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Showing papers on "X chromosome published in 2015"


Journal ArticleDOI
09 Jul 2015-Nature
TL;DR: This model implies that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites, and deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary.
Abstract: The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (∼1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.

738 citations


Journal ArticleDOI
17 Jul 2015-Science
TL;DR: These findings demonstrate that, while Xist attracts repressive complexes to the Xi, it actively repels chromosomal architectural factors such as the cohesins from the Xi.
Abstract: INTRODUCTION The mammal has evolved an epigenetic mechanism to silence one of two X chromosomes in the XX female to equalize gene dosages with the XY male. Once established, the inactivated X chromosome (Xi) is extremely stable and is maintained through the lifetime of the female mammal. The principal regulator, Xist, is a long noncoding RNA that orchestrates the silencing process along the Xi. Xist is believed to operate as a scaffold to recruit and spread repressive complexes, such as Polycomb Repressive Complex 2, along the X chromosome. The identities of crucial interacting factors, however, have remained largely unknown. RATIONALE Although the Xi’s epigenetic stability is a necessary homeostatic property, an ability to unlock this epigenetic state is of great current interest. The X chromosome is home to nearly 1000 genes, at least 50 of which have been implicated in X-linked diseases, such as Rett syndrome and fragile X syndrome. The Xi is therefore a reservoir of functional genes that could be tapped to replace expression of a disease allele on the active X (Xa). A major gap in current understanding is the lack of a comprehensive Xist interactome. Progress toward a full interactome would advance knowledge of epigenetic regulation by long noncoding RNA and potentially inform treatment of X-linked diseases. RESULTS We have developed an RNA-centric proteomic method called iDRiP (identification of direct RNA-interacting proteins). Using iDRiP, we identified 80 to 200 proteins in the Xist interactome. The interactors fall into several functional categories, including cohesins, condensins, topoisomerases, RNA helicases, chromatin remodelers, histone modifiers, DNA methyltransferases, nucleoskeletal factors, and nuclear matrix proteins. Targeted inhibition demonstrates that Xi silencing can be destabilized by disrupting multiple components of the interactome, consistent with the idea that these factors synergistically repress Xi transcription. Triple-drug treatments lead to a net increase of Xi expression and up-regulation of ~100 to 200 Xi genes. We then carry out a focused study of X-linked cohesin sites. Chromatin immunoprecipitation sequencing analysis demonstrates three types of cohesin sites on the X chromosome: Xi-specific sites, Xa-specific sites, and biallelic sites. We find that the Xa-specific binding sites represent a default state. Ablating Xist results in restoration of Xa-specific sites on the Xi. These findings demonstrate that, while Xist attracts repressive complexes to the Xi, it actively repels chromosomal architectural factors such as the cohesins from the Xi. Finally, we examine how Xist and the repulsion of cohesins affect Xi chromosome structure. In wild-type cells, the Xa is characterized by ~112 topologically associated domains (TADs) and the Xi by two megadomains. Intriguingly, loss of Xist and restoration of cohesin binding result in a reversion of the Xi to an Xa-like chromosome conformation. Hi-C analysis shows that TADs return to the Xi in a manner correlated with the reappearance of cohesins and with a transcriptionally permissive state. CONCLUSION Our study unveils many layers of Xi repression and demonstrates a central role for RNA in the topological organization of mammalian chromosomes. Our study also supports a model in which Xist RNA simultaneously acts as (i) a scaffold for the recruitment of repressive complexes to establish and maintain the inactive state and (ii) a repulsion mechanism to extrude architectural factors such as cohesins to avoid acquisition of a transcription-favorable chromatin conformation. Finally, our findings indicate that the stability of the Xi can be perturbed by targeted inhibition of multiple components of the Xist interactome.

417 citations


Journal ArticleDOI
TL;DR: Using a binomial model to assess allelic expression, a continuum between complete silencing and expression from the inactive X (Xi) is demonstrated, and common escape genes and genes with significant differences in XCI status between tissues were identified.
Abstract: X chromosome inactivation (XCI) silences most genes on one X chromosome in female mammals, but some genes escape XCI. To identify escape genes in vivo and to explore molecular mechanisms that regulate this process we analyzed the allele-specific expression and chromatin structure of X-linked genes in mouse tissues and cells with skewed XCI and distinguishable alleles based on single nucleotide polymorphisms. Using a binomial model to assess allelic expression, we demonstrate a continuum between complete silencing and expression from the inactive X (Xi). The validity of the RNA-seq approach was verified using RT-PCR with species-specific primers or Sanger sequencing. Both common escape genes and genes with significant differences in XCI status between tissues were identified. Such genes may be candidates for tissue-specific sex differences. Overall, few genes (3–7%) escape XCI in any of the mouse tissues examined, suggesting stringent silencing and escape controls. In contrast, an in vitro system represented by the embryonic-kidney-derived Patski cell line showed a higher density of escape genes (21%), representing both kidney-specific escape genes and cell-line specific escape genes. Allele-specific RNA polymerase II occupancy and DNase I hypersensitivity at the promoter of genes on the Xi correlated well with levels of escape, consistent with an open chromatin structure at escape genes. Allele-specific CTCF binding on the Xi clustered at escape genes and was denser in brain compared to the Patski cell line, possibly contributing to a more compartmentalized structure of the Xi and fewer escape genes in brain compared to the cell line where larger domains of escape were observed.

