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X chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is an iconic example of epigenetic regulation. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI remains contested. Here, we examine genome-wide signatures of DNA methylation across fives tissues from a male and female koala (Phascolarctos cinereus), and present the first whole-genome, multi-tissue marsupial 'methylome atlas'. Using these novel data, we elucidate divergent versus common features of representative marsupial and eutherian DNA methylation. First, tissue-specific differential DNA methylation in koalas primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx, the primary lncRNA associated with marsupial XCI, is consistent with the epigenetic regulation of female-specific (and presumably inactive X chromosome-specific) expression. Finally, we use the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5%) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to functionally conserved patterns of XCI in two deep branches of mammals. [ABSTRACT FROM AUTHOR]

Les femmes représentent 80 % des personnes affectées par les maladies auto-immunes. Le rôle des hormones sexuelles sur la réponse immune est bien documenté, résultant en particulier de la fixation des œstrogènes sur leurs récepteurs nucléaires exprimés par nombre de cellules immunitaires. En plus de l'action des hormones stéroïdes sexuelles, des preuves s'accumulent en faveur d'un rôle des facteurs génétiques liés au chromosome X. Chez la femme, suite aux mécanismes d'inactivation de l'X (ICX), un des deux chromosomes X est inactivé de manière aléatoire, générant ainsi une mosaïque de cellules exprimant des gènes de l'X hérités, soit de la mère, soit du père portés par l'X actif. Cependant, 15 à 23 % de gènes de l'X inactif (Xi) échappent à l'ICX, contribuant à l'émergence d'une autre population hétérogène de cellules exprimant le même gène à partir des deux chromosomes X. Parmi ces gènes, certains gènes de l'immunité ont été récemment identifiés. Si la contribution directe de ce mécanisme de dosage génétique dans la susceptibilité à l'auto-immunité reste encore à être explorée en profondeur, le mosaïsme cellulaire résultant de l'ICX et de l'échappement à l'ICX induit une plasticité fonctionnelle unique au sein des cellules immunitaires de femmes. 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 involved in autoimmunity, recent data demonstrate that female sex hormones are not the only cause of the female predisposition. In addition to the action of sex steroid hormones, there is growing evidence for a role of genetic factors linked to the X chromosome. In female mammals, following the mechanisms of X chromosome inactivation (ICX), one of the two X chromosomes is randomly inactivated, resulting in a cellular mosaicism, where about one-half of the cells in a tissue express the maternal X chromosome and the other half the paternal one. However, 15–23% of inactive X (Xi) genes escape XCI, contributing to the emergence of a female specific heterogeneous population of cells expressing the same gene from both X chromosomes. Among these genes, some immune-related genes have recently been identified. Although the direct contribution of this genetic mechanism in the female susceptibility to autoimmunity remains to be fully established, the cellular mosaicism resulting from XCI and escape from XCI is likely to create a unique functional plasticity within female immune cells. [ABSTRACT FROM AUTHOR]

During embryogenesis in mammals, the 3‐dimensional (3D) genome organization changes globally in parallel with transcription changes in a cell‐type specific manner. This involves the progressive formation of heterochromatin, the best example of which is the inactive X chromosome (Xi) in females, originally discovered as a compact 3D structure at the nuclear periphery known as the Barr body. The heterochromatin formation on the autosomes and the Xi is tightly associated with the differentiation state and the developmental potential of cells, making it an ideal readout of the cellular epigenetic state. At a glance, the heterochromatin appears to be uniform. However, recent studies are beginning to reveal a more complex picture, with multiple hierarchical levels co‐existing within the heterochromatin compartment. Such hierarchical levels appear to exist in the heterochromatin compartment on autosomes as well as on the Xi. Here, we review recent progress in our understanding of the 3D genome organization changes during the period of differentiation surrounding pluripotency in vivo and in vitro, with a focus on the heterochromatin compartment. We first look at the whole genome, then focus on the Xi, and discuss their differences and similarities. Finally, we present a unified view of how the heterochromatin compartment is formed and regulated during early development. In particular, we emphasize that there are multiple layers within the heterochromatic compartment on both the autosomes and the Xi, with regulatory mechanisms common and specific to each layer. [ABSTRACT FROM AUTHOR]

The long non-coding RNA (lncRNA) Xist is crucially involved in a process called X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female mammals to achieve X dosage compensation between the sexes. Because Xist RNA silences the X chromosome from which it is transcribed, the activation of Xist transcription marks the initiation of the XCI process and thus, mechanisms and players that activate this gene are of central importance to the XCI process. During female mouse embryogenesis, XCI occurs in two steps. At the 2-4 cell stages imprinted XCI (iXCI) silences exclusively the paternally inherited X chromosome (Xp). While extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo random XCI (rXCI) around implantation. Both iXCI and rXCI are dependent on Xist. Rlim, also known as Rnf12, is an X-linked E3 ubiquitin ligase that is involved in the transcriptional activation of Xist. However, while data on the crucial involvement of Rlim during iXCI appear clear, its role in rXCI has been controversial. This review discusses data leading to this disagreement and recent evidence for a regulatory switch of Xist transcription in epiblasts of implanting embryos, partially reconciling the roles of Rlim during Xist activation., (© 2024 The Author(s).)

Sex differences are widespread during neurodevelopment and play a role in neuropsychiatric conditions such as autism, which is more prevalent in males than females. In humans, males have been shown to have larger brain volumes than females with development of the hippocampus and amygdala showing prominent sex differences. Mechanistically, sex steroids and sex chromosomes drive these differences in brain development, which seem to peak during prenatal and pubertal stages. Animal models have played a crucial role in understanding sex differences, but the study of human sex differences requires an experimental model that can recapitulate complex genetic traits. To fill this gap, human induced pluripotent stem cell-derived brain organoids are now being used to study how complex genetic traits influence prenatal brain development. For example, brain organoids from individuals with autism and individuals with X chromosome-linked Rett syndrome and fragile X syndrome have revealed prenatal differences in cell proliferation, a measure of brain volume differences, and excitatory-inhibitory imbalances. Brain organoids have also revealed increased neurogenesis of excitatory neurons due to androgens. However, despite growing interest in using brain organoids, several key challenges remain that affect its validity as a model system. In this review, we discuss how sex steroids and the sex chromosomes each contribute to sex differences in brain development. Then, we examine the role of X chromosome inactivation as a factor that drives sex differences. Finally, we discuss the combined challenges of modeling X chromosome inactivation and limitations of brain organoids that need to be taken into consideration when studying sex differences., (© 2024 Published by Elsevier Inc on behalf of Society of Biological Psychiatry.)

