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![Xist/Tsix inversions lead to ectopic Xist expression, Xist RNA coating and X-linked gene silencing in male mESCs
a, nCounter RNA expression levels in 40-kb inversion (red), 70-kb inversion (blue) and wild type (gray). Each inversion has its corresponding wild type comparison because they were processed in different batches. Bars depict the mean of two independent experiments, with dots depicting the independent experiments. b, Xist RNA accumulation analyzed by RNA–FISH in male wild type (gray), 40-kb inversion (red) and 70-kb inversion (blue) mESCs. Each inversion has its corresponding wild type comparison because they were processed in different batches. Bars represent mean percentage of counted cells with either a Xist pinpoint, Xist accumulation or full Xist cloud. Dots represent independent experiments. n = 3 independent experiments, each experiment counting 100–400 cells (Supplementary Table 6 for exact sample size details). Statistical analysis was performed on independent experiments, using a two-sided Fisher’s exact test, mutant versus wild type. *P < 0.05 in all three experiments, see Supplementary Table 8 for exact P values. Scale bar, 2 μm, images at same scale. c, Violin plots indicating Xist RAP enrichment at the X chromosome. Substantial and significant enrichment in the 40-kb inversion (red), and slight but significant enrichment in the 70-kb inversion (blue) compared to wild type (gray). The x axis depicts mean RAP signal normalized to input. n = 2 independent experiments). Box corresponds to 25th and 75th percentiles, black line to the median, whiskers to 1.5× the interquantile range (IQR) and dots to individual datapoints beyond the 1.5× IQR. Statistical analysis was performed using a two-sided Wilcoxon Rank Sum test. d, Xist RAP signal (normalized to input, mean of two replicates) along the entire X chromosome in wild type (gray), 40-kb [Tsix-Xist] inversion (red) and 70-kb [Xite-Jpx] inversion (blue) male mESCs, in comparison to Xist RAP signal in female mouse lymphatic fibroblasts (MLF) (dotted line). e, X-linked gene silencing represented by the mRNA expression ratio between X-linked genes and bootstrapped autosomal genes (n = 1,000). Box plots represent the median (black line), 25–75% (box), 1.5× IQR (whiskers) and outliers (dots) of the bootstrap ratios. Statistical analysis was performed using a two-sided Wilcoxon Rank Sum test. f, X-linked gene silencing specific for Xist-bound genes, as shown by the difference in log2 fold-change in mRNA expression levels between inversion and wild type cells, for genes not bound by Xist RNA (labeled as no Xist) versus genes that are bound by Xist RNA (labeled as Xist). Box plots represent the median (black line), 25–75% (box) and 1.5× IQR (whiskers). Genes were classified as bound by Xist RNA when RAP signal at the TSS > 7.5, n = number of genes in each group. Statistical analysis was performed using a two-sided Wilcoxon Rank Sum test. NS, not significant. g, Schematic illustration of the effects of inverting Tsix-Xist (40 kb) and Xite-Jpx (70 kb) on transcriptional activity in male mESCs. Note that each inversion has been generated in two independent cell lines. Results for the second clones are shown in Supplementary Fig. 6.](publication/333407218/figure/fig4/AS:763398266564609@1559019738102/Xist-Tsix-inversions-lead-to-ectopic-Xist-expression-Xist-RNA-coating-and-X-linked-gene.png)
Xist/Tsix inversions lead to ectopic Xist expression, Xist RNA coating and X-linked gene silencing in male mESCs a, nCounter RNA expression levels in 40-kb inversion (red), 70-kb inversion (blue) and wild type (gray). Each inversion has its corresponding wild type comparison because they were processed in different batches. Bars depict the mean of two independent experiments, with dots depicting the independent experiments. b, Xist RNA accumulation analyzed by RNA–FISH in male wild type (gray), 40-kb inversion (red) and 70-kb inversion (blue) mESCs. Each inversion has its corresponding wild type comparison because they were processed in different batches. Bars represent mean percentage