiated at the 4-cell stage; however, the X chromosome is reactivated in the inner cell mass cells of blastocysts, and random XCI is subsequently initiated in epiblast cells. However, recent findings show that the patterns of XCI are not conserved among mammals. In this study, we used quantitative RT-PCR and RNA in situ hybridization combined with immunofluorescence to investigate the pattern of XCI during bovine embryo development. Expression of XIST (X-inactive specific transcript) RNA was significantly upregulated at the morula stage. For the first time, we demonstrate that XIST accumulation in bovine embryos starts in nuclei of female morulae, but its colocalization with histone H3 lysine 27 trimethylation was first detected in day 7 blastocysts. Both in the inner cell mass and in putative epiblast precursors, we observed a proportion of cells with XIST RNA and H3K27me3 colocalization. Surprisingly, the onset of XCI did not lead to a global downregulation of X-linked genes, even in day 9 blastocysts. Together, our findings confirm that diverse patterns of XCI initiation exist among developing mammalian embryos.

Aneuploid rescue precedes X-chromosome inactivation and increases the incidence of its skewness by reducing the size of the embryonic progenitor cell pool.

Do monosomy rescue (MR) and trisomy rescue (TR) in preimplantation human embryos affect other developmental processes, such as X-chromosome inactivation (XCI)? Aneuploid rescue precedes XCI and increases the incidence of XCI skewness by reducing the size of the embryonic progenitor cell pools. More than half of preimplantation human embryos harbor aneuploid cells, some of which can be spontaneously corrected through MR or TR. XCI in females is an indispensable process, which is predicted to start at the early-blastocyst phase. We examined the frequency of XCI skewness in young females who carried full uniparental disomy (UPD) resulting from MR or TR/gamete complementation (GC). The results were statistically analyzed using a theoretical model in which XCI involves various numbers of embryonic progenitor cells. We studied 39 children and young adults ascertained by imprinting disorders. XCI ratios were determined by DNA methylation analysis of a polymorphic locus in the androgen receptor gene. We used Bayesian approach to assess the probability of the occurrence of extreme XCI skewness in the MR and TR/GC groups using a theoretical model of 1-12 cell pools. A total of 12 of 39 individuals (31%) showed skewed XCI. Extreme skewness was observed in 3 of 15 MR cases (20%) and 1 of 24 TR/GC cases (4.2%). Statistical analysis indicated that XCI in the MR group was likely to have occurred when the blastocyst contained three or four euploid embryonic progenitor cells. The estimated size of the embryonic progenitor cell pools was approximately one-third or one-fourth of the predicted size of normal embryos. The TR/GC group likely had a larger pool size at the onset of XCI, although the results remained inconclusive. This is an observational study and needs to be validated by experimental analyses. This study provides evidence that the onset of XCI is determined by an intrinsic clock, irrespectively of the number of embryonic progenitor cells. Our findings can also be applied to individuals without UPD or imprinting disorders. This study provides a clue to understand chromosomal and cellular dynamics in the first few days of human development, their effects on XCI skewing and the possible implications for the expression of X-linked diseases in females. This study was supported by the Grants-in-aid for Scientific Research on Innovative Areas (17H06428) and for Scientific Research (B) (17H03616) from Japan Society for the Promotion of Science (JSPS), and grants from Japan Agency for Medical Research and Development (AMED) (18ek0109266h0002 and 18ek0109278h0002), National Center for Child Health and Development and Takeda Science Foundation. The authors declare no conflict of interest. Not applicable.

A symmetric toggle switch explains the onset of random X inactivation in different mammals.

Gene-regulatory networks control establishment and maintenance of alternative gene expression states during development. A particular challenge is the acquisition of opposing states by two copies of the same gene, as it is the case in mammals for Xist at the onset of random X-chromosome inactivation (XCI). The regulatory principles that lead to stable mono-allelic expression of Xist remain unknown. Here, we uncovered the minimal Xist regulatory network, by combining mathematical modeling and experimental validation of central model predictions. We identified a symmetric toggle switch as the basis for random mono-allelic Xist up-regulation, which reproduces data from several mutant, aneuploid and polyploid murine cell lines with various Xist expression patterns. Moreover, this toggle switch explains the diversity of strategies employed by different species at the onset of XCI. In addition to providing a unifying conceptual framework to explore X-chromosome inactivation across mammals, our study sets the stage for identifying the molecular mechanisms required to initiate random XCI.

