RESUMO
PURPOSE: Transient Bartter syndrome related to pathogenic variants of MAGED2 is the most recently described antenatal Bartter syndrome. Despite its transient nature, it is the most severe form of Bartter syndrome in the perinatal period. Our aim was to describe 14 new cases and to try to explain the incomplete penetrance in women. METHODS: We report on 14 new cases, including 3 females, and review the 40 cases described to date. We tested the hypothesis that MAGED2 is transcriptionally regulated by differential methylation of its CpG-rich promotor by pyrosequencing of DNA samples extracted from fetal and adult leukocytes and kidney samples. RESULTS: Analysis of the data from 54 symptomatic patients showed spontaneous resolution of symptoms in 27% of cases, persistent complications in 41% of cases and fatality in 32% of cases. Clinical anomalies were reported in 76% of patients, mostly renal anomalies (52%), cardiovascular anomalies (29%) and dysmorphic features (13%). A developmental delay was reported in 24% of patients. Variants were found in all regions of the gene. Methylation analysis of the MAGED2 CpG-rich promotor showed a correlation with gender, independent of age, tissue or presence of symptoms, excluding a role for this mechanism in the incomplete penetrance in women. CONCLUSION: This work enriches the phenotypic and genetic description of this recently described disease, and deepens our understanding of the pathophysiological role and regulation of MAGED2. Finally, by describing the wide range of outcomes in patients, this work opens the discussion on genetic counseling offered to families.
RESUMO
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.
RESUMO
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.
Assuntos
Autoimunidade , Macrófagos , Receptor 7 Toll-Like , Inativação do Cromossomo X , Animais , Feminino , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Autoimunidade/genética , Camundongos , Masculino , Macrófagos/metabolismo , Macrófagos/imunologia , RNA Longo não Codificante/genética , Transdução de Sinais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologiaRESUMO
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.
Assuntos
Trofoblastos , Humanos , Trofoblastos/metabolismo , Trofoblastos/citologia , Metilação de DNA , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Modelos Biológicos , Implantação do Embrião , Diferenciação Celular , Epigênese Genética , Transcriptoma/genética , Proteômica/métodosRESUMO
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.
Assuntos
Placenta , RNA Longo não Codificante , Gravidez , Humanos , Feminino , Camundongos , Animais , Placenta/metabolismo , Inativação do Cromossomo X/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Mamíferos/genética , Embrião de Mamíferos/metabolismoRESUMO
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.
Assuntos
Células-Tronco Pluripotentes , Animais , Feminino , Humanos , Hibridização in Situ Fluorescente , RNA Longo não Codificante/genética , Cromossomo X , Inativação do Cromossomo X/genéticaRESUMO
Characterizing X chromosome inactivation in nonhuman primates reveals some surprises.
Assuntos
Inativação do Cromossomo XRESUMO
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.
Assuntos
Inativação do Cromossomo X/fisiologia , Animais , Feminino , Inativação Gênica/fisiologia , Genética/tendências , Humanos , Mamíferos/genética , Cromossomo X/genéticaAssuntos
Genoma Humano , Neoplasias/genética , RNA não Traduzido , Animais , DNA/genética , Humanos , CamundongosAssuntos
Tolnaftato , Inativação do Cromossomo X , Cromossomos Humanos X , Feminino , Células Germinativas , HumanosRESUMO
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.
Assuntos
Células-Tronco Embrionárias Humanas , Inativação do Cromossomo X , Comunicação Autócrina , Cromossomos Humanos X , Camadas Germinativas , Humanos , Inativação do Cromossomo X/genéticaRESUMO
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.
Assuntos
Cromossomos Humanos X , Desenvolvimento Embrionário/genética , Inativação do Cromossomo X , Animais , Humanos , Processos de Determinação Sexual/genéticaRESUMO
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.
Assuntos
Retrovirus Endógenos/genética , Elementos Facilitadores Genéticos , Primatas/virologia , RNA Longo não Codificante/genética , Retroviridae/genética , Animais , Elementos de DNA Transponíveis , Retrovirus Endógenos/metabolismo , Evolução Molecular , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno , Humanos , Filogenia , Primatas/classificação , Primatas/genética , RNA Longo não Codificante/metabolismo , Retroviridae/metabolismo , Especificidade da EspécieRESUMO
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.