of counted cells with either a Xist pinpoint, Xist accumulation or full Xist cloud. Dots represent independent experiments. n = 3 independent experiments, each experiment counting 100–400 cells (Supplementary Table 6 for exact sample size details). Statistical analysis was performed on independent experiments, using a two-sided Fisher’s exact test, mutant versus wild type. *P < 0.05 in all three experiments, see Supplementary Table 8 for exact P values. Scale bar, 2 μm, images at same scale. c, Violin plots indicating Xist RAP enrichment at the X chromosome. Substantial and significant enrichment in the 40-kb inversion (red), and slight but significant enrichment in the 70-kb inversion (blue) compared to wild type (gray). The x axis depicts mean RAP signal normalized to input. n = 2 independent experiments). Box corresponds to 25th and 75th percentiles, black line to the median, whiskers to 1.5× the interquantile range (IQR) and dots to individual datapoints beyond the 1.5× IQR. Statistical analysis was performed using a two-sided Wilcoxon Rank Sum test. d, Xist RAP signal (normalized to input, mean of two replicates) along the entire X chromosome in wild type (gray), 40-kb [Tsix-Xist] inversion (red) and 70-kb [Xite-Jpx] inversion (blue) male mESCs, in comparison to Xist RAP signal in female mouse lymphatic fibroblasts (MLF) (dotted line). e, X-linked gene silencing represented by the mRNA expression ratio between X-linked genes and bootstrapped autosomal genes (n = 1,000). Box plots represent the median (black line), 25–75% (box), 1.5× IQR (whiskers) and outliers (dots) of the bootstrap ratios. Statistical analysis was performed using a two-sided Wilcoxon Rank Sum test. f, X-linked gene silencing specific for Xist-bound genes, as shown by the difference in log2 fold-change in mRNA expression levels between inversion and wild type cells, for genes not bound by Xist RNA (labeled as no Xist) versus genes that are bound by Xist RNA (labeled as Xist). Box plots represent the median (black line), 25–75% (box) and 1.5× IQR (whiskers). Genes were classified as bound by Xist RNA when RAP signal at the TSS > 7.5, n = number of genes in each group. Statistical analysis was performed using a two-sided Wilcoxon Rank Sum test. NS, not significant. g, Schematic illustration of the effects of inverting Tsix-Xist (40 kb) and Xite-Jpx (70 kb) on transcriptional activity in male mESCs. Note that each inversion has been generated in two independent cell lines. Results for the second clones are shown in Supplementary Fig. 6.
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The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically asso...
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Regulation of gene expression by chromatin structure has been under intensive investigation, establishing nuclear organization and genome architecture as a potent and effective means of regulating developmental processes. The substantial growth in our knowledge of the molecular mechanisms underlying retinogenesis has been powered by several genome-...
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... Though XIST is used in XCI by all eutherians, the precise mechanisms deployed are variable, but a unifying regulatory system is emerging for initiating and maintaining random XCI [21,22]. As illustrated by studies in the mouse, the chromatin organization of the active and inactive X become distinct [23]. The precise timing for the initiation of inactivation is thought to be very early, taking place in the epiblast, before implantation [19]. ...
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’.
... These regulatory requirements are integrated by non-coding actors flanking the XIST gene within the X-inactivation centre (XIC). These include cREs [34][35][36][37] and a compendium of LRGs 38-42 the action of which is delimited by topological chromatin domains [43][44][45] . Significant differences have been reported in the way XIST is regulated by XIC actors between humans and mice, which may underly the divergence in timing and pattern of XIST expression during evolution [46][47][48][49] . ...