RNA-FISH and Immunofluorescence of Mouse Preimplantation and Postimplantation Embryos.

There are two modes of X chromosome inactivation (XCI) in the mouse. One mode is imprinted XCI: it is initiated at around the four-cell stage in favor of the paternal X chromosome, and is maintained in the extraembryonic tissues. The other mode is random XCI, which takes place in the epiblast lineage at the periimplantation stage. X-linked noncoding Xist RNA, which becomes upregulated on the X chromosome to be inactivated at the onset of XCI and plays a critical role in both imprinted and random XCI, and its accumulation in the nucleus have been referred to as one of the hallmarks of the presence of the inactivated X chromosome. RNA-FISH has therefore been an invaluable method for the study of XCI. As XCI status changes dynamically during periimplantation development in the mouse, analysis using samples from these developmental stages is absolutely necessary for elucidation of the molecular basis of XCI mechanisms. However, dissection of the embryos at around the periimplantation stages is not easy, and this impedes in vivo analysis of the kinetics of XCI. Here, we describe our methods for dissecting the periimplantation stage embryo and subsequent procedures for RNA-FISH and immunostaining.

High-resolution RNA allelotyping along the inactive X chromosome: evidence of RNA polymerase III in regulating chromatin configuration

We carried out padlock capture, a high-resolution RNA allelotyping method, to study X chromosome inactivation (XCI). We examined the gene reactivation pattern along the inactive X (Xi), after Xist (X-inactive specific transcript), a prototype long non-coding RNA essential for establishing X chromosome inactivation (XCI) in early embryos, is conditionally deleted from Xi in somatic cells (Xi∆Xist). We also monitored the behaviors of X-linked non-coding transcripts before and after XCI. In each mutant cell line, gene reactivation occurs to ~6% genes along Xi∆Xist in a recognizable pattern. Genes with upstream regions enriched for SINEs are prone to be reactivated. SINE is a class of retrotransposon transcribed by RNA polymerase III (Pol III). Intriguingly, a significant fraction of Pol III transcription from non-coding regions is not subjected to Xist-mediated transcriptional silencing. Pol III inhibition affects gene reactivation status along Xi∆Xist, alters chromatin configuration and interferes with the establishment XCI during in vitro differentiation of ES cells. These results suggest that Pol III transcription is involved in chromatin structure re-organization during the onset of XCI and functions as a general mechanism regulating chromatin configuration in mammalian cells.

Thoughts about SLC16A2, TSIX and XIST gene like sites in the human genome and a potential role in cellular chromosome counting.

BackgroundChromosome counting is a process in which cells determine somehow their intrinsic chromosome number(s). The best-studied cellular mechanism that involves chromosome counting is ‘chromosome-kissing’ and X-chromosome inactivation (XCI) mechanism. It is necessary for the well-known dosage compensation between the genders in mammals to balance the number of active X-chromosomes (Xa) with regard to diploid set of autosomes. At the onset of XCI, two X-chromosomes are coming in close proximity and pair physically by a specific segment denominated X-pairing region (Xpr) that involves the SLC16A2 gene.ResultsAn Ensembl BLAST search for human and mouse SLC16A2/Slc16a2 homologues revealed, that highly similar sequences can be found at almost each chromosome in the corresponding genomes. Additionally, a BLAST search for SLC16A2/TSIX/XIST (genes responsible for XCI) reveled that “SLC16A2/TSIX/XIST like sequences” cover equally all chromosomes, too. With respect to this we provide following hypotheses.HypothesesIf a single genomic region containing the SLC16A2 gene on X-chromosome is responsible for maintaining “balanced” active copy numbers, it is possible that similar sequences or gene/s have the same function on other chromosomes (autosomes). SLC16A2 like sequences on autosomes could encompass evolutionary older, but functionally active key regions for chromosome counting in early embryogenesis. Also SLC16A2 like sequence on autosomes could be involved in inappropriate chromosomes pairing and, thereby be involved in aneuploidy formation during embryogenesis and cancer development. Also, “SLC16A2/TSIX/XIST gene like sequence combinations” covering the whole genome, could be important for the determination of X:autosome ratio in cells and chromosome counting.ConclusionsSLC16A2 and/or SLC16A2/TSIX/XIST like sequence dispersed across autosomes and X-chromosome(s) could serve as bases for a counting mechanism to determine X:autosome ratio and could potentially be a mechanism by which a cell also counts its autosomes. It could also be that such specific genomic regions have the same function for each specific autosome. As errors during the obviously existing process of chromosome counting are one if not the major origin of germline/somatic aneuploidy the here presented hypotheses should further elaborated and experimentally tested.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-016-0271-7) contains supplementary material, which is available to authorized users.

