ABSTRACT
X inactivation (XCI) is triggered by upregulation of XIST, which coats the chromosome in cis, promoting formation of a heterochromatic domain (Xi). XIST role beyond initiation of XCI is only beginning to be elucidated. Here, we demonstrate that XIST loss impairs differentiation of human mammary stem cells (MaSCs) and promotes emergence of highly tumorigenic and metastatic carcinomas. On the Xi, XIST deficiency triggers epigenetic changes and reactivation of genes overlapping Polycomb domains, including Mediator subunit MED14. MED14 overdosage results in increased Mediator levels and hyperactivation of the MaSC enhancer landscape and transcriptional program, making differentiation less favorable. We further demonstrate that loss of XIST and Xi transcriptional instability is common among human breast tumors of poor prognosis. We conclude that XIST is a gatekeeper of human mammary epithelium homeostasis, thus unveiling a paradigm in the control of somatic cell identity with potential consequences for our understanding of gender-specific malignancies.
Subject(s)
Mediator Complex/metabolism , Neoplastic Stem Cells/metabolism , RNA, Long Noncoding/metabolism , Breast Neoplasms/metabolism , Cell Differentiation , Epigenesis, Genetic , Humans , RNA, Long Noncoding/genetics , X Chromosome InactivationABSTRACT
The lncRNA Xist forms â¼50 diffraction-limited foci to transcriptionally silence one X chromosome. How this small number of RNA foci and interacting proteins regulate a much larger number of X-linked genes is unknown. We show that Xist foci are locally confined, contain â¼2 RNA molecules, and nucleate supramolecular complexes (SMACs) that include many copies of the critical silencing protein SPEN. Aggregation and exchange of SMAC proteins generate local protein gradients that regulate broad, proximal chromatin regions. Partitioning of numerous SPEN molecules into SMACs is mediated by their intrinsically disordered regions and essential for transcriptional repression. Polycomb deposition via SMACs induces chromatin compaction and the increase in SMACs density around genes, which propagates silencing across the X chromosome. Our findings introduce a mechanism for functional nuclear compartmentalization whereby crowding of transcriptional and architectural regulators enables the silencing of many target genes by few RNA molecules.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Mitochondrial Proteins/metabolism , RNA, Long Noncoding/metabolism , X Chromosome/metabolism , Animals , Cell Line , Embryonic Stem Cells , Fibroblasts , Gene Silencing , Humans , Mice , Protein Binding , X Chromosome InactivationABSTRACT
Facultative heterochromatin (fHC) concerns the developmentally regulated heterochromatinization of different regions of the genome and, in the case of the mammalian X chromosome and imprinted loci, of only one allele of a homologous pair. The formation of fHC participates in the timely repression of genes, by resisting strong trans activators. In this review, we discuss the molecular mechanisms underlying the establishment and maintenance of fHC in mammals using a mouse model. We focus on X-chromosome inactivation (XCI) as a paradigm for fHC but also relate it to genomic imprinting and homeobox (Hox) gene cluster repression. A vital role for noncoding transcription and/or transcripts emerges as the general principle of triggering XCI and canonical imprinting. However, other types of fHC are established through an unknown mechanism, independent of noncoding transcription (Hox clusters and noncanonical imprinting). We also extensively discuss polycomb-group repressive complexes (PRCs), which frequently play a vital role in fHC maintenance.
Subject(s)
Gene Expression Regulation, Developmental , Genomic Imprinting , Heterochromatin/metabolism , Polycomb-Group Proteins/genetics , X Chromosome Inactivation , X Chromosome/metabolism , Animals , Chromatin Assembly and Disassembly , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Embryo, Mammalian , Female , Gene Silencing , Heterochromatin/chemistry , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Histones/genetics , Histones/metabolism , Humans , Male , Oocytes/cytology , Oocytes/growth & development , Oocytes/metabolism , Polycomb-Group Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Spermatozoa/cytology , Spermatozoa/growth & development , Spermatozoa/metabolism , X Chromosome/chemistryABSTRACT
X-chromosome inactivation (XCI) is the epigenetic mechanism that ensures X-linked dosage compensation between cells of females (XX karyotype) and males (XY). XCI is essential for female embryos to survive through development and requires the accurate spatiotemporal regulation of many different factors to achieve remarkable chromosome-wide gene silencing. As a result of XCI, the active and inactive X chromosomes are functionally and structurally different, with the inactive X chromosome undergoing a major conformational reorganization within the nucleus. In this Review, we discuss the multiple layers of genetic and epigenetic regulation that underlie initiation of XCI during development and then maintain it throughout life, in light of the most recent findings in this rapidly advancing field. We discuss exciting new insights into the regulation of X inactive-specific transcript (XIST), the trigger and master regulator of XCI, and into the mechanisms and dynamics that underlie the silencing of nearly all X-linked genes. Finally, given the increasing interest in understanding the impact of chromosome organization on gene regulation, we provide an overview of the factors that are thought to reshape the 3D structure of the inactive X chromosome and of the relevance of such structural changes for XCI establishment and maintenance.
Subject(s)
Epigenesis, Genetic , RNA, Long Noncoding , Epigenesis, Genetic/genetics , Female , Gene Silencing , Humans , Male , RNA, Long Noncoding/genetics , X Chromosome/genetics , X Chromosome Inactivation/geneticsABSTRACT
During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing.
Subject(s)
Chromatin/metabolism , X Chromosome Inactivation/genetics , X Chromosome Inactivation/physiology , Acetylation , Animals , Chromatin/genetics , Embryonic Stem Cells , Epigenomics/methods , Female , Gene Silencing , Histone Deacetylases/metabolism , Histones/metabolism , Mice , Polycomb-Group Proteins/metabolism , Protein Processing, Post-Translational , RNA, Long Noncoding/metabolism , Transcription, Genetic , Ubiquitination , X Chromosome/metabolismABSTRACT
Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing.
Subject(s)
Mass Spectrometry/methods , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/analysis , RNA-Binding Proteins/metabolism , Animals , Chromatin/metabolism , Female , Gene Silencing , Humans , Mice , RNA-Binding Proteins/genetics , Ribonucleoproteins/analysisABSTRACT
Since the human genome was sequenced, the term "epigenetics" is increasingly being associated with the hope that we are more than just the sum of our genes. Might what we eat, the air we breathe, or even the emotions we feel influence not only our genes but those of descendants? The environment can certainly influence gene expression and can lead to disease, but transgenerational consequences are another matter. Although the inheritance of epigenetic characters can certainly occur-particularly in plants-how much is due to the environment and the extent to which it happens in humans remain unclear.
Subject(s)
Epigenesis, Genetic , Gene-Environment Interaction , Animals , DNA Methylation , Gene Expression Regulation , Germ Cells , Humans , Plants/geneticsABSTRACT
A new level of chromosome organization, topologically associating domains (TADs), was recently uncovered by chromosome conformation capture (3C) techniques. To explore TAD structure and function, we developed a polymer model that can extract the full repertoire of chromatin conformations within TADs from population-based 3C data. This model predicts actual physical distances and to what extent chromosomal contacts vary between cells. It also identifies interactions within single TADs that stabilize boundaries between TADs and allows us to identify and genetically validate key structural elements within TADs. Combining the model's predictions with high-resolution DNA FISH and quantitative RNA FISH for TADs within the X-inactivation center (Xic), we dissect the relationship between transcription and spatial proximity to cis-regulatory elements. We demonstrate that contacts between potential regulatory elements occur in the context of fluctuating structures rather than stable loops and propose that such fluctuations may contribute to asymmetric expression in the Xic during X inactivation.
