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We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Endonucleasas/metabolismo , Enzimas Multifuncionales/metabolismo , ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Microscopía Fluorescente , Mitocondrias/genética , ARN/química , ARN Mensajero/química , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
Although population-level analyses revealed significant roles for CTCF and cohesin in mammalian genome organization, their contributions at the single-cell level remain incompletely understood. Here, we used a super-resolution microscopy approach to measure the effects of removal of CTCF or cohesin in mouse embryonic stem cells. Single-chromosome traces revealed cohesin-dependent loops, frequently stacked at their loop anchors forming multi-way contacts (hubs), bridging across TAD boundaries. Despite these bridging interactions, chromatin in intervening TADs was not intermixed, remaining separated in distinct loops around the hub. At the multi-TAD scale, steric effects from loop stacking insulated local chromatin from ultra-long range (>4 Mb) contacts. Upon cohesin removal, the chromosomes were more disordered and increased cell-cell variability in gene expression. Our data revise the TAD-centric understanding of CTCF and cohesin and provide a multi-scale, structural picture of how they organize the genome on the single-cell level through distinct contributions to loop stacking.
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Cromatina , Cromosomas , Animales , Ratones , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Cromosomas/genética , Cromosomas/metabolismo , Cromatina/genética , Cromatina/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mamíferos/metabolismoRESUMEN
The establishment of cell types during development requires precise interactions between genes and distal regulatory sequences. We have a limited understanding of how these interactions look in three dimensions, vary across cell types in complex tissue, and relate to transcription. Here we describe optical reconstruction of chromatin architecture (ORCA), a method that can trace the DNA path in single cells with nanoscale accuracy and genomic resolution reaching two kilobases. We used ORCA to study a Hox gene cluster in cryosectioned Drosophila embryos and labelled around 30 RNA species in parallel. We identified cell-type-specific physical borders between active and Polycomb-repressed DNA, and unexpected Polycomb-independent borders. Deletion of Polycomb-independent borders led to ectopic enhancer-promoter contacts, aberrant gene expression, and developmental defects. Together, these results illustrate an approach for high-resolution, single-cell DNA domain analysis in vivo, identify domain structures that change with cell identity, and show that border elements contribute to the formation of physical domains in Drosophila.
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Cromatina/química , ADN/análisis , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Conformación de Ácido Nucleico , ARN/análisis , Análisis de la Célula Individual , Animales , Cromatina/genética , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , Drosophila melanogaster/citología , Elementos de Facilitación Genéticos/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/genética , Genoma de los Insectos/genética , Masculino , Familia de Multigenes/genética , Especificidad de Órganos , Proteínas del Grupo Polycomb/genética , Regiones Promotoras Genéticas/genética , ARN/genética , ARN/metabolismo , Transcripción GenéticaRESUMEN
Polycomb-group proteins play critical roles in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction. This process is essential for embryonic stem cell (ESC) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling progressive spreading of H3K27me3 on the linear genome. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remain unclear. Here, we apply H3K27me3 HiChIP, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes. Genetic deletion of H3K27me3 loop anchors results in disruption of spatial contact between distant loci and altered H3K27me3 in cis, both locally and megabases away on the same chromosome. In mouse embryos, loop anchor deletion leads to ectopic activation of the partner gene, suggesting that Polycomb-associated loops control gene silencing during development. Further, we find that alterations in PRC2 occupancy resulting from an RNA bindingdeficient EZH2 mutant are accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 uses RNA binding to enhance long-range chromosome folding and H3K27me3 spreading. Developmental gene loci have unique roles in Polycomb spreading, emerging as important architectural elements of the epigenome.