A prominent and conserved role for YY1 in Xist transcriptional activation

Accumulation of the non-coding RNA Xist on one X chromosome in female cells is a hallmark of X-chromosome inactivation in eutherians. Here, we uncovered an essential function for the ubiquitous autosomal transcription factor Yin-Yang 1 (YY1) in the transcriptional activation of Xist in both human and mouse. We show that loss of YY1 prevents Xist up-regulation during the initiation and maintenance of X-inactivation, and that YY1 binds directly the Xist 5′ region to trigger the activity of the Xist promoter. Binding of YY1 to the Xist 5′ region prior to X-chromosome inactivation competes with the Xist repressor REX1 while DNA methylation controls mono-allelic fixation of YY1 to Xist at the onset of X-chromosome inactivation. YY1 is thus the first autosomal activating factor involved in a fundamental and conserved pathway of Xist regulation that ensures the asymmetric transcriptional up-regulation of the master regulator of X-chromosome inactivation.

Jarid2 Is Implicated in the Initial Xist-Induced Targeting of PRC2 to the Inactive X Chromosome

During X chromosome inactivation (XCI), the Polycomb Repressive Complex 2 (PRC2) is thought to participate in the early maintenance of the inactive state. Although Xist RNA is essential for the recruitment of PRC2 to the X chromosome, the precise mechanism remains unclear. Here, we demonstrate that the PRC2 cofactor Jarid2 is an important mediator of Xist-induced PRC2 targeting. The region containing the conserved B and F repeats of Xist is critical for Jarid2 recruitment via its unique N-terminal domain. Xist-induced Jarid2 recruitment occurs chromosome-wide independently of a functional PRC2 complex, unlike at other parts of the genome, such as CG-rich regions, where Jarid2 and PRC2 binding are interdependent. Conversely, we show that Jarid2 loss prevents efficient PRC2 and H3K27me3 enrichment to Xist-coated chromatin. Jarid2 thus represents an important intermediate between PRC2 and Xist RNA for the initial targeting of the PRC2 complex to the X chromosome during onset of XCI.

Jpx RNA Activates Xist by Evicting CTCF

In mammals, dosage compensation between XX and XY individuals occurs through X chromosome inactivation (XCI). The noncoding Xist RNA is expressed and initiates XCI only when more than one X chromosome is present. Current models invoke a dependency on the X-to-autosome ratio (X:A), but molecular factors remain poorly defined. Here, we demonstrate that molecular titration between an X-encoded RNA and an autosomally encoded protein dictates Xist induction. In pre-XCI cells, CTCF protein represses Xist transcription. At the onset of XCI, Jpx RNA is upregulated, binds CTCF, and extricates CTCF from one Xist allele. We demonstrate that CTCF is an RNA-binding protein and is titrated away from the Xist promoter by Jpx RNA. Thus, Jpx activates Xist by evicting CTCF. The functional antagonism via molecular titration reveals a role for long noncoding RNA in epigenetic regulation.