Subject(s)
Chromosomes/chemistry , Transcription, Genetic , X Chromosome Inactivation , Animals , Chromatin/chemistry , Female , In Situ Hybridization, Fluorescence , Male , Mice , Models, Biological , Models, Molecular , RNA, Long Noncoding/metabolismABSTRACT
In mammals, the process of X-chromosome inactivation ensures equivalent levels of X-linked gene expression between males and females through the silencing of one of the two X chromosomes in female cells. The process is established early in development and is initiated by a unique locus, which produces a long noncoding RNA, Xist. The Xist transcript triggers gene silencing in cis by coating the future inactive X chromosome. It also induces a cascade of chromatin changes, including posttranslational histone modifications and DNA methylation, and leads to the stable repression of all X-linked genes throughout development and adult life. We review here recent progress in our understanding of the molecular mechanisms involved in the initiation of Xist expression, the propagation of the Xist RNA along the chromosome, and the cis-elements and trans-acting factors involved in the maintenance of the repressed state. We also describe the diverse strategies used by nonplacental mammals for X-chromosome dosage compensation and highlight the common features and differences between eutherians and metatherians, in particular regarding the involvement of long noncoding RNAs.
Subject(s)
Gene Silencing , RNA, Long Noncoding/genetics , X Chromosome Inactivation/genetics , Animals , Chromatin/genetics , Chromatin/ultrastructure , Chromosome Mapping , Chromosomes, Human, X/genetics , Embryonic Stem Cells/ultrastructure , Evolution, Molecular , Female , Genomic Imprinting , Humans , Long Interspersed Nucleotide Elements , Male , Marsupialia/genetics , Mice , Sex Determination Processes , Transcription Factors/genetics , X Chromosome/genetics , X Chromosome/ultrastructureABSTRACT
cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.
Subject(s)
Conserved Sequence/genetics , RNA, Long Noncoding/genetics , X Chromosome/genetics , Animals , Cell Line , Enhancer Elements, Genetic/genetics , Mice , Promoter Regions, Genetic/genetics , RNA, Antisense/genetics , Silencer Elements, Transcriptional/genetics , Transcription, Genetic/geneticsABSTRACT
In somatic nuclei of female therian mammals, the two X chromosomes display very different chromatin states: One X is typically euchromatic and transcriptionally active, and the other is mostly silent and forms a cytologically detectable heterochromatic structure termed the Barr body. These differences, which arise during female development as a result of X-chromosome inactivation (XCI), have been the focus of research for many decades. Initial approaches to define the structure of the inactive X chromosome (Xi) and its relationship to gene expression mainly involved microscopy-based approaches. More recently, with the advent of genomic techniques such as chromosome conformation capture, molecular details of the structure and expression of the Xi have been revealed. Here, we review our current knowledge of the 3D organization of the mammalian X-chromosome chromatin and discuss its relationship with gene activity in light of the initiation, spreading, and maintenance of XCI, as well as escape from gene silencing.
Subject(s)
Chromatin/genetics , Gene Expression Regulation/genetics , X Chromosome Inactivation/genetics , X Chromosome/genetics , Animals , Female , Gene Silencing , Humans , Mammals , RNA, Long Noncoding/geneticsABSTRACT
Random X inactivation represents a paradigm for monoallelic gene regulation during early ES cell differentiation. In mice, the choice of X chromosome to inactivate in XX cells is ensured by monoallelic regulation of Xist RNA via its antisense transcription unit Tsix/Xite. Homologous pairing events have been proposed to underlie asymmetric Tsix expression, but direct evidence has been lacking owing to their dynamic and transient nature. Here we investigate the live-cell dynamics and outcome of Tsix pairing in differentiating mouse ES cells. We find an overall increase in genome dynamics including the Xics during early differentiation. During pairing, however, Xic loci show markedly reduced movements. Upon separation, Tsix expression becomes transiently monoallelic, providing a window of opportunity for monoallelic Xist upregulation. Our findings reveal the spatiotemporal choreography of the X chromosomes during early differentiation and indicate a direct role for pairing in facilitating symmetry-breaking and monoallelic regulation of Xist during random X inactivation.
