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 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, i.e. not catenated. Simulations show that even low levels of random strand passage, e.g. 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.
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.
SummaryDNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 regulates chromatin levels of condensins and topo II. 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 likely through shortening the average loop size and reducing DNA amount in each layer of mitotic loops. Furthermore, H1.8-mediated suppression of condensins and topo II binding to chromatin limits chromosome individualization by preventing resolution of interchromosomal linkages. While linker histones locally compact DNA by clustering nucleosomes, we propose that H1.8 controls chromosome morphology and topological organization through restricting the loading of condensins and topo II on chromatin.
The topological state of chromosomes is an important factor controlling their mechanical properties, dynamics and function. Recent work has shown that interphase chromosomes are largely free of entanglements. How cycling cells establish this state from a dense mitotic chromosome and maintain it through interphase in the presence of Topoisomerase II activity remains mysterious. Using multi-contact 3C, Hi-C, and polymer simulations, we find that mitotic chromosomes are intrachromosomally entangled, while interphase chromosomes are not, pointing to cell cycle-dependent control of chromosome topology. The majority of mitotic entanglements are removed during anaphase/telophase, with most of the remaining ones removed during early G1, in a process that requires Topoisomerase II. Polymer simulations show that swelling and decondensation of mitotic chromosomes during mitotic exit produce entropic forces that bias Topoisomerase II activity towards decatenation of condensin loops. This is followed by prevention of formation of new entanglements by lower Topoisomerase II activity in G1, and, in heterochromatic B compartments, by interplay between cohesin and Topoisomerase II. Our results show how cells control chromosome topology during mitotic exit and G1 employing both biophysical and molecular mechanisms to modulate Topoisomerase II activity and directionality.
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.
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