227 citations


Journal ArticleDOI
TL;DR: The X-linked lncRNA Firre helps to position the inactive X chromosome near the nucleolus and to preserve one of its main epigenetic features, H3K27me3, demonstrating a role in maintenance of an important epigenetic feature of the inactiveX chromosome.
Abstract: Background In mammals, X chromosome genes are present in one copy in males and two in females. To balance the dosage of X-linked gene expression between the sexes, one of the X chromosomes in females is silenced. X inactivation is initiated by upregulation of the lncRNA (long non-coding RNA) Xist and recruitment of specific chromatin modifiers. The inactivated X chromosome becomes heterochromatic and visits a specific nuclear compartment adjacent to the nucleolus.

220 citations


Journal ArticleDOI
TL;DR: The categorization of genes that escape from X inactivation provides candidates for sex-specific differences in disease and a model of predominately cis-acting influences on inactivation status was supported.
Abstract: X-chromosome inactivation (XCI) achieves dosage compensation between males and females through the silencing of the majority of genes on one of the female X chromosomes. Thus, the female X chromosomes provide a unique opportunity to study euchromatin and heterochromatin of allelic regions within the same nuclear environment. We examined the interplay of DNA methylation (DNAm) with CpG density, transcriptional activity and chromatin state at genes on the X chromosome using over 1800 female samples analysed with the Illumina Infinium Human Methylation450 BeadChip. DNAm was used to predict an inactivation status for 63 novel transcription start sites (TSSs) across 27 tissues. There was high concordance of inactivation status across tissues, with 62% of TSSs subject to XCI in all 27 tissues examined, whereas 9% escaped from XCI in all tissues, and the remainder showed variable escape from XCI between females in subsets of tissues. Inter-female and twin data supported a model of predominately cis-acting influences on inactivation status. The level of expression from the inactive X relative to the active X correlated with the amount of female promoter DNAm to a threshold of ∼30%, beyond which genes were consistently subject to inactivation. The inactive X showed lower DNAm than the active X at intragenic and intergenic regions for genes subject to XCI, but not at genes that escape from inactivation. Our categorization of genes that escape from X inactivation provides candidates for sex-specific differences in disease.

213 citations


Journal ArticleDOI
Lot Snijders Blok1, Erik C. Madsen2, Jane Juusola3, Christian Gilissen1, Diana Baralle4, Margot R.F. Reijnders1, Hanka Venselaar1, Céline Helsmoortel5, Megan T. Cho3, Alexander Hoischen1, Lisenka E.L.M. Vissers1, Tom S. Koemans1, Willemijn M. Wissink-Lindhout1, Evan E. Eichler6, Evan E. Eichler7, Corrado Romano, Hilde Van Esch8, Connie T.R.M. Stumpel9, Maaike Vreeburg9, E. Smeets9, Karin Oberndorff, Bregje W.M. van Bon1, Bregje W.M. van Bon10, Marie Shaw10, Jozef Gecz10, Eric Haan10, M Bienek11, C Jensen11, Bart Loeys5, Anke Van Dijck5, A. Micheil Innes12, Hilary Racher12, Sascha Vermeer13, Nataliya Di Donato14, Andreas Rump14, Katrina Tatton-Brown15, Michael Parker16, Alex Henderson17, Sally Ann Lynch16, Alan Fryer, Alison Ross, Pradeep Vasudevan18, Usha Kini19, Ruth Newbury-Ecob20, Kate Chandler21, Alison Male22, Sybe Dijkstra, Jolanda H. Schieving1, Jacques C. Giltay23, Koen L.I. van Gassen23, Janneke H M Schuurs-Hoeijmakers1, Perciliz L. Tan2, Igor Pediaditakis2, Stefan A. Haas11, Kyle Retterer3, Patrick Reed3, Kristin G. Monaghan3, Eden Haverfield3, Marvin R. Natowicz24, Angela Myers, Michael C. Kruer16, Quinn Stein16, Kevin A. Strauss25, Karlla W. Brigatti25, Katherine G. Keating26, Barbara K. Burton26, Katherine H. Kim26, Joel Charrow26, Jennifer Norman, Audrey Foster-Barber27, Antonie D. Kline28, Amy S. Kimball28, Elaine H. Zackai29, Margaret H. Harr29, Joyce Fox, Julie McLaughlin, Kristin Lindstrom16, Katrina Haude30, Kees E. P. van Roozendaal9, Han G. Brunner9, Wendy K. Chung31, R. Frank Kooy5, Rolph Pfundt1, Vera M. Kalscheuer11, Sarju G. Mehta, Nicholas Katsanis2, Tjitske Kleefstra1 
TL;DR: A consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway is demonstrated, and a differential effect by gender is shown, possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDx3X mutations.
Abstract: Intellectual disability (ID) affects approximately 1%–3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems, corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%–3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants, we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo. We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations.

212 citations


Journal ArticleDOI
TL;DR: A novel Hi-C method is applied to map allelic chromatin contacts of the mouse inactive X chromosome to discover a specific bipartite organization that probably plays an important role in maintenance of gene silencing.
Abstract: In mammals, one of the female X chromosomes and all imprinted genes are expressed exclusively from a single allele in somatic cells. To evaluate structural changes associated with allelic silencing, we have applied a recently developed Hi-C assay that uses DNase I for chromatin fragmentation to mouse F1 hybrid systems. We find radically different conformations for the two female mouse X chromosomes. The inactive X has two superdomains of frequent intrachromosomal contacts separated by a boundary region. Comparison with the recently reported two-superdomain structure of the human inactive X shows that the genomic content of the superdomains differs between species, but part of the boundary region is conserved and located near the Dxz4/DXZ4 locus. In mouse, the boundary region also contains a minisatellite, Ds-TR, and both Dxz4 and Ds-TR appear to be anchored to the nucleolus. Genes that escape X inactivation do not cluster but are located near the periphery of the 3D structure, as are regions enriched in CTCF or RNA polymerase. Fewer short-range intrachromosomal contacts are detected for the inactive alleles of genes subject to X inactivation compared with the active alleles and with genes that escape X inactivation. This pattern is also evident for imprinted genes, in which more chromatin contacts are detected for the expressed allele. By applying a novel Hi-C method to map allelic chromatin contacts, we discover a specific bipartite organization of the mouse inactive X chromosome that probably plays an important role in maintenance of gene silencing.

212 citations


Journal ArticleDOI
TL;DR: This analysis aggregated three published studies that have examined X chromosome inactivation status of genes across the X chromosome, generating consensus calls and identifying discordancies to compile a comprehensive list of X-chromosome inactivation statuses for genes.
Abstract: X chromosome inactivation is the epigenetic silencing of the majority of the genes on one of the X chromosomes in XX therian mammals. In humans, approximately 15 % of genes consistently escape from this inactivation and another 15 % of genes vary between individuals or tissues in whether they are subject to, or escape from, inactivation. Multiple studies have provided inactivation status calls for a large subset of the genes on the X chromosome; however, these studies vary in which genes they were able to make calls for and in some cases which call they give a specific gene. This analysis aggregated three published studies that have examined X chromosome inactivation status of genes across the X chromosome, generating consensus calls and identifying discordancies. The impact of expression level and chromosomal location on X chromosome inactivation status was also assessed. Overall, we assigned a consensus XCI status 639 genes, including 78 % of protein-coding genes expressed outside of the testes, with a lower frequency for non-coding RNA and testis-specific genes. Study-specific discordancies suggest that there may be instability of XCI during cell culture and also highlight study-specific variations in call type. We observe an enrichment of discordant genes at boundaries between genes subject to and escaping from inactivation. This study has compiled a comprehensive list of X-chromosome inactivation statuses for genes and also discovered some biases which will help guide future studies examining X-chromosome inactivation.

167 citations


Journal ArticleDOI
TL;DR: In this paper, two non-coding RNAs are key for MSL assembly and spreading to active genes along the length of the X chromosome along with other noncodingRNAs are found to play a fundamental role in the increased transcriptional output of the male X.
Abstract: Dosage compensation in Drosophila increases the transcription of genes on the single X chromosome in males to equal that of both X chromosomes in females. Site-specific histone acetylation by the male-specific lethal (MSL) complex is thought to play a fundamental role in the increased transcriptional output of the male X. Nucleation and sequence-independent spreading of the complex to active genes serves as a model for understanding the targeting and function of epigenetic chromatin-modifying complexes. Interestingly, two noncoding RNAs are key for MSL assembly and spreading to active genes along the length of the X chromosome.

