DNA damage reduces heterogeneity and coherence of chromatin motions.

Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.

Polymer models reveal how chromatin modification can modulate force at the kinetochore.

A key feature of chromosome segregation is the ability to sense tension between sister kinetochores. DNA between sister kinetochores must be packaged in a way that sustains tension propagation from one kinetochore to its sister, approximately 1 micron away. A molecular bottlebrush consisting of a primary axis populated with a crowded array of side chains provides a means to build tension over length scales considerably larger than the stiffness of the individual elements, that is, DNA polymer. Evidence for the bottlebrush organization of chromatin between sister kinetochores comes from genetic, cell biological, and polymer modeling of the budding yeast centromere. In this study, we have used polymer dynamic simulations of the bottlebrush to recapitulate experimental observations of kinetochore structure. Several aspects of the spatial distribution of kinetochore proteins and their response to perturbation lack a mechanistic understanding. Changes in physical parameters of bottlebrush, DNA stiffness, and DNA loops directly impact the architecture of the inner kinetochore. This study reveals that the bottlebrush is an active participant in building tension between sister kinetochores and proposes a mechanism for chromatin feedback to the kinetochore.

Shaping centromeres to resist mitotic spindle forces.

The centromere serves as the binding site for the kinetochore and is essential for the faithful segregation of chromosomes throughout cell division. The point centromere in yeast is encoded by a ∼115 bp specific DNA sequence, whereas regional centromeres range from 6-10 kbp in fission yeast to 5-10 Mbp in humans. Understanding the physical structure of centromere chromatin (pericentromere in yeast), defined as the chromatin between sister kinetochores, will provide fundamental insights into how centromere DNA is woven into a stiff spring that is able to resist microtubule pulling forces during mitosis. One hallmark of the pericentromere is the enrichment of the structural maintenance of chromosome (SMC) proteins cohesin and condensin. Based on studies from population approaches (ChIP-seq and Hi-C) and experimentally obtained images of fluorescent probes of pericentromeric structure, as well as quantitative comparisons between simulations and experimental results, we suggest a mechanism for building tension between sister kinetochores. We propose that the centromere is a chromatin bottlebrush that is organized by the loop-extruding proteins condensin and cohesin. The bottlebrush arrangement provides a biophysical means to transform pericentromeric chromatin into a spring due to the steric repulsion between radial loops. We argue that the bottlebrush is an organizing principle for chromosome organization that has emerged from multiple approaches in the field.

Simulating Dynamic Chromosome Compaction: Methods for Bridging In Silico to In Vivo.

The application of polymer models to chromosome structure and dynamics is a powerful approach for dissecting functional properties of the chromosome. The models are based on well-established bead-spring models of polymers and are distinct from molecular dynamics studies used in structural biology. In this work, we outline a polymer dynamics model that simulates budding yeast chromatin fibers in a viscous environment inside the nucleus using DataTank as a user interface for the C++ simulation. We highlight features for creating the nucleolus, a dynamic region of chromatin with protein-mediated, transient chromosomal cross-links, providing a predictive, stochastic polymer-physics model for versatile analyses of chromosome spatiotemporal organization. DataTank provides real-time visualization and data analytics methods during simulation. The simulation pipeline provides insights into the entangled chromosome milieu in the nucleus and creates simulated chromosome data, both structural and dynamic, that can be directly compared to experimental observations of live cells in interphase and mitosis.

The rDNA is biomolecular condensate formed by polymer-polymer phase separation and is sequestered in the nucleolus by transcription and R-loops.

The nucleolus is the site of ribosome biosynthesis encompassing the ribosomal DNA (rDNA) locus in a phase separated state within the nucleus. In budding yeast, we find the rDNA locus and Cdc14, a protein phosphatase that co-localizes with the rDNA, behave like a condensate formed by polymer-polymer phase separation, while ribonucleoproteins behave like a condensate formed by liquid-liquid phase separation. The compaction of the rDNA and Cdc14's nucleolar distribution are dependent on the concentration of DNA cross-linkers. In contrast, ribonucleoprotein nucleolar distribution is independent of the concentration of DNA cross-linkers and resembles droplets in vivo upon replacement of the endogenous rDNA locus with high-copy plasmids. When ribosomal RNA is transcribed from the plasmids by Pol II, the rDNA-binding proteins and ribonucleoprotein signals are weakly correlated, but upon repression of transcription, ribonucleoproteins form a single, stable droplet that excludes rDNA-binding proteins from its center. Degradation of RNA-DNA hybrid structures, known as R-loops, by overexpression of RNase H1 results in the physical exclusion of the rDNA locus from the nucleolar center. Thus, the rDNA locus is a polymer-polymer phase separated condensate that relies on transcription and physical contact with RNA transcripts to remain encapsulated within the nucleolus.

