RESUMO
The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.
Assuntos
Cromossomos , DNA Topoisomerases Tipo II , DNA Topoisomerases Tipo II/genética , Cromossomos/genética , Mitose/genética , Interfase/genética , PolímerosRESUMO
Over the last several years enormous progress has been made in identifying the molecular machines, including condensins and topoisomerases that fold mitotic chromosomes. The discovery that condensins generate chromatin loops through loop extrusion has revolutionized, and energized, the field of chromosome folding. To understand how these machines fold chromosomes with the appropriate dimensions, while disentangling sister chromatids, it needs to be determined how they are regulated and deployed. Here, we outline the current understanding of how these machines and factors are regulated through cell cycle dependent expression, chromatin localization, activation and inactivation through post-translational modifications, and through associations with each other, with other factors and with the chromatin template itself. There are still many open questions about how condensins and topoisomerases are regulated but given the pace of progress in the chromosome folding field, it seems likely that many of these will be answered in the years ahead.
Assuntos
Cromátides , Cromatina , Cromatina/genética , Mitose/genéticaRESUMO
During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to scale coordinately with mitotic spindles, but the underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog Xenopus laevis to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle, and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be reset by cytoplasmic factors from earlier developmental stages. In vitro, increasing nuclear-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin α scales mitotic chromosomes to cell surface area/volume ratio (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and Hi-C data suggest that mitotic chromosomes shrink during embryogenesis through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter chromosome axis. Together, our findings demonstrate how mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.
Assuntos
Núcleo Celular , Cromossomos , Animais , Xenopus laevis/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Fuso Acromático/metabolismo , Tamanho Celular , MitoseRESUMO
DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.
Assuntos
Cromatina/metabolismo , Cromossomos/genética , DNA Topoisomerases Tipo II/genética , Histonas/genética , Adenosina Trifosfatases/metabolismo , Animais , Extratos Celulares/química , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Feminino , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Oócitos/química , Oócitos/metabolismo , Fuso Acromático/genética , Fuso Acromático/patologia , Fuso Acromático/ultraestrutura , Xenopus laevisRESUMO
During interphase, the eukaryotic genome is organized into chromosome territories that are spatially segregated into compartment domains. The extent to which interacting domains or chromosomes are entangled is not known. We analyze series of co-occurring chromatin interactions using multi-contact 3C (MC-3C) in human cells to provide insights into the topological entanglement of chromatin. Multi-contact interactions represent percolation paths (C-walks) through three-dimensional (3D) chromatin space. We find that the order of interactions within C-walks that occur across interfaces where chromosomes or compartment domains interact is not random. Polymer simulations show that such C-walks are consistent with distal domains being topologically insulated, that is, not catenated. Simulations show that even low levels of random strand passage, for example by topoisomerase II, would result in entanglements, increased mixing at domain interfaces and an order of interactions within C-walks not consistent with experimental MC-3C data. Our results indicate that, during interphase, entanglements between chromosomes and chromosomal domains are rare.