166 citations


Journal ArticleDOI
TL;DR: Recent advances in knowledge of Xist-mediated silencing are discussed, focusing on Xist spreading, the nuclear organization of the inactive X chromosome, recruitment of the polycomb complex and the role of the nuclear matrix in the process of X chromosome inactivation.
Abstract: In female mammals, one of the two X chromosomes in each cell is transcriptionally silenced in order to achieve dosage compensation between the genders in a process called X chromosome inactivation. The master regulator of this process is the long non-coding RNA Xist. During X-inactivation, Xist accumulates in cis on the future inactive X chromosome, triggering a cascade of events that provoke the stable silencing of the entire chromosome, with relatively few genes remaining active. How Xist spreads, what are its binding sites, how it recruits silencing factors and how it induces a specific topological and nuclear organization of the chromatin all remain largely unanswered questions. Recent studies have improved our understanding of Xist localization and the proteins with which it interacts, allowing a reappraisal of ideas about Xist function. We discuss recent advances in our knowledge of Xist-mediated silencing, focusing on Xist spreading, the nuclear organization of the inactive X chromosome, recruitment of the polycomb complex and the role of the nuclear matrix in the process of X chromosome inactivation.

147 citations


Journal ArticleDOI
TL;DR: This study provides the first integrated analysis of the inactive X chromosome in the context of breast cancer and establishes that epigenetic erosion of the active X can lead to the disappearance of the Barr body in breast cancer cells.
Abstract: Disappearance of the Barr body is considered a hallmark of cancer, although whether this corresponds to genetic loss or to epigenetic instability and transcriptional reactivation is unclear. Here we show that breast tumors and cell lines frequently display major epigenetic instability of the inactive X chromosome, with highly abnormal 3D nuclear organization and global perturbations of heterochromatin, including gain of euchromatic marks and aberrant distributions of repressive marks such as H3K27me3 and promoter DNA methylation. Genome-wide profiling of chromatin and transcription reveal modified epigenomic landscapes in cancer cells and a significant degree of aberrant gene activity from the inactive X chromosome, including several genes involved in cancer promotion. We demonstrate that many of these genes are aberrantly reactivated in primary breast tumors, and we further demonstrate that epigenetic instability of the inactive X can lead to perturbed dosage of X-linked factors. Taken together, our study provides the first integrated analysis of the inactive X chromosome in the context of breast cancer and establishes that epigenetic erosion of the inactive X can lead to the disappearance of the Barr body in breast cancer cells. This work offers new insights and opens up the possibility of exploiting the inactive X chromosome as an epigenetic biomarker at the molecular and cytological levels in cancer.

Journal ArticleDOI
TL;DR: The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome, and the association of escape regions with TADs, in mouse and human, suggests that Tads are the primary targets during propagation of XCI over the X chromosome.
Abstract: Background: During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. Results: Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. Conclusions: The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of escape regions with TADs, in mouse and human, suggests that TADs are the primary targets during propagation of XCI over the X chromosome.

Journal ArticleDOI
TL;DR: A review of advances in the field of X inactivation, which results in random silencing of one X chromosome in female mammals, dedicated to Mary Lyon who passed away last year.
Abstract: X-chromosome inactivation, which was discovered by Mary Lyon in 1961 results in random silencing of one X chromosome in female mammals. This review is dedicated to Mary Lyon, who passed away last year. She predicted many of the features of X inactivation, for e.g., the existence of an X inactivation center, the role of L1 elements in spreading of silencing and the existence of genes that escape X inactivation. Starting from her published work here we summarize advances in the field.

Journal ArticleDOI
TL;DR: XWAS will provide the tools and incentive for others to incorporate the X chromosome into GWAS and similar studies in any species with an XX/XY system, hence enabling discoveries of novel loci implicated in many diseases and in their sexual dimorphism.
Abstract: XWAS is a new software suite for the analysis of the X chromosome in association studies and similar genetic studies. The X chromosome plays an important role in human disease and traits of many species, especially those with sexually dimorphic characteristics. Special attention needs to be given to its analysis due to the unique inheritance pattern, which leads to analytical complications that have resulted in the majority of genome-wide association studies (GWAS) either not considering X or mishandling it with toolsets that had been designed for non-sex chromosomes. We hence developed XWAS to fill the need for tools that are specially designed for analysis of X. Following extensive, stringent, and X-specific quality control, XWAS offers an array of statistical tests of association, including: 1) the standard test between a SNP (single nucleotide polymorphism) and disease risk, including after first stratifying individuals by sex, 2) a test for a differential effect of a SNP on disease between males and females, 3) motivated by X-inactivation, a test for higher variance of a trait in heterozygous females as compared with homozygous females, and 4) for all tests, a version that allows for combining evidence from all SNPs across a gene. We applied the toolset analysis pipeline to 16 GWAS datasets of immune-related disorders and 7 risk factors of coronary artery disease, and discovered several new X-linked genetic associations. XWAS will provide the tools and incentive for others to incorporate the X chromosome into GWAS and similar studies in any species with an XX/XY system, hence enabling discoveries of novel loci implicated in many diseases and in their sexual dimorphism.

