Biology subjects

Gerlich, D. W.

Publications and source records attributed to Gerlich, D. W..

17 recordsLinked to original sources

bioRxiv2026

Nested TAD hierarchy defines cohesion zones on replicated chromosomes

During each cell cycle, cells must not only duplicate DNA sequence but also preserve the three-dimensional genome architecture required for gene regulation and DNA repair. After replication, this requires coordination between cohesin-mediated loop extrusion and sister-chromatid cohesion for homology-directed DNA repair. Because extrusion promotes sister resolution whereas cohesion can impede extrusion, how both activities coexist on replicated chromosomes is unclear. Here we show that cohesin function is partitioned within the nested TAD hierarchy: cohesive cohesin accumulates at high-level boundaries to form "cohesion zones," while loop-extruding cohesin occupies boundaries across all hierarchical levels. Our data support a model in which this segregation emerges from dynamic interplay between cohesin pools and semi-permeable CTCF barriers, which act in cis and in trans to constrain sister-sister misalignment. Thus, a CTCF-defined boundary framework enables sister tethering while preserving dynamic loop folding, allowing replicated genomes to simultaneously support gene regulation and faithful DNA repair.

genomics↗

bioRxiv2026

Electrostatic control of chromatin compaction safeguards against apoptotic DNA release

Apoptosis involves extensive intracellular reorganisation to facilitate the clearance of dying cells. A key step in this process is the destruction of the genome through fragmentation by caspase-activated endonuclease (CAD). Rather than dispersing after CAD-mediated cleavage, DNA fragments are compacted into a dense chromatin compartment. However, the underlying mechanism and biological relevance of this compaction remain unknown. Here we show that global deacetylation of histone tails promotes chromatin compaction during apoptosis, preventing DNA release into apoptotic extracellular vesicles. Using synthetic effectors that modulate nucleosome electrostatics independently of histone modifications, we demonstrate that electrostatic attraction alone is sufficient to compact and sequester fragmented chromatin. These findings reveal a mechanism by which global reprogramming of histone modifications coordinates fragmentation of the genome with its physical sequestration during apoptosis. Furthermore, our synthetic approach provides a tool to probe the role of physical forces in genome organisation across diverse biological contexts.

cell biology↗

bioRxiv2025

An electrostatic repulsion model of centromere organisation

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.

cell biology↗

bioRxiv2025

Conformational asymmetry of replicated human chromosomes

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.

molecular biology↗

bioRxiv2025

Cohesin guides homology search during DNA repairvia loops and sister chromatid linkages

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 non-homologous bases remains unclear. Here, we introduce sister-pore-C, a high-resolution method for mapping intra- and trans-molecular interactions in replicated chromosomes. We show that DSBs reshape chromosome architecture by recruiting two functionally distinct pools of cohesin. Loop-forming cohesin accumulates across a megabase-scale domain to control homology sampling within topologically associating domains (TADs) surrounding the break site, while cohesive cohesin concentrates at the break site to tether broken ends to the sister chromatid. This dual mechanism restricts the homology search space, highlighting how chromosome conformation helps preserve genomic integrity.

cell biology↗

bioRxiv2023

PP2A-B55alpha,delta phosphatase counteracts Ki67-dependent chromosome individualization during mitosis

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 (B55 and B55{delta}), we report that PP2A-B55 /{delta} complexes display overlapping roles in controlling the dynamics of proper chromosome individualization and clustering during mitosis. In the absence of PP2A-B55/{delta} 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.

cell biology↗

bioRxiv2023

Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2 phase

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 cohesins 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.

cell biology↗

bioRxiv2022

Cohesin mediates DNA loop extrusion and sister chromatid cohesion by distinct mechanisms

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, which can extrude DNA into loops but is unable to mediate cohesion. Our results suggest that the latter defect arises during cohesion establishment. The observation that cohesins cohesion and loop extrusion activities can be 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 wildtype cohesin. This suggests that cohesion establishment and cohesins 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.

cell biology↗

bioRxiv2022

HiCognition: a visual exploration and hypothesis testing tool for 3D genomics

The 3D organization of the genome and epigenetic marks play important roles in gene expression, DNA repair, and chromosome segregation. Understanding how structure and composition of the chromatin fiber contribute to function requires integrated analysis of multiple genomics datasets from various techniques, experimental conditions, and cell states. Genome browsers facilitate such analysis, yet currently visualize only a few regions at a time and lack statistical functions that are often necessary to extract meaningful information. Here, we present HiCognition, a visual exploration and machine-learning tool based on a new genomic region set concept, which enables detection of patterns and associations between 3D chromosome conformation and collections of 1D genomics profiles of any type. By revealing how transcriptional activity and cohesin subunit isoforms contribute to chromosome conformation, we showcase how the flexible user interface and machine learning tools of HiCognition can help understand the relationship between structure and function of the genome.

bioinformatics↗

bioRxiv2021

In Diverse Conditions Intrinsic Chromatin Condensates Have Liquid-like Material Properties

