Different relative scalings between transient forces and thermal fluctuations tune regimes of chromatin organization.

Within the nucleus, structural maintenance of chromosome protein complexes, namely condensin and cohesin, create an architecture to facilitate the organization and proper function of the genome. Condensin, in addition to performing loop extrusion, creates localized clusters of chromatin in the nucleolus through transient crosslinks. Large-scale simulations revealed three different dynamic behaviors as a function of timescale: slow crosslinking leads to no clusters, fast crosslinking produces rigid slowly changing clusters, while intermediate timescales produce flexible clusters that mediate gene interaction. By mathematically analyzing different relative scalings of the two sources of stochasticity, thermal fluctuations and the force induced by the transient crosslinks, we predict these three distinct regimes of cluster behavior. Standard time-averaging that takes the fluctuations of the transient crosslink force to zero predicts the existence of rigid clusters. Accounting for the interaction of both fluctuations from the crosslinks and thermal noise with an effective energy landscape predicts the timescale-dependent lifetimes of flexible clusters. No clusters are predicted when the fluctuations of the transient crosslink force are taken to be large relative to thermal fluctuations. This mathematical perturbation analysis illuminates the importance of accounting for stochasticity in local incoherent transient forces to predict emergent complex biological behavior.

Chromosome segregation: Brushing up on centromeres.

Turning centromere DNA into a mechanical spring is central to the fidelity of chromosome segregation. A recent study shows how centromere DNA loops and partitioning cohesin and condensin convert centromeres and pericentromeres into bipartite bottlebrushes.

Transient crosslinking controls the condensate formation pathway within chromatin networks.

The network structure of densely packed chromatin within the nucleus of eukaryotic cells acts in concert with nonequilibrium processes. Using statistical physics simulations, we explore the control provided by transient crosslinking of the chromatin network by structural-maintenance-of-chromosome (SMC) proteins over (i) the physical properties of the chromatin network and (ii) condensate formation of embedded molecular species. We find that the density and lifetime of transient SMC crosslinks regulate structural relaxation modes and tune the sol-vs-gel state of the chromatin network, which imparts control over the kinetic pathway to condensate formation. Specifically, lower density, shorter-lived crosslinks induce sollike networks and a droplet-fusion pathway, whereas higher density, longer-lived crosslinks induce gellike networks and an Ostwald-ripening pathway.

Dicentric chromosomes are resolved through breakage and repair at their centromeres.

Chromosomes with two centromeres provide a unique opportunity to study chromosome breakage and DNA repair using completely endogenous cellular machinery. Using a conditional transcriptional promoter to control the second centromere, we are able to activate the dicentric chromosome and follow the appearance of DNA repair products. We find that the rate of appearance of DNA repair products resulting from homology-based mechanisms exceeds the expected rate based on their limited centromere homology (340 bp) and distance from one another (up to 46.3 kb). In order to identify whether DNA breaks originate in the centromere, we introduced 12 single-nucleotide polymorphisms (SNPs) into one of the centromeres. Analysis of the distribution of SNPs in the recombinant centromeres reveals that recombination was initiated with about equal frequency within the conserved centromere DNA elements CDEII and CDEIII of the two centromeres. The conversion tracts range from about 50 bp to the full length of the homology between the two centromeres (340 bp). Breakage and repair events within and between the centromeres can account for the efficiency and distribution of DNA repair products. We propose that in addition to providing a site for kinetochore assembly, the centromere may be a point of stress relief in the face of genomic perturbations.

Polymer Modeling Reveals Interplay between Physical Properties of Chromosomal DNA and the Size and Distribution of Condensin-Based Chromatin Loops.

Transient DNA loops occur throughout the genome due to thermal fluctuations of DNA and the function of SMC complex proteins such as condensin and cohesin. Transient crosslinking within and between chromosomes and loop extrusion by SMCs have profound effects on high-order chromatin organization and exhibit specificity in cell type, cell cycle stage, and cellular environment. SMC complexes anchor one end to DNA with the other extending some distance and retracting to form a loop. How cells regulate loop sizes and how loops distribute along chromatin are emerging questions. To understand loop size regulation, we employed bead-spring polymer chain models of chromatin and the activity of an SMC complex on chromatin. Our study shows that (1) the stiffness of the chromatin polymer chain, (2) the tensile stiffness of chromatin crosslinking complexes such as condensin, and (3) the strength of the internal or external tethering of chromatin chains cooperatively dictate the loop size distribution and compaction volume of induced chromatin domains. When strong DNA tethers are invoked, loop size distributions are tuned by condensin stiffness. When DNA tethers are released, loop size distributions are tuned by chromatin stiffness. In this three-way interaction, the presence and strength of tethering unexpectedly dictates chromatin conformation within a topological domain.

Misregulation of cell cycle-dependent methylation of budding yeast CENP-A contributes to chromosomal instability.

Centromere (CEN) identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved CEN-specific histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle-dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation. We generated a custom antibody that specifically recognizes methylated Cse4-R37 and showed that methylation of Cse4 is cell cycle regulated with maximum levels of methylated Cse4-R37 and its enrichment at the CEN chromatin occur in the mitotic cells. Methyl-mimic cse4-R37F mutant exhibits synthetic lethality with kinetochore mutants, reduced levels of CEN-associated kinetochore proteins and chromosome instability (CIN), suggesting that mimicking the methylation of Cse4-R37 throughout the cell cycle is detrimental to faithful chromosome segregation. Our results showed that SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37 and overexpression of UPA1 leads to CIN phenotype. In summary, our studies have defined a role for cell cycle-regulated methylation of Cse4 in high-fidelity chromosome segregation and highlight an important role of epigenetic modifications such as methylation of kinetochore proteins in preventing CIN, an important hallmark of human cancers.

Adaptive partitioning of a gene locus to the nuclear envelope in Saccharomyces cerevisiae is driven by polymer-polymer phase separation.

Partitioning of active gene loci to the nuclear envelope (NE) is a mechanism by which organisms increase the speed of adaptation and metabolic robustness to fluctuating resources in the environment. In the yeast Saccharomyces cerevisiae, adaptation to nutrient depletion or other stresses, manifests as relocalization of active gene loci from nucleoplasm to the NE, resulting in more efficient transport and translation of mRNA. The mechanism by which this partitioning occurs remains a mystery. Here, we demonstrate that the yeast inositol depletion-responsive gene locus INO1 partitions to the nuclear envelope, driven by local histone acetylation-induced polymer-polymer phase separation from the nucleoplasmic phase. This demixing is consistent with recent evidence for chromatin phase separation by acetylation-mediated dissolution of multivalent histone association and fits a physical model where increased bending stiffness of acetylated chromatin polymer causes its phase separation from de-acetylated chromatin. Increased chromatin spring stiffness could explain nucleation of transcriptional machinery at active gene loci.

The power of weak, transient interactions across biology: A paradigm of emergent behavior.

A growing list of diverse biological systems and their equally diverse functionalities provides realizations of a paradigm of emergent behavior. In each of these biological systems, pervasive ensembles of weak, short-lived, spatially local interactions act autonomously to convey functionalities at larger spatial and temporal scales. In this article, a range of diverse systems and functionalities are presented in a cursory manner with literature citations for further details. Then two systems and their properties are discussed in more detail: yeast chromosome biology and human respiratory mucus.

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.

Mechanisms of DNA Mobilization and Sequestration.

The entire genome becomes mobilized following DNA damage. Understanding the mechanisms that act at the genome level requires that we embrace experimental and computational strategies to capture the behavior of the long-chain DNA polymer, which is the building block for the chromosome. Long-chain polymers exhibit constrained, sub-diffusive motion in the nucleus. Cross-linking proteins, including cohesin and condensin, have a disproportionate effect on genome organization in their ability to stabilize transient interactions. Cross-linking proteins can segregate the genome into sub-domains through polymer-polymer phase separation (PPPS) and can drive the formation of gene clusters through small changes in their binding kinetics. Principles from polymer physics provide a means to unravel the mysteries hidden in the chains of life.

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.

Cdc7-mediated phosphorylation of Cse4 regulates high-fidelity chromosome segregation in budding yeast.

Faithful chromosome segregation maintains chromosomal stability as errors in this process contribute to chromosomal instability (CIN), which has been observed in many diseases including cancer. Epigenetic regulation of kinetochore proteins such as Cse4 (CENP-A in humans) plays a critical role in high-fidelity chromosome segregation. Here we show that Cse4 is a substrate of evolutionarily conserved Cdc7 kinase, and that Cdc7-mediated phosphorylation of Cse4 prevents CIN. We determined that Cdc7 phosphorylates Cse4 in vitro and interacts with Cse4 in vivo in a cell cycle-dependent manner. Cdc7 is required for kinetochore integrity as reduced levels of CEN-associated Cse4, a faster exchange of Cse4 at the metaphase kinetochores, and defects in chromosome segregation, are observed in a cdc7-7 strain. Phosphorylation of Cse4 by Cdc7 is important for cell survival as constitutive association of a kinase-dead variant of Cdc7 (cdc7-kd) with Cse4 at the kinetochore leads to growth defects. Moreover, phospho-deficient mutations of Cse4 for consensus Cdc7 target sites contribute to CIN phenotype. In summary, our results have defined a role for Cdc7-mediated phosphorylation of Cse4 in faithful chromosome segregation.

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.

Behavior of dicentric chromosomes in budding yeast.

DNA double-strand breaks arise in vivo when a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms.

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.

R-loops at centromeric chromatin contribute to defects in kinetochore integrity and chromosomal instability in budding yeast.

R-loops, the byproduct of DNA-RNA hybridization and the displaced single-stranded DNA (ssDNA), have been identified in bacteria, yeasts, and other eukaryotic organisms. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric (CEN) chromatin in wild-type budding yeast. Here we used an hpr1∆ strain that accumulates R-loops to investigate the consequences of R-loops at CEN chromatin and chromosome segregation. We show that Hpr1 interacts with the CEN-histone H3 variant, Cse4, and prevents the accumulation of R-loops at CEN chromatin for chromosomal stability. DNA-RNA immunoprecipitation (DRIP) analysis showed an accumulation of R-loops at CEN chromatin that was reduced by overexpression of RNH1 in hpr1∆ strains. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3, and mislocalization of histone H3 at CEN chromatin were observed in hpr1∆ strains. We determined that accumulation of R-loops at CEN chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN) and these phenotypes are suppressed by RNH1 overexpression in hpr1∆ strains. In summary, our studies provide mechanistic insights into how accumulation of R-loops at CEN contributes to defects in kinetochore integrity and CIN.

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.