How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. Although PRC2 binds G-tracts with the potential to form RNA G-quadruplexes (rG4s), whether rG4s fold extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model in mouse embryonic stem cells, here we identify multiple folded rG4s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4s. High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity, and stabilizing rG4 in vivo antagonizes H3 at lysine 27 (H3K27me3) enrichment on the inactive X chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of chromosome-wide gene silencing., Competing Interests: Declaration of interests J.T.L. is a cofounder of Fulcrum Therapeutics, a scientific adviser to Skyhawk Therapeutics, and a Non-Executive Director of GSK., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: To analyze the clinical features and genetic etiology of 17 Chinese pedigrees affected with X-linked intellectual disability (XLID)., Methods: Seventeen pedigrees affected with unexplained intellectual disability which had presented at Henan Provincial People's Hospital from May 2021 to May 2023 were selected as the study subjects. Clinical data of the probands and their pedigree members were collected. Trio-whole exome sequencing (Trio-WES), Sanger sequencing and X chromosome inactivation (XCI) analysis were carried out. Pathogenicity of candidate variants was predicted based on the guidelines from the American College of Medical Genetics and Genomics and co-segregation analysis., Results: The 17 probands, including 9 males and 8 females with an age ranging from 0.6 to 8 years old, had all shown mental retardation and developmental delay. Fourteen variants were detected by genetic testing, which included 4 pathogenic variants (MECP2: c.502C>T, MECP2: c.916C>T/c.806delG, IQSEC2: c.1417G>T), 4 likely pathogenic variants (MECP2: c.1157_1197del/c.925C>T, KDM5C: c.2128A>T, SLC6A8: c.1631C>T) and 6 variants of uncertain significance (KLHL15: c.26G>C, PAK3: c.970A>G/c.1520G>A, GRIA3: c.2153C>G, TAF1: c.2233T>G, HUWE1: c.10301T>A). The PAK3: c.970A>G, GRIA3: c.2153C>G and TAF1: c.2233T>G variants were considered as the genetic etiology for pedigrees 12, 14 and 15 by co-segregation analysis, respectively. The proband of pedigree 13 was found to have non-random XCI (81:19). Therefore, the PAK3: c.1520G>A variant may underlie its pathogenesis., Conclusion: Trio-WES has attained genetic diagnosis for the 17 XLID pedigrees. Sanger sequencing and XCI assay can provide auxiliary tests for the diagnosis of XLID.

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.

Development from fertilized egg to functioning multi-cellular organism requires precision. There is no precision, and often no survival, without plasticity. Plasticity is conferred partly by stochastic variation, present inherently in all biological systems. Gene expression levels fluctuate ubiquitously through transcription, alternative splicing, translation and turnover. Small differences in gene expression are exploited to trigger early differentiation, conferring distinct function on selected individual cells and setting in motion regulatory interactions. Non-selected cells then acquire new functions along the spatio-temporal developmental trajectory. The differentiation process has many stochastic components. Meiotic segregation, mitochondrial partitioning, X-inactivation and the dynamic DNA binding of transcription factor assemblies-all exhibit randomness. Non-random X-inactivation generally signals deleterious X-linked mutations. Correct neural wiring, such as retina to brain, arises through repeated confirmatory activity of connections made randomly. In immune system development, both B-cell antibody generation and the emergence of balanced T-cell categories begin through stochastic trial and error followed by functional selection. Aberrant selection processes lead to immune dysfunction. DNA sequence variants also arise through stochastic events: some involving environmental fluctuation (radiation or presence of pollutants), or genetic repair system malfunction. The phenotypic outcome of mutations is also fluid. Mutations may be advantageous in some circumstances, deleterious in others. This article is part of a discussion meeting issue 'Causes and consequences of stochastic processes in development and disease'.

Background: X-chromosome inactivation (XCI) is an epigenetic process that occurs during early development in mammalian females by randomly silencing one of two copies of the X chromosome in each cell. The preferential inactivation of either the maternal or paternal copy of the X chromosome in a majority of cells results in a skewed or non-random pattern of X inactivation and is observed in over 25% of adult females. Identifying skewed X inactivation is of clinical significance in patients with suspected rare genetic diseases due to the possibility of biased expression of disease-causing genes present on the active X chromosome. The current clinical test for the detection of skewed XCI relies on the methylation status of the methylation-sensitive restriction enzyme (Hpall) binding site present in proximity of short tandem polymorphic repeats on the androgen receptor (AR) gene. This approach using one locus results in uninformative or inconclusive data for 10-20% of tests. Further, recent studies have shown inconsistency between methylation of the AR locus and the state of inactivation of the X chromosome. Herein, we develop a method for estimating X inactivation status, using exome and transcriptome sequencing data derived from blood in 227 female samples. We built a reference model for evaluation of XCI in 135 females from the GTEx consortium. We tested and validated the model on 11 female individuals with different types of undiagnosed rare genetic disorders who were clinically tested for X-skew using the AR gene assay and compared results to our outlier-based analysis technique., Results: In comparison to the AR clinical test for identification of X inactivation, our method was concordant with the AR method in 9 samples, discordant in 1, and provided a measure of X inactivation in 1 sample with uninformative clinical results. We applied this method on an additional 81 females presenting to the clinic with phenotypes consistent with different hereditary disorders without a known genetic diagnosis., Conclusions: This study presents the use of transcriptome and exome sequencing data to provide an accurate and complete estimation of X-inactivation and skew status in a cohort of female patients with different types of suspected rare genetic disease., (© 2024. The Author(s).)

Competing Interests: FA has received consulting fees and/or royalties from Celgene, Inova, Advise Connect Inspire, and Hillstar Bio, Inc. FA is an inventor on a licensed patent (US patent no. 14/617,412) and licensed provisional patent (US patent no. 62/481,158). The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

X inactivation is the means of equalizing the dosage of X chromosomal genes in male and female eutherian mammals, so that only one X is active in each cell. The XIST locus (in cis) on each additional X chromosome initiates the transcriptional silence of that chromosome, making it an inactive X. How the active X in both males and females is protected from inactivation by its own XIST locus is not well understood in any mammal. Previous studies of autosomal duplications suggest that gene(s) on the short arm of human chromosome 19 repress XIST on the active X. Here, we examine the time of transcription of some candidate genes in preimplantation embryos using single-cell RNA sequencing data from human embryos and qRT-PCR from bovine embryos. The candidate genes assayed are those transcribed from 19p13.3-13.2, which are widely expressed and can remodel chromatin. Our results confirm that XIST is expressed at low levels from the future active X in embryos of both sexes; they also show that the XIST locus is repressed in both sexes when pluripotency factors are being upregulated, during the 4-8 cell and morula stages in human and bovine embryos - well before the early blastocyst (E5) when XIST on the inactive X in females starts to be upregulated. Our data suggest a role for DNMT1, UHRF1, SAFB and SAFB2 in XIST repression; they also exclude XACT and other 19p candidate genes and provide the transcriptional timing for some genes not previously assayed in human or bovine preimplantation embryos., (© 2022. The Author(s), under exclusive licence to European Society of Human Genetics.)

Female human induced pluripotent stem cell (hiPSC) lines exhibit variability in X-inactivation status. The majority of hiPSC lines maintain one transcriptionally active X (Xa) and one inactive X (Xi) chromosome from donor cells. However, at low frequency, hiPSC lines with two Xas are produced, suggesting that epigenetic alterations of the Xi occur sporadically during reprogramming. We show here that X-inactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated by the Kyoto method (retroviral or episomal reprogramming), which uses leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Early passage Xa/Xi hiPSC lines generated on non-SNL feeders were converted into Xa/Xa hiPSC lines after several passages on SNL feeders, and supplementation with recombinant LIF caused reactivation of some of X-linked genes. Thus, feeders are a significant factor affecting X-inactivation status. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and -inactivation.

Abstract Background Incontinentia pigmenti (IP) is a rare X‐linked disorder affecting the skin and other ectodermal tissues that is caused by mutation of the IKBKG/NEMO gene. Previous studies have reported that the overall mutation detection rate in IP is ~75%. We hypothesized that a low‐level mosaicism existed in the remaining cases. Methods Genomic variations in the IKBKG gene were examined in 30 IP probands and their family members. Standard mutational analyses were performed to detect common deletions, nucleotide alterations, and copy number variations. To assess skewing of the X chromosome inactivation (XCI) pattern, a HUMARA assay was performed. We compared the results of this analysis with phenotype severity. Results Pathogenic variants were identified in 20 probands (66.7%), the rate of detection was suboptimal. The remaining 10 probands tended to manifest a mild phenotype with no skewed X chromosome inactivation that is generally observed in IP patients. Quantitative nested PCR and digital droplet PCR were performed for the 10 patients and mosaicism of the common IKBKG deletion were identified in five patients. Conclusion Overall, we detected 25 IKBKG mutations (83.3%). Determination of the XCI value in advance of mutational analyses for IP could improve the mutation detection rate. Our improved detection rate for these mutations, particularly those with a low‐level mosaicism, may present opportunities for appropriate genetic counseling.

Meiosis is a specialized form of cellular division that results in the precise halving of the genome to produce gametes for sexual reproduction. Checkpoints function during meiosis to detect errors and subsequently to activate a signaling cascade that prevents the formation of aneuploid gametes. Indeed, asynapsis of a homologous chromosome pair elicits a checkpoint response that can in turn trigger germline apoptosis. In a heterogametic germ line, however, sex chromosomes proceed through meiosis with unsynapsed regions and are not recognized by checkpoint machinery. We conducted a directed RNAi screen in Caenorhabditis elegans to identify regulatory factors that prevent recognition of heteromorphic sex chromosomes as unpaired and uncovered a role for the SET domain histone H3 lysine 9 histone methyltransferase (HMTase) MET-2 and two additional HMTases in shielding the male X from checkpoint machinery. We found that MET-2 also mediates the transcriptional silencing program of meiotic sex chromosome inactivation (MSCI) but not meiotic silencing of unsynapsed chromatin (MSUC), suggesting that these processes are distinct. Further, MSCI and checkpoint shielding can be uncoupled, as double-strand breaks targeted to an unpaired, transcriptionally silenced extra-chromosomal array induce checkpoint activation in germ lines depleted for met-2. In summary, our data uncover a mechanism by which repressive chromatin architecture enables checkpoint proteins to distinguish between the partnerless male X chromosome and asynapsed chromosomes thereby shielding the lone X from inappropriate activation of an apoptotic program.

Abstract Background Rett syndrome (RTT) is a neurodevelopmental disorder that predominantly affects girls, resulting from a loss‐of‐function variant in X‐linked MECP2. Here, we report a rare case of a girl with RTT with an X chromosome mosaic karyotype (46,XX/47,XXX). Methods Fluorescent in situ hybridization (FISH) was carried out to confirm the mosaic karyotype. Sanger sequencing was carried out to genetically diagnose RTT. Furthermore, we assessed the X chromosome inactivation (XCI) pattern. MECP2 expression levels were examined via RT‐PCR. Results The patient presented with preserved speech variant, the milder form of RTT. Genetic examination revealed a de novo, heterozygous, truncating variant of MECP2. FISH revealed mosaicism in the 47,XXX karyotype in 6% of her cells. The XCI assay revealed unbalanced inactivation with skewing in favor of the paternal X chromosome. MECP2 was downregulated to only 84% of the control, indicating that the patient's variant was probably of paternal origin. Unbalanced XCI in this patient might have contributed to the alleviation of the phenotype. However, her supernumerary X chromosome was derived from maternal X chromosome harboring the wild‐type allele and might have had no preferential effect on her RTT‐related phenotype. Conclusion The present results indicate that phenotypic effects of X chromosome aneuploidy depend on the nature of the supernumerary X chromosome, the pattern of mosaicism, and XCI status.

Recent efforts in mapping spatial genome organization have revealed three evocative and conserved structural features of the inactive X in female mammals. First, the chromosomal conformation of the inactive X reveals a loss of topologically associated domains (TADs) present on the active X. Second, the macrosatellite DXZ4 emerges as a singular boundary that suppresses physical interactions between two large TAD-depleted “megadomains.” Third, DXZ4 reaches across several megabases to form “superloops” with two other X-linked tandem repeats, FIRRE and ICCE, which also loop to each other. Although all three structural features are conserved across rodents and primates, deletion of mouse and human orthologs of DXZ4 and FIRRE from the inactive X have revealed limited impact on X chromosome inactivation (XCI) and escape in vitro. In contrast, loss of Xist or SMCHD1 have been shown to impair TAD erasure and gene silencing on the inactive X. In this perspective, we summarize these results in the context of new research describing disruption of X-linked tandem repeats in vivo, and discuss their possible molecular roles through the lens of evolutionary conservation and clinical genetics. As a null hypothesis, we consider whether the conservation of some structural features on the inactive X may reflect selection for X-linked tandem repeats on account of necessary cis- and trans-regulatory roles they may play on the active X, rather than the inactive X. Additional hypotheses invoking a role for X-linked tandem repeats on X reactivation, for example in the germline or totipotency, remain to be assessed in multiple developmental models spanning mammalian evolution.

Abstract Background X chromosome inactivation (XCI) is an important gene regulation mechanism in females to equalize the expression levels of X chromosome between two sexes. Generally, one of two X chromosomes in females is randomly chosen to be inactivated. Nonrandom XCI (XCI skewing) is also observed in females, which has been reported to play an important role in many X-linked diseases. However, there is no statistical measure available for the degree of the XCI skewing based on family data in population genetics. Results In this article, we propose a statistical approach to measure the degree of the XCI skewing based on family trios, which is represented by a ratio of two genotypic relative risks in females. The point estimate of the ratio is obtained from the maximum likelihood estimates of two genotypic relative risks. When parental genotypes are missing in some family trios, the expectation-conditional-maximization algorithm is adopted to obtain the corresponding maximum likelihood estimates. Further, the confidence interval of the ratio is derived based on the likelihood ratio test. Simulation results show that the likelihood-based confidence interval has an accurate coverage probability under the situations considered. Also, we apply our proposed method to the rheumatoid arthritis data from USA for its practical use, and find out that a locus, rs2238907, may undergo the XCI skewing against the at-risk allele. But this needs to be further confirmed by molecular genetics. Conclusions The proposed statistical measure for the skewness of XCI is applicable to complete family trio data or family trio data with some paternal genotypes missing. The likelihood-based confidence interval has an accurate coverage probability under the situations considered. Therefore, our proposed statistical measure is generally recommended in practice for discovering the potential loci which undergo the XCI skewing.

Abstract Background The product of dosage compensation in female mammals is the inactive X chromosome (Xi). Xi facultative heterochromatin is organized into two different types, one of which is defined by histone H3 trimethylated at lysine 9 (H3K9me3). The rationale for this study was to assess SET domain bifurcated 1 (SETDB1) as a candidate for maintaining this repressive modification at the human Xi. Results Here, we show that loss of SETDB1 does not result in large-scale H3K9me3 changes at the Xi, but unexpectedly we observed striking decompaction of the Xi territory. Close examination revealed a 0.5 Mb region of the Xi that transitioned from H3K9me3 heterochromatin to euchromatin within the 3′ end of the IL1RAPL1 gene that is part of a common chromosome fragile site that is frequently deleted or rearranged in patients afflicted with intellectual disability and other neurological ailments. Centrally located within this interval is a powerful enhancer adjacent to an ERVL-MaLR element. In the absence of SETDB1, the enhancer is reactivated on the Xi coupled with bidirectional transcription from the ERVL-MaLR element. Xa deletion of the enhancer/ERVL-MaLR resulted in loss of full-length IL1RAPL1 transcript in cis, coupled with trans decompaction of the Xi chromosome territory, whereas Xi deletion increased detection of full-length IL1RAPL1 transcript in trans, but did not impact Xi compaction. Conclusions These data support a critical role for SETDB1 in maintaining the ERVL-MaLR element and adjacent enhancer in the 3′ end of the IL1RAPL1 gene in a silent state to facilitate Xi compaction.

Background: Incontinentia pigmenti (IP) is a rare X‐linked disorder affecting the skin and other ectodermal tissues that is caused by mutation of the IKBKG/NEMO gene. Previous studies have reported that the overall mutation detection rate in IP is ~75%. We hypothesized that a low‐level mosaicism existed in the remaining cases. Methods: Genomic variations in the IKBKG gene were examined in 30 IP probands and their family members. Standard mutational analyses were performed to detect common deletions, nucleotide alterations, and copy number variations. To assess skewing of the X chromosome inactivation (XCI) pattern, a HUMARA assay was performed. We compared the results of this analysis with phenotype severity. Results: Pathogenic variants were identified in 20 probands (66.7%), the rate of detection was suboptimal. The remaining 10 probands tended to manifest a mild phenotype with no skewed X chromosome inactivation that is generally observed in IP patients. Quantitative nested PCR and digital droplet PCR were performed for the 10 patients and mosaicism of the common IKBKG deletion were identified in five patients. Conclusion: Overall, we detected 25 IKBKG mutations (83.3%). Determination of the XCI value in advance of mutational analyses for IP could improve the mutation detection rate. Our improved detection rate for these mutations, particularly those with a low‐level mosaicism, may present opportunities for appropriate genetic counseling. [ABSTRACT FROM AUTHOR]

46,XX testicular disorder of sex development (46,XX TDSD) is a relatively rare condition characterised by the presence of testicular tissue with 46,XX karyotype. The present study aims to reveal the phenotype to genotype correlation in a series of sex‐determining region Y (SRY)‐positive 46,XX TDSD cases. We present the clinical findings, hormone profiles and genetic test results of six patients with SRY‐positive 46,XX TDSD and give the details and follow‐up findings of our three of previously published patients. All patients presented common characteristics such as azoospermia, hypergonadotropic hypogonadism and an SRY gene translocated on the terminal part of the short arm of one of the X chromosomes. Mean ± standard deviation (SD) height of the patients was 164.78 ± 8.0 cm. Five patients had decreased secondary sexual characteristics, and three patients had gynaecomastia with varying degrees. Five of the seven patients revealed a translocation between protein kinase X (PRKX) and inverted protein kinase Y (PRKY) genes, and the remaining two patients showed a translocation between the pseudoautosomal region 1 (PAR1) of X chromosome and the differential region of Y chromosome. X chromosome inactivation (XCI) analysis results demonstrated random and skewed XCI in 5 cases and 1 case, respectively. In brief, we delineate the phenotypic spectrum of patients with SRY‐positive 46,XX TDSD and the underlying mechanisms of Xp;Yp translocations. [ABSTRACT FROM AUTHOR]