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Cromosomas , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Histonas , Complejo Represivo Polycomb 2 , Animales , Inmunoprecipitación de Cromatina/métodos , Cromosomas/química , Cromosomas/metabolismo , Embrión de Mamíferos , Proteína Potenciadora del Homólogo Zeste 2/genética , Histonas/genética , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lisina/metabolismo , Metilación , Ratones , Conformación de Ácido Nucleico , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/metabolismoRESUMEN
Chromosomal dynamics plays a central role in a number of critical biological processes, such as transcriptional regulation, genetic recombination, and DNA replication. However, visualization of chromatin is generally limited to live imaging of a few fluorescently labeled chromosomal loci or high-resolution reconstruction of multiple loci from a single time frame. To aid in mapping the underlying chromosomal structure based on parsimonious experimental measurements, we present an exact analytical expression for the evolution of the polymer configuration based on a flexible-polymer model, and we propose an algorithm that tracks the polymer configuration from live images of chromatin marked with several fluorescent marks. Our theory identifies the resolution of microscopy needed to achieve high-accuracy tracking for a given spacing of markers, establishing the statistical confidence in the assignment of genome identity to the visualized marks. We then leverage experimental data of locus-tracking measurements to demonstrate the validity of our modeling approach and to establish a basis for the design of experiments with a desired resolution. Altogether, this work provides a computational approach founded on polymer physics that vastly improves the interpretation of in vivo measurements of biopolymer dynamics.
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Cromatina , Polímeros , Cromosomas , Replicación del ADN , AlgoritmosRESUMEN
Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression. Here we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive or Polycomb-repressed states, and observed distinct chromatin organizations for each state. All three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed domains show the densest packing and most intriguing chromatin folding behaviour, in which chromatin packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins play an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly relevant to genome regulation.
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Ensamble y Desensamble de Cromatina , Cromatina/genética , Cromatina/metabolismo , Drosophila melanogaster/genética , Epigénesis Genética , Animales , Línea Celular , Posicionamiento de Cromosoma , Drosophila melanogaster/citología , Represión Epigenética , Fractales , Genoma/genética , Proteínas del Grupo Polycomb/metabolismo , Transcripción GenéticaRESUMEN
Segmentation of the Drosophila embryo begins with the establishment of spatially restricted gap gene-expression patterns in response to broad gradients of maternal transcription factors, such as Bicoid. Numerous studies have documented the fidelity of these expression patterns, even when embryos are subjected to genetic or environmental stress, but the underlying mechanisms for this transcriptional precision are uncertain. Here we present evidence that every gap gene contains multiple enhancers with overlapping activities to produce authentic patterns of gene expression. For example, a recently identified hunchback (hb) enhancer (located 5-kb upstream of the classic enhancer) ensures repression at the anterior pole. The combination of intronic and 5' knirps (kni) enhancers produces a faithful expression pattern, even though the intronic enhancer alone directs an abnormally broad expression pattern. We present different models for "enhancer synergy," whereby two enhancers with overlapping activities produce authentic patterns of gene expression.
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Drosophila melanogaster/embriología , Inducción Embrionaria , Elementos de Facilitación Genéticos/fisiología , Proteínas Activadoras de GTPasa/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Animales , Tipificación del Cuerpo , Drosophila melanogaster/genética , Embrión no Mamífero , Factores de Transcripción/genéticaRESUMEN
Although promoters and their enhancers are frequently contained within a topologically associating domain (TAD), some developmentally important genes have their promoter and enhancers within different TADs. Hypotheses about molecular mechanisms enabling cross-TAD interactions remain to be assessed. To test these hypotheses, we used optical reconstruction of chromatin architecture to characterize the conformations of the Pitx1 locus on single chromosomes in developing mouse limbs. Our data support a model in which neighboring boundaries are stacked as a result of loop extrusion, bringing boundary-proximal cis-elements into contact. This stacking interaction also contributes to the appearance of architectural stripes in the population average maps. Through molecular dynamics simulations, we found that increasing boundary strengths facilitates the formation of the stacked boundary conformation, counter-intuitively facilitating border bypass. This work provides a revised view of the TAD borders' function, both facilitating and preventing cis-regulatory interactions, and introduces a framework to distinguish border-crossing from border-respecting enhancer-promoter pairs.
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Cromatina , Elementos de Facilitación Genéticos , Animales , Ratones , Elementos de Facilitación Genéticos/genética , Cromatina/genética , Cromosomas , Regiones Promotoras Genéticas/genética , Elementos AisladoresRESUMEN
Regulation of gene expression hinges on the interplay between enhancers and promoters, traditionally explored through pairwise analyses. Recent advancements in mapping genome folding, like GAM, SPRITE, and multi-contact Hi-C, have uncovered multi-way interactions among super-enhancers (SEs), spanning megabases, yet have not measured their frequency in single cells or the relationship between clustering and transcription. To close this gap, here we used multiplexed imaging to map the 3D positions of 376 SEs across thousands of mammalian nuclei. Notably, our single-cell images reveal that while SE-SE contacts are rare, SEs often form looser associations we termed "communities". These communities, averaging 4-5 SEs, assemble cooperatively under the combined effects of genomic tethers, Pol2 clustering, and nuclear compartmentalization. Larger communities are associated with more frequent and larger transcriptional bursts. Our work provides insights about the SE interactome in single cells that challenge existing hypotheses on SE clustering in the context of transcriptional regulation.
