RESUMEN
Cell cycle progression is regulated by the orderly balance between kinase and phosphatase activities. PP2A phosphatase holoenzymes containing the B55 family of regulatory B subunits function as major CDK1-counteracting phosphatases during mitotic exit in mammals. However, the identification of the specific mitotic roles of these PP2A-B55 complexes has been hindered by the existence of multiple B55 isoforms. Here, through the generation of loss-of-function genetic mouse models for the two ubiquitous B55 isoforms (B55α and B55δ), we report that PP2A-B55α and PP2A-B55δ complexes display overlapping roles in controlling the dynamics of proper chromosome individualization and clustering during mitosis. In the absence of PP2A-B55 activity, mitotic cells display increased chromosome individualization in the presence of enhanced phosphorylation and perichromosomal loading of Ki-67. These data provide experimental evidence for a regulatory mechanism by which the balance between kinase and PP2A-B55 phosphatase activity controls the Ki-67-mediated spatial organization of the mass of chromosomes during mitosis.
Asunto(s)
Antígeno Ki-67 , Mitosis , Proteína Fosfatasa 2 , Animales , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/genética , Ratones , Antígeno Ki-67/metabolismo , Fosforilación , Cromosomas de los Mamíferos/metabolismo , Cromosomas de los Mamíferos/genética , Cromosomas/metabolismoRESUMEN
Eukaryotic genomes are organized by loop extrusion and sister chromatid cohesion, both mediated by the multimeric cohesin protein complex. Understanding how cohesin holds sister DNAs together, and how loss of cohesion causes age-related infertility in females, requires knowledge as to cohesin's stoichiometry in vivo. Using quantitative super-resolution imaging, we identified two discrete populations of chromatin-bound cohesin in postreplicative human cells. Whereas most complexes appear dimeric, cohesin that localized to sites of sister chromatid cohesion and associated with sororin was exclusively monomeric. The monomeric stoichiometry of sororin:cohesin complexes demonstrates that sister chromatid cohesion is conferred by individual cohesin rings, a key prediction of the proposal that cohesion arises from the co-entrapment of sister DNAs.
Asunto(s)
Proteínas de Ciclo Celular , Cromátides , Cohesinas , Intercambio de Cromátides Hermanas , Humanos , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Cromatina/metabolismo , Cohesinas/metabolismo , ADN/genética , ADN/metabolismo , Línea Celular TumoralRESUMEN
We describe u-track3D, a software package that extends the versatile u-track framework established in 2D to address the specific challenges of 3D particle tracking. First, we present the performance of the new package in quantifying a variety of intracellular dynamics imaged by multiple 3D microcopy platforms and on the standard 3D test dataset of the particle tracking challenge. These analyses indicate that u-track3D presents a tracking solution that is competitive to both conventional and deep-learning-based approaches. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability that automatically defines a score for every trajectory, thereby overcoming the challenges of trajectory validation by visual inspection. With these combined strategies, u-track3D provides a complete framework for unbiased studies of molecular processes in complex volumetric sequences.
Asunto(s)
Algoritmos , Imagenología Tridimensional , Imagenología Tridimensional/métodos , Examen FísicoRESUMEN
Cohesin connects CTCF-binding sites and other genomic loci in cis to form chromatin loops and replicated DNA molecules in trans to mediate sister chromatid cohesion. Whether cohesin uses distinct or related mechanisms to perform these functions is unknown. Here, we describe a cohesin hinge mutant that can extrude DNA into loops but is unable to mediate cohesion in human cells. Our results suggest that the latter defect arises during cohesion establishment. The observation that cohesin's cohesion and loop extrusion activities can be partially separated indicates that cohesin uses distinct mechanisms to perform these two functions. Unexpectedly, the same hinge mutant can also not be stopped by CTCF boundaries as well as wild-type cohesin. This suggests that cohesion establishment and cohesin's interaction with CTCF boundaries depend on related mechanisms and raises the possibility that both require transient hinge opening to entrap DNA inside the cohesin ring.
Asunto(s)
Proteínas de Ciclo Celular , Cromátides , Humanos , Cromátides/genética , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , CohesinasRESUMEN
Genome browsers facilitate integrated analysis of multiple genomics datasets yet visualize only a few regions at a time and lack statistical functions for extracting meaningful information. We present HiCognition, a visual exploration and machine-learning tool based on a new genomic region set concept, enabling detection of patterns and associations between 3D chromosome conformation and collections of 1D genomics profiles of any type. By revealing how transcription and cohesion subunit isoforms contribute to chromosome conformation, we showcase how the flexible user interface and machine learning tools of HiCognition help to understand the relationship between the structure and function of the genome.