Journal ArticleDOI
TL;DR: It is shown that dosage compensation has not evolved in threespine sticklebacks through upregulation of the X chromosome in males, and results suggest that dosage imbalances may have been avoided at haploinsufficient genes by retaining function of the Y chromosome allele through strong purifying selection.
Abstract: Sex chromosomes are subject to unique evolutionary forces that cause suppression of recombination, leading to sequence degeneration and the formation of heteromorphic chromosome pairs (i.e., XY or ZW). Although progress has been made in characterizing the outcomes of these evolutionary processes on vertebrate sex chromosomes, it is still unclear how recombination suppression and sequence divergence typically occur and how gene dosage imbalances are resolved in the heterogametic sex. The threespine stickleback fish (Gasterosteus aculeatus) is a powerful model system to explore vertebrate sex chromosome evolution, as it possesses an XY sex chromosome pair at relatively early stages of differentiation. Using a combination of whole-genome and transcriptome sequencing, we characterized sequence evolution and gene expression across the sex chromosomes. We uncovered two distinct evolutionary strata that correspond with known structural rearrangements on the Y chromosome. In the oldest stratum, only a handful of genes remain, and these genes are under strong purifying selection. By comparing sex-linked gene expression with expression of autosomal orthologs in an outgroup, we show that dosage compensation has not evolved in threespine sticklebacks through upregulation of the X chromosome in males. Instead, in the oldest stratum, the genes that still possess a Y chromosome allele are enriched for genes predicted to be dosage sensitive in mammals and yeast. Our results suggest that dosage imbalances may have been avoided at haploinsufficient genes by retaining function of the Y chromosome allele through strong purifying selection.

Journal ArticleDOI
TL;DR: This complex system of chromosomes in mammals continues to provide important insights into mechanisms of epigenetic regulation.
Abstract: Many organisms show major chromosomal differences between sexes. In mammals, females have two copies of a large, gene-rich chromosome, the X, whereas males have one X and a small, gene-poor Y. The imbalance in expression of several hundred genes is lethal if not dealt with by dosage compensation. The male-female difference is addressed by silencing of genes on one female X early in development. However, both males and females now have only one active X chromosome. This is compensated by twofold up-regulation of genes on the active X. This complex system continues to provide important insights into mechanisms of epigenetic regulation.

Journal ArticleDOI
TL;DR: Handedness in both sexes is associated with the AR CAG-repeat length, with longer repeats being related to a higher incidence of non-right-handedness and differences in AR signaling in the developing brain might be one of the factors that determine individual differences in brain lateralization.
Abstract: Prenatal androgen exposure has been suggested to be one of the factors influencing handedness, making the androgen receptor gene (AR) a likely candidate gene for individual differences in handedness. Here, we examined the relationship between the length of the CAG-repeat in AR and different handedness phenotypes in a sample of healthy adults of both sexes (n = 1057). Since AR is located on the X chromosome, statistical analyses in women heterozygous for CAG-repeat lengths are complicated by X chromosome inactivation. We thus analyzed a sample of women that were homozygous for the CAG-repeat length (n = 77). Mixed-handedness in men was significantly associated with longer CAG-repeat blocks and women homozygous for longer CAG-repeats showed a tendency for stronger left-handedness. These results suggest that handedness in both sexes is associated with the AR CAG-repeat length, with longer repeats being related to a higher incidence of non-right-handedness. Since longer CAG-repeat blocks have been linked to less efficient AR function, these results implicate that differences in AR signaling in the developing brain might be one of the factors that determine individual differences in brain lateralization.

Journal ArticleDOI
TL;DR: The findings implicate RAB39B, an essential regulator of vesicular-trafficking, in clinically typical PD, in multi-incident U.S. kindred with Parkinson’s disease (PD).
Abstract: To identify the causal gene in a multi-incident U.S. kindred with Parkinson’s disease (PD). We characterized a family with a classical PD phenotype in which 7 individuals (5 males and 2 females) were affected with a mean age at onset of 46.1 years (range, 29-57 years). We performed whole exome sequencing on 4 affected and 1 unaffected family members. Sanger-sequencing was then used to verify and genotype all candidate variants in the remainder of the pedigree. Cultured cells transfected with wild-type or mutant constructs were used to characterize proteins of interest. We identified a missense mutation (c.574G > A; p.G192R) in the RAB39B gene that closely segregated with disease and exhibited X-linked dominant inheritance with reduced penetrance in females. The mutation occurred in a highly conserved amino acid residue and was not observed among 87,725 X chromosomes in the Exome Aggregation Consortium dataset. Sequencing of the RAB39B coding region in 587 familial PD cases yielded two additional mutations (c.428C > G [p.A143G] and c.624_626delGAG [p.R209del]) that were predicted to be deleterious in silico but occurred in families that were not sufficiently informative to assess segregation with disease. Experiments in PC12 and SK-N-BE(2)C cells demonstrated that p.G192R resulted in mislocalization of the mutant protein, possibly by altering the structure of the hypervariable C-terminal domain which mediates intracellular targeting. Our findings implicate RAB39B, an essential regulator of vesicular-trafficking, in clinically typical PD. Further characterization of normal and aberrant RAB39B function might elucidate important mechanisms underlying neurodegeneration in PD and related disorders.

Journal ArticleDOI
TL;DR: The authors' data provides a set of genes with epigenetic alteration likely to be indicators of autoimmunity and emphasizes the role of CXCR3 in the natural history of PBC.
Abstract: Although the etiology of primary biliary cirrhosis (PBC) remains enigmatic, there are several pieces of data supporting the thesis that a strong genetic predisposition and environmental factors interact to produce a selective loss of tolerance. The striking female predominance of PBC has suggested that this sex predisposition may be secondary to epigenetic alterations on the X chromosome. In the present study, we rigorously defined the X chromosome methylation profile of CD4, CD8, and CD14 cells from 30 PBC patients and 30 controls. Genomic DNA from sorted CD4, CD8, and CD14 subpopulations was isolated, sonicated, and immunoprecipitated for analysis of methylation. All products were hybridized to a custom-tiled four-plex array containing 27,728 CpG islands annotated by UCSC and 22,532 well-characterized RefSeq promoter regions. Furthermore, bisulfite sequencing was then used for validation on a subsequent group of independent samples from PBC patients and controls. Thence, expression levels of selected X-linked genes were evaluated by quantitative real-time PCR with cDNA samples from all subjects. We report herein that a total of 20, 15, and 19 distinct gene promoters reflected a significant difference in DNA methylation in CD4+ T, CD8+ T, and CD14+ cells in patients with PBC. Interestingly, there was hypermethylation of FUNDC2 in CD8+ T cells and a striking demethylation of CXCR3 in CD4+ T cells, which inversely correlated with CXCR3 expression levels in CD4+ T cells from early-stage PBC patients. Our data provides a set of genes with epigenetic alteration likely to be indicators of autoimmunity and emphasizes the role of CXCR3 in the natural history of PBC.

Journal ArticleDOI
TL;DR: The pattern of gene expression is altered in KS, and the degree of differential gene expression in blood cells is associated with the clinical phenotype, which contributes to a number of pathologies in KS.
Abstract: Context: Klinefelter syndrome (KS) is the most common chromosome disorder in men (47,XXY), exhibiting a phenotype with marked variation and increased morbidity. The pathophysiological link between the supernumerary X chromosome and the clinical phenotype remains unknown. Objective: To elucidate whether differential gene expression patterns can be detected in KS patients and whether these are related to inherent clinical features. Design, Setting, Participants: EXAKT (Epigenetics, X-chromosomal Features and Clinical Applications in Klinefelter Syndrome Trial) is a Munster-based prospective project involving 132 Klinefelter men and their parents. A range of cardiovascular, inflammatory, and metabolic factors, in comparison to age-matched male (n = 50)/female controls (n = 50) and in relation to genetic features, is assessed. Main Outcomes and Measures: Our predefined hypothesis was that differential gene expression patterns in blood cells exist in KS patients vs male controls and are related to the clinical...

Journal ArticleDOI
TL;DR: It is shown that the extent to which diversity is reduced is incompatible with direct selection or the action of background selection and soft selective sweeps alone, and therefore, it is suggested that very strong selective sweeps have independently targeted these specific regions in several species.
Abstract: The unique inheritance pattern of the X chromosome exposes it to natural selection in a way that is different from that of the autosomes, potentially resulting in accelerated evolution. We perform a comparative analysis of X chromosome polymorphism in 10 great ape species, including humans. In most species, we identify striking megabase-wide regions, where nucleotide diversity is less than 20% of the chromosomal average. Such regions are found exclusively on the X chromosome. The regions overlap partially among species, suggesting that the underlying targets are partly shared among species. The regions have higher proportions of singleton SNPs, higher levels of population differentiation, and a higher nonsynonymous-to-synonymous substitution ratio than the rest of the X chromosome. We show that the extent to which diversity is reduced is incompatible with direct selection or the action of background selection and soft selective sweeps alone, and therefore, we suggest that very strong selective sweeps have independently targeted these specific regions in several species. The only genomic feature that we can identify as strongly associated with loss of diversity is the location of testis-expressed ampliconic genes, which also have reduced diversity around them. We hypothesize that these genes may be responsible for selective sweeps in the form of meiotic drive caused by an intragenomic conflict in male meiosis.

