RESUMO
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.
Assuntos
Cromossomos/química , Transcrição Gênica , Inativação do Cromossomo X , Animais , Cromatina/química , Feminino , Hibridização in Situ Fluorescente , Masculino , Camundongos , Modelos Biológicos , Modelos Moleculares , RNA Longo não Codificante/metabolismoRESUMO
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.
Assuntos
Sequência Conservada/genética , RNA Longo não Codificante/genética , Cromossomo X/genética , Animais , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Camundongos , Regiões Promotoras Genéticas/genética , RNA Antissenso/genética , Elementos Silenciadores Transcricionais/genética , Transcrição Gênica/genéticaRESUMO
Changes in gene dosage can have tremendous evolutionary potential (e.g. whole-genome duplications), but without compensatory mechanisms, they can also lead to gene dysregulation and pathologies. Sex chromosomes are a paradigmatic example of naturally occurring gene dosage differences and their compensation. In species with chromosome-based sex determination, individuals within the same population necessarily show 'natural' differences in gene dosage for the sex chromosomes. In this Review, we focus on the mammalian X chromosome and discuss recent new insights into the dosage-compensation mechanisms that evolved along with the emergence of sex chromosomes, namely X-inactivation and X-upregulation. We also discuss the evolution of the genetic loci and molecular players involved, as well as the regulatory diversity and potentially different requirements for dosage compensation across mammalian species.
Assuntos
Mecanismo Genético de Compensação de Dose , Mamíferos , Inativação do Cromossomo X , Cromossomo X , Animais , Humanos , Cromossomo X/genética , Mamíferos/genética , Inativação do Cromossomo X/genética , Dosagem de Genes , Evolução MolecularRESUMO
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.
Assuntos
Cromatina/genética , Regulação da Expressão Gênica/genética , Inativação do Cromossomo X/genética , Cromossomo X/genética , Animais , Feminino , Inativação Gênica , Humanos , Mamíferos , RNA Longo não Codificante/genéticaRESUMO
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.
Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Fertilização/genética , Células Germinativas/citologia , Pais , Alelos , Animais , Cromatina/química , Cromatina/genética , Posicionamento Cromossômico , Cromossomos de Mamíferos/química , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genoma/genética , Impressão Genômica , Células Germinativas/metabolismo , Histonas/química , Histonas/metabolismo , Masculino , Metilação , Camundongos , Proteínas do Grupo Polycomb/metabolismo , Análise de Célula Única , Inativação do Cromossomo X/genéticaRESUMO
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.
Assuntos
RNA Longo não Codificante/genética , Inativação do Cromossomo X/genética , Cromossomo X/genética , Animais , Linhagem Celular , Feminino , Células HEK293 , Humanos , Mamíferos/genética , Camundongos , Transcrição Gênica/genéticaRESUMO
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.
Assuntos
RNA Longo não Codificante , Inativação do Cromossomo X , Animais , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina , Comunicação , Expressão Gênica , Genoma , Camundongos , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genéticaRESUMO
How does the folding of the genome relate to the regulation of gene expression? Using fly embryos and quantitative live imaging, a recent study by Yokoshi et al. reveals that the answer might depend on whether enhancer-promoter communication occurs inside or in-between topological domains.
Assuntos
Comunicação , Elementos Facilitadores Genéticos , Regiões Promotoras GenéticasRESUMO
Developmental enhancers drive gene expression in specific cell types during animal development. They integrate signals from many different sources mediated through the binding of transcription factors, producing specific responses in gene expression. Transcription factors often bind low-affinity sequences for only short durations. How brief, low-affinity interactions drive efficient transcription and robust gene expression is a central question in developmental biology. Localized high concentrations of transcription factors have been suggested as a possible mechanism by which to use these enhancer sites effectively. Here, we discuss the evidence for such transcriptional microenvironments, mechanisms for their formation and the biological consequences of such sub-nuclear compartmentalization for developmental decisions and evolution.
Assuntos
Núcleo Celular/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The authors wish to make the following changes to their paper [...].