Eukaryotic nuclear DNA is wrapped around histone proteins to form nucleosomes, which further assemble to package and regulate the genome. Understanding of the physical mechanisms that contribute to higher order chromatin organization is limited. Previously, we reported the intrinsic capacity of chromatin to undergo phase separation and form dynamic liquid-like condensates, which can be regulated by cellular factors. Recent work from Hansen, Hendzel, and colleagues suggested these intrinsic chromatin condensates are solid in all but a specific set of conditions. Here we show that intrinsic chromatin condensates are fluid in diverse solutions, without need for specific buffering components. Exploring experimental differences in sample preparation and imaging between these two studies, we suggest what may have led Hansen, Hendzel, and colleagues to mischaracterize the innate properties of chromatin condensates. We also describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained movement of chromatin in cells.

biophysics↗

bioRxiv2021

A chromatin phase transition protects mitotic chromosomes against microtubule perforation

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. While condensin confers mechanical resistance towards spindle pulling forces14-16, 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.

cell biology↗

bioRxiv2020

u-track 3D: measuring and interrogating intracellular dynamics in three dimensions.

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.

cell biology↗

bioRxiv2020

Sister-chromatid-sensitive Hi-C reveals the conformation of replicated human chromosomes

The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C)1-3 has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells4-11 yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine. Genome-wide conformation maps of human chromosomes revealed 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, characterized by facultative heterochromatin, as well as 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 dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes.

cell biology↗

bioRxiv2018

Augmin-mediated amplification of long-lived spindle microtubules directs plus-ends to kinetochores

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 non-centrosomal 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 microtubules. By ultra-fast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth towards individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore-fiber assembly.

cell biology↗

bioRxiv2018

Dynamics of sister chromatid resolution during cell cycle progression

Faithful genome transmission in dividing cells requires that the two copies of each chromosomes DNA package into separate, but physically linked, sister chromatids. The linkage between sister chromatids is mediated by cohesin, yet where sister chromatids are linked and how they resolve during cell cycle progression has remained unclear. Here, we investigated sister chromatid organization in live human cells using dCas9-mEGFP labelling of endogenous genomic loci. We detected substantial sister locus separation during G2 phase, irrespective of the proximity to cohesin enrichment sites. Almost all sister loci separated within a few hours after their respective replication, and then rapidly equilibrated their average distances within dynamic chromatin polymers. Our findings explain why the topology of sister chromatid resolution in G2 largely reflects the DNA replication program. Further, these data suggest that cohesin enrichment sites are not persistent cohesive sites in human cells. Rather, cohesion might occur at variable genomic positions within the cell population.

cell biology↗

bioRxiv2017

PARP inhibition causes premature loss of cohesion in cancer cells

Poly(ADP-ribose) polymerases (PARPs) regulate various aspects of cellular function including mitotic progression. Although PARP inhibitors have been undergoing various clinical trials and the PARP1/2 inhibitor olaparib was approved as monotherapy for BRCA-mutated ovarian cancer, their mode of action in killing tumour cells is not fully understood. We investigated the effect of PARP inhibition on mitosis in cancerous (cervical, ovary, breast and osteosarcoma) and non-cancerous cells by live-cell imaging. The clinically relevant inhibitor olaparib induced strong perturbations in mitosis, including problems with chromosome alignment at the metaphase plate, anaphase delay, and premature loss of cohesion (cohesion fatigue) after a prolonged metaphase arrest, resulting in sister chromatid scattering. PARP1 and PARP2 depletion suppressed the phenotype while PARP2 overexpression enhanced it, suggesting that olaparib-bound PARP1 and PARP2 rather than the lack of catalytic activity causes this phenotype. Olaparib-induced mitotic chromatid scattering was observed in various cancer cell lines with increased protein levels of PARP1 and PARP2, but not in non-cancer or cancer cell lines that expressed lower levels of PARP1 or PARP2. Interestingly, the sister chromatid scattering phenotype occurred only when olaparib was added during the S-phase preceding mitosis, suggesting that PARP1 and PARP2 entrapment at replication forks impairs sister chromatid cohesion. Clinically relevant DNA-damaging agents that impair replication progression such as topoisomerase inhibitors and cisplatin were also found to induce sister chromatid scattering and metaphase plate alignment problems, suggesting that these mitotic phenotypes are a common outcome of replication perturbation.

cell biology↗

bioRxiv2017

A Deep Learning And Novelty Detection Framework For Rapid Phenotyping In High-Content Screening

Supervised machine learning is a powerful and widely used method to analyze high-content screening data. Despite its accuracy, efficiency, and versatility, supervised machine learning has drawbacks, most notably its dependence on a priori knowledge of expected phenotypes and time-consuming classifier training. We provide a solution to these limitations with CellCognition Explorer, a generic novelty detection and deep learning framework. Application to several large-scale screening data sets on nuclear and mitotic cell morphologies demonstrates that CellCognition Explorer enables discovery of rare phenotypes without user training, which has broad implications for improved assay development in high-content screening.

cell biology↗