Session Q06: Physics of Genome Organization: Phase Separation

Chromatin constrains the formation, diffusion, and coarsening of phase-separated condensates

In the nucleus of a eukaryotic cell, DNA is organized into chromatin – a complex polymeric material which stores information and controls gene expression. An emerging mechanism for biological organization, including within the nucleus, is biomolecular phase separation into condensed droplets of protein and nucleic acids. We utilized an optogenetic strategy to examine how chromatin influences droplet coarsening in the nucleus. We found that droplet growth dynamics are directly inhibited by the chromatin-dense environment, which gives rise to an anomalously slow coarsening exponent, , contrasting with the classical prediction of. This slowdown of growth could arise due to subdiffusion of individual droplets, which would predict , where  is the subdiffusive exponent. Tracking the fluctuating motion of droplets within chromatin revealed a subdiffusive exponent, , which combined with a lack of observable Ostwald ripening accounts for the anomalous coarsening behavior. We further combine theory and molecular dynamics simulations to show that cross-linked chromatin can not only mechanically suppress droplets’ coalescence and ripening but also quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on the regulation of biomolecular condensates and demonstrate the use of condensate emulsions to probe the intracellular environment.    

Chromatin organization: the role of microphase separation

Chromatin has been broadly classified into two different types, active (euchromatin) which is accessible to the transcription machinery and inactive (heterochromatin) which is not. Having two types of constituents in chromatin, can lead to microphase separation into compartments that are enriched in either active or inactive chromatin. However, how such microphase separation is reflected in the global organization of chromatin is still elusive. Additionally, some regions of chromatin interact with soluble molecules in the nucleoplasm which can further regulate the solvent quality experienced by the chromatin blocks. Based on recent experiments and simulations showing that chromatin phase separates from the nucleoplasm, we propose a minimal, block copolymer model for chromatin in which each type of block experiences different solvent conditions which can also be regulated by soluble molecules. We analytically predict the length scales of the microphases and their dependence on the copolymer characteristics and interactions with the soluble molecules. We then compare our analytical results with Brownian dynamics simulations of multiblock copolymers in the presence of such soluble molecules and discuss the relevance of our results to recent experiments on chromatin organization.   

The polymer model of constitutive heterochromatin

Chromatin structure is regulated on multiple levels, yet the mechanisms by which this occurs are not yet understood. In recent studies, phase separation has been shown to play a key role in a wide range of cellular processes including, some features of chromatin organization. We develop a simple model containing spherical particles and a single chain polymer to represent proteins and chromatin. By examining the interplay between protein-protein and protein-chromatin interactions, we investigate the constitutive-heterochromatin(c-Het) organization through liquid-liquid phase separation and/or polymer-polymer phase separations mechanisms. We construct a phase diagram for a multivalent protein that includes globule polymer-stable protein droplet, globule polymer-unstable protein droplet, coil polymer-unstable droplet, and coil-like polymer-stable droplet. We show that c-Het resides in the globule polymer-unstable protein droplet phase, in which the protein condensate forms due to interactions between the proteins and the polymer. In contrast to a standard phase separating system, both our analytical and simulations results show that the total protein concentration in the system controls the protein concentration in a droplet. Furthermore, the transition from the compact polymer to the coil state is accompanied by hysteresis: this suggests that the c-Het is metastable, and that the transition from regular c-Het domains to disturbed domains is likely irreversible. Finally, we find that protein-protein interactions are a key factor contributing to the mechanical properties of chromatin domains, shedding light on the nature of chromatin-protein condensates in the nucleus.   

Modeling the competition between the RAD 51 and the RPA in binding to the single-stranded DNA using a one-dimensional lattice model.

A crucial step of homologous recombination in the DNA repair mechanism is the search for homology between DNA molecules closely followed by strand exchange. This step is governed by the nucleoprotein RAD51, which competes with Replication Protein A (RPA) to saturate single-stranded DNA (ssDNA) overhangs. Such interactions are stochastic in nature. Therefore, we have developed a dynamic lattice model to model the competition between RAD51 and RPA molecules binding to the single-stranded DNA. This will assist us in better understanding the real-time dynamics observed by single-molecule microscopy. With this model, we will study various binding parameters' effects on each protein’s saturation level.

    

Mesoscale modeling of the dynamics of phase-separated chromatin compartments in eukaryotic nucleus

The latest super-resolution imaging experiments have revealed a surprisingly dynamic and stochastic nature of chromatin in eukaryotic nuclei which is reminiscent of multi-phase fluid behavior. As a result, the concepts from the theory of complex fluids such as phase separation, viscoelasticity, and droplet nucleation have found widespread utility in understanding salient features of nuclear organization. In order to understand and disentangle the complex interplay of forces that contribute to the emergent patterns of organization and dynamics, we have devised a phenomenological field-theoretic model of nucleus as a multi-phase condensate of liquid chromatin types. Armed with a mesoscopic model of nuclear chromatin, we have shed light on the distinct dynamical and structural contributions of chromatin type interactions intermingling of chromosomal territories and lamina binding. We also shed light on the dynamical heterogeneity and coherent motions of chromatin domains which are fully captured by an interplay of micro-phase separation of chromatin types and lamina binding.   

Mechanical force derived from transcriptional activity can induce local order and frozen motion in interphase chromosome

Recent experiments reported that dynamics of human interphase chromosome is accelerated by inhibiting transcription. This result is counterintuitive as transcription involves the elongation process of RNA polymerase which is known to exert force along DNA. To understand this phenomenon, we investigated, using polymer simulations, how mechanical force derived from transcriptional activity affects the dynamic properties of interphase chromosome. In our simulations, a pair of active forces is exerted on chromosome loci in an extensile manner to mimic the effect of transcriptional elongation. We observe that the dynamics of the active polymer chain becomes slower than that of a passive one, which is already in a glassy regime. We demonstrate that the active force gives rise to local crystallization along with frozen motion in a particular range of the force magnitude. By noting the extent of agreement between our results and experimental observations, we suggest the possibility of local order in interphase chromosome induced by transcriptional activity and its contribution to chromatin structure and dynamics.   

Resolving chromatin landscapes around nuclear condensates

Many biomolecular condensates associate with specific chromatin loci in the cell nucleus. Transcription condensates are linked to super-enhancer chromatin domains that are characterized by extended accumulations of transcription factor and coactivator binding sites as well as stereