Targeted Mutagenesis of Tsix Leads to Nonrandom X Inactivation

AbstractDuring X inactivation, mammalian female cells make the selection of one active and one inactive X chromosome. X chromosome choice occurs randomly and results in Xist upregulation on the inactive X. We have hypothesized that the antisense gene, Tsix, controls Xist expression. Here, we create a targeted deletion of Tsix in female and male mouse cells. Despite a deficiency of Tsix RNA, X chromosome counting remains intact: female cells still inactivate one X, while male cells block X inactivation. However, heterozygous female cells show skewed Xist expression and primary nonrandom inactivation of the mutant X. The ability of the mutant X to block Xist accumulation is compromised. We conclude that Tsix regulates Xist in cis and determines X chromosome choice without affecting silencing. Therefore, counting, choice, and silencing are genetically separable. Contrasting effects in XX and XY cells argue that negative and positive factors are involved in choosing active and inactive Xs

Retinoic acid accelerates downregulation of the Xist repressor, Oct4, and increases the likelihood of Xist activation when Tsix is deficient

<p>Abstract</p> <p>Background</p> <p>Imbalances in X-linked gene dosage between the sexes are resolved by transcriptionally silencing one of two X-chromosomes in female cells of the early mammalian embryo. X-inactivation is triggered by expression of the non-coding <it>Xist </it>gene. In turn, <it>Xist </it>is dually regulated by the antisense Tsix RNA and by the Oct4 pluripotency factor. Although there is general agreement that <it>Tsix </it>is an inhibitor of <it>Xist</it>, some laboratories have observed ectopic <it>Xist </it>induction in differentiating male ES cells when <it>Tsix </it>is mutated, whereas we have not observed significant changes in <it>Xist</it>. These observational differences have led to fundamentally diverse models of X-chromosome counting. Here, we investigate if different methods of cell differentiation and use of all <it>-trans </it>retinoic acid (RA) could be causative factors and how they might impact <it>Xist </it>expression.</p> <p>Results</p> <p>We compared suspension and cell-adhesion cultures in the presence or absence of RA and find that RA significantly impacts <it>Xist </it>expression in <it>Tsix</it>-mutant male cells. Whereas the standard embryoid body method infrequently leads to ectopic Xist expression, adding RA generates a significant number of Xist-positive male cells. However, while normal Xist clouds in wild-type female cells are robust and well-circumscribed, those found in the RA-treated mutant males are loosely dispersed. Furthermore, ectopic Xist expression does not generally lead to complete gene silencing. We attribute the effect of RA on <it>Xist </it>to RA's repressive influence on Oct4, a pluripotency factor recently shown to regulate <it>Tsix </it>and <it>Xist</it>. RA-treated ES cells exhibit accelerated decreases in Oct4 RNA levels and also display accelerated loss of binding to <it>Xist </it>intron 1. When <it>Tsix </it>is deficient, the faster kinetics of Oct4 loss tip the equilibrium towards <it>Xist </it>expression. However, the aberrant Xist clusters are unlikely to explain elevated cell death, as X-linked silencing does not necessarily correlate with the qualitatively aberrant Xist clusters.</p> <p>Conclusions</p> <p>We conclude that RA treatment leads to premature downregulation of Oct4 and partial derepression of <it>Xist </it>irrespective of X-chromosome counting. RA-induced Xist clusters in male cells do not result in global or stable silencing, and excess cell death is not observed. These data and RA's known pleiotropic effects on ES transcription networks suggest that RA differentation bypasses normal X-inactivation controls and should be used judiciously. We propose that the likelihood of <it>Xist </it>expression is determined by a balance of multiple <it>Xist </it>activators and repressors, and that levels of Oct4 and Tsix are crucial toward achieving this balance.</p

X-chromosome hyperactivation in mammals via nonlinear relationships between chromatin states and transcription

Dosage compensation in mammals occurs at two levels. In addition to balancing X-chromosome dosage between males and females via X-inactivation, mammals also balance dosage of Xs and autosomes. It has been proposed that X-autosome equalization occurs by upregulation of Xa (active X). To investigate mechanism, we perform allele-specific ChIP-seq for chromatin epitopes and analyze RNA-seq data. The hypertranscribed Xa demonstrates enrichment of active chromatin marks relative to autosomes. We derive predictive models for relationships among POL-II, active mark densities, and gene expression, and suggest that Xa upregulation involves increased transcription initiation and elongation. Enrichment of active marks on Xa does not scale proportionally with transcription output, a disparity explained by nonlinear quantitative dependencies among active histone marks, POL-II occupancy, and transcription. Significantly, the trend of nonlinear upregulation also occurs on autosomes. Thus, Xa upregulation involves combined increases of active histone marks and POL-II occupancy, without invoking X-specific dependencies between chromatin states and transcription