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While critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we bypass the need for sequence-specific transcription factors and recruit activators directly using CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible by single dCas9. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mESCs, where neither gene is expressed. We demonstrate that while activator recruitment to any tested region results in transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence or presence of the repressive chromatin marks at the locus.
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Repressive chromatin modifications are thought to compact chromatin to silence transcription. However, it is unclear how chromatin structure changes during silencing and epigenetic memory formation. We measured gene expression and chromatin structure in single cells after recruitment and release of repressors at a reporter gene. Chromatin structure is heterogeneous, with open and compact conformations present in both active and silent states. Recruitment of repressors associated with epigenetic memory produces chromatin compaction across 10-20 kilobases, while reversible silencing does not cause compaction at this scale. Chromatin compaction is inherited, but changes molecularly over time from histone methylation (H3K9me3) to DNA methylation. The level of compaction at the end of silencing quantitatively predicts epigenetic memory weeks later. Similarly, chromatin compaction at the Nanog locus predicts the degree of stem-cell fate commitment. These findings suggest that the chromatin state across tens of kilobases, beyond the gene itself, is important for epigenetic memory formation.
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Recent whole genome polymerase binding assays in the Drosophila embryo have shown that a substantial proportion of uninduced genes have pre-assembled RNA polymerase-II transcription initiation complex (PIC) bound to their promoters. These constitute a subset of promoter proximally paused genes for which mRNA elongation instead of promoter access is regulated. This difference can be described as a rearrangement of the regulatory topology to control the downstream transcriptional process of elongation rather than the upstream transcriptional initiation event. It has been shown experimentally that genes with the former mode of regulation tend to induce faster and more synchronously, and that promoter-proximal pausing is observed mainly in metazoans, in accord with a posited impact on synchrony. However, it has not been shown whether or not it is the change in the regulated step per se that is causal. We investigate this question by proposing and analyzing a continuous-time Markov chain model of PIC assembly regulated at one of two steps: initial polymerase association with DNA, or release from a paused, transcribing state. Our analysis demonstrates that, over a wide range of physical parameters, increased speed and synchrony are functional consequences of elongation control. Further, we make new predictions about the effect of elongation regulation on the consistent control of total transcript number between cells. We also identify which elements in the transcription induction pathway are most sensitive to molecular noise and thus possibly the most evolutionarily constrained. Our methods produce symbolic expressions for quantities of interest with reasonable computational effort and they can be used to explore the interplay between interaction topology and molecular noise in a broader class of biochemical networks. We provide general-purpose code implementing these methods.
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Elementos de Facilitación Genéticos , Modelos Genéticos , Regiones Promotoras Genéticas , Transcripción Genética , Activación Transcripcional , Animales , Drosophila , Embrión no Mamífero , Cadenas de Markov , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Patterning of the terminal regions of the Drosophila embryo relies on the gradient of phosphorylated ERK/MAPK (dpERK), which is controlled by the localized activation of the Torso receptor tyrosine kinase [1-4]. This model is supported by a large amount of data, but the gradient itself has never been quantified. We present the first measurements of the dpERK gradient and establish a new intracellular layer of its regulation. Based on the quantitative analysis of the spatial pattern of dpERK in mutants with different levels of Torso as well as the dynamics of the wild-type dpERK pattern, we propose that the terminal-patterning gradient is controlled by a cascade of diffusion-trapping modules. A ligand-trapping mechanism establishes a sharply localized pattern of the Torso receptor occupancy on the surface of the embryo. Inside the syncytial embryo, nuclei play the role of traps that localize diffusible dpERK. We argue that the length scale of the terminal-patterning gradient is determined mainly by the intracellular module.
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Tipificación del Cuerpo , Núcleo Celular/metabolismo , Drosophila/embriología , Proteínas Tirosina Quinasas/metabolismo , Animales , Drosophila/anatomía & histología , Drosophila/genética , Drosophila/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de SeñalRESUMEN
Understanding the environmental factors influencing animal movements is fundamental to theoretical and applied research in the field of movement ecology. Studies relating fine-scale movement paths to spatiotemporally structured landscape data, such as vegetation productivity or human activity, are particularly lacking despite the obvious importance of such information to understanding drivers of animal movement. In part, this may be because few approaches provide the sophistication to characterize the complexity of movement behavior and relate it to diverse, varying environmental stimuli. We overcame this hurdle by applying, for the first time to an ecological question, a finite impulse-response signal-filtering approach to identify human and natural environmental drivers of movements of 13 free-ranging African elephants (Loxodonta africana) from distinct social groups collected over seven years. A minimum mean-square error (MMSE) estimation criterion allowed comparison of the predictive power of landscape and ecological model inputs. We showed that a filter combining vegetation dynamics, human and physical landscape features, and previous movement outperformed simpler filter structures, indicating the importance of both dynamic and static landscape features, as well as habit, on movement decisions taken by elephants. Elephant responses to vegetation productivity indices were not uniform in time or space, indicating that elephant foraging strategies are more complex than simply gravitation toward areas of high productivity. Predictions were most frequently inaccurate outside protected area boundaries near human settlements, suggesting that human activity disrupts typical elephant movement behavior. Successful management strategies at the human-elephant interface, therefore, are likely to be context specific and dynamic. Signal processing provides a promising approach for elucidating environmental factors that drive animal movements over large time and spatial scales.
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Conducta Animal , Ecosistema , Elefantes/fisiología , Modelos Biológicos , Movimiento , Animales , Dinámica PoblacionalRESUMEN
Animal genomes are organized into topologically associated domains (TADs). TADs are thought to contribute to gene regulation by facilitating enhancer-promoter (E-P) contacts within a TAD and preventing these contacts across TAD borders. However, the absolute difference in contact frequency across TAD boundaries is usually less than 2-fold, even though disruptions of TAD borders can change gene expression by 10-fold. Existing models fail to explain this hypersensitive response. Here, we propose a futile cycle model of enhancer-mediated regulation that can exhibit hypersensitivity through bistability and hysteresis. Consistent with recent experiments, this regulation does not exhibit strong correlation between E-P contact and promoter activity, even though regulation occurs through contact. Through mathematical analysis and stochastic simulation, we show that this system can create an illusion of E-P biochemical specificity and explain the importance of weak TAD boundaries. It also offers a mechanism to reconcile apparently contradictory results from recent global TAD disruption with local TAD boundary deletion experiments. Together, these analyses advance our understanding of cis-regulatory contacts in controlling gene expression and suggest new experimental directions.
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Cromatina/química , Biología Computacional/métodos , Regulación de la Expresión Génica , Transcripción Genética , Animales , Genoma , Humanos , Hipersensibilidad , Ratones , Regiones Promotoras GenéticasRESUMEN
Chromatin conformation capture (3C) methods and fluorescent in situ hybridization (FISH) microscopy have been used to investigate the spatial organization of the genome. Although powerful, both techniques have limitations. Hi-C is challenging for low cell numbers and requires very deep sequencing to achieve its high resolution. In contrast, FISH can be done on small cell numbers and capture rare cell populations, but typically targets pairs of loci at a lower resolution. Here we detail a protocol for optical reconstruction of chromatin architecture (ORCA), a microscopy approach to trace the 3D DNA path within the nuclei of fixed tissues and cultured cells with a genomic resolution as fine as 2 kb and a throughput of ~10,000 cells per experiment. ORCA can identify structural features with comparable resolution to Hi-C while providing single-cell resolution and multimodal measurements characteristic of microscopy. We describe how to use this DNA labeling in parallel with multiplexed labeling of dozens of RNAs to relate chromatin structure and gene expression in the same cells. Oligopaint probe design, primary probe making, sample collection, cryosectioning and RNA/DNA primary probe hybridization can be completed in 1.5 weeks, while automated RNA/DNA barcode hybridization and RNA/DNA imaging typically takes 2-6 d for data collection and 2-7 d for the automated steps of image analysis.
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Hibridación Fluorescente in Situ/métodos , Microscopía Fluorescente/métodos , Mapeo de Restricción Óptica/métodos , Línea Celular , Núcleo Celular/genética , Células Cultivadas , Cromatina/metabolismo , Inmunoprecipitación de Cromatina/métodos , Cromosomas/genética , ADN/química , ADN/genética , Sondas de ADN , Colorantes Fluorescentes/química , Técnicas Genéticas , Genoma/genética , Genómica/métodos , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , ARN/química , ARN/genéticaRESUMEN
Chromatin architecture plays an important role in gene regulation. Recent advances in super-resolution microscopy have made it possible to measure chromatin 3D structure and transcription in thousands of single cells. However, leveraging these complex data sets with a computationally unbiased method has been challenging. Here, we present a deep learning-based approach to better understand to what degree chromatin structure relates to transcriptional state of individual cells. Furthermore, we explore methods to "unpack the black box" to determine in an unbiased manner which structural features of chromatin regulation are most important for gene expression state. We apply this approach to an Optical Reconstruction of Chromatin Architecture dataset of the Bithorax gene cluster in Drosophila and show it outperforms previous contact-focused methods in predicting expression state from 3D structure. We find the structural information is distributed across the domain, overlapping and extending beyond domains identified by prior genetic analyses. Individual enhancer-promoter interactions are a minor contributor to predictions of activity.
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ADN/genética , Aprendizaje Profundo , Drosophila melanogaster/genética , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Transcripción Genética , Algoritmos , Animales , Cromatina/genética , Simulación por Computador , Regulación de la Expresión Génica , Silenciador del Gen , Genoma de los Insectos , Familia de Multigenes , Redes Neurales de la ComputaciónRESUMEN
The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.
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Ensamble y Desensamble de Cromatina/fisiología , Represión Epigenética/fisiología , Regulación de la Expresión Génica/fisiología , Complejos Multiproteicos/fisiología , Proteínas del Grupo Polycomb/fisiología , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/fisiología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Represión Epigenética/genética , Edición Génica , Regulación de la Expresión Génica/genética , Genes Homeobox , Genoma , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Mutación con Pérdida de Función , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteolisis , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
The Bicoid gradient in the Drosophila embryo provided the first example of a morphogen gradient studied at the molecular level. The exponential shape of the Bicoid gradient had always been interpreted within the framework of the localized production, diffusion, and degradation model. We propose an alternative mechanism, which assumes no Bicoid degradation. The medium where the Bicoid gradient is formed and interpreted is very dynamic. Most notably, the number of nuclei changes over three orders of magnitude from fertilization, when Bicoid synthesis is initiated, to nuclear cycle 14 when most of the measurements were taken. We demonstrate that a model based on Bicoid diffusion and nucleocytoplasmic shuttling in the presence of the growing number of nuclei can account for most of the properties of the Bicoid concentration profile. Consistent with experimental observations, the Bicoid gradient in our model is established before nuclei migrate to the periphery of the embryo and remains stable during subsequent nuclear divisions.
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Núcleo Celular/metabolismo , Drosophila/embriología , Proteínas de Homeodominio/metabolismo , Modelos Biológicos , Transactivadores/metabolismo , Algoritmos , Animales , Tipificación del Cuerpo , División Celular , Drosophila/metabolismo , Proteínas de Drosophila , Proteínas de Homeodominio/química , Transactivadores/químicaRESUMEN
The spatial organization of chromatin is pivotal for regulating genome functions. We report an imaging method for tracing chromatin organization with kilobase- and nanometer-scale resolution, unveiling chromatin conformation across topologically associating domains (TADs) in thousands of individual cells. Our imaging data revealed TAD-like structures with globular conformation and sharp domain boundaries in single cells. The boundaries varied from cell to cell, occurring with nonzero probabilities at all genomic positions but preferentially at CCCTC-binding factor (CTCF)- and cohesin-binding sites. Notably, cohesin depletion, which abolished TADs at the population-average level, did not diminish TAD-like structures in single cells but eliminated preferential domain boundary positions. Moreover, we observed widespread, cooperative, multiway chromatin interactions, which remained after cohesin depletion. These results provide critical insight into the mechanisms underlying chromatin domain and hub formation.