Unravelling how gene regulatory networks are remodelled during evolution is crucial to understand how species adapt to environmental changes. We addressed this question for X-chromosome inactivation, a process essential to female development that is governed, in eutherians, by the XIST lncRNA and its cis-regulators. To reach high resolution, we studied closely related primate species, spanning 55 million years of evolution. We show that the XIST regulatory circuitry has diversified extensively over such an evolutionary timeframe. The insertion of a HERVK transposon has reshuffled XIST 3D interaction network in macaque embryonic stem cells (ESC) and XIST expression is maintained by the additive effects of the JPX lncRNA gene and a macaque specific enhancer. In contrast, JPX is the main contributor to XIST expression in human ESCs but is not significantly involved in XIST regulation in marmoset ESCs. None of these entities are however under purifying selection, which suggests that neutrally evolving non-coding elements harbour high adaptive potentials.
... The Xist-containing TAD is highly conserved in human; however, the Tsix TAD, like the gene itself, is less conserved (Bonev et al., 2017;Galupa and Heard, 2018;Rao et al., 2014). Topological partitioning of positive and negative controllers of XCI helps facilitate their differential regulation, with an inversion of the TAD boundary that places the Xist and Tsix promoters in their opposing TADs, leading to a switch in promoter interactions and mis-regulation of their expression (van Bemmel et al., 2019). ...
In mammals, the second X chromosome in females is silenced to enable dosage compensation between XX females and XY males. This essential process involves the formation of a dense chromatin state on the inactive X (Xi) chromosome. There is a wealth of information about the hallmarks of Xi chromatin and the contribution each makes to silencing, leaving the tantalising possibility of learning from this knowledge to potentially remove silencing to treat X-linked diseases in females. Here, we discuss the role of each chromatin feature in the establishment and maintenance of the silent state, which is of crucial relevance for such a goal.
... 55 In most cases, TAD formation precedes gene expression. 56 In the human genome, TADs emerge during the eight-cell stage when the interactions between regulatory elements within TAD are low and gradually increase throughout embryo development. ...
... This alteration in the regulatory environment enables enhancers to interact with nontarget promoters, causing promoters that should regulate Tsix gene expression to be ectopically activated within TAD-E, ultimately leading to aberrant Xist expression in male mESCs. 56 The HoxD gene clusters locate between two adjacent TADs: the telomeric side (T-DOM) and the centromeric side (C-DOM). During mammalian limb development, HoxD genes are sequentially regulated by spatiotemporalspecific enhancers within these two TADs. ...
Linear DNA undergoes a series of compression and folding events, forming various three-dimensional (3D) structural units in mammalian cells, including chromosomal territory, compartment, topologically associating domain, and chromatin loop. These structures play crucial roles in regulating gene expression, cell differentiation, and disease progression. Deciphering the principles underlying 3D genome folding and the molecular mechanisms governing cell fate determination remains a challenge. With advancements in high-throughput sequencing and imaging techniques, the hierarchical organization and functional roles of higher-order chromatin structures have been gradually illuminated. This review systematically discussed the structural hierarchy of the 3D genome, the effects and mechanisms of cis-regulatory elements interaction in the 3D genome for regulating spatiotemporally specific gene expression, the roles and mechanisms of dynamic changes in 3D chromatin conformation during embryonic development, and the pathological mechanisms of diseases such as congenital developmental abnormalities and cancer, which are attributed to alterations in 3D genome organization and aberrations in key structural proteins. Finally, prospects were made for the research about 3D genome structure, function, and genetic intervention, and the roles in disease development, prevention, and treatment, which may offer some clues for precise diagnosis and treatment of related diseases.
... Hi-C creates contact matrices that sample all interactions from all regions of the genome (all versus all). Hi-C has been pivotal in elucidating key principles in the organization of the genome, such as chromatin territories 44 , TADs 10 , X-inactivation 45,46 and even interchromosmal trans-interactions 47 . The resolution has markedly improved with optimization of the technique and increased sequencing depth 34,48 . ...
Micro Capture-C (MCC) is a chromatin conformation capture (3C) method for visualizing reproducible three-dimensional contacts of specified regions of the genome at base pair resolution. These methods are an established family of techniques that use proximity ligation to assay the topology of chromatin. MCC can generate data at substantially higher resolution than previous techniques through multiple refinements of the 3C method. Using a sequence agnostic nuclease, the maintenance of cellular integrity and full sequencing of the ligation junctions, MCC achieves subnucleosomal levels of resolution, which can be used to reveal transcription factor binding sites analogous to DNAse I footprinting. Gene dense regions, close-range enhancer-promoter contacts, individual enhancers within super-enhancers and multiple other types of loci or regulatory regions that were previously challenging to assay with conventional 3C techniques, are readily observed using MCC. MCC requires training in common molecular biology techniques and bioinformatics to perform the experiment and analyze the data. The protocol can be expected to be completed in a 3 week timeframe for experienced molecular biologists.
... Replacing the IDR of the mammalian orthologue of MSL2 with that of the D. melanogaster protein and expression of roX2 is sufficient to nucleate ectopic X chromosome dosage compensation in mammalian cells, showing that the roX-MSL2 IDR interaction is the primary determinant of compartmentalization of the X chromosome and that such interactions are preserved over vast evolutionary distances 109 . Similar processes are involved in the regulation of X chromosome dosage compensation in placental mammals by XIST, which performs several functions, including repulsion of euchromatic factors, scaffolding of new heterochromatic factors and reorganization of chromosome structure [110][111][112][113] . ...
Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term ‘lncRNAs’ encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease. This Consensus Statement addresses the definition, nomenclature and classification of long non-coding RNAs, and provides a shared viewpoint on their features and functions. The authors also discuss research challenges and provide recommendations to advance our understanding of long non-coding RNAs.
... [104,125,126] When switching the orientation and swapping Xist and Tsix in opposite TADs, Xist became regulated like Tsix normally would and vice versa, indicating that the TAD boundary has an important function in shielding developmentally regulated genes such as Xist and Tsix from inappropriate regulatory inputs from enhancers in the neighboring TADs. [127,128] In sum, TADs might increase the precision of gene regulation, in particular when it comes to developmental transition contexts [124,127] or during specific cellular responses as in the case of inflammation in macrophages. [123] Once again, the study of X inactivation led to key insight into the relationships between TADs and transcription. ...
... [104,125,126] When switching the orientation and swapping Xist and Tsix in opposite TADs, Xist became regulated like Tsix normally would and vice versa, indicating that the TAD boundary has an important function in shielding developmentally regulated genes such as Xist and Tsix from inappropriate regulatory inputs from enhancers in the neighboring TADs. [127,128] In sum, TADs might increase the precision of gene regulation, in particular when it comes to developmental transition contexts [124,127] or during specific cellular responses as in the case of inflammation in macrophages. [123] Once again, the study of X inactivation led to key insight into the relationships between TADs and transcription. ...
The spatial organization of genomes is becoming increasingly understood. In mammals, where it is most investigated, this organization ties in with transcription, so an important research objective is to understand whether gene activity is a cause or a consequence of genome folding in space. In this regard, the phenomena of X‐chromosome inactivation and reactivation open a unique window of investigation because of the singularities of the inactive X chromosome. Here we focus on the cause–consequence nexus between genome conformation and transcription and explain how recent results about the structural changes associated with inactivation and reactivation of the X chromosome shed light on this problem. The cause–consequence nexus between genome conformation and transcription is essential for our understanding of gene regulation. Uniquely addressed by the phenomena of X inactivation and reactivation in female mammals, where transcription and 3D structure change chromosome‐wide, we highlight how recent insights can help us understand gene regulation in 3D.
... Indeed, from the data, the packing density for the large-scale structure is high, although large-scale structures were shown to be on top of each other in certain cases, possibly reflecting the familiar TADs (16,17). Tangles, knots, or complicated looping configurations were not observed, as discussed in the literature (46,47). The large-scale structure is also seen to be smoothly aligned and associating with one another. ...
Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome structure, which is present throughout the nucleus. This study further documents and analyzes these chromosome structures. The paper is divided into four parts: 1) evidence (preliminary) for a unified interphase chromosome structure; 2) a proposed unified interphase chromosome architecture; 3) organization as chromosome territories (e.g., fitting the 46 human chromosomes into a 10-μm-diameter nucleus); and 4) structure unification into a polytene chromosome architecture and lampbrush chromosomes. Finally, the paper concludes with a living light microscopy cell study showing that the G1 nucleus contains very similar structures throughout. The main finding is that this chromosome structure appears to coil the 11-nm nucleosome fiber into a defined hollow structure, analogous to a Slinky helical spring [ https://en.wikipedia.org/wiki/Slinky ; motif used in Bowerman et al. , eLife 10, e65587 (2021)]. This Slinky architecture can be used to build chromosome territories, extended to the polytene chromosome structure, as well as to the structure of lampbrush chromosomes.
... Examples include the acquisition of Topologically Associated Domains (TADs) during embryogenesis and the role of transcription in defining local Chromosome Interaction Domains (CIDs) in bacteria and archaea [8][9][10][11] . Pathological consequences have also been documented in mammals, caused by tertiary structure disruption with transcriptional consequences [12][13][14][15] . A growing body of data has linked tertiary structure with primary sequence evolution. ...
In all organisms, the DNA sequence and the structural organization of chromosomes affect gene expression. The extremely thermophilic crenarchaeon Sulfolobus has one circular chromosome with three origins of replication. We previously revealed that this chromosome has defined A and B compartments that have high and low gene expression, respectively. As well as higher levels of gene expression, the A compartment contains the origins of replication. To evaluate the impact of three-dimensional organization on genome evolution, we characterized the effect of replication origins and compartmentalization on primary sequence evolution in eleven Sulfolobus species. Using single-nucleotide polymorphism analyses, we found that distance from an origin of replication was associated with increased mutation rates in the B but not in the A compartment. The enhanced polymorphisms distal to replication origins suggest that replication termination may have a causal role in their generation. Further mutational analyses revealed that the sequences in the A compartment are less likely to be mutated, and that there is stronger purifying selection than in the B compartment. Finally, we applied the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to show that the B compartment is less accessible than the A compartment. Taken together, our data suggest that compartmentalization of chromosomal DNA can influence chromosome evolution in Sulfolobus. We propose that the A compartment serves as a haven for stable maintenance of gene sequences, while sequences in the B compartment can be diversified. Analysis of eleven Sulfolobus strains reveals that chromosome organization affects mutation rates in this species.
... These two loci overlap one another in a sense and antisense orientation but are in distinct topologically associating domains (TADs); Tsix is the antisense repressor of Xist, whose up-regulation leads to X inactivation (81). The bipartite structure of the locus in two TADs facilitates partitioning of the X inactivation center (XIC) and supports appropriate timing of X inactivation through Xist transcription in early development (82). Moreover, an early step in the formation of heterochromatin across the inactive X is the silencing of LINEs and SINEs within the Xist RNA compartment (77). ...
Mobile elements and repetitive genomic regions are sources of lineage-specific genomic innovation and uniquely fingerprint individual genomes. Comprehensive analyses of such repeat elements, including those found in more complex regions of the genome, require a complete, linear genome assembly. We present a de novo repeat discovery and annotation of the T2T-CHM13 human reference genome. We identified previously unknown satellite arrays, expanded the catalog of variants and families for repeats and mobile elements, characterized classes of complex composite repeats, and located retroelement transduction events. We detected nascent transcription and delineated CpG methylation profiles to define the structure of transcriptionally active retroelements in humans, including those in centromeres. These data expand our insight into the diversity, distribution, and evolution of repetitive regions that have shaped the human genome.