Human X-chromosome inactivation pattern distributions fit a model of genetically influenced choice better than models of completely random choice

In eutherian mammals, one X-chromosome in every XX somatic cell is transcriptionally silenced through the process of X-chromosome inactivation (XCI). Females are thus functional mosaics, where some cells express genes from the paternal X, and the others from the maternal X. The relative abundance of the two cell populations (X-inactivation pattern, XIP) can have significant medical implications for some females. In mice, the 'choice' of which X to inactivate, maternal or paternal, in each cell of the early embryo is genetically influenced. In humans, the timing of XCI choice and whether choice occurs completely randomly or under a genetic influence is debated. Here, we explore these questions by analysing the distribution of XIPs in large populations of normal females. Models were generated to predict XIP distributions resulting from completely random or genetically influenced choice. Each model describes the discrete primary distribution at the onset of XCI, and the continuous secondary distribution accounting for changes to the XIP as a result of development and ageing. Statistical methods are used to compare models with empirical data from Danish and Utah populations. A rigorous data treatment strategy maximises information content and allows for unbiased use of unphased XIP data. The Anderson-Darling goodness-of-fit statistics and likelihood ratio tests indicate that a model of genetically influenced XCI choice better fits the empirical data than models of completely random choice.

Tissue-specific differences in the proportion of mosaic large NF1 deletions are suggestive of a selective growth advantage of hematopoietic del(+/−) stem cells

Type-2 NF1 deletions spanning 1.2 Mb are frequently of postzygotic origin and hence tend to be associated with mosaicism for normal cells and those harboring the deletion (del(+/-) cells). Eleven patients with mosaic type-2 deletions were investigated by FISH and high proportions (94-99%) of del(+/-) cells were detected both in whole blood and in isolated CD3+, CD14+, CD15+, and CD19+ leukocytes. Significantly lower proportions of del(+/-) cells (24-82%) were however noted in urine-derived epithelial cells. A patient harboring an atypical large NF1 deletion with nonrecurrent breakpoints was also found to have a much higher proportion of del(+/-) cells in blood (96%) than in urine (51%). The tissue-specific differences in the proportions of del(+/-) cells as well as the X chromosome inactivation (XCI) patterns observed in these mosaic patients suggest that the majority of the deletions had occurred before or during the preimplantation blastocyst stage before the onset of XCI. We postulate that hematopoietic del(+/-) stem cells present at an early developmental stage are characterized by a selective growth advantage over normal cells lacking the deletion, leading to a high proportion of del(+/-) cells in peripheral blood from the affected patients.

Mechanics and Dynamics of X-Chromosome Pairing at X Inactivation

At the onset of X-chromosome inactivation, the vital process whereby female mammalian cells equalize X products with respect to males, the X chromosomes are colocalized along their Xic (X-inactivation center) regions. The mechanism inducing recognition and pairing of the X's remains, though, elusive. Starting from recent discoveries on the molecular factors and on the DNA sequences (the so-called “pairing sites”) involved, we dissect the mechanical basis of Xic colocalization by using a statistical physics model. We show that soluble DNA-specific binding molecules, such as those experimentally identified, can be indeed sufficient to induce the spontaneous colocalization of the homologous chromosomes but only when their concentration, or chemical affinity, rises above a threshold value as a consequence of a thermodynamic phase transition. We derive the likelihood of pairing and its probability distribution. Chromosome dynamics has two stages: an initial independent Brownian diffusion followed, after a characteristic time scale, by recognition and pairing. Finally, we investigate the effects of DNA deletion/insertions in the region of pairing sites and compare model predictions to available experimental data.

Symmetry-Breaking Model forX-Chromosome Inactivation

In mammals, dosage compensation of X linked genes in female cells is achieved by inactivation of one of their two X chromosomes which is randomly chosen. The earliest steps in X-chromosome inactivation (XCI), namely, the mechanism whereby cells count their X chromosomes and choose between two equivalent X chromosomes, remain mysterious. Starting from the recent discovery of X chromosome colocalization at the onset of X-chromosome inactivation, we propose a statistical mechanics model of XCI, which is investigated by computer simulations and checked against experimental data. Our model describes how a "blocking factor" complex is self-assembled and why only one is formed out of many diffusible molecules, resulting in a spontaneous symmetry breaking in the binding to two identical chromosomes. These results are used to derive a scenario of biological implications.