Journal ArticleDOI
TL;DR: Activating mutations of the fibroblast growth factor 23 (FGF-23) gene and inactivating mutations in the phosphate regulating gene (PHEX gene with homologies to endopeptidases on the X chromosome) have been identified and have been implicated in the pathogenesis of these disturbances.
Abstract: Hypophosphatemic rickets (HR) is a genetic disorder, which prevents sufficient reabsorption of phosphate in the proximal renal tubule, with increased phosphate excretion, resulting in rickets. The more common form of HR is an X-linked inherited trait, with a prevalence of 1/20,000. The defective gene is located on the X chromosome, but females may present with a wide variety of clinical manifestations. The less common form of HR is caused by autosomal-dominant transmission. Activating mutations of the fibroblast growth factor 23 (FGF-23) gene and inactivating mutations in the phosphate regulating gene (PHEX gene with homologies to endopeptidases on the X chromosome), involved in the regulation of FGF-23, have been identified and have been implicated in the pathogenesis of these disturbances. A review of etiopathogenesis and clinical, differential diagnostic and therapeutic aspects of HR, with a particular emphasis on bone impairment, is reported.

Journal ArticleDOI
TL;DR: The finding of disease-specific variants occurring in complete linkage disequilibrium raises new insights and intriguing questions about the origin of the disease haplotype, the existence of phenocopies and of reduced penetrance, and the causative genetic alteration in XDP.
Abstract: X-linked recessive dystonia-parkinsonism is a rare movement disorder that is highly prevalent in Panay Island in the Philippines. Earlier studies identified seven different genetic alterations within a 427-kb disease locus on the X chromosome; however, the exact disease-causing variant among these is still not unequivocally determined. To further investigate the genetic cause of this disease, we sequenced all previously reported genetic alterations in 166 patients and 473 Filipino controls. Singly occurring variants in our ethnically matched controls would have allowed us to define these as polymorphisms, but none were found. Instead, we identified five patients carrying none of the disease-associated variants, and one male control carrying all of them. In parallel, we searched for novel single-nucleotide variants using next-generation sequencing. We did not identify any shared variants in coding regions of the X chromosome. However, by validating intergenic variants discovered via genome sequencing, we were able to define the boundaries of the disease-specific haplotype and narrow the disease locus to a 294-kb region that includes four known genes. Using microarray-based analyses, we ruled out the presence of disease-linked copy number variants within the implicated region. Finally, we utilized in silico analysis and detected no strong evidence of regulatory regions surrounding the disease-associated variants. In conclusion, our finding of disease-specific variants occurring in complete linkage disequilibrium raises new insights and intriguing questions about the origin of the disease haplotype, the existence of phenocopies and of reduced penetrance, and the causative genetic alteration in XDP.

Journal ArticleDOI
TL;DR: It is proposed that the sex-independent, three-dimensional conformation of the X chromosome poises it for exploitation by the MSL complex, thereby facilitating spreading in males.

Journal ArticleDOI
TL;DR: The Sry transgene in FCG mice is present in multiple copies at one locus on chromosome 3, which does not interrupt known genes, suggesting that differences between XX and XY mice with the same type of gonad are not caused by difference in prenatal androgen levels.
Abstract: The “four core genotypes” (FCG) mouse model has emerged as a major model testing if sex differences in phenotypes are caused by sex chromosome complement (XX vs. XY) or gonadal hormones or both. The model involves deletion of the testis-determining gene Sry from the Y chromosome and insertion of an Sry transgene onto an autosome. It produces XX and XY mice with testes, and XX and XY mice with ovaries, so that XX and XY mice with the same type of gonad can be compared to assess phenotypic effects of sex chromosome complement in cells and tissues. We used PCR to amplify the Sry transgene and adjacent genomic sequences, to resolve the location of the Sry transgene to chromosome 3 and confirmed this location by fluorescence in situ hybridization (FISH) of the Sry construct to metaphase chromosomes. Using quantitative PCR, we estimate that 12–14 copies of the transgene were inserted. The anogenital distance (AGD) of FCG pups at 27–29 days after birth was not different in XX vs. XY males, or XX vs. XY females, suggesting that differences between XX and XY mice with the same type of gonad are not caused by difference in prenatal androgen levels. The Sry transgene in FCG mice is present in multiple copies at one locus on chromosome 3, which does not interrupt known genes. XX and XY mice with the same type of gonad do not show evidence of different androgen levels prenatally.

Journal ArticleDOI
TL;DR: It is shown that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes, which exist as tripartite ring structures, and that a parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome.
Abstract: The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the facioscapulohumeral muscular dystrophy locus. Although homology with canonical SMC family proteins suggests a role in chromosome organization, the mechanisms underlying SMCHD1 function and target site selection remain poorly understood. Here we show that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes, which exist as tripartite ring structures. Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins. We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms. A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome. Together, our results provide key insights into SMCHD1 function and target site selection.

