The Ftx Noncoding Locus Controls X Chromosome Inactivation Independently of Its RNA Products.

Accumulation of the Xist long noncoding RNA (lncRNA) on one X chromosome is the trigger for X chromosome inactivation (XCI) in female mammals. Xist expression, which needs to be tightly controlled, involves a cis-acting region, the X-inactivation center (Xic), containing many lncRNA genes that evolved concomitantly to Xist from protein-coding ancestors through pseudogeneization and loss of coding potential. Here, we uncover an essential role for the Xic-linked noncoding gene Ftx in the regulation of Xist expression. We show that Ftx is required in cis to promote Xist transcriptional activation and establishment of XCI. Importantly, we demonstrate that this function depends on Ftx transcription and not on the RNA products. Our findings illustrate the multiplicity of layers operating in the establishment of XCI and highlight the diversity in the modus operandi of the noncoding players.

Species-specific regulation of XIST by the JPX/FTX orthologs.

X chromosome inactivation (XCI) is an essential process, yet it initiates with remarkable diversity in various mammalian species. XIST, the main trigger of XCI, is controlled in the mouse by an interplay of lncRNA genes (LRGs), some of which evolved concomitantly to XIST and have orthologues across all placental mammals. Here, we addressed the functional conservation of human orthologues of two such LRGs, FTX and JPX. By combining analysis of single-cell RNA-seq data from early human embryogenesis with various functional assays in matched human and mouse pluripotent stem- or differentiated post-XCI cells, we demonstrate major functional differences for these orthologues between species, independently of primary sequence conservation. While the function of FTX is not conserved in humans, JPX stands as a major regulator of XIST expression in both species. However, we show that different entities of JPX control the production of XIST at various steps depending on the species. Altogether, our study highlights the functional versatility of LRGs across evolution, and reveals that functional conservation of orthologous LRGs may involve diversified mechanisms of action. These findings represent a striking example of how the evolvability of LRGs can provide adaptative flexibility to constrained gene regulatory networks.

X chromosome inactivation in human development.

X chromosome inactivation (XCI) is a key developmental process taking place in female mammals to compensate for the imbalance in the dosage of X-chromosomal genes between sexes. It is a formidable example of concerted gene regulation and a paradigm for epigenetic processes. Although XCI has been substantially deciphered in the mouse model, how this process is initiated in humans has long remained unexplored. However, recent advances in the experimental capacity to access human embryonic-derived material and in the laws governing ethical considerations of human embryonic research have allowed us to enlighten this black box. Here, we will summarize the current knowledge of human XCI, mainly based on the analyses of embryos derived from in vitro fertilization and of pluripotent stem cells, and highlight any unanswered questions.

Enlightening the contribution of the dark matter to the X chromosome inactivation process in mammals.

X-chromosome inactivation (XCI) in mammals represents an exceptional example of transcriptional co-regulation occurring at the level of an entire chromosome. XCI is considered as a means to compensate for gene dosage imbalance between sexes, yet the largest part of the chromosome is composed of repeated elements of different nature and origins. Here we consider XCI from a repeat point of view, interrogating the mechanisms for inactivating X chromosome-derived repeated sequences and discussing the contribution of repetitive elements to the silencing process itself and to its evolution.

A subset of the histone H3 lysine 9 methyltransferases Suv39h1, G9a, GLP, and SETDB1 participate in a multimeric complex.

Lysine 9 of histone 3 (H3K9) can be mono-, di-, or trimethylated, inducing distinct effects on gene expression and chromatin compaction. H3K9 methylation can be mediated by several histone methyltransferases (HKMTs) that possess mono-, di-, or trimethylation activities. Here we provide evidence that a subset of each of the main H3K9 HKMTs, G9a/KMT1C, GLP/KMT1D, SETDB1/KMT1E, and Suv39h1/KMT1A, coexist in the same megacomplex. Moreover, in Suv39h or G9a null cells, the remaining HKMTs are destabilized at the protein level, indicating that the integrity of these HKMTs is interdependent. The four HKMTs are recruited to major satellite repeats, a known Suv39h1 genomic target, but also to multiple G9a target genes. Moreover, we report a functional cooperation between the four H3K9 HKMTs in the regulation of known G9a target genes. Altogether, our data identify a H3K9 methylation multimeric complex.

Establishment of X chromosome inactivation and epigenomic features of the inactive X depend on cellular contexts.

X chromosome inactivation (XCI) is an essential epigenetic process that ensures X-linked gene dosage equilibrium between sexes in mammals. XCI is dynamically regulated during development in a manner that is intimately linked to differentiation. Numerous studies, which we review here, have explored the dynamics of X inactivation and reactivation in the context of development, differentiation and diseases, and the phenotypic and molecular link between the inactive status, and the cellular context. Here, we also assess whether XCI is a uniform mechanism in mammals by analyzing epigenetic signatures of the inactive X (Xi) in different species and cellular contexts. It appears that the timing of XCI and the epigenetic signature of the inactive X greatly vary between species. Surprisingly, even within a given species, various Xi configurations are found across cellular states. We discuss possible mechanisms underlying these variations, and how they might influence the fate of the Xi.

Molecular coupling of Tsix regulation and pluripotency.

The reprogramming of X-chromosome inactivation during the acquisition of pluripotency in vivo and in vitro is accompanied by the repression of Xist, the trigger of X-inactivation, and the upregulation of its antisense counterpart Tsix. We have shown that key factors supporting pluripotency-Nanog, Oct4 and Sox2-bind within Xist intron 1 in undifferentiated embryonic stem cells (ESC) to repress Xist transcription. However, the relationship between transcription factors of the pluripotency network and Tsix regulation has remained unclear. Here we show that Tsix upregulation in embryonic stem cells depends on the recruitment of the pluripotent marker Rex1, and of the reprogramming-associated factors Klf4 and c-Myc, by the DXPas34 minisatellite associated with the Tsix promoter. Upon deletion of DXPas34, binding of the three factors is abrogated and the transcriptional machinery is no longer efficiently recruited to the Tsix promoter. Additional analyses including knockdown experiments further demonstrate that Rex1 is critically important for efficient transcription elongation of Tsix. Hence, distinct embryonic-stem-cell-specific complexes couple X-inactivation reprogramming and pluripotency, with Nanog, Oct4 and Sox2 repressing Xist to facilitate the reactivation of the inactive X, and Klf4, c-Myc and Rex1 activating Tsix to remodel Xist chromatin and ensure random X-inactivation upon differentiation. The holistic pattern of Xist/Tsix regulation by pluripotent factors that we have identified suggests a general direct governance of complex epigenetic processes by the machinery dedicated to pluripotency.

How Many Non-coding RNAs Does It Take to Compensate Male/Female Genetic Imbalance?

Genetic sex determination in mammals relies on dimorphic sex chromosomes that confer phenotypic/physiologic differences between males and females. In this heterogametic system, X and Y chromosomes diverged from an ancestral pair of autosomes, creating a genetic disequilibrium between XX females and XY males. Dosage compensation mechanisms alleviate intrinsic gene dosage imbalance, leading to equal expression levels of most X-linked genes in the two sexes. In therian mammals, this is achieved through inactivation of one of the two X chromosomes in females. Failure to undergo X-chromosome inactivation (XCI) results in developmental arrest and death. Although fundamental for survival, a surprising loose conservation in the mechanisms to achieve XCI during development in therian lineage has been, and continues, to be uncovered. XCI involves the concerted action of non-coding RNAs (ncRNAs), including the well-known Xist RNA, and has thus become a classical paradigm to study the mode of action of this particular class of transcripts. In this chapter, we will describe the processes coping with sex chromosome genetic imbalance and how ncRNAs underlie dosage compensation mechanisms and influence male-female differences in mammals. Moreover, we will discuss how ncRNAs have been tinkered with during therian evolution to adapt XCI mechanistic to species-specific constraints.

Altered X-chromosome inactivation predisposes to autoimmunity.

In mammals, males and females show marked differences in immune responses. Males are globally more sensitive to infectious diseases, while females are more susceptible to systemic autoimmunity. X-chromosome inactivation (XCI), the epigenetic mechanism ensuring the silencing of one X in females, may participate in these sex biases. We perturbed the expression of the trigger of XCI, the noncoding RNA Xist, in female mice. This resulted in reactivation of genes on the inactive X, including members of the Toll-like receptor 7 (TLR7) signaling pathway, in monocyte/macrophages and dendritic and B cells. Consequently, female mice spontaneously developed inflammatory signs typical of lupus, including anti-nucleic acid autoantibodies, increased frequencies of age-associated and germinal center B cells, and expansion of monocyte/macrophages and dendritic cells. Mechanistically, TLR7 signaling is dysregulated in macrophages, leading to sustained expression of target genes upon stimulation. These findings provide a direct link between maintenance of XCI and female-biased autoimmune manifestations and highlight altered XCI as a cause of autoimmunity.

Unraveling hallmark suitability for staging pre- and post-implantation stem cell models.

The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias.

XIST dampens X chromosome activity in a SPEN-dependent manner during early human development.

XIST (X-inactive specific transcript) long noncoding RNA (lncRNA) is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female preimplantation embryos without triggering X chromosome silencing. The XACT (X-active coating transcript) lncRNA coaccumulates with XIST on active X chromosomes and may antagonize XIST function. Here, we used human embryonic stem cells in a naive state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during preimplantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and dampens the transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional before XCI, confirms the existence of a transient process of X chromosome dosage compensation and reveals that XCI and dampening rely on the same set of factors.

Ftx is a non-coding RNA which affects Xist expression and chromatin structure within the X-inactivation center region.

X chromosome inactivation (XCI) is an essential epigenetic process which involves several non-coding RNAs (ncRNAs), including Xist, the master regulator of X-inactivation initiation. Xist is flanked in its 5' region by a large heterochromatic hotspot, which contains several transcription units including a gene of unknown function, Ftx (five prime to Xist). In this article, we describe the characterization and functional analysis of murine Ftx. We present evidence that Ftx produces a conserved functional long ncRNA, and additionally hosts microRNAs (miR) in its introns. Strikingly, Ftx partially escapes X-inactivation and is upregulated specifically in female ES cells at the onset of X-inactivation, an expression profile which closely follows that of Xist. We generated Ftx null ES cells to address the function of this gene. In these cells, only local changes in chromatin marks are detected within the hotspot, indicating that Ftx is not involved in the global maintenance of the heterochromatic structure of this region. The Ftx mutation, however, results in widespread alteration of transcript levels within the X-inactivation center (Xic) and particularly important decreases in Xist RNA levels, which were correlated with increased DNA methylation at the Xist CpG island. Altogether our results indicate that Ftx is a positive regulator of Xist and lead us to propose that Ftx is a novel ncRNA involved in XCI.

Long non-coding RNAs and human X-chromosome regulation: a coat for the active X chromosome.

In mammals, the genic disequilibrium between males (XY) and females (XX) is resolved through the inactivation of one of the X-chromosomes in females. X-chromosome inactivation (XCI) takes place in all mammalian species, but has mainly been studied in the mouse model where it was shown to be controlled by the interplay of several long non-coding RNA (lncRNA). However, recent data point toward the existence of species divergences among mammals in the strategies used to achieve XCI. The recent discovery of XACT, a novel lncRNA that coats the active X-chromosome specifically in human pluripotent cells, further highlights the existence of human-specific mechanisms of X-chromosome regulation. Here, we discuss the roles of lncRNAs in defining species-specific mechanisms controlling X-inactivation and explore the potential role of large lncRNAs in gene activation.

Study of X Chromosome Activity Status in Human Naive Pluripotent Stem Cells Using RNA-FISH.

X chromosome activity is a defining attribute of naive pluripotency, with naive pluripotency being a rare context in which both X chromosomes of females are active. RNA-fluorescence in situ hybridization (RNA-FISH) is a powerful tool to determine the transcriptional status of specific genes with allelic and single-cell resolution and has been widely used in the context of X chromosome inactivation, the process ensuring dosage compensation for X-linked genes between sexes in mammals. RNA-FISH using genomic or intronic probes allows the detection of newly synthesized transcripts at the site of transcription. This technique is invaluable for appreciating the putative heterogeneity in the expression profiles within cell populations. RNA-FISH has the added advantage of allowing the visualization of gene transcription in a spatial perspective. Here, we provide a detailed protocol describing the application of RNA-FISH to detect nascent X-linked transcripts in female naive human embryonic stem cells to assess their X chromosome status, along with another complementary technique, DNA-FISH.

Digging into X chromosome inactivation.

Characterizing X chromosome inactivation in nonhuman primates reveals some surprises.

[Latest insights on X-chromosome inactivation: When general principles should be revisited]. / Dernières nouvelles du chromosome X - Des principes généraux nuancés.

The inactivation of one of the two X chromosomes of female mammals is a vital process and a paradigm for epigenetic regulations. X-inactivation is triggered, early during embryo development, by the accumulation of a peculiar noncoding RNA, XIST, which interacts with a plethora of molecular complexes and ultimately protects the coated chromosome from the expression machinery. Once installed, the inactive state is locked by multiple layers of chromatin modifications, ensuring its stable perpetuation across cell divisions. However, recent discoveries made in various model organisms urge us to revisit some of the general principles of the X-inactivation process.

TITLE: Dernières nouvelles du chromosome X - Des principes généraux nuancés. ABSTRACT: L'inactivation d'un des deux chromosomes X des femelles mammifères est un processus vital et emblématique des régulations épigénétiques. Elle est déclenchée par l'accumulation d'un ARN non codant, XIST, qui isole le chromosome concerné de la machinerie transcriptionnelle ; l'état inactif persiste ensuite de manière stable au cours des divisions cellulaires successives. Cependant, des découvertes récentes conduisent à revisiter certains principes généraux de l'inactivation du chromosome X initialement établis. Ainsi le chercheur, tout comme le poète**, est-il invité à « vingt fois sur le métier remettre son ouvrage ¼.

Straight to the X: Modeling Human X Chromosome Inactivation in hESCs by FGF Signal Blockade.

In this issue of Cell Stem Cell, An et al. (2020) provide a model to study human X chromosome inactivation. They followed X chromosome activity and traced cellular heterogeneity in naive hESCS, showed that it is caused by incomplete blockade of FGF-signaling, and in doing so isolated cells resembling pre-implantation epiblasts.

Nuclear mRNA degradation pathway(s) are implicated in Xist regulation and X chromosome inactivation.

A critical step in X-chromosome inactivation (XCI), which results in the dosage compensation of X-linked gene expression in mammals, is the coating of the presumptive inactive X chromosome by the large noncoding Xist RNA, which then leads to the recruitment of other factors essential for the heterochromatinisation of the inactive X and its transcriptional silencing. In an approach aimed at identifying genes implicated in the X-inactivation process by comparative transcriptional profiling of female and male mouse gastrula, we identified the Eif1 gene involved in translation initiation and RNA degradation. We show here that female embryonic stem cell lines, silenced by RNA interference for the Eif1 gene, are unable to form Xist RNA domains upon differentiation and fail to undergo X-inactivation. To probe further an effect involving RNA degradation pathways, the inhibition by RNA interference of Rent1, a factor essential for nonsense-mediated decay and Exosc10, a specific nuclear component of the exosome, was analysed and shown to similarly impair Xist upregulation and XCI. In Eif1-, Rent1-, and Exosc10-interfered clones, Xist spliced form(s) are strongly downregulated, while the levels of unspliced form(s) of Xist and the stability of Xist RNA remain comparable to that of the control cell lines. Our data suggests a role for mRNA nuclear degradation pathways in the critical regulation of spliced Xist mRNA levels and the onset of the X-inactivation process.

A primate-specific retroviral enhancer wires the XACT lncRNA into the core pluripotency network in humans.

Transposable elements (TEs) have been proposed to play an important role in driving the expansion of gene regulatory networks during mammalian evolution, notably by contributing to the evolution and function of long non-coding RNAs (lncRNAs). XACT is a primate-specific TE-derived lncRNA that coats active X chromosomes in pluripotent cells and may contribute to species-specific regulation of X-chromosome inactivation. Here we explore how different families of TEs have contributed to shaping the XACT locus and coupling its expression to pluripotency. Through a combination of sequence analysis across primates, transcriptional interference, and genome editing, we identify a critical enhancer for the regulation of the XACT locus that evolved from an ancestral group of mammalian endogenous retroviruses (ERVs), prior to the emergence of XACT. This ERV was hijacked by younger hominoid-specific ERVs that gave rise to the promoter of XACT, thus wiring its expression to the pluripotency network. This work illustrates how retroviral-derived sequences may intervene in species-specific regulatory pathways.