Asunto(s)
Genoma Humano , Genómica , Programas Informáticos , Humanos , Genómica/métodos , Cromosomas Humanos , Aprendizaje AutomáticoRESUMEN
Genetic information is stored in linear DNA molecules, which are highly folded inside cells. DNA replication along the folded template path yields two sister chromatids that initially occupy the same nuclear region in an intertwined arrangement. Dividing cells must disentangle and condense the sister chromatids into separate bodies such that a microtubule-based spindle can move them to opposite poles. While the spindle-mediated transport of sister chromatids has been studied in detail, the chromosome-intrinsic mechanics presegregating sister chromatids have remained elusive. Here, we show that human sister chromatids resolve extensively already during interphase, in a process dependent on the loop-extruding activity of cohesin, but not that of condensins. Increasing cohesin's looping capability increases sister DNA resolution in interphase nuclei to an extent normally seen only during mitosis, despite the presence of abundant arm cohesion. That cohesin can resolve sister chromatids so extensively in the absence of mitosis-specific activities indicates that DNA loop extrusion is a generic mechanism for segregating replicated genomes, shared across different Structural Maintenance of Chromosomes (SMC) protein complexes in all kingdoms of life.
Asunto(s)
Cromátides , Proteínas Cromosómicas no Histona , Humanos , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mitosis , ADN , Fase G2 , CohesinasRESUMEN
Chromosomes transform during the cell cycle, allowing transcription and replication during interphase and chromosome segregation during mitosis. Morphological changes are thought to be driven by the combined effects of DNA loop extrusion and a chromatin solubility phase transition. By extruding the chromatin fibre into loops, condensins enrich at an axial core and provide resistance to spindle pulling forces. Mitotic chromosomes are further compacted by deacetylation of histone tails, rendering chromatin insoluble and resistant to penetration by microtubules. Regulation of surface properties by Ki-67 allows independent chromosome movement in early mitosis and clustering during mitotic exit. Recent progress has provided insight into how the extraordinary material properties of chromatin emerge from these activities, and how these properties facilitate faithful chromosome segregation.
Asunto(s)
Cromatina , Cromosomas , Mitosis , Histonas/genética , Segregación CromosómicaRESUMEN
Nuclear DNA in eukaryotes is wrapped around histone proteins to form nucleosomes on a chromatin fiber. Dynamic folding of the chromatin fiber into loops and variations in the degree of chromatin compaction regulate essential processes such as transcription, recombination, and mitotic chromosome segregation. Our understanding of the physical properties that allow chromatin to be dynamically remodeled even in highly compacted states is limited. Previously, we reported that chromatin has an intrinsic capacity to phase separate and form dynamic liquid-like condensates, which can be regulated by cellular factors [B. A. Gibson et al., Cell 179, 470-484.e421 (2019)]. Recent contradictory reports claim that a specific set of solution conditions is required for fluidity in condensates that would otherwise be solid [J. C. Hansen, K. Maeshima, M. J. Hendzel, Epigenetics Chromatin 14, 50 (2021); H. Strickfaden et al., Cell 183, 1772-1784.e1713 (2020)]. We sought to resolve these discrepancies, as our ability to translate with confidence these biophysical observations to cells requires their precise characterization. Moreover, whether chromatin assemblies are dynamic or static affects how processes such as transcription, loop extrusion, and remodeling will engage them inside cells. Here, we show in diverse conditions and without specific buffering components that chromatin fragments form phase separated fluids in vitro. We also explore how sample preparation and imaging affect the experimental observation of chromatin condensate dynamics. Last, we describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained chromatin movement observed in cells.
Asunto(s)
Cromatina , Histonas , Histonas/metabolismo , Nucleosomas , ADN/metabolismo , Ensamble y Desensamble de CromatinaRESUMEN
Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming daughter cells1-3. Assembly of mitotic chromosomes involves DNA looping by condensin4-8 and chromatin compaction by global histone deacetylation9-13. Although condensin confers mechanical resistance to spindle pulling forces14-16, it is not known how histone deacetylation affects material properties and, as a consequence, segregation mechanics of mitotic chromosomes. Here we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with the physical characteristics necessary for their precise movement during cell division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. By contrast, hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin phase separation to genome segregation in dividing cells.