Female hemophilia is an intriguing rare disorder and few larger reports on its genetic etiology are available. While historically the diagnosis was satisfactorily reached by factor VIII activity assays, the clinical and potentially therapeutic heterogeneity of female hemophilia calls for comprehensive molecular diagnosis in each case. Currently, the genetic investigations are not a part of routine, state-funded, diagnostics in Poland, and thus molecular epidemiological data are missing. We set out to perform a comprehensive genetic analysis of Polish females with hemophilia A. Eighteen females with hemophilia A (including 2 with severe and 5 with moderate hemophilia phenotype) consented for genetic diagnostics. To establish F8 mutations, we used next-generation sequencing of a panel of genes associated with hematological disorders, standard assays for recurrent intragenic F8 inversions and MLPA when deletions were suspected. When appropriate we also used karyotyping, genomic microarrays and X chromosome inactivation assays. While abnormally skewed X-chromosome inactivation combined with a F8 variant on the active allele was, as expected, the most common genetic etiology, a number of other genetic scenarios were unraveled. This included: misdiagnosis (molecular diagnosis of vWd), Turner syndrome, compound heterozygosity and androgen insensitivity syndrome (a phenotypical 46,XY female with a novel androgen receptor gene mutation). We report 3 novel F8 mutations. Every case of female hemophilia warrants full genomic diagnostics, as this may change the diagnosis or reveal broader morbidity than a coagulation disorder (Turner syndrome, androgen insensitivity, or cardiovascular morbidity that we described previously in a SHAM syndrome carrier). • Congenital hemophilia A in females is an ultra rare disorder. • Genetic diagnostics may uncover misdiagnosis or associated syndromes. • The molecular patterns leading to hemophilia phenotype in females are widely varied. [ABSTRACT FROM AUTHOR]

Intellectual disability (ID) affects 30% more males than females. This sex bias can be attributed to the enrichment of genes on the X chromosome playing essential roles in the central nervous system and their hemizygous state on males. Moreover, as a result of X chromosome inactivation (XCI), most genes on one of the X chromosomes in female somatic cells are epigenetically silenced, so that females carrying X-linked variants are not expected to be so severely affected as males. Consequently, the knowledge about X-linked ID (XLID) in females is still scarce. Herein, we used extreme XCI skewing (≥ 90%) to predict X-linked variants in females with idiopathic ID. XCI profiles from 53 probands were estimated from blood and buccal mucosa through a methylation-sensitive AR/RP2 assay. DNA samples with extreme XCI skewing were then submitted to array-comparative genomic hybridization and whole-exome sequencing. Seven females (13.2%) exhibited extreme XCI skewing, a percentage significantly higher than expected for healthy females in our population. XLID-potentially related variants were identified in five patients with extreme XCI skewing, including one pathogenic rstructural rearrangement [der(X) chromosome from a t(X;2)] and four single nucleotide variants in NLGN4X, HDAC8, TAF1, and USP9X genes, two of which affecting XCI escape genes. XCI skewing showed to be an outstanding approach for the characterization of molecular mechanisms underlying XLID in females. Beyond expanding the spectrum of variants/phenotypes associated with ID, our results pointed to compensatory biological pathways underlying XCI and uncover new insights into the involvement of escape genes on XLID, impacting genetic counseling. [ABSTRACT FROM AUTHOR]

A heterozygous deletion at Xq27.3q28 including FMR1, AFF2, and IDS causing intellectual disability and characteristic facial features is very rare in females, with only 10 patients having been reported. Here, we examined two female patients with different clinical features harboring the Xq27.3q28 deletion and determined the chromosomal breakpoints. Moreover, we assessed the X chromosome inactivation (XCI) in peripheral blood from both patients. Both patients had an almost overlapping deletion at Xq27.3q28, however, the more severe patient (Patient 1) showed skewed XCI of the normal X chromosome (79:21) whereas the milder patient (Patient 2) showed random XCI. Therefore, deletion at Xq27.3q28 critically affected brain development, and the ratio of XCI of the normal X chromosome greatly affected the clinical characteristics of patients with deletion at Xq27.3q28. As the chromosomal breakpoints were determined, we analyzed a change in chromatin domains termed topologically associated domains (TADs) using published Hi‐C data on the Xq27.3q28 region, and found that only patient 1 had a possibility of a drastic change in TADs. The altered chromatin topologies on the Xq27.3q28 region might affect the clinical features of patient 1 by changing the expression of genes just outside the deletion and/or the XCI establishment during embryogenesis resulting in skewed XCI. [ABSTRACT FROM AUTHOR]

Background: X chromosome inactivation (XCI) is the mechanism by which the X-linked gene dosage is adjusted between the sexes. Evidence shows that many sex-specific diseases have their basis in X chromosome biology. While female schizophrenia patients often have a delayed age of disease onset and clinical phenotypes that are different from those of males, it is unknown whether the sex differences in schizophrenia are associated with X-linked gene dosage and the choice of X chromosome silencing in female cells. Previous studies demonstrated that sex chromosome aneuploidies may be related to the pathogeneses of some psychiatric diseases. Here, we examined the changes in skewed XCI in patients with schizophrenia. Methods: A total of 109 female schizophrenia (SCZ) patients and 80 age- and sex-matched healthy controls (CNTLs) were included in this study. We evaluated clinical features including disease onset age, disease duration, clinical symptoms by the Positive and Negative Syndrome Scale (PANSS) and antipsychotic treatment dosages. The XCI skewing patterns were analyzed by the methylation profile of the HUMARA gene found in DNA isolated from SCZ patient and CNTL leukocytes in the three age groups. Results: First, we found that the frequency of skewed XCI in SCZ patients was 4 times more than that in the age- and sex-matched CNTLs (p < 0.01). Second, we found an earlier onset of severe XCI skewing in the SCZ patients than in CNTLs. Third, we demonstrated a close relationship between the severity of skewed XCI and schizophrenic symptoms (PANSS score ≥ 90) as well as the age of disease onset. Fourth, we demonstrated that the skewed XCI in SCZ patients was not transmitted from the patients' mothers. Limitations: The XCI skewing pattern might differ depending on tissues or organs. Although this is the first study to explore skewed XCI in SCZ, in the future, samples from different tissues or cells in SCZ patients might be important for understanding the impact of skewed XCI in this disease. Conclusion: Our study, for the first time, investigated skewed XCI in female SCZ patients and presented a potential mechanism for the sex differences in SCZ. Our data also suggested that XCI might be a potential target for the development of female-specific interventions for SCZ. [ABSTRACT FROM AUTHOR]

