Session W06: Physics of Genome Organization: From DNA to Chromatin

Chromatin network retards droplet coalescence

    Nuclear bodies are membraneless condensates that may form via liquid-liquid phase separation. The viscoelastic chromatin network could impact their stability and may hold the key for understanding experimental observations that defy predictions of classical theories. However, quantitative studies on the role of the chromatin network in phase separation have remained challenging. Using a diploid human genome model parameterized with chromosome conformation capture (Hi-C) data, we study the thermodynamics and kinetics of nucleoli formation. Dynamical simulations predict the formation of multiple droplets for nucleolar particles that experience specific interactions with nucleolus-associated domains (NADs). Coarsening dynamics, surface tension, and coalescence kinetics of the simulated droplets are all in quantitative agreement with experimental measurements for nucleoli. Free energy calculations further support that a two-droplet state, often observed for nucleoli in somatic cells, is metastable and separated from the single-droplet state with an entropic barrier. Our study suggests that nucleoli-chromatin interactions facilitate droplets' nucleation but hinder their coarsening due to the coupled motion between droplets and the chromatin network: as droplets coalesce, the chromatin network becomes increasingly constrained. Therefore, the chromatin network supports a nucleation and arrest mechanism to stabilize the multi-droplet state for nucleoli and possibly for other nuclear bodies.   

Rheological Properties of Chromatin and Nuclear Microenvironment in Living Cells

Genome organization and dynamics are essential in gene regulation and cellular functions. Chromosomal DNA wraps around histone octamers to form the basic functional unit, nucleosomes. Chromatin, a complex of multiple nucleosomes and proteins, is organized into a hierarchical structure which allows reconfigurable processes to occur during transcriptional regulation. The nuclear environment is a heterogeneous and viscoelastic medium, which constrains the transport of biological macromolecules, impacts the efficiency of enzymatic reactions, and influences chromatin folding and dynamics.  Investigation of chromatin and nuclear microenvironment viscoelasticity under physiological conditions has been a major challenge in the field. In this work, we use genomic-locus tracking and polymer models to characterize the viscoelasticity of chromatin and the surrounding nuclear environment. Specific genomic loci were imaged and tracked by CRISPR-based live-cell imaging techniques [1.2]. Elucidating the viscoelasticity of chromatin and its local microenvironment is a key to improving our understanding of human genome stability and nuclear function. This work demonstrates a new non-invasive approach to studying the local rheological properties of chromatin and nucleoplasm in the cell nucleus.   

Active Dynamics of Chromosome Polymers

Mechanical manipulation of chromosomes, driven via the forces applied by microscopic motor proteins, is an active process essential for biological function. The enormous length of the chromatin polymer makes coarse-graining an important aspect in modeling chromosomes. Effective-equilibrium descriptions of coarse-grained chromosomes have been useful in understanding structural features, however, these models are limited by their equilibrium-like dynamics and a lack of connection to microscopic activity. Here, we propose an active polymer model for chromosomes, where microscopic activity leads to non-equilibrium fluctuations at the coarse-grained length scales. Our model predicts various experimentally realizable signatures, such as the scaling of mean-squared displacement with time for various levels of activity. In all, we construct an active chromosome model and utilize it to directly probe the consequences of microscopic activity on structure and dynamics.   

Phase separation and correlated motions in motorized genome

The eukaryotic genome is organized non-randomly in the three-dimensional space, where the ATP-consuming processes, like transcription, actively impact the spatial organization of chromosomes. Here, we studied the impact of non-equilibrium activities on the structural dynamics and organization of the human genome. A potential energy function derived from chromosome conformation capture data was used to account for effective interactions that fold individual chromosomes and drive their phase separation. Non-equilibrium activities were introduced to active genomic regions through the introduction of time-correlated active forces. Molecular dynamics simulations revealed a striking impact of active forces on genomic phase separation. We explored how the balance between the two forces may dictate the formation of normal or inverted chromosome distribution in the nuclei, and contribute to the genome dynamics in the view of long-range coherent motions.   

When motors collide: how transcription and loop-extruding cohesins organize chromatin

The SMC complex cohesin organizes interphase chromatin into loops by a process known as "loop extrusion," through which cohesin progressively reels in DNA and extrudes it as a loop. Extrusion can generate distinctive patterns in genomic spatial organization through interactions with stationary "boundary elements," such as CTCF proteins. Less is known about whether and how extrusion is impeded by other elements on the molecularly crowded genome, such as translocating RNA polymerase motors. We analyze chromosome conformation capture (Hi-C) data near active genes, including in conditions in which cohesin dynamics or transcription are perturbed. We develop a model in which RNA polymerases act as "moving barriers,"  impeding and pushing translocating cohesins as they continue to extrude loops. Simulations of the model reproduce experimentally observed cohesin accumulation and genomic contact patterns. We develop a theory that explains how cohesin accumulation patterns result from the probability of encounter with polymerase and cohesin lifetimes on chromatin. Our analysis thus demonstrates how transcriptional activity shapes the genome by modulating the activity of loop-extruding cohesin motors.   

Limited spreading enzyme stabilizes epigenetic memory

The epigenetic state of a cell is characterized by patterns of covalent histone modifications ("marks") across the genome, with different marks typical of active (euchromatic) and silenced (heterochromatic) regions. These mark patterns can be stable over many cell generations—a form of epigenetic memory—despite continuous loss due to replication. Here, we investigate the possibility that the increased density of heterochromatin and its phase separation could contribute to this stability. We introduce a minimal biophysical model stylizing chromatin and its dynamics through the cell cycle, in which modifying enzymes "spread" marks between nearby histones and marked histones experience self-attraction. We find that marks localize sharply and stably to denser regions, but if the density differences are allowed to arise endogenously from the self-attraction, the model generically exhibits uncontrolled spread or global loss of heterochromatin. Limitation of the modifying enzymes relative to their histone substrates—a plausible but oft-neglected element—totally changes this picture, yielding a memory system, stable for hundreds of cell generations, that depends on self-attraction and dense heterochromatin, suggesting a functional role for this hallmark of nuclear organization.   