Assuntos
RNA Longo não Codificante/genética , Inativação do Cromossomo X/genética , Cromossomo X/genética , Animais , Linhagem Celular , Feminino , Células HEK293 , Humanos , Mamíferos/genética , Camundongos , Transcrição Gênica/genéticaRESUMO
Induced pluripotent stem cells (iPSCs) have considerably impacted human developmental biology and regenerative medicine, notably because they circumvent the use of cells of embryonic origin and offer the potential to generate patient-specific pluripotent stem cells. However, conventional reprogramming protocols produce developmentally advanced, or primed, human iPSCs (hiPSCs), restricting their use to post-implantation human development modeling. Hence, there is a need for hiPSCs resembling preimplantation naive epiblast. Here, we develop a method to generate naive hiPSCs directly from somatic cells, using OKMS overexpression and specific culture conditions, further enabling parallel generation of their isogenic primed counterparts. We benchmark naive hiPSCs against human preimplantation epiblast and reveal remarkable concordance in their transcriptome, dependency on mitochondrial respiration and X-chromosome status. Collectively, our results are essential for the understanding of pluripotency regulation throughout preimplantation development and generate new opportunities for disease modeling and regenerative medicine.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Blastocisto/metabolismo , Células Cultivadas , Reprogramação Celular/genética , Técnicas de Reprogramação Celular , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Camadas Germinativas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , TranscriptomaRESUMO
The human blastocyst forms 5 days after one of the smallest human cells (the sperm) fertilizes one of the largest human cells (the egg). Depending on the sex-chromosome contribution from the sperm, the resulting embryo will either be female, with two X chromosomes (XX), or male, with an X and a Y chromosome (XY). In early development, one of the major differences between XX female and XY male embryos is the conserved process of X-chromosome inactivation (XCI), which compensates gene expression of the two female X chromosomes to match the dosage of the single X chromosome of males. Most of our understanding of the pre-XCI state and XCI establishment is based on mouse studies, but recent evidence from human pre-implantation embryo research suggests that many of the molecular steps defined in the mouse are not conserved in human. Here, we will discuss recent advances in understanding the control of X-chromosome dosage compensation in early human embryonic development and compare it to that of the mouse.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'.
Assuntos
Cromossomos Humanos X/genética , Mecanismo Genético de Compensação de Dose/genética , Desenvolvimento Embrionário , Inativação do Cromossomo X/genética , Animais , Desenvolvimento Embrionário/genética , Feminino , Humanos , Masculino , Camundongos , Cromossomo X/genéticaRESUMO
Sex chromosome dosage compensation is essential in most metazoans, but the developmental timing and underlying mechanisms vary significantly, even among placental mammals. Here we identify human-specific mechanisms regulating X chromosome activity in early embryonic development. Single-cell RNA sequencing and imaging revealed co-activation and accumulation of the long noncoding RNAs (lncRNAs) XACT and XIST on active X chromosomes in both early human pre-implantation embryos and naive human embryonic stem cells. In these contexts, the XIST RNA adopts an unusual, highly dispersed organization, which may explain why it does not trigger X chromosome inactivation at this stage. Functional studies in transgenic mouse cells show that XACT influences XIST accumulation in cis. Our findings therefore suggest a mechanism involving antagonistic activity of XIST and XACT in controlling X chromosome activity in early human embryos, and they highlight the contribution of rapidly evolving lncRNAs to species-specific developmental mechanisms.
Assuntos
Cromossomos Humanos X/genética , Desenvolvimento Embrionário/genética , RNA Longo não Codificante/genética , RNA não Traduzido/genética , Animais , Blastocisto/metabolismo , Bases de Dados como Assunto , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Células-Tronco Pluripotentes/metabolismo , RNA Longo não Codificante/metabolismo , RNA não Traduzido/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , TransgenesRESUMO
Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome.
Assuntos
Reprogramação Celular/genética , Cromatina/genética , Cromossomos Humanos X/genética , Histonas/genética , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genética , Animais , Fusão Celular/métodos , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Epigênese Genética , Feminino , Fibroblastos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Mitose , Células-Tronco Embrionárias Murinas/metabolismoRESUMO
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.