X chromosome inactivation (XCI) is the process whereby one of the X chromosomes in female mammalian cells is silenced to equalize X-linked gene expression with males. XCI depends on the long noncoding RNA Xist, which coats the inactive X chromosome in cis and triggers a cascade of events that ultimately lead to chromosome-wide transcriptional silencing that is stable for the lifetime of an organism. In recent years, the discovery of proteins that interact with Xist have led to new insights into how the initiation of XCI occurs. Nevertheless, there are still various unknowns about the mechanisms by which Xist orchestrates and maintains stable X-linked silencing. Here, we review recent work elucidating the role of Xist and its protein partners in mediating chromosome-wide transcriptional repression, as well as discuss a model by which Xist may compartmentalize proteins across the inactive X chromosome to enable both the initiation and maintenance of XCI. [ABSTRACT FROM AUTHOR]

X chromosome structural abnormalities are relatively common in Turner syndrome patients, in particular X isochromosomes. Reports over the last five decades examining asynchronous DNA replication between the normal X and isochromosome have clearly established that the structurally abnormal chromosome is the inactive X chromosome (Xi). Here the organization of chromatin at a deleted X chromosome, an Xq isochromosome, and two isodicentric chromosomes were examined. Consistent with previous differential staining methods, at interphase, the X isochromosome and isodicentric X chromosomes frequently formed bipartite Barr bodies, observed by fluorescence microscopy using numerous independent bona fide markers of Xi heterochromatin. At metaphase, with the exception of the pseudoautosomal region and the duplicated locus of the macrosatellite DXZ4 (if present on the abnormal X chromosome based on break points), euchromatin markers were absent from the Xi, whereas histone variant macroH2A formed reproducible banded mirror-image chromosomes. Unexpectedly, the isodicentric chromosome in 46,X,idic(X)(q28) cells, which carry a near full-length q-arm-to-q-arm fused chromosome, showed at interphase very rare instances of Xi chromatin bodies that were separated by large distances in the nucleus. Further examination using immunofluorescence and FISH support the possibility that these rare cells may represent ones in which one half of the isodicentric chromosome is active and the other half is inactive. [ABSTRACT FROM AUTHOR]

Objective: To develop a mouse artificial chromosome (MAC) carrying the mouse Xist gene (X-inactive specific transcript; Xist-MAC) as a systematic in vitro approach for investigating Xist RNA-mediated chromosome inactivation. Results: Ectopic expression of the Xist gene in CHO cells led to the accumulation of Xist RNA in cis on the MAC. In addition, the introduction of Xist-MAC to embryonic stem cells from male mice via microcell-mediated chromosome transfer resulted in the accumulation of Xist RNA in cis on the MAC. Chromosomal inactivation was observed in the differentiated state. Moreover, this phenomenon was accompanied by the epigenetic modification of H3K27 trimethylation. Conclusions: We successfully generated a novel chromosome inactivation model, Xist-MAC, which will provide a valuable tool for the screening and functional analysis of X chromosome inactivation-related genes and proteins. [ABSTRACT FROM AUTHOR]

This year's Annual Review Issue of The Journal of Pathology contains 18 invited reviews on current research areas in pathology. The subject areas reflect the broad range of topics covered by the journal and this year encompass the development and application of software in digital histopathology, implementation of biomarkers in pathology practice; genetics and epigenetics, and stromal influences in disease. The reviews are authored by experts in their field and provide comprehensive updates in the chosen areas, in which there has been considerable recent progress in our understanding of disease. [ABSTRACT FROM AUTHOR]

Our genomes contain the blueprint of what makes us human and many indications as to why we develop disease. Until the last 10 years, most studies had focussed on protein-coding genes, more specifically DNA sequences coding for proteins. However, this represents less than 5% of our genomes. The other 95% is referred to as the 'dark matter' of our genomes, our understanding of which is extremely limited. Part of this 'dark matter' includes regions that give rise to RNAs that do not code for proteins. A subset of these non-coding RNAs are long non-coding RNAs (lncRNAs), which in particular are beginning to be dissected and their importance to human health revealed. To improve our understanding and treatment of disease it is vital that we understand the molecular and cellular function of lncRNAs, and how their misregulation can contribute to disease. It is not yet clear what proportion of lncRNAs is actually functional; conservation during evolution is being used to understand the biological importance of lncRNA. Here, we present key themes within the field of lncRNAs, emphasising the importance of their roles in both the nucleus and the cytoplasm of cells, as well as patterns in their modes of action. We discuss their potential functions in development and disease using examples where we have the greatest understanding. Finally, we emphasise why lncRNAs can serve as biomarkers and discuss their emerging potential for therapy. [ABSTRACT FROM AUTHOR]

The process of dosage compensation of genes occurs in the female mammalian cells during early embryonic development: one of the two X chromosomes becomes transcriptionally inactive and is subsequently inherited in the inactive state in all somatic cells. Embryonic stem cells (ESCs) together with induced pluripotent stem cells (iPSCs) have become the models for studying early development. In human female iPSCs and ESCs, one of the X chromosomes is always active, while the state of the other X chromosome can vary: it can be completely active or partially or completely inactivated as in somatic cells. Determining the state of inactivation of the X chromosome in human pluripotent stem cells (PSC) appears to be a complicated and state-of the-art question. However, various methods for determining the inactivation state frequently result in the contradictory findings. The analysis of the AR gene methylation, which is widely used in clinical laboratories, makes it possible to determine the ratio of alleles of the inactivated X chromosome in a patient's blood cells and tissues. Previously, this approach was suggested to determine the status of X chromosome inactivation in human iPSCs. In the present study, we analyzed the inactivation state of the X chromosome in the human iPSC line by standard methods (XIST gene expression, XIST promoter methylation, histone modification, etc.), as well as by the level of methylation of the AR gene via fragment analysis. The methylation of the AR locus failed to correlate with the X chromosome inactivation state in female iPSCs. Thus, we assume that the methylation of the AR gene cannot be used as an indicator of X chromosome inactivation in human female iPSC lines. [ABSTRACT FROM AUTHOR]