4D Chromosome Organization: Combining Polymer Physics, Knot Theory and High Performance Computing

Self-organization is a universal concept spanning numerous disciplines including mathematics, physics and biology. Chromosomes are self-organizing polymers that fold into orderly, origami-like dynamic structures. In the past decade, advances in experimental biology have provided a means to reveal information about chromosome connectivity,  allowing us to directly use this information from experiments to generate 3D models of individual genes, chromosomes and even genomes. In this talk I will present a novel data-driven modeling approach, 4DHiC, and demonstrate a number of possibilities that this method holds. X chromosome inactivation is a dynamic process whereby genes on one of the female X chromosomes are turned off. I will discuss a detailed 3D modeling study of the time-evolution of X chromosome inactivation, highlighting both global and local properties of chromosomes that result in topology-driven dynamical arrest.   

Nucleosome-scale structure drives linker histone enrichment in heterochromatin in silico

Linker histones have been recognized as dominant determinants of short-range chromatin structure, causing condensation of a 30-nm fiber in vitro, and have been demonstrated both to have important roles in regulating topologically-associating domain (TAD) structure and to be strongly enriched in heterochromatin. However, studies to-date have found great difficulty elucidating the mechanisms by which linker histones selectively partition into heterochromatin, though it has been postulated that H1 may bind to specific histone PTMs or induce modifications of core histones. In this work we introduce a multi-scale polymer model for chromatin which captures both megabase-scale phase separation and nucleosome-scale physics, explicitly accounting for the effects of both DNA linker length and H1 binding. We demonstrate that phase separation induces changes in nucleosome-scale structure which are sufficient to cause co-precipitation of linker histones in heterochromatin at least as great as the enrichment observed in vivo. Our work demonstrates that specific histone-code recognition is not necessary to induce linker histone enrichment in heterochromatin, and illustrates the potential for mesoscale models to answer key questions in biological systems which are experimentally intractable.   

Differential regulation of alternative promoters emerges from unified kinetics of enhancer-promoter interaction

Many eukaryotic genes contain alternative promoters with distinct expression patterns. How these promoters are differentially regulated remains elusive. Here, we apply single-molecule imaging to quantify the transcriptional regulation of two alternative promoters (P1 and P2) of the Bicoid (Bcd) target gene hunchback in syncytial blastoderm Drosophila embryos. Contrary to the previous notion that Bcd only activates P2, we find that Bcd activates both promoters via the same two enhancers. P1 activation is less frequent and requires binding of more Bcd molecules than P2 activation. Using a theoretical model to relate promoter activity to enhancer states, we show that the two promoters follow common transcription kinetics driven by sequential Bcd binding at the two enhancers. Bcd binding at either enhancer primarily activates P2, while P1 activation relies more on Bcd binding at both enhancers. These results provide a quantitative framework for understanding the dynamics of complex eukaryotic gene regulation.   

Mesoscale mixing of chromosomes

It is not well studied why chromosome mixing is a very slow process. In a previous study, the chromosome was assumed to be an infinitely long polymer and modeled as a ring polymer. This study suggests that topological constraints can slow down chromosome mixing dynamics. In this talk, we present our simulations of how the extent of chromosome mixing dynamics changes over time, as a function of the chromosome volume fraction, self-interactions, and binding to the lamina. We simulate a multi-chromosome nucleus using a bead-spring model with 4 polymer chains. We calculate the contact map and chromosome mixing index from our simulations and compare it with its maximal theoretical value for different chromosomal volume fractions and interactions. We consider two initial conditions: (i) when all chromosomes are separated and (ii) all chromosomes are mixed. Initial condition (i) predicts that the chromatin mixing index increases as a power law of the time, while initial condition (ii) predicts a steady-state or metastable, average value of the chromosome mixing index for which chromosomes are partially mixed. Using this, we estimate the time scale and the extent of chromosome mixing which can vary depending on the volume fraction, interactions, and chromosome size.   

Impact of cytosolic crowders on the morphology of bacterial chromosomes

Recent experimental studies on E. coli elaborated the role of macromolecular crowding on chromosome organization. Motivated by such observation, we consider a "feather-boa" type model of chromosome in the presence of non-additive crowders, encapsulated inside cylindrical confinement. Using molecular dynamic simulations, we thoroughly investigated the impact of the size and density variation of such crowders on the spatial organization of a model chromosome in cylindrical cellular confinement. Our results show, in the regime of small crowder size, the depletion effect leads to chromosome localization on the cylindrical surface of the cell, while the crowders occupy the bulk volume. Increasing the crowder size to intermediate values actuates the occurrence of transverse segregation between the crowders and the chromosome leading to a complimentary helical organization of the two. Further size-increment leads to longitudinal segregation causing the complete spatial expulsion of crowders from the central chromosomal region while the segregated crowders further compress the chromosome from the two ends. The increase in crowder size, spatial segregation of crowders and the chromosome, and the augmentation of helical turns in the model chromosome are found to be concomitant. Using crowders of intermediate size, we show how the variation of crowder density recaptures the spatial organization of the chromosome and crowders.