RESUMO
Developmental enhancers bind transcription factors and dictate patterns of gene expression during development. Their molecular evolution can underlie phenotypical evolution, but the contributions of the evolutionary pathways involved remain little understood. Here, using mutation libraries in Drosophila melanogaster embryos, we observed that most point mutations in developmental enhancers led to changes in gene expression levels but rarely resulted in novel expression outside of the native pattern. In contrast, random sequences, often acting as developmental enhancers, drove expression across a range of cell types; random sequences including motifs for transcription factors with pioneer activity acted as enhancers even more frequently. Our findings suggest that the phenotypic landscapes of developmental enhancers are constrained by enhancer architecture and chromatin accessibility. We propose that the evolution of existing enhancers is limited in its capacity to generate novel phenotypes, whereas the activity of de novo elements is a primary source of phenotypic novelty.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Evolução Molecular , Fenótipo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Early in development, placental and marsupial mammals harbouring at least two X chromosomes per nucleus are faced with a choice that affects the rest of their lives: which of those X chromosomes to transcriptionally inactivate. This choice underlies phenotypical diversity in the composition of tissues and organs and in their response to the environment, and can determine whether an individual will be healthy or affected by an X-linked disease. Here, we review our current understanding of the process of choice during X-chromosome inactivation and its implications, focusing on the strategies evolved by different mammalian lineages and on the known and unknown molecular mechanisms and players involved.
Assuntos
Mecanismo Genético de Compensação de Dose , Marsupiais , Animais , Feminino , Mamíferos/genética , Marsupiais/genética , Placenta , Gravidez , Cromossomo X/genética , Inativação do Cromossomo X/genéticaRESUMO
How histone modifications affect animal development remains difficult to ascertain. Despite the prevalence of histone 3 lysine 4 monomethylation (H3K4me1) on enhancers, hypomethylation appears to have minor effects on phenotype and viability. Here, we genetically reduce H3K4me1 deposition in Drosophila melanogaster and find that hypomethylation reduces transcription factor enrichment in nuclear microenvironments, disrupts gene expression, and reduces phenotypic robustness. Using a developmental phenomics approach, we find changes in morphology, metabolism, behavior, and offspring production. However, many phenotypic changes are only detected when hypomethylated flies develop outside of standard laboratory environments or with specific genetic backgrounds. Therefore, quantitative phenomics measurements can unravel how pleiotropic modulators of gene expression affect developmental robustness under conditions resembling the natural environments of a species.
Assuntos
Drosophila melanogaster , Elementos Facilitadores Genéticos , Animais , Drosophila melanogaster/metabolismo , Fenômica , Histonas/metabolismo , FenótipoRESUMO
The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, and repair. Understanding the exact causality between genome topology and function is therefore crucial and increasingly the subject of intensive research. Chromosome conformation capture technologies (3C) allow inferring the 3D structure of chromatin by measuring the frequency of interactions between any region of the genome. Here we describe a fast and simple protocol to perform Capture Hi-C, a 3C-based target enrichment method that characterizes the allele-specific 3D organization of megabased-sized genomic targets at high-resolution. In Capture Hi-C, target regions are captured by an array of biotinylated probes before downstream high-throughput sequencing. Thus, higher resolution and allele-specificity are achieved while improving the time-effectiveness and affordability of the technology. To demonstrate its strengths, the Capture Hi-C protocol was applied to the mouse X-inactivation center (Xic), the master regulatory locus of X-chromosome inactivation (XCI).
Assuntos
Cromatina , Cromossomos , Camundongos , Animais , Mapeamento Cromossômico/métodos , Cromatina/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Genômica/métodosRESUMO
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 associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other's TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Longo não Codificante/genética , Inativação do Cromossomo X , Animais , Diferenciação Celular/genética , Expressão Ectópica do Gene , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Inativação Gênica , Loci Gênicos , Masculino , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Inversão de Sequência , Transcrição GênicaRESUMO
The packaging of genetic material into chromatin and chromosomes has been recognized for more than a century, thanks to microscopy and biochemical approaches. This was followed by the progressive realization that chromatin organization is critical for genome functions such as transcription and DNA replication and repair. The recent discovery that chromosomes are partitioned at the submegabase scale into topologically associating domains (TADs) has implications for our understanding of gene regulation during developmental processes such as X-chromosome inactivation, as well as for evolution and for the search for disease-associated loci. Here we discuss our current knowledge about this recently recognized level of mammalian chromosome organization, with a special emphasis on the potential role of TADs as a structural basis for the function and evolution of mammalian regulatory landscapes.
RESUMO
X-chromosome inactivation is established during early development. In mice, transcriptional repression of the paternal X-chromosome (Xp) and enrichment in epigenetic marks such as H3K27me3 is achieved by the early blastocyst stage. X-chromosome inactivation is then reversed in the inner cell mass. The mechanisms underlying Xp reactivation remain enigmatic. Using in vivo single-cell approaches (allele-specific RNAseq, nascent RNA-fluorescent in situ hybridization and immunofluorescence), we show here that different genes are reactivated at different stages, with more slowly reactivated genes tending to be enriched in H3meK27. We further show that in UTX H3K27 histone demethylase mutant embryos, these genes are even more slowly reactivated, suggesting that these genes carry an epigenetic memory that may be actively lost. On the other hand, expression of rapidly reactivated genes may be driven by transcription factors. Thus, some X-linked genes have minimal epigenetic memory in the inner cell mass, whereas others may require active erasure of chromatin marks.
Assuntos
Massa Celular Interna do Blastocisto/metabolismo , Epigênese Genética , Fatores de Transcrição/farmacocinética , Inativação do Cromossomo X/genética , Animais , Feminino , Genes Ligados ao Cromossomo X , Histonas/metabolismo , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Modelos Genéticos , Gravidez , RNA Longo não Codificante/genética , Análise de Sequência de RNA , Análise de Célula ÚnicaRESUMO
The long noncoding RNA Xist is expressed from only the paternal X chromosome in mouse preimplantation female embryos and mediates transcriptional silencing of that chromosome. In females, absence of Xist leads to postimplantation lethality. Here, through single-cell RNA sequencing of early preimplantation mouse embryos, we found that the initiation of imprinted X-chromosome inactivation absolutely requires Xist. Lack of paternal Xist leads to genome-wide transcriptional misregulation in the early blastocyst and to failure to activate the extraembryonic pathway that is essential for postimplantation development. We also demonstrate that the expression dynamics of X-linked genes depends on the strain and parent of origin as well as on the location along the X chromosome, particularly at the first 'entry' sites of Xist. This study demonstrates that dosage-compensation failure has an effect as early as the blastocyst stage and reveals genetic and epigenetic contributions to orchestrating transcriptional silencing of the X chromosome during early embryogenesis.
Assuntos
Desenvolvimento Embrionário/genética , Impressão Genômica , RNA Longo não Codificante/genética , Inativação do Cromossomo X/genética , Alelos , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular/genética , Mecanismo Genético de Compensação de Dose , Implantação do Embrião/genética , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Inativação Gênica , Genes Ligados ao Cromossomo X , Cinética , Masculino , Camundongos Endogâmicos C57BL , Mutação/genética , RNA Longo não Codificante/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Fatores de Tempo , Cromossomo X/genéticaRESUMO
X-chromosome inactivation (XCI) is a developmentally associated process that evolved in mammals to enable gene dosage compensation between XX and XY individuals. In placental mammals, it is triggered by the long noncoding RNA Xist, which is produced from a complex regulatory locus, the X-inactivation centre (Xic). Recent insights into the regulatory landscape of the Xic, including its partitioning into topological associating domains (TADs) and its genetic dissection, have important implications for the monoallelic regulation of Xist. Here, we present some of the latest studies on X inactivation with a special focus on the regulation of Xist, its various functions and the putative role of chromosome conformation in regulating the dynamics of this locus during development and differentiation.