Background: Rett syndrome (RTT) is a neurodevelopmental disorder that predominantly affects girls, resulting from a loss‐of‐function variant in X‐linked MECP2. Here, we report a rare case of a girl with RTT with an X chromosome mosaic karyotype (46,XX/47,XXX). Methods: Fluorescent in situ hybridization (FISH) was carried out to confirm the mosaic karyotype. Sanger sequencing was carried out to genetically diagnose RTT. Furthermore, we assessed the X chromosome inactivation (XCI) pattern. MECP2 expression levels were examined via RT‐PCR. Results: The patient presented with preserved speech variant, the milder form of RTT. Genetic examination revealed a de novo, heterozygous, truncating variant of MECP2. FISH revealed mosaicism in the 47,XXX karyotype in 6% of her cells. The XCI assay revealed unbalanced inactivation with skewing in favor of the paternal X chromosome. MECP2 was downregulated to only 84% of the control, indicating that the patient's variant was probably of paternal origin. Unbalanced XCI in this patient might have contributed to the alleviation of the phenotype. However, her supernumerary X chromosome was derived from maternal X chromosome harboring the wild‐type allele and might have had no preferential effect on her RTT‐related phenotype. Conclusion: The present results indicate that phenotypic effects of X chromosome aneuploidy depend on the nature of the supernumerary X chromosome, the pattern of mosaicism, and XCI status. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) have been identified in all eukaryotes and are most abundant in the human genome. However, the functional importance and mechanisms of action for human lncRNAs are largely unknown. Using comparative sequence, structural, and functional analyses, we characterize the evolution and molecular function of human lncRNA JPX. We find that human JPX and its mouse homolog, lncRNA Jpx , have deep divergence in their nucleotide sequences and RNA secondary structures. Despite such differences, both lncRNAs demonstrate robust binding to CTCF, a protein that is central to Jpx 's role in X chromosome inactivation. In addition, our functional rescue experiment using Jpx -deletion mutant cells shows that human JPX can functionally complement the loss of Jpx in mouse embryonic stem cells. Our findings support a model for functional conservation of lncRNAs independent from sequence and structural divergence. This study provides mechanistic insight into the evolution of lncRNA function. Image 1 • Comparison of human JPX with its mouse homolog reveals rapid sequence evolution. • Human JPX shows RNA secondary structure divergent from mouse Jpx. • Human JPX RNA and mouse Jpx RNA both bind CTCF. • Human JPX rescues Jpx -deletion defects in mouse ES cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

X-chromosome inactivation occurs randomly for one of the two X chromosomes in female cells during development. Inactivation occurs when RNA transcribed from the Xist gene on the X chromosome from which it is expressed spreads to coat the whole X chromosome. In the first issue of Epigenetics and Chromatin, Nesterova and colleagues investigate the role of the RNA interference pathway enzyme Dicer in DNA methylation of the Xist promoter.

X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans -acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting. [Display omitted] • XIST localizes across the X and spreads to autosomal regions in female naive hPSCs • XIST dampens the expression of X-linked and autosomal genes in female naive hPSCs • XIST mediates chromatin changes at target regions on the X and autosomes • Xist spreads to autosomal regions during XCI initiation in differentiating mouse PSCs During X chromosome dampening, when XIST expression is uncoupled from complete gene silencing and displays a non-typical dispersed configuration, XIST spreads beyond the X chromosome to downregulate gene expression on autosomes. [ABSTRACT FROM AUTHOR]

A significant number of human X-linked genes escape X chromosome inactivation and are thus expressed from both the active and inactive X chromosomes. The basis for escape from inactivation and the potential role of the X chromosome primary DNA sequence in determining a gene's X inactivation status is unclear. Using a combination of the X chromosome sequence and a comprehensive X inactivation profile of more than 600 genes, two independent yet complementary approaches were used to systematically investigate the relationship between X inactivation and DNA sequence features. First, statistical analyses revealed that a number of repeat features, including long interspersed nuclear element (LINE) and mammalian-wide interspersed repeat repetitive elements, are significantly enriched in regions surrounding transcription start sites of genes that are subject to inactivation, while Alu repetitive elements and short motifs containing ACG/CGT are significantly enriched in those that escape inactivation. Second, linear support vector machine classifiers constructed using primary DNA sequence features were used to correctly predict the X inactivation status for >80% of all X-linked genes. We further identified a small set of features that are important for accurate classification, among which LINE-1 and LINE-2 content show the greatest individual discriminatory power. Finally, as few as 12 features can be used for accurate support vector machine classification. Taken together, these results suggest that features of the underlying primary DNA sequence of the human X chromosome may influence the spreading and/or maintenance of X inactivation.

The Polycomb group (PcG) encodes an evolutionarily conserved set of chromatin-modifying proteins that are thought to maintain cellular transcriptional memory by stably silencing gene expression. In mouse embryos that are mutated for the PcG protein Eed, X-chromosome inactivation (XCI) is not stably maintained in extra-embryonic tissues. Eed is a component of a histone-methyltransferase complex that is thought to contribute to stable silencing in undifferentiated cells due to its enrichment on the inactive X-chromosome in cells of the early mouse embryo and in stem cells of the extra-embryonic trophectoderm lineage. Here, we demonstrate that the inactive X-chromosome in Eed(-/-) trophoblast stem cells and in cells of the trophectoderm-derived extra-embryonic ectoderm in Eed(-/-) embryos remain transcriptionally silent, despite lacking the PcG-mediated histone modifications that normally characterize the facultative heterochromatin of the inactive X-chromosome. Whereas undifferentiated Eed(-/-) trophoblast stem cells maintained XCI, reactivation of the inactive X-chromosome occurred when these cells were differentiated. These results indicate that PcG complexes are not necessary to maintain transcriptional silencing of the inactive X-chromosome in undifferentiated stem cells. Instead, PcG proteins seem to propagate cellular memory by preventing transcriptional activation of facultative heterochromatin during differentiation.

