RESUMO
Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional structure to accomplish their primary function is unclear. Using super-resolution imaging, capture-C, and polymer modeling, we show that vertebrate centromeres are partitioned by condensins into two subdomains during mitosis. The bipartite structure is found in human, mouse, and chicken cells and is therefore a fundamental feature of vertebrate centromeres. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores, with each subdomain binding a distinct microtubule bundle. Cohesin links the centromere subdomains, limiting their separation in response to spindle forces and avoiding merotelic kinetochore-spindle attachments. Lagging chromosomes during cancer cell divisions frequently have merotelic attachments in which the centromere subdomains are separated and bioriented. Our work reveals a fundamental aspect of vertebrate centromere biology with implications for understanding the mechanisms that guarantee faithful chromosome segregation.
Assuntos
Centrômero , Coesinas , Cinetocoros , Mitose , Animais , Humanos , Camundongos , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Galinhas , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Segregação de Cromossomos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismoRESUMO
The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.
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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.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Mitocondriais/metabolismo , RNA Longo não Codificante/metabolismo , Cromossomo X/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias , Fibroblastos , Inativação Gênica , Humanos , Camundongos , Ligação Proteica , Inativação do Cromossomo XRESUMO
Eukaryotic chromatin is highly condensed but dynamically accessible to regulation and organized into subdomains. We demonstrate that reconstituted chromatin undergoes histone tail-driven liquid-liquid phase separation (LLPS) in physiologic salt and when microinjected into cell nuclei, producing dense and dynamic droplets. Linker histone H1 and internucleosome linker lengths shared across eukaryotes promote phase separation of chromatin, tune droplet properties, and coordinate to form condensates of consistent density in manners that parallel chromatin behavior in cells. Histone acetylation by p300 antagonizes chromatin phase separation, dissolving droplets in vitro and decreasing droplet formation in nuclei. In the presence of multi-bromodomain proteins, such as BRD4, highly acetylated chromatin forms a new phase-separated state with droplets of distinct physical properties, which can be immiscible with unmodified chromatin droplets, mimicking nuclear chromatin subdomains. Our data suggest a framework, based on intrinsic phase separation of the chromatin polymer, for understanding the organization and regulation of eukaryotic genomes.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteína p300 Associada a E1A/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Escherichia coli/genética , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Células Sf9RESUMO
Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination (HR). How it operates in cells remains elusive. We developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembling extensive NPFs. DSB end-tethering promotes coordinated search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology search that is orchestrated by chromatin organizers, long-range resection, end-tethering, and specialized genetic elements and that exploits the stiff NPF structure conferred by Rad51 oligomerization.
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Chromatin loops between gene pairs have been observed in diverse contexts in both flies and vertebrates. Combining high-resolution Capture-C, DNA fluorescence in situ hybridization, and genetic perturbations, we dissect the functional role of three loops between genes with related function during Drosophila embryogenesis. By mutating the loop anchor (but not the gene) or the gene (but not loop anchor), we disentangle loop formation and gene expression and show that the 3D proximity of paralogous gene loci supports their co-regulation. Breaking the loop leads to either an attenuation or enhancement of expression and perturbs their relative levels of expression and cross-regulation. Although many loops appear constitutive across embryogenesis, their function can change in different developmental contexts. Taken together, our results indicate that chromatin gene-gene loops act as architectural scaffolds that can be used in different ways in different contexts to fine-tune the coordinated expression of genes with related functions and sustain their cross-regulation.
Assuntos
Cromatina , Cromossomos , Animais , Hibridização in Situ Fluorescente , Cromatina/genética , Drosophila/genéticaRESUMO
Animal development depends on not only the linear genome sequence that embeds millions of cis-regulatory elements, but also the three-dimensional (3D) chromatin architecture that orchestrates the interplay between cis-regulatory elements and their target genes. Compared to our knowledge of the cis-regulatory sequences, the understanding of the 3D genome organization in human and other eukaryotes is still limited. Recent advances in technologies to map the 3D genome architecture have greatly accelerated the pace of discovery. Here, we review emerging concepts of chromatin organization in mammalian cells, discuss the dynamics of chromatin conformation during development, and highlight important roles for chromatin organization in cancer and other human diseases.
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Genoma , Mamíferos/genética , Animais , Doença/genética , Regulação da Expressão Gênica , Humanos , Neoplasias/genéticaRESUMO
Alternative precursor messenger RNA (pre-mRNA) splicing plays a pivotal role in the flow of genetic information from DNA to proteins by expanding the coding capacity of genomes. Regulation of alternative splicing is as important as regulation of transcription to determine cell- and tissue-specific features, normal cell functioning, and responses of eukaryotic cells to external cues. Its importance is confirmed by the evolutionary conservation and diversification of alternative splicing and the fact that its deregulation causes hereditary disease and cancer. This review discusses the multiple layers of cotranscriptional regulation of alternative splicing in which chromatin structure, DNA methylation, histone marks, and nucleosome positioning play a fundamental role in providing a dynamic scaffold for interactions between the splicing and transcription machineries. We focus on evidence for how the kinetics of RNA polymerase II (RNAPII) elongation and the recruitment of splicing factors and adaptor proteins to chromatin components act in coordination to regulate alternative splicing.
Assuntos
Processamento Alternativo , Cromatina/metabolismo , Transcrição Gênica , Animais , Metilação de DNA , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Modelos Genéticos , Nucleossomos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
This article discusses three reviews on the theme of nuclear organization.
Assuntos
Núcleo Celular/química , Núcleo Celular/genética , Chironomidae/citologia , Células Eucarióticas/metabolismo , Animais , Chironomidae/genética , Puffs Cromossômicos , Células Eucarióticas/citologia , Lâmina Nuclear/química , Ribossomos/química , Ribossomos/metabolismo , Transcrição GênicaRESUMO
Allele-specific expression of imprinted gene clusters is governed by gametic DNA methylation at master regulators called imprinting control regions (ICRs). Non-gametic or secondary differentially methylated regions (DMRs) at promoters and exonic regions reinforce monoallelic expression but do not control an entire cluster. Here, we unveil an unconventional secondary DMR that is indispensable for tissue-specific imprinting of two previously unlinked genes, Grb10 and Ddc. Using polymorphic mice, we mapped an intronic secondary DMR at Grb10 with paternal-specific CTCF binding (CBR2.3) that forms contacts with Ddc. Deletion of paternal CBR2.3 removed a critical insulator, resulting in substantial shifting of chromatin looping and ectopic enhancer-promoter contacts. Destabilized gene architecture precipitated abnormal Grb10-Ddc expression with developmental consequences in the heart and muscle. Thus, we redefine the Grb10-Ddc imprinting domain by uncovering an unconventional intronic secondary DMR that functions as an insulator to instruct the tissue-specific, monoallelic expression of multiple genes-a feature previously ICR exclusive.
Assuntos
Impressão Genômica , RNA Longo não Codificante , Alelos , Animais , Cromatina/genética , Metilação de DNA , Proteína Adaptadora GRB10/genética , Coração , CamundongosRESUMO
The long-range interactions of cis-regulatory elements (cREs) play a central role in gene regulation. cREs can be characterized as accessible chromatin sequences. However, it remains technically challenging to comprehensively identify their spatial interactions. Here, we report a new method HiCAR (Hi-C on accessible regulatory DNA), which utilizes Tn5 transposase and chromatin proximity ligation, for the analysis of open-chromatin-anchored interactions with low-input cells. By applying HiCAR in human embryonic stem cells and lymphoblastoid cells, we demonstrate that HiCAR identifies high-resolution chromatin contacts with an efficiency comparable with that of in situ Hi-C over all distance ranges. Interestingly, we found that the "poised" gene promoters exhibit silencer-like function to repress the expression of distal genes via promoter-promoter interactions. Lastly, we applied HiCAR to 30,000 primary human muscle stem cells and demonstrated that HiCAR is capable of analyzing chromatin accessibility and looping using low-input primary cells and clinical samples.