Subject(s)
Cell Differentiation , Chromosome Pairing , Embryonic Stem Cells/metabolism , X Chromosome Inactivation , X Chromosome/metabolism , Animals , Embryonic Stem Cells/cytology , Female , Mice , RNA, Long Noncoding , RNA, Untranslated/genetics , Time-Lapse ImagingABSTRACT
Paternal and maternal epigenomes undergo marked changes after fertilization1. Recent epigenomic studies have revealed the unusual chromatin landscapes that are present in oocytes, sperm and early preimplantation embryos, including atypical patterns of histone modifications2-4 and differences in chromosome organization and accessibility, both in gametes5-8 and after fertilization5,8-10. However, these studies have led to very different conclusions: the global absence of local topological-associated domains (TADs) in gametes and their appearance in the embryo8,9 versus the pre-existence of TADs and loops in the zygote5,11. The questions of whether parental structures can be inherited in the newly formed embryo and how these structures might relate to allele-specific gene regulation remain open. Here we map genomic interactions for each parental genome (including the X chromosome), using an optimized single-cell high-throughput chromosome conformation capture (HiC) protocol12,13, during preimplantation in the mouse. We integrate chromosome organization with allelic expression states and chromatin marks, and reveal that higher-order chromatin structure after fertilization coincides with an allele-specific enrichment of methylation of histone H3 at lysine 27. These early parental-specific domains correlate with gene repression and participate in parentally biased gene expression-including in recently described, transiently imprinted loci14. We also find TADs that arise in a non-parental-specific manner during a second wave of genome assembly. These de novo domains are associated with active chromatin. Finally, we obtain insights into the relationship between TADs and gene expression by investigating structural changes to the paternal X chromosome before and during X chromosome inactivation in preimplantation female embryos15. We find that TADs are lost as genes become silenced on the paternal X chromosome but linger in regions that escape X chromosome inactivation. These findings demonstrate the complex dynamics of three-dimensional genome organization and gene expression during early development.
Subject(s)
Blastocyst/cytology , Blastocyst/metabolism , Chromatin/metabolism , Embryonic Development/genetics , Fertilization/genetics , Germ Cells/cytology , Parents , Alleles , Animals , Chromatin/chemistry , Chromatin/genetics , Chromosome Positioning , Chromosomes, Mammalian/chemistry , Chromosomes, Mammalian/genetics , Chromosomes, Mammalian/metabolism , Female , Gene Expression Regulation, Developmental , Genome/genetics , Genomic Imprinting , Germ Cells/metabolism , Histones/chemistry , Histones/metabolism , Male , Methylation , Mice , Polycomb-Group Proteins/metabolism , Single-Cell Analysis , X Chromosome Inactivation/geneticsABSTRACT
Xist represents a paradigm for the function of long non-coding RNA in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Several proteins that bind to Xist RNA have recently been identified, including the transcriptional repressor SPEN1-3, the loss of which has been associated with deficient XCI at multiple loci2-6. Here we show in mice that SPEN is a key orchestrator of XCI in vivo and we elucidate its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and in embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly decreases the expression of genes that escape XCI. We show that SPEN is immediately recruited to the X chromosome upon the upregulation of Xist, and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, suggesting that active transcription is required to tether SPEN to chromatin. We define the SPOC domain as a major effector of the gene-silencing function of SPEN, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify the protein partners of SPOC, including NCoR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in the regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for the initiation of XCI, bridging Xist RNA with the transcription machinery-as well as with nucleosome remodellers and histone deacetylases-at active enhancers and promoters.