Human pluripotent stem cells (hPSCs) are not only a promising tool to investigate differentiation to many cell types, including the germline, but are also a potential source of cells to use for regenerative medicine purposes in the future. However, current in vitro models to generate human primordial germ cell-like cells (hPGCLCs) have revealed high variability regarding differentiation efficiency depending on the hPSC lines used. Here, we investigated whether differences in X chromosome inactivation (XCI) in female hPSCs could contribute to the variability of hPGCLC differentiation efficiency during embryoid body (EB) formation. For this, we first characterized the XCI state in different hPSC lines by investigating the expression of XIST and H3K27me3, followed by differentiation and quantification of hPGCLCs. We observed that the XCI state did not influence the efficiency to differentiate to hPGCLCs; rather, hPSCs derived from cells isolated from urine showed an increased trend towards hPGCLCs differentiation compared to skin-derived hPSCs. In addition, we also characterized the XCI state in the generated hPGCLCs. Interestingly, we observed that independent of the XCI state of the hPSCs used, both hPGCLCs and soma cells in the EBs acquired XIST expression, indicative of an inactive X chromosome. In fact, culture conditions for EB formation seemed to promote XIST expression. Together, our results contribute to understanding how epigenetic properties of hPSCs influence differentiation and to optimize differentiation methods to obtain higher numbers of hPGCLCs, the first step to achieve human in vitro gametogenesis.

During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.

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.

Recent evidence has determined that the conserved X chromosome mega-structures controlled by the Firre and Dxz4 loci are not required for X chromosome inactivation (XCI) in cell lines. Here, we examined the in vivo contribution of these loci by generating mice carrying a single or double deletion of Firre and Dxz4. We found that these mutants are viable, fertile and show no defect in random or imprinted XCI. However, the lack of these elements results in many dysregulated genes on autosomes in an organ-specific manner. By comparing the dysregulated genes between the single and double deletion, we identified superloop, megadomain, and Firre locus-dependent gene sets. The largest transcriptional effect was observed in all strains lacking the Firre locus, indicating that this locus is the main driver for these autosomal expression signatures. Collectively, these findings suggest that these X-linked loci are involved in autosomal gene regulation rather than XCI biology.

Dosage compensation between XX female and XY male cells is achieved by a process known as X chromosome inactivation (XCI) in mammals. XCI is initiated early during development in female cells and is subsequently stably maintained in most somatic cells. Despite its stability, the robust transcriptional silencing of XCI is reversible, in the embryo and also in a number of reprogramming settings. Although XCI has been intensively studied, the dynamics, factors, and mechanisms of X chromosome reactivation (XCR) remain largely unknown. In this review, we discuss how new sequencing technologies and reprogramming approaches have enabled recent advances that revealed the timing of transcriptional activation during XCR. We also discuss the factors and chromatin features that might be important to understand the dynamics and mechanisms of the erasure of transcriptional gene silencing on the inactive X chromosome (Xi).

X chromosome inactivation (XCI) is a process that equalizes the expression of X-linked genes between males and females. It relies on Xist, continuously expressed in somatic cells during XCI maintenance. However, how Xist impacts XCI maintenance and its functional motifs remain unclear. In this study, we conducted a comprehensive analysis of Xist, using rabbits as an ideal non-primate model. Homozygous knockout of exon 1, exon 6, and repeat A in female rabbits resulted in embryonic lethality. However, X ∆ReA X females, with intact X chromosome expressing Xist, showed no abnormalities. Interestingly, there were no significant differences between females with homozygous knockout of exons 2-5 and wild-type rabbits, suggesting that exons 2, 3, 4, and 5 are less important for XCI. These findings provide evolutionary insights into Xist function., (© 2024. The Author(s).)

Objective: To explore the correlation between skewed X chromosome inactivation (XCI) and clinical phenotype of a Chinese pedigree with loss of heterozygosity at Xq22.1q22.3., Methods: A pedigree diagnosed at Taizhou Hospital on November 10, 2021 was selected as the study subject. G-banded chromosomal karyotyping and copy number variation sequencing (CNV-seq) were carried out to analyze the amniotic fluid and peripheral blood samples from the couple. XCI was detected by PCR amplification of CAG repeats in exon 1 of androgen receptor gene before and after the digestion with methylation-sensitive restriction enzyme Hpa II. Correlation between the genotype and clinical phenotype was analyzed., Results: The karyotypes of the pregnant woman and the fetus were both determined as 46,X,del(X)(q22), and the result of CNV-seq was seq[hg19]del(X)(q22.1q22.3) chrX: g.10046000&#95;105740000del, suggesting that both had harbored a 5.28 Mb deletion on the X chromosome. No obvious abnormality was found in the husband. XCI analysis showed that the activity ratio of the two X chromosomes of the pregnant woman and her fetus was 0 : 100. The X chromosome harboring the q22.1q22.3 deletion was completely inactivated, and the inactivated X chromosome of the fetus was derived from its mother., Conclusion: The fetus has harbored a maternally derived inactivated X chromosome del(X)(q22) , and its phenotype is closely associated with the activity of the abnormal X chromosome. Pedigree XCI analysis combined with the clinical phenotype has facilitated recognition of the maternal phenotype and prognosis of female fetus with loss of heterozygosity at Xq22.1q22.3.

Ninety percent of congenital nephrogenic diabetes insipidus (NDI) are X-linked inherited and are caused by mutations in the vasopressin type 2 receptor gene (AVPR2). Most affected individuals are males. Only sporadic female cases have been reported. Here, we first reported a female monozygotic twin with discordant phenotypes for NDI carrying a missense variant c.845T>C (p.Leu282Pro) in exon 4 of AVPR2. Intracellular cAMP concentrations in COS7 cells transfected with AVPR2-L282P were significantly decreased by about 60% compared with those in wild-type AVPR2 plasmid transfected cells, suggesting this variation was pathogenic. The X-inactivation pattern was investigated in peripheral leukocytes and urine sediments in both the unaffected and affected pair. Results showed that the affected pair had a skewed X chromosome inactivation (XCI) pattern in urine sediments and a random XCI pattern in leukocytes, while the unaffected pair showed a random XCI pattern both in leukocytes and urine sediments. This was the first report of monozygotic twins who developed different phenotypes of NDI. Our study suggested that the development of NDI symptoms is more closely associated with the XCI pattern in urine sediments compared with the XCI pattern in peripheral leukocytes. Analysis of XCI in peripheral leukocytes may not be enough to explore possible mechanisms., (© 2023 Wiley Periodicals LLC.)