The DXPas34 Repeat Regulates Random and Imprinted X Inactivation

X chromosome inactivation (XCI) is initiated by expression of the noncoding Xist RNA in the female embryo. Tsix, the antisense noncoding partner of Xist, serves as its regulator during both imprinted and random XCI. Here, we show that Tsix in part acts through a 34mer repeat, DXPas34. DXPas34 contains bidirectional promoter activity, producing overlapping forward and reverse transcripts. We generate three new Tsix alleles in mouse embryonic stem cells and show that, while the Tsix promoter is unexpectedly dispensable, DXPas34 plays dual positive-negative functions. At the onset of XCI, DXPas34 stimulates Tsix expression through its enhancer activity. Once XCI is established, DXPas34 becomes repressive and stably silences Tsix. Germline transmission of the DXPas34 mutation demonstrates its necessity for both random and imprinted XCI in mice. Intriguingly, sequence analysis suggests that DXPas34 could potentially have descended from an ancient retrotransposon. We hypothesize that DXPas34 was acquired by Tsix to regulate antisense function.

A Transient Heterochromatic State in Xist Preempts X Inactivation Choice without RNA Stabilization

X chromosome inactivation (XCI) depends on a noncoding sense-antisense transcript pair, Xist and Tsix. At the onset of XCI, Xist RNA accumulates on one of two Xs, coating and silencing the chromosome in cis. The molecular basis for monoallelic Xist upregulation is not known, though evidence predominantly supports a posttranscriptional mechanism through RNA stabilization. Here, we test whether Tsix RNA destabilizes Xist RNA. Unexpectedly, we find that Xist upregulation is not based on transcript stabilization at all but is instead controlled by transcription in a sex-specific manner. Tsix directly regulates its transcription. On the future inactive X, Tsix downregulation induces a transient heterochromatic state in Xist, followed paradoxically by high-level Xist expression. A Tsix-deficient X chromosome adopts the heterochromatic state in pre-XCI cells. This state persists through XCI establishment and "reverts" to a euchromatic state during XCI maintenance. We have therefore identified chromatin marks that preempt and predict asymmetric Xist expression.

Functional intergenic transcription: a case study of the X-inactivation centre.

Long known to be riddled with repetitive elements and regarded as 'junk', intergenic regions in the mammalian genome now appear to be more than incidental spacers between coding sequences. Here, I review the example of Xite, an intergenic region at the X-inactivation centre which was recently shown to regulate the X-chromosome choice decision. Xite contains a series of DNaseI-hypersensitive sites and harbours two intergenic transcription start sites. These intergenic transcription elements act at the onset of X-chromosome inactivation (XCI) to bias the selection of the active X. It has been proposed that Xite acts in cis on Tsix by promoting its persistence during XCI. Xite has also been proposed to be a candidate for the X-controlling element, a naturally occurring modifier of XCI ratios in mice and possibly also in humans. It seems likely that intergenic transcription will turn out to be a widespread phenomenon in mammals and that, more importantly, it will emerge as a significant regulatory mechanism for the expression of coding sequences.

Xite, X-Inactivation Intergenic Transcription Elements that Regulate the Probability of Choice

Allelic expression differences contribute to phenotypic variation. In X chromosome inactivation (XCI), unfavorable XCI ratios promote X-linked disease penetrance in females. During XCI, one X is randomly silenced by Xist. X chromosome choice is determined by asymmetric expression of Tsix whose antisense action represses Xist. Here, we discover a cis element in the mouse X-inactivation center that regulates Tsix. Xite harbors intergenic transcription start sites and DNaseI hypersensitive sites with allelic differences. At the onset of XCI, deleting Xite downregulates Tsix in cis and skews XCI ratios, suggesting that Xite promotes Tsix persistence on the active X. Truncating Xite RNA is inconsequential, indicating that Xite action does not require intact transcripts. We propose that allele-specific Xite action promotes Tsix asymmetry and generates X chromosome inequality. Therefore, Xite is a candidate for the Xce, the classical modifier of XCI ratios.