Performance of deep learning restoration methods for the extraction of particle dynamics in noisy microscopy image sequences.

Particle tracking in living systems requires low light exposure and short exposure times to avoid phototoxicity and photobleaching and to fully capture particle motion with high-speed imaging. Low-excitation light comes at the expense of tracking accuracy. Image restoration methods based on deep learning dramatically improve the signal-to-noise ratio in low-exposure data sets, qualitatively improving the images. However, it is not clear whether images generated by these methods yield accurate quantitative measurements such as diffusion parameters in (single) particle tracking experiments. Here, we evaluate the performance of two popular deep learning denoising software packages for particle tracking, using synthetic data sets and movies of diffusing chromatin as biological examples. With synthetic data, both supervised and unsupervised deep learning restored particle motions with high accuracy in two-dimensional data sets, whereas artifacts were introduced by the denoisers in three-dimensional data sets. Experimentally, we found that, while both supervised and unsupervised approaches improved tracking results compared with the original noisy images, supervised learning generally outperformed the unsupervised approach. We find that nicer-looking image sequences are not synonymous with more precise tracking results and highlight that deep learning algorithms can produce deceiving artifacts with extremely noisy images. Finally, we address the challenge of selecting parameters to train convolutional neural networks by implementing a frugal Bayesian optimizer that rapidly explores multidimensional parameter spaces, identifying networks yielding optimal particle tracking accuracy. Our study provides quantitative outcome measures of image restoration using deep learning. We anticipate broad application of this approach to critically evaluate artificial intelligence solutions for quantitative microscopy.

Statistical mechanics of chromosomes: in vivo and in silico approaches reveal high-level organization and structure arise exclusively through mechanical feedback between loop extruders and chromatin substrate properties.

The revolution in understanding higher order chromosome dynamics and organization derives from treating the chromosome as a chain polymer and adapting appropriate polymer-based physical principles. Using basic principles, such as entropic fluctuations and timescales of relaxation of Rouse polymer chains, one can recapitulate the dominant features of chromatin motion observed in vivo. An emerging challenge is to relate the mechanical properties of chromatin to more nuanced organizational principles such as ubiquitous DNA loops. Toward this goal, we introduce a real-time numerical simulation model of a long chain polymer in the presence of histones and condensin, encoding physical principles of chromosome dynamics with coupled histone and condensin sources of transient loop generation. An exact experimental correlate of the model was obtained through analysis of a model-matching fluorescently labeled circular chromosome in live yeast cells. We show that experimentally observed chromosome compaction and variance in compaction are reproduced only with tandem interactions between histone and condensin, not from either individually. The hierarchical loop structures that emerge upon incorporation of histone and condensin activities significantly impact the dynamic and structural properties of chromatin. Moreover, simulations reveal that tandem condensin-histone activity is responsible for higher order chromosomal structures, including recently observed Z-loops.

Polymer perspective of genome mobilization.

Chromosome motion is an intrinsic feature of all DNA-based metabolic processes and is a particularly well-documented response to both DNA damage and repair. By using both biological and polymer physics approaches, many of the contributing factors of chromatin motility have been elucidated. These include the intrinsic properties of chromatin, such as stiffness, as well as the loop modulators condensin and cohesin. Various biological factors such as external tethering to nuclear domains, ATP-dependent processes, and nucleofilaments further impact chromatin motion. DNA damaging agents that induce double-stranded breaks also cause increased chromatin motion that is modulated by recruitment of repair and checkpoint proteins. Approaches that integrate biological experimentation in conjunction with models from polymer physics provide mechanistic insights into the role of chromatin dynamics in biological function. In this review we discuss the polymer models and the effects of both DNA damage and repair on chromatin motion as well as mechanisms that may underlie these effects.

AI-Assisted Forward Modeling of Biological Structures.

The rise of machine learning and deep learning technologies have allowed researchers to automate image classification. We describe a method that incorporates automated image classification and principal component analysis to evaluate computational models of biological structures. We use a computational model of the kinetochore to demonstrate our artificial-intelligence (AI)-assisted modeling method. The kinetochore is a large protein complex that connects chromosomes to the mitotic spindle to facilitate proper cell division. The kinetochore can be divided into two regions: the inner kinetochore, including proteins that interact with DNA; and the outer kinetochore, comprised of microtubule-binding proteins. These two kinetochore regions have been shown to have different distributions during metaphase in live budding yeast and therefore act as a test case for our forward modeling technique. We find that a simple convolutional neural net (CNN) can correctly classify fluorescent images of inner and outer kinetochore proteins and show a CNN trained on simulated, fluorescent images can detect difference in experimental images. A polymer model of the ribosomal DNA locus serves as a second test for the method. The nucleolus surrounds the ribosomal DNA locus and appears amorphous in live-cell, fluorescent microscopy experiments in budding yeast, making detection of morphological changes challenging. We show a simple CNN can detect subtle differences in simulated images of the ribosomal DNA locus, demonstrating our CNN-based classification technique can be used on a variety of biological structures.