Asunto(s)
Cromatina , Microtúbulos , Mitosis , Acetilación , Cromatina/metabolismo , Segregación Cromosómica , ADN/metabolismo , Histonas/metabolismo , Microtúbulos/metabolismo , Transición de Fase , Huso Acromático/metabolismoRESUMEN
Chromosome conformation capture (Hi-C) techniques map the 3D organization of entire genomes. How sister chromatids fold in replicated chromosomes, however, cannot be determined with conventional Hi-C because of the identical DNA sequences of sister chromatids. Here, we present a protocol for sister chromatid-sensitive Hi-C (scsHi-C) that enables the distinction of DNA contacts within individual sister chromatids (cis sister contacts) from those between sister chromatids (trans sister contacts), thereby allowing investigation of the organization of replicated genomes. scsHi-C is based on live-cell labeling of nascent DNA by the synthetic nucleoside 4-thio-thymidine (4sT), which incorporates into a distinct DNA strand on each sister chromatid because of semi-conservative DNA replication. After purification of genomic DNA and in situ Hi-C library preparation, 4sT is chemically converted into 5-methyl-cytosine in the presence of OsO4/NH4Cl to introduce T-to-C signature point mutations on 4sT-labeled DNA. The Hi-C library is then sequenced, and ligated fragments are assigned to sister chromatids on the basis of strand orientation and the presence of signature mutations. The ensemble of scsHi-C contacts thereby represents genome-wide contact probabilities within and across sister chromatids. scsHi-C can be completed in 2 weeks, has been successfully applied in HeLa cells and can potentially be established for any cell type that allows proper cell cycle synchronization and incorporation of sufficient amounts of 4sT. The genome-wide maps of replicated chromosomes detected by scsHi-C enable investigation of the molecular mechanisms shaping sister chromatid topologies and the relevance of sister chromatid conformation in crucial processes like DNA repair, mitotic chromosome formation and potentially other biological processes.
Asunto(s)
Cromátides , Replicación del ADN , Cromátides/genética , Reparación del ADN , Células HeLa , HumanosRESUMEN
The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1,2 analysis has revealed a complex genomic landscape of internal chromosomal structures in vertebrate cells3-7, but the identical sequence of sister chromatids has made it difficult to determine how they topologically interact in replicated chromosomes. Here we describe sister-chromatid-sensitive Hi-C (scsHi-C), which is based on labelling of nascent DNA with 4-thio-thymidine and nucleoside conversion chemistry. Genome-wide conformation maps of human chromosomes reveal that sister-chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister-chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired and are characterized by facultative heterochromatin and insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister-chromatid topologies and our scsHi-C technology will make it possible to investigate how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.
Asunto(s)
Cromátides/química , Emparejamiento Cromosómico , Replicación del ADN , Genoma Humano/genética , Conformación de Ácido Nucleico , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/análisis , ADN/biosíntesis , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , CohesinasRESUMEN
Gene expression in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation1. This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores2. During mitosis, however, the nuclear envelope in animal and plant cells disassembles, allowing cytoplasmic and nuclear components to intermix3. When the nuclear envelope is reformed, cytoplasmic components are removed from the nucleus by receptor-mediated transport through nuclear pores2. These pores have a size limit of 39 nanometres4-7, which raises the question of how larger cytoplasmic molecules are cleared from the nucleus. Here we show in HeLa cells that large cytoplasmic components are displaced before nuclear envelope assembly by the movement of chromosomes to a dense cluster. This clustering occurs when chromosomes approach the poles of anaphase spindles, and is mediated by a microtubule-independent mechanism that involves the surfactant-like protein Ki-67. Ki-67 forms repulsive molecular brushes during the early stages of mitosis8, but during mitotic exit the brushes collapse and Ki-67 promotes chromosome clustering. We show that the exclusion of mature ribosomes from the nucleus after mitosis depends on Ki-67-regulated chromosome clustering. Thus, our study reveals that chromosome mechanics help to re-establish the compartmentalization of eukaryotic cells after open mitosis.
Asunto(s)
Posicionamiento de Cromosoma , Cromosomas Humanos/metabolismo , Citoplasma/metabolismo , Antígeno Ki-67/metabolismo , Membrana Nuclear/metabolismo , Transporte Biológico , Células HeLa , Humanos , Mitosis , Ribosomas/metabolismo , Huso AcromáticoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Histonas/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Escherichia coli/genética , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Células Sf9RESUMEN
During mitosis, replicated chromosomes segregate such that each daughter cell receives one copy of the genome. Faithful mechanical transport during mitosis requires that chromosomes undergo extensive structural changes as the cell cycle progresses, resulting in the formation of compact, cylindrical bodies. Such structural changes encompass a range of different activities, including longitudinal condensation of the chromosome axis, global chromatin compaction, resolution of sister chromatids, and individualisation of chromosomes into separate bodies. After mitosis, chromosomes undergo further reorganisation to rebuild interphase cell nuclei. Here we review the requirements for mitotic chromosomes to successfully transmit genetic information to daughter cells and the biophysical principles that underpin such requirements.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Cromosomas/genética , Cromosomas/ultraestructura , Animales , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Reparación del ADN , Replicación del ADN , Proteínas Fúngicas/metabolismo , Genoma , Humanos , Mitosis , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle pole-derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at noncentrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubule lattices. By ultrafast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth toward individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore fiber assembly.