Assuntos
Cromatina , Sequências Reguladoras de Ácido Nucleico , Cromatina/genética , DNA , Regulação da Expressão Gênica , Humanos , Regiões Promotoras GenéticasRESUMO
Protein acetylation is conserved across phylogeny and has been recognized as one of the most prominent post-translational modifications since its discovery nearly 60 years ago. Histone acetylation is an active mark characteristic of open chromatin, but acetylation on specific lysine residues and histone variants occurs in different biological contexts and can confer various outcomes. The significance of acetylation events is indicated by the associations of lysine acetyltransferases, deacetylases, and acetyl-lysine readers with developmental disorders and pathologies. Recent advances have uncovered new roles of acetylation regulators in chromatin-centric events, which emphasize the complexity of these functional networks. In this review, we discuss mechanisms and dynamics of acetylation in chromatin organization and DNA-templated processes, including gene transcription and DNA repair and replication.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Animais , Cromatina/genética , Reparo do DNA , Replicação do DNA , Instabilidade Genômica , Histonas/genética , Humanos , Lisina , Transcrição GênicaRESUMO
During self-renewal, cell-type-defining features are drastically perturbed in mitosis and must be faithfully reestablished upon G1 entry, a process that remains largely elusive. Here, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. We captured distinct waves of transcriptional reactivation with rapid induction of stem cell genes and transient activation of lineage-specific genes. Topological reorganization at different hierarchical levels also occurred in an asynchronous manner and showed partial coordination with transcriptional resetting. Globally, rapid transcriptional and architectural resetting associated with mitotic retention of H3K27 acetylation, supporting a bookmarking function. Indeed, mitotic depletion of H3K27ac impaired the early reactivation of bookmarked, stem-cell-associated genes. However, 3D chromatin reorganization remained largely unaffected, suggesting that these processes are driven by distinct forces upon mitotic exit. This study uncovers principles and mediators of PSC molecular resetting during self-renewal.
Assuntos
Cromatina/genética , Código das Histonas/genética , Histonas/genética , Mitose/genética , Células-Tronco Pluripotentes/fisiologia , Acetilação , Animais , Linhagem Celular , Drosophila/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transcrição Gênica/genética , Ativação Transcricional/genéticaRESUMO
MYOD-directed fibroblast trans-differentiation into skeletal muscle provides a unique model to investigate how one transcription factor (TF) reconfigures the three-dimensional chromatin architecture to control gene expression, which is otherwise achieved by the combinatorial activities of multiple TFs. Integrative analysis of genome-wide high-resolution chromatin interactions, MYOD and CTCF DNA-binding profile, and gene expression, revealed that MYOD directs extensive re-wiring of interactions involving cis-regulatory and structural genomic elements, including promoters, enhancers, and insulated neighborhoods (INs). Re-configured INs were hot-spots of differential interactions, whereby MYOD binding to highly constrained sequences at IN boundaries and/or inside INs led to alterations of promoter-enhancer interactions to repress cell-of-origin genes and to activate muscle-specific genes. Functional evidence shows that MYOD-directed re-configuration of chromatin interactions temporally preceded the effect on gene expression and was mediated by direct MYOD-DNA binding. These data illustrate a model whereby a single TF alters multi-loop hubs to drive somatic cell trans-differentiation.
Assuntos
Transdiferenciação Celular , Reprogramação Celular , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Fibroblastos/metabolismo , Desenvolvimento Muscular , Proteína MyoD/metabolismo , Mioblastos Esqueléticos/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Transdiferenciação Celular/genética , Cromatina/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Desenvolvimento Muscular/genética , Proteína MyoD/genética , Conformação de Ácido Nucleico , Fenótipo , Ligação Proteica , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
The function of the CCCTC-binding factor (CTCF) in the organization of the genome has become an important area of investigation, but the mechanisms by which CTCF dynamically contributes to genome organization are not clear. We previously discovered that CTCF binds to large numbers of endogenous RNAs, promoting its self-association. In this regard, we now report two independent features that disrupt CTCF association with chromatin: inhibition of transcription and disruption of CTCF-RNA interactions through mutations of 2 of its 11 zinc fingers that are not required for CTCF binding to its cognate DNA site: zinc finger 1 (ZF1) or zinc finger 10 (ZF10). These mutations alter gene expression profiles as CTCF mutants lose their ability to form chromatin loops and thus the ability to insulate chromatin domains and to mediate CTCF long-range genomic interactions. Our results point to the importance of CTCF-mediated RNA interactions as a structural component of genome organization.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , RNA/metabolismo , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/genética , Linhagem Celular , Cromatina/química , Cromatina/genética , Camundongos , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA/química , RNA/genética , Relação Estrutura-Atividade , Transcrição Gênica , Dedos de ZincoRESUMO
Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.