Xist RNA Is a Potent Suppressor of Hematologic Cancer in Mice

SummaryX chromosome aneuploidies have long been associated with human cancers, but causality has not been established. In mammals, X chromosome inactivation (XCI) is triggered by Xist RNA to equalize gene expression between the sexes. Here we delete Xist in the blood compartment of mice and demonstrate that mutant females develop a highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome (mixed MPN/MDS) with 100% penetrance. Significant disease components include primary myelofibrosis, leukemia, histiocytic sarcoma, and vasculitis. Xist-deficient hematopoietic stem cells (HSCs) show aberrant maturation and age-dependent loss. Reconstitution experiments indicate that MPN/MDS and myelofibrosis are of hematopoietic rather than stromal origin. We propose that Xist loss results in X reactivation and consequent genome-wide changes that lead to cancer, thereby causally linking the X chromosome to cancer in mice. Thus, Xist RNA not only is required to maintain XCI but also suppresses cancer in vivo

Dynamic Regulation of ARGONAUTE4 within Multiple Nuclear Bodies in Arabidopsis thaliana

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent—disruption of one class does not affect the other—suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis

A Mammal-Specific Doublesex Homolog Associates with Male Sex Chromatin and Is Required for Male Meiosis

Gametogenesis is a sexually dimorphic process requiring profound differences in germ cell differentiation between the sexes. In mammals, the presence of heteromorphic sex chromosomes in males creates additional sex-specific challenges, including incomplete X and Y pairing during meiotic prophase. This triggers formation of a heterochromatin domain, the XY body. The XY body disassembles after prophase, but specialized sex chromatin persists, with further modification, through meiosis. Here, we investigate the function of DMRT7, a mammal-specific protein related to the invertebrate sexual regulators Doublesex and MAB-3. We find that DMRT7 preferentially localizes to the XY body in the pachytene stage of meiotic prophase and is required for male meiosis. In Dmrt7 mutants, meiotic pairing and recombination appear normal, and a transcriptionally silenced XY body with appropriate chromatin marks is formed, but most germ cells undergo apoptosis during pachynema. A minority of mutant cells can progress to diplonema, but many of these escaping cells have abnormal sex chromatin lacking histone H3K9 di- and trimethylation and heterochromatin protein 1β accumulation, modifications that normally occur between pachynema and diplonema. Based on the localization of DMRT7 to the XY body and the sex chromatin defects observed in Dmrt7 mutants, we conclude that DMRT7 plays a role in the sex chromatin transformation that occurs between pachynema and diplonema. We suggest that DMRT7 may help control the transition from meiotic sex chromosome inactivation to postmeiotic sex chromatin in males. In addition, because it is found in all branches of mammals, but not in other vertebrates, Dmrt7 may shed light on evolution of meiosis and of sex chromatin

Large-Scale Population Study of Human Cell Lines Indicates that Dosage Compensation Is Virtually Complete

X chromosome inactivation in female mammals results in dosage compensation of X-linked gene products between the sexes. In humans there is evidence that a substantial proportion of genes escape from silencing. We have carried out a large-scale analysis of gene expression in lymphoblastoid cell lines from four human populations to determine the extent to which escape from X chromosome inactivation disrupts dosage compensation. We conclude that dosage compensation is virtually complete. Overall expression from the X chromosome is only slightly higher in females and can largely be accounted for by elevated female expression of approximately 5% of X-linked genes. We suggest that the potential contribution of escape from X chromosome inactivation to phenotypic differences between the sexes is more limited than previously believed

Allele-Specific Chromatin Immunoprecipitation Studies Show Genetic Influence on Chromatin State in Human Genome

Several recent studies have shown a genetic influence on gene expression variation, including variation between the two chromosomes within an individual and variation between individuals at the population level. We hypothesized that genetic inheritance may also affect variation in chromatin states. To test this hypothesis, we analyzed chromatin states in 12 lymphoblastoid cells derived from two Centre d'Etude du Polymorphisme Humain families using an allele-specific chromatin immunoprecipitation (ChIP-on-chip) assay with Affymetrix 10K SNP chip. We performed the allele-specific ChIP-on-chip assays for the 12 lymphoblastoid cells using antibodies targeting at RNA polymerase II and five post-translation modified forms of the histone H3 protein. The use of multiple cell lines from the Centre d'Etude du Polymorphisme Humain families allowed us to evaluate variation of chromatin states across pedigrees. These studies demonstrated that chromatin state clustered by family. Our results support the idea that genetic inheritance can determine the epigenetic state of the chromatin as shown previously in model organisms. To our knowledge, this is the first demonstration in humans that genetics may be an important factor that influences global chromatin state mediated by histone modification, the hallmark of the epigenetic phenomena

ATRX Directs Binding of PRC2 to Xist RNA and Polycomb Targets

X chromosome inactivation (XCI) depends on the long noncoding RNA Xist and its recruitment of Polycomb Repressive Complex 2 (PRC2). PRC2 is also targeted to other sites throughout the genome to effect transcriptional repression. Using XCI as a model, we apply an unbiased proteomics approach to isolate Xist and PRC2 regulators and identified ATRX. ATRX unexpectedly functions as a high-affinity RNA-binding protein that directly interacts with RepA/Xist RNA to promote loading of PRC2 in vivo. Without ATRX, PRC2 cannot load onto Xist RNA nor spread in cis along the X chromosome. Moreover, epigenomic profiling reveals that genome-wide targeting of PRC2 depends on ATRX, as loss of ATRX leads to spatial redistribution of PRC2 and derepression of Polycomb responsive genes. Thus, ATRX is a required specificity determinant for PRC2 targeting and function