PyMEGABASE: Predicting Cell-Type-Specific Structural Annotations of Chromosomes Using the Epigenome.

The folding patterns of interphase genomes in higher eukaryotes, as obtained from DNA-proximity-ligation or Hi-C experiments, are used to classify loci into structural classes called compartments and subcompartments. These structurally annotated (sub) compartments are known to exhibit specific epigenomic characteristics and cell-type-specific variations. To explore the relationship between genome structure and the epigenome, we present PyMEGABASE (PYMB), a maximum-entropy-based neural network model that predicts (sub) compartment annotations of a locus based solely on the local epigenome, such as ChIP-Seq of histone post-translational modifications. PYMB builds upon our previous model while improving robustness, capability to handle diverse inputs and user-friendly implementation. We employed PYMB to predict subcompartments for over a hundred human cell types available in ENCODE, shedding light on the links between subcompartments, cell identity, and epigenomic signals. The fact that PYMB, trained on data for human cells, can accurately predict compartments in mice suggests that the model is learning underlying physicochemical principles transferable across cell types and species. Reliable at higher resolutions (up to 5 kbp), PYMB is used to investigate compartment-specific gene expression. Not only can PYMB generate (sub) compartment information without Hi-C experiments, but its predictions are also interpretable. Analyzing PYMB's trained parameters, we explore the importance of various epigenomic marks in each subcompartment prediction. Furthermore, the predictions of the model can be used as input for OpenMiChroM software, which has been calibrated to generate three-dimensional structures of the genome. Detailed documentation of PYMB is available at https://pymegabase.readthedocs.io, including an installation guide using pip or conda, and Jupyter/Colab notebook tutorials.

Structural reorganization and relaxation dynamics of axially stressed chromosomes.

Chromosomes endure mechanical stresses throughout the cell cycle; for example, resulting from the pulling of chromosomes by spindle fibers during mitosis or deformation of the nucleus during cell migration. The response to physical stress is closely related to chromosome structure and function. Micromechanical studies of mitotic chromosomes have revealed them to be remarkably extensible objects and informed early models of mitotic chromosome organization. We use a data-driven, coarse-grained polymer modeling approach to explore the relationship between the spatial organization of individual chromosomes and their emergent mechanical properties. In particular, we investigate the mechanical properties of our model chromosomes by axially stretching them. Simulated stretching led to a linear force-extension curve for small strain, with mitotic chromosomes behaving about 10-fold stiffer than interphase chromosomes. Studying their relaxation dynamics, we found that chromosomes are viscoelastic solids with a highly liquid-like, viscous behavior in interphase that becomes solid-like in mitosis. This emergent mechanical stiffness originates from lengthwise compaction, an effective potential capturing the activity of loop-extruding SMC complexes. Chromosomes denature under large strains via unraveling, which is characterized by opening of large-scale folding patterns. By quantifying the effect of mechanical perturbations on the chromosome's structural features, our model provides a nuanced understanding of in vivo mechanics of chromosomes.

Shaping the genome via lengthwise compaction, phase separation, and lamina adhesion.

The link between genomic structure and biological function is yet to be consolidated, it is, however, clear that physical manipulation of the genome, driven by the activity of a variety of proteins, is a crucial step. To understand the consequences of the physical forces underlying genome organization, we build a coarse-grained polymer model of the genome, featuring three fundamentally distinct classes of interactions: lengthwise compaction, i.e., compaction of chromosomes along its contour, self-adhesion among epigenetically similar genomic segments, and adhesion of chromosome segments to the nuclear envelope or lamina. We postulate that these three types of interactions sufficiently represent the concerted action of the different proteins organizing the genome architecture and show that an interplay among these interactions can recapitulate the architectural variants observed across the tree of life. The model elucidates how an interplay of forces arising from the three classes of genomic interactions can drive drastic, yet predictable, changes in the global genome architecture, and makes testable predictions. We posit that precise control over these interactions in vivo is key to the regulation of genome architecture.

DNA supercoiling-mediated collective behavior of co-transcribing RNA polymerases.

Multiple RNA polymerases (RNAPs) transcribing a gene have been known to exhibit collective group behavior, causing the transcription elongation rate to increase with the rate of transcription initiation. Such behavior has long been believed to be driven by a physical interaction or 'push' between closely spaced RNAPs. However, recent studies have posited that RNAPs separated by longer distances may cooperate by modifying the DNA segment under transcription. Here, we present a theoretical model incorporating the mechanical coupling between RNAP translocation and the DNA torsional response. Using stochastic simulations, we demonstrate DNA supercoiling-mediated long-range cooperation between co-transcribing RNAPs. We find that inhibiting transcription initiation can slow down the already recruited RNAPs, in agreement with recent experimental observations, and predict that the average transcription elongation rate varies non-monotonically with the rate of transcription initiation. We further show that while RNAPs transcribing neighboring genes oriented in tandem can cooperate, those transcribing genes in divergent or convergent orientations can act antagonistically, and that such behavior holds over a large range of intergenic separations. Our model makes testable predictions, revealing how the mechanical interplay between RNAPs and the DNA they transcribe can govern transcriptional dynamics.

3D genomics across the tree of life reveals condensin II as a determinant of architecture type.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

Coarse-grained modelling of DNA plectoneme pinning in the presence of base-pair mismatches.

