DNA replication creates two sister chromatids that must acquire specific three-dimensional conformations to support genome function and stability. This organization is largely mediated by cohesin complexes, which extrude intra-chromosomal loops and link two chromatids, thus forming “chromatid cohesion”. Although sister chromatids are genetically identical, the replication process is intrinsically asymmetric: each chromatid inherits a different parental DNA strand, while the new strands are synthesized using distinct “leading” and “lagging” mechanisms of the replication fork. Whether and how this molecular asymmetry impacts higher-order chromatin organization remains unknown. Using sister-chromatid-sensitive Hi-C, strand-specific FISH, and polymer modeling, we reveal a consistent, genome-wide shift in sister chromatid alignment, biased along the 5′-3′ direction of the inherited strands. This shift persists without loop extrusion but is lost upon disruption of cohesion, implicating cohesive cohesins in maintaining the displacement. Polymer simulations indicate that a modest (∼100 kb) misalignment of “cohesive” cohesins is responsible for the observed asymmetry. We propose two mechanistic models that explain how this displacement arises from replication fork asymmetry: either through the dislocation of cohesin during replication or through the asymmetric anchoring and subsequent random sliding of cohesin pairs. These findings reveal a previously unrecognized chromosome-scale asymmetry in sister chromatid organization, which has implications for homology search during DNA repair. ### Competing Interest Statement The authors have declared no competing interest. FWF Austrian Science Fund, SFB F 8804-B “Meiosis” European Research Council, 101163751, 101019039, Marie Curie Skłodowska Grant Agreement Nr. 847548, Marie Skłodowska-Curie Individual Fellowship Nr. 101103258, Marie Sklodowska-Curie grant agreement Nr. 101033347 Vienna Science and Technology Fund, LS19-001 European Molecular Biology Organization, ALTF 866-2022 Boehringer Ingelheim Fonds Austrian Academy of Sciences Vienna International Postdoc Program

During cell division, chromosomes reorganise into compact bodies in which centromeres localise precisely at the chromatin surface1-4 to enable kinetochore-microtubule interactions essential for genome segregation5-8. The physical principles guiding this centromere positioning remain unknown. Here, we reveal that human core centromeres are directed to the chromatin surface by repulsion of centromere-associated proteins - independent of condensin-mediated loop extrusion and microtubule engagement. Using cellular perturbations, biochemical reconstitution, and multiscale molecular dynamics simulations, we show that chromatin surface localisation emerges from repulsion between condensed chromatin and both the kinetochore and the highly negatively charged centromere protein, CENP-B. Together, these elements form a centromeric region composed of two domains with opposing affinities, one favouring integration within the mitotic chromosome and the other favouring exposure to the surrounding cytoplasm, thereby driving surface positioning. Tethering synthetic negatively charged proteins to chromatin was sufficient to recapitulate this surface localisation in cells and in vitro, indicating that electrostatic repulsion is a key determinant of surface localisation. These findings demonstrate that centromere layering is not hardwired by chromatin folding patterns but instead emerges from phase separation in chromatin. Our work uncovers electrostatic polarity as a general and programmable mechanism to spatially organise chromatin.

Accurate repair of DNA double-strand breaks (DSBs) is essential for genome stability, and defective repair underlies diseases such as cancer. Homologous recombination uses an intact homologous sequence to faithfully restore damaged DNA, yet how broken DNA ends find homologous sites in a genome containing billions of bases remains unclear. Here, we introduce sister-pore-C, a high-resolution method to map intra- and intermolecular interactions in replicated chromosomes. We show how DSBs remodel chromosome architecture using two functionally distinct pools of cohesin. Loop-extruding cohesin accumulates across megabase-scale domains surrounding DSBs to control local homology sampling, whereas cohesive cohesin concentrates at break sites to tether DNA ends to the sister chromatid. This mechanism restricts the homology-sampling space, highlighting how chromosome conformation helps to preserve genomic integrity.

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.

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.

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.

Genetic information is stored in linear DNA molecules, which fold extensively inside cells. DNA replication along the folded template path yields two sister chromatids that initially occupy the same nuclear region in a highly 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 pre-segregating 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.

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.

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. Gibsonet al.,Cell179, 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 Chromatin14, 50 (2021); H. Strickfadenet al.,Cell183, 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.

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.

ABSTRACT 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 (PP2A-B55) 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-B55 α/δ complexes display overlapping roles in controlling the dynamics of proper chromosome individualization and clustering during mitosis. In the absence of PP2A-B55α/δ 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 new 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.

Chromosomes transform during the cell cycle, allowing tran-scription 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.

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 cells 1 – 3 . Assembly of mitotic chromosomes involves DNA looping by condensin 4 – 8 and chromatin compaction by global histone deacetylation 9 – 13 . Although condensin confers mechanical resistance to spindle pulling forces 14 – 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.

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 OsO 4 /NH 4 Cl 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.

Developments in microscopy and visualization techniques have long pushed the boundaries of our understanding of cells. With powerful machine-learning techniques, we’re now facing a paradigm shift in image analysis for cell biology. Conventionally, image-based phenotypes have been described using a selected set of features we can easily intuit, such as shape or marker intensity. In the deep-learning era, neural network models can automatically learn representations of cellular phenotypes. These deep-learning features are data-driven, scalable, and sensitive to the representation of sophisticated or hidden patterns in the images.

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 cells 1–3 . Assembly of mitotic chromosomes involves DNA looping by condensin 4–8 and chromatin compaction by global histone deacetylation 9–13 . While condensin confers mechanical resistance towards spindle pulling forces 14–16 , it is not known how histone deacetylation affects material properties and 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 specific characteristics necessary for their precise movement during cellular 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. 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-intrinsic phase separation to genome segregation in dividing cells.

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. Modified chromosome conformation capture (Hi-C) technology is used to characterize the interactions between sister chromatids, despite their identical DNA sequences.

Particle tracking is a ubiquitous task in the study of dynamic molecular and cellular processes through microscopy. Light-sheet microscopy has opened a path to acquiring complete cell volumes for investigation in 3-dimensions (3D). However, hypothesis formulation and quantitative analysis have remained difficult due to fundamental challenges in the visualization and the verification of large and dense sets of 3D particle trajectories. Here we describe u-track 3D, a software package that addresses these two challenges. Building on the established framework of particle association in space and time implemented for 2D time-lapse sequences, we first report a complete and versatile pipeline for particle tracking in 3D. 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 which defines a score for every trajectory, thereby overcoming the challenges of trajectory validation by visual inspection. With these combined strategies, u-track 3D provides a framework for the unbiased study of molecular processes in complex volumetric sequences.