The regulation of chromosome segregation via centromere loops.

Biophysical studies of the yeast centromere have shown that the organization of the centromeric chromatin plays a crucial role in maintaining proper tension between sister kinetochores during mitosis. While centromeric chromatin has traditionally been considered a simple spring, recent work reveals the centromere as a multifaceted, tunable shock absorber. Centromeres can differ from other regions of the genome in their heterochromatin state, supercoiling state, and enrichment of structural maintenance of chromosomes (SMC) protein complexes. Each of these differences can be utilized to alter the effective stiffness of centromeric chromatin. In budding yeast, the SMC protein complexes condensin and cohesin stiffen chromatin by forming and cross-linking chromatin loops, respectively, into a fibrous structure resembling a bottlebrush. The high density of the loops compacts chromatin while spatially isolating the tension from spindle pulling forces to a subset of the chromatin. Paradoxically, the molecular crowding of chromatin via cohesin and condensin also causes an outward/poleward force. The structure allows the centromere to act as a shock absorber that buffers the variable forces generated by dynamic spindle microtubules. Based on the distribution of SMCs from bacteria to human and the conserved distance between sister kinetochores in a wide variety of organisms (0.4 to 1 micron), we propose that the bottlebrush mechanism is the foundational principle for centromere function in eukaryotes.

Transient crosslinking kinetics optimize gene cluster interactions.

Our understanding of how chromosomes structurally organize and dynamically interact has been revolutionized through the lens of long-chain polymer physics. Major protein contributors to chromosome structure and dynamics are condensin and cohesin that stochastically generate loops within and between chains, and entrap proximal strands of sister chromatids. In this paper, we explore the ability of transient, protein-mediated, gene-gene crosslinks to induce clusters of genes, thereby dynamic architecture, within the highly repeated ribosomal DNA that comprises the nucleolus of budding yeast. We implement three approaches: live cell microscopy; computational modeling of the full genome during G1 in budding yeast, exploring four decades of timescales for transient crosslinks between 5kbp domains (genes) in the nucleolus on Chromosome XII; and, temporal network models with automated community (cluster) detection algorithms applied to the full range of 4D modeling datasets. The data analysis tools detect and track gene clusters, their size, number, persistence time, and their plasticity (deformation). Of biological significance, our analysis reveals an optimal mean crosslink lifetime that promotes pairwise and cluster gene interactions through "flexible" clustering. In this state, large gene clusters self-assemble yet frequently interact (merge and separate), marked by gene exchanges between clusters, which in turn maximizes global gene interactions in the nucleolus. This regime stands between two limiting cases each with far less global gene interactions: with shorter crosslink lifetimes, "rigid" clustering emerges with clusters that interact infrequently; with longer crosslink lifetimes, there is a dissolution of clusters. These observations are compared with imaging experiments on a normal yeast strain and two condensin-modified mutant cell strains. We apply the same image analysis pipeline to the experimental and simulated datasets, providing support for the modeling predictions.

Three-Dimensional Thermodynamic Simulation of Condensin as a DNA-Based Translocase.

Chromatin dynamics and organization can be altered by condensin complexes. In turn, the molecular behavior of a condensin complex changes based on the tension of the substrate to which condensin is bound. This interplay between chromatin organization and condensin behavior demonstrates the need for tools that allows condensin complexes to be observed on a variety of chromatin organizations. We provide a method for simulating condensin complexes on a dynamic polymer substrate using the polymer dynamics simulator ChromoShake and the condensin simulator RotoStep. These simulations can be converted into simulated fluorescent images that are able to be directly compared to experimental images of condensin and fluorescently labeled chromatin. Our pipeline enables users to explore how changes in condensin behavior alters chromatin dynamics and vice versa while providing simulated image datasets that can be directly compared to experimental observations.

tRNA Genes Affect Chromosome Structure and Function via Local Effects.

The genome is packaged and organized in an ordered, nonrandom manner, and specific chromatin segments contact nuclear substructures to mediate this organization. tRNA genes (tDNAs) are binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the roles of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacked any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromatin architecture or chromosome tethering and mobility. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long-range HML-HMR heterochromatin clustering with concomitant effects on gene silencing. We propose that the tDNAs primarily affect local chromatin structure, which results in effects on long-range chromosome architecture.

Fork pausing allows centromere DNA loop formation and kinetochore assembly.

De novo kinetochore assembly, but not template-directed assembly, is dependent on COMA, the kinetochore complex engaged in cohesin recruitment. The slowing of replication fork progression by treatment with phleomycin (PHL), hydroxyurea, or deletion of the replication fork protection protein Csm3 can activate de novo kinetochore assembly in COMA mutants. Centromere DNA looping at the site of de novo kinetochore assembly can be detected shortly after exposure to PHL. Using simulations to explore the thermodynamics of DNA loops, we propose that loop formation is disfavored during bidirectional replication fork migration. One function of replication fork stalling upon encounters with DNA damage or other blockades may be to allow time for thermal fluctuations of the DNA chain to explore numerous configurations. Biasing thermodynamics provides a mechanism to facilitate macromolecular assembly, DNA repair, and other nucleic acid transactions at the replication fork. These loop configurations are essential for sister centromere separation and kinetochore assembly in the absence of the COMA complex.

Geometric partitioning of cohesin and condensin is a consequence of chromatin loops.

SMC (structural maintenance of chromosomes) complexes condensin and cohesin are crucial for proper chromosome organization. Condensin has been reported to be a mechanochemical motor capable of forming chromatin loops, while cohesin passively diffuses along chromatin to tether sister chromatids. In budding yeast, the pericentric region is enriched in both condensin and cohesin. As in higher-eukaryotic chromosomes, condensin is localized to the axial chromatin of the pericentric region, while cohesin is enriched in the radial chromatin. Thus, the pericentric region serves as an ideal model for deducing the role of SMC complexes in chromosome organization. We find condensin-mediated chromatin loops establish a robust chromatin organization, while cohesin limits the area that chromatin loops can explore. Upon biorientation, extensional force from the mitotic spindle aggregates condensin-bound chromatin from its equilibrium position to the axial core of pericentric chromatin, resulting in amplified axial tension. The axial localization of condensin depends on condensin's ability to bind to chromatin to form loops, while the radial localization of cohesin depends on cohesin's ability to diffuse along chromatin. The different chromatin-tethering modalities of condensin and cohesin result in their geometric partitioning in the presence of an extensional force on chromatin.

RotoStep: A Chromosome Dynamics Simulator Reveals Mechanisms of Loop Extrusion.

ChromoShake is a three-dimensional simulator designed to explore the range of configurational states a chromosome can adopt based on thermodynamic fluctuations of the polymer chain. Here, we refine ChromoShake to generate dynamic simulations of a DNA-based motor protein such as condensin walking along the chromatin substrate. We model walking as a rotation of DNA-binding heat-repeat proteins around one another. The simulation is applied to several configurations of DNA to reveal the consequences of mechanical stepping on taut chromatin under tension versus loop extrusion on single-tethered, floppy chromatin substrates. These simulations provide testable hypotheses for condensin and other DNA-based motors functioning along interphase chromosomes. Our model reveals a novel mechanism for condensin enrichment in the pericentromeric region of mitotic chromosomes. Increased condensin dwell time at centromeres results in a high density of pericentric loops that in turn provide substrate for additional condensin.

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus.

Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks.

Microtubule dynamics drive enhanced chromatin motion and mobilize telomeres in response to DNA damage.

Chromatin exhibits increased mobility on DNA damage, but the biophysical basis for this behavior remains unknown. To explore the mechanisms that drive DNA damage-induced chromosome mobility, we use single-particle tracking of tagged chromosomal loci during interphase in live yeast cells together with polymer models of chromatin chains. Telomeres become mobilized from sites on the nuclear envelope and the pericentromere expands after exposure to DNA-damaging agents. The magnitude of chromatin mobility induced by a single double-strand break requires active microtubule function. These findings reveal how relaxation of external tethers to the nuclear envelope and internal chromatin-chromatin tethers, together with microtubule dynamics, can mobilize the genome in response to DNA damage.

Entropy gives rise to topologically associating domains.

We investigate chromosome organization within the nucleus using polymer models whose formulation is closely guided by experiments in live yeast cells. We employ bead-spring chromosome models together with loop formation within the chains and the presence of nuclear bodies to quantify the extent to which these mechanisms shape the topological landscape in the interphase nucleus. By investigating the genome as a dynamical system, we show that domains of high chromosomal interactions can arise solely from the polymeric nature of the chromosome arms due to entropic interactions and nuclear confinement. In this view, the role of bio-chemical related processes is to modulate and extend the duration of the interacting domains.

SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin.

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1 since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization compared to either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.