Subject(s)
DNA-Binding Proteins/metabolism , Epigenesis, Genetic , Gene Silencing , RNA-Binding Proteins/metabolism , Transcription, Genetic , X Chromosome Inactivation/genetics , X Chromosome/genetics , Animals , Blastocyst/cytology , Blastocyst/metabolism , Chromatin/genetics , Chromatin/metabolism , DNA-Binding Proteins/chemistry , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryonic Stem Cells/metabolism , Enhancer Elements, Genetic/genetics , Female , Histone Deacetylases/metabolism , Male , Methylation , Mice , Promoter Regions, Genetic/genetics , Protein Domains , RNA, Long Noncoding/genetics , RNA-Binding Proteins/chemistryABSTRACT
Accumulation of the Xist long noncoding RNA (lncRNA) on one X chromosome is the trigger for X chromosome inactivation (XCI) in female mammals. Xist expression, which needs to be tightly controlled, involves a cis-acting region, the X-inactivation center (Xic), containing many lncRNA genes that evolved concomitantly to Xist from protein-coding ancestors through pseudogeneization and loss of coding potential. Here, we uncover an essential role for the Xic-linked noncoding gene Ftx in the regulation of Xist expression. We show that Ftx is required in cis to promote Xist transcriptional activation and establishment of XCI. Importantly, we demonstrate that this function depends on Ftx transcription and not on the RNA products. Our findings illustrate the multiplicity of layers operating in the establishment of XCI and highlight the diversity in the modus operandi of the noncoding players.
Subject(s)
RNA, Long Noncoding/genetics , X Chromosome Inactivation/genetics , X Chromosome/genetics , Animals , Cell Line , Female , HEK293 Cells , Humans , Mammals/genetics , Mice , Transcription, Genetic/geneticsABSTRACT
The interplay between the topological organization of the genome and the regulation of gene expression remains unclear. Depletion of molecular factors (e.g. CTCF) underlying topologically associating domains (TADs) leads to modest alterations in gene expression, whereas genomic rearrangements involving TAD boundaries disrupt normal gene expression and can lead to pathological phenotypes. Here, we targeted the TAD neighboring that of the noncoding transcript Xist, which controls X-chromosome inactivation. Inverting 245â kb within the TAD led to expected rearrangement of CTCF-based contacts but revealed heterogeneity in the 'contact' potential of different CTCF sites. Expression of most genes therein remained unaffected in mouse embryonic stem cells and during differentiation. Interestingly, expression of Xist was ectopically upregulated. The same inversion in mouse embryos led to biased Xist expression. Smaller inversions and deletions of CTCF clusters led to similar results: rearrangement of contacts and limited changes in local gene expression, but significant changes in Xist expression in embryos. Our study suggests that the wiring of regulatory interactions within a TAD can influence the expression of genes in neighboring TADs, highlighting the existence of mechanisms of inter-TAD communication.
Subject(s)
RNA, Long Noncoding , X Chromosome Inactivation , Animals , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Chromatin , Communication , Gene Expression , Genome , Mice , RNA, Long Noncoding/genetics , X Chromosome Inactivation/geneticsABSTRACT
Molecular biology holds a vast potential for tackling climate change and biodiversity loss. Yet, it is largely absent from the current strategies. We call for a community-wide action to bring molecular biology to the forefront of climate change solutions.
Subject(s)
Biodiversity , Climate Change , Ecosystem , Molecular BiologyABSTRACT
During X chromosome inactivation (XCI), Xist RNA coats and silences one of the two X chromosomes in female cells. Little is known about how XCI spreads across the chromosome, although LINE-1 elements have been proposed to play a role. Here we show that LINEs participate in creating a silent nuclear compartment into which genes become recruited. A subset of young LINE-1 elements, however, is expressed during XCI, rather than being silenced. We demonstrate that such LINE expression requires the specific heterochromatic state induced by Xist. These LINEs often lie within escape-prone regions of the X chromosome, but close to genes that are subject to XCI, and are associated with putative endo-siRNAs. LINEs may thus facilitate XCI at different levels, with silent LINEs participating in assembly of a heterochromatic nuclear compartment induced by Xist, and active LINEs participating in local propagation of XCI into regions that would otherwise be prone to escape.