Damaged or mismatched DNA bases result in the formation of physical defects in double-stranded DNA. In vivo, defects in DNA must be rapidly and efficiently repaired to maintain cellular function and integrity. Defects can also alter the mechanical response of DNA to bending and twisting constraints, both of which are important in defining the mechanics of DNA supercoiling. Here, we use coarse-grained molecular dynamics (MD) simulation and supporting statistical-mechanical theory to study the effect of mismatched base pairs on DNA supercoiling. Our simulations show that plectoneme pinning at the mismatch site is deterministic under conditions of relatively high force (>2 pN) and high salt concentration (>0.5 M NaCl). Under physiologically relevant conditions of lower force (0.3 pN) and lower salt concentration (0.2 M NaCl), we find that plectoneme pinning becomes probabilistic and the pinning probability increases with the mismatch size. These findings are in line with experimental observations. The simulation framework, validated with experimental results and supported by the theoretical predictions, provides a way to study the effect of defects on DNA supercoiling and the dynamics of supercoiling in molecular detail.

Chromosome disentanglement driven via optimal compaction of loop-extruded brush structures.

Eukaryote cell division features a chromosome compaction-decompaction cycle that is synchronized with their physical and topological segregation. It has been proposed that lengthwise compaction of chromatin into mitotic chromosomes via loop extrusion underlies the compaction-segregation/resolution process. We analyze this disentanglement scheme via considering the chromosome to be a succession of DNA/chromatin loops-a polymer "brush"-where active extrusion of loops controls the brush structure. Given type-II DNA topoisomerase (Topo II)-catalyzed topology fluctuations, we find that interchromosome entanglements are minimized for a certain "optimal" loop that scales with the chromosome size. The optimal loop organization is in accord with experimental data across species, suggesting an important structural role of genomic loops in maintaining a less entangled genome. Application of the model to the interphase genome indicates that active loop extrusion can maintain a level of chromosome compaction with suppressed entanglements; the transition to the metaphase state requires higher lengthwise compaction and drives complete topological segregation. Optimized genomic loops may provide a means for evolutionary propagation of gene-expression patterns while simultaneously maintaining a disentangled genome. We also find that compact metaphase chromosomes have a densely packed core along their cylindrical axes that explains their observed mechanical stiffness. Our model connects chromosome structural reorganization to topological resolution through the cell cycle and highlights a mechanism of directing Topo II-mediated strand passage via loop extrusion-driven lengthwise compaction.

DNA Mechanics and Topology.

We review the current understanding of the mechanics of DNA and DNA-protein complexes, from scales of base pairs up to whole chromosomes. Mechanics of the double helix as revealed by single-molecule experiments will be described, with an emphasis on the role of polymer statistical mechanics. We will then discuss how topological constraints- entanglement and supercoiling-impact physical and mechanical responses. Models for protein-DNA interactions, including effects on polymer properties of DNA of DNA-bending proteins will be described, relevant to behavior of protein-DNA complexes in vivo. We also discuss control of DNA entanglement topology by DNA-lengthwise-compaction machinery acting in concert with topoisomerases. Finally, the chapter will conclude with a discussion of relevance of several aspects of physical properties of DNA and chromatin to oncology.

Defect-facilitated buckling in supercoiled double-helix DNA.

We present a statistical-mechanical model for stretched twisted double-helix DNA, where thermal fluctuations are treated explicitly from a Hamiltonian without using any scaling hypotheses. Our model applied to defect-free supercoiled DNA describes the coexistence of multiple plectoneme domains in long DNA molecules at physiological salt concentrations (≈0.1M Na^{+}) and stretching forces (≈1pN). We find a higher (lower) number of domains at lower (higher) ionic strengths and stretching forces, in accord with experimental observations. We use our model to study the effect of an immobile point defect on the DNA contour that allows a localized kink. The degree of the kink is controlled by the defect size, such that a larger defect further reduces the bending energy of the defect-facilitated kinked end loop. We find that a defect can spatially pin a plectoneme domain via nucleation of a kinked end loop, in accord with experiments and simulations. Our model explains previously reported magnetic tweezer experiments [A. Dittmore et al., Phys. Rev. Lett. 119, 147801 (2017)PRLTAO0031-900710.1103/PhysRevLett.119.147801] showing two buckling signatures: buckling and "rebuckling" in supercoiled DNA with a base-unpaired region. Comparing with experiments, we find that under 1 pN force, a kinked end loop nucleated at a base-mismatched site reduces the bending energy by ≈0.7 k_{B}T per unpaired base. Our model predicts the coexistence of three states at the buckling and rebuckling transitions, which warrants new experiments.

Nucleation of Multiple Buckled Structures in Intertwined DNA Double Helices.

We study the statistical-mechanical properties of intertwined double-helical DNAs (DNA braids). In magnetic tweezers experiments, we find that torsionally stressed stretched braids supercoil via an abrupt buckling transition, which is associated with the nucleation of a braid end loop, and that the buckled braid is characterized by a proliferation of multiple domains. Differences between the mechanics of DNA braids and supercoiled single DNAs can be understood as an effect of the increased bulkiness in the structure of the former. The experimental results are in accord with the predictions of a statistical-mechanical model.

Supercoiling DNA Locates Mismatches.

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.6 pN force and show that under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

Torque and buckling in stretched intertwined double-helix DNAs.

We present a statistical-mechanical model for the behavior of intertwined DNAs, with a focus on their torque and extension as a function of their catenation (linking) number and applied force, as studied in magnetic tweezers experiments. Our model produces results in good agreement with available experimental data and predicts a catenation-dependent effective twist modulus distinct from what is observed for twisted individual double-helix DNAs. We find that buckling occurs near the point where experiments have observed a kink in the extension versus linking number, and that the subsequent "supercoiled braid" state corresponds to a proliferation of multiple small plectoneme structures. We predict a discontinuity in extension at the buckling transition corresponding to nucleation of the first plectoneme domain. We also find that buckling occurs for lower linking number at lower salt; the opposite trend is observed for supercoiled single DNAs.