Journal ArticleDOI
TL;DR: Collectively in this cohort of 19 novel cases of SRY-negative 46,XX DSD, the duplications upstream of SOX9 account for ~10.5% of the cases, and are responsible for the disease phenotype, even when inherited from a normal father.
Abstract: Duplications in the ~2 Mb desert region upstream of SOX9 at 17q24.3 may result in familial 46,XX disorders of sex development (DSD) without any effects on the XY background. A balanced translocation with its breakpoint falling within the same region has also been described in one XX DSD subject. We analyzed, by conventional and molecular cytogenetics, 19 novel SRY-negative unrelated 46,XX subjects both familial and sporadic, with isolated DSD. One of them had a de novo reciprocal t(11;17) translocation. Two cases carried partially overlapping 17q24.3 duplications ~500 kb upstream of SOX9, both inherited from their normal fathers. Breakpoints cloning showed that both duplications were in tandem, whereas the 17q in the reciprocal translocation was broken at ~800 kb upstream of SOX9, which is not only close to a previously described 46,XX DSD translocation, but also to translocations without any effects on the gonadal development. A further XX male, ascertained because of intellectual disability, carried a de novo cryptic duplication at Xq27.1, involving SOX3. CNVs involving SOX3 or its flanking regions have been reported in four XX DSD subjects. Collectively in our cohort of 19 novel cases of SRY-negative 46,XX DSD, the duplications upstream of SOX9 account for ~10.5% of the cases, and are responsible for the disease phenotype, even when inherited from a normal father. Translocations interrupting this region may also affect the gonadal development, possibly depending on the chromatin context of the recipient chromosome. SOX3 duplications may substitute SRY in some XX subjects.

Journal ArticleDOI
TL;DR: Evidence is provided that, in Silene latifolia, this largely involved losses of Y-linked genes, and not suppressed expression ofY-linked alleles, or gene additions to the X chromosome, which suggests that chromosome-wide dosage compensation does not occur in this plant.

Journal ArticleDOI
TL;DR: DNA methylation changes were widespread on all autosomal chromosomes in 45,X and in 47,XXY individuals, with Turner individuals presenting five times more affected loci, which suggests a certain stochastic/random contribution to the methylationChanges at each locus.
Abstract: Abnormal sex chromosome numbers in humans are observed in Turner (45,X) and Klinefelter (47,XXY) syndromes. Both syndromes are associated with several clinical phenotypes, whose molecular mechanisms are obscure, and show a range of inter-individual penetrance. In order to understand the effect of abnormal numbers of X chromosome on the methylome and its correlation to the variable clinical phenotype, we performed a genome-wide methylation analysis using MeDIP and Illumina’s Infinium assay on individuals with four karyotypes: 45,X, 46,XY, 46,XX, and 47,XXY. DNA methylation changes were widespread on all autosomal chromosomes in 45,X and in 47,XXY individuals, with Turner individuals presenting five times more affected loci. Differentially methylated CpGs, in most cases, have intermediate methylation levels and tend to occur outside CpG islands, especially in individuals with Turner syndrome. The X inactivation process appears to be less effective in Klinefelter syndrome as methylation on the X was decreased compared to normal female samples. In a large number of individuals, we verified several loci by pyrosequencing and observed only weak inter-loci correlations between the verified regions. This suggests a certain stochastic/random contribution to the methylation changes at each locus. Interestingly, methylation patterns on some PAR2 loci differ between male and Turner syndrome individuals and between female and Klinefelter syndrome individuals, which possibly contributed to this distinguished and unique autosomal methylation patterns in Turner and Klinefelter syndrome individuals. The presented data clearly show that gain or loss of an X chromosome results in different epigenetic effects, which are not necessary opposite.

Journal ArticleDOI
TL;DR: The findings expand the clinical spectrum of NAA10 and suggest that the proposed correlation between mutant Naa10 enzyme activity and phenotype severity is more complex than anticipated; the p.Tyr43Ser mutant enzyme has less catalytic activity than thep.Ser37Pro mutant associated with lethal Ogden syndrome but results in a milder phenotype.
Abstract: We report two brothers from a non-consanguineous Irish family presenting with a novel syndrome characterised by intellectual disability, facial dysmorphism, scoliosis and long QT. Their mother has a milder phenotype including long QT. X-linked inheritance was suspected. Whole exome sequencing identified a novel missense variant (c.128 A > C; p.Tyr43Ser) in NAA10 (X chromosome) as the cause of the family’s disorder. Sanger sequencing confirmed that the mutation arose de novo in the carrier mother. NAA10 encodes the catalytic subunit of the major human N-terminal acetylation complex NatA. In vitro assays for the p.Tyr43Ser mutant enzyme showed a significant decrease in catalytic activity and reduced stability compared to wild-type Naa10 protein. NAA10 has previously been associated with Ogden syndrome, Lenz microphthalmia syndrome and non-syndromic developmental delay. Our findings expand the clinical spectrum of NAA10 and suggest that the proposed correlation between mutant Naa10 enzyme activity and phenotype severity is more complex than anticipated; the p.Tyr43Ser mutant enzyme has less catalytic activity than the p.Ser37Pro mutant associated with lethal Ogden syndrome but results in a milder phenotype. Importantly, we highlight the need for cardiac assessment in males and females with NAA10 variants as both patients and carriers can have long QT.