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Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/química , DNA/genética , Complexos Multiproteicos/química , Proteínas de Ligação a Poli-ADP-Ribose/química , Quebra Cromossômica , Cromossomos/genética , DNA/química , Reparo do DNA/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Etoposídeo/química , Conversão Gênica/genética , Células HCT116 , Humanos , Cinética , Complexos Multiproteicos/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Torção Mecânica , Transcrição Gênica , Translocação Genética/genéticaRESUMO
In mammalian cells, the cohesin protein complex is believed to translocate along chromatin during interphase to form dynamic loops through a process called active loop extrusion. Chromosome conformation capture and imaging experiments have suggested that chromatin adopts a compact structure with limited interpenetration between chromosomes and between chromosomal sections. We developed a theory demonstrating that active loop extrusion causes the apparent fractal dimension of chromatin to cross-over between two and four at contour lengths on the order of 30 kilo-base pairs. The anomalously high fractal dimension [Formula: see text] is due to the inability of extruded loops to fully relax during active extrusion. Compaction on longer contour length scales extends within topologically associated domains (TADs), facilitating gene regulation by distal elements. Extrusion-induced compaction segregates TADs such that overlaps between TADs are reduced to less than 35% and increases the entanglement strand of chromatin by up to a factor of 50 to several Mega-base pairs. Furthermore, active loop extrusion couples cohesin motion to chromatin conformations formed by previously extruding cohesins and causes the mean square displacement of chromatin loci during lag times ([Formula: see text]) longer than tens of minutes to be proportional to [Formula: see text]. We validate our results with hybrid molecular dynamics-Monte Carlo simulations and show that our theory is consistent with experimental data. This work provides a theoretical basis for the compact organization of interphase chromatin, explaining the physical reason for TAD segregation and suppression of chromatin entanglements which contribute to efficient gene regulation.
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Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Coesinas , Interfase , Cromatina/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Humanos , Animais , Segregação de Cromossomos/fisiologiaRESUMO
Advances in chromatin mapping have exposed the complex chromatin hierarchical organization in mammals, including topologically associating domains (TADs) and their substructures, yet the functional implications of this hierarchy in gene regulation and disease progression are not fully elucidated. Our study delves into the phenomenon of shared TAD boundaries, which are pivotal in maintaining the hierarchical chromatin structure and regulating gene activity. By integrating high-resolution Hi-C data, chromatin accessibility, and DNA double-strand breaks (DSBs) data from various cell lines, we systematically explore the complex regulatory landscape at high-level TAD boundaries. Our findings indicate that these boundaries are not only key architectural elements but also vibrant hubs, enriched with functionally crucial genes and complex transcription factor binding site-clustered regions. Moreover, they exhibit a pronounced enrichment of DSBs, suggesting a nuanced interplay between transcriptional regulation and genomic stability. Our research provides novel insights into the intricate relationship between the 3D genome structure, gene regulation, and DNA repair mechanisms, highlighting the role of shared TAD boundaries in maintaining genomic integrity and resilience against perturbations. The implications of our findings extend to understanding the complexities of genomic diseases and open new avenues for therapeutic interventions targeting the structural and functional integrity of TAD boundaries.
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Cromatina , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Regulação da Expressão Gênica , Humanos , Cromatina/metabolismo , Cromatina/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Animais , Genômica/métodos , Instabilidade Genômica , Montagem e Desmontagem da CromatinaRESUMO
Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.
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Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Genoma Humano , Proteína HMGB2/metabolismo , Fator de Ligação a CCCTC/genética , Proliferação de Células , Senescência Celular , Cromatina/genética , Proteína HMGB2/genética , Células Endoteliais da Veia Umbilical Humana , HumanosRESUMO
The active loop extrusion hypothesis proposes that chromatin threads through the cohesin protein complex into progressively larger loops until reaching specific boundary elements. We build upon this hypothesis and develop an analytical theory for active loop extrusion which predicts that loop formation probability is a nonmonotonic function of loop length and describes chromatin contact probabilities. We validate our model with Monte Carlo and hybrid Molecular Dynamics-Monte Carlo simulations and demonstrate that our theory recapitulates experimental chromatin conformation capture data. Our results support active loop extrusion as a mechanism for chromatin organization and provide an analytical description of chromatin organization that may be used to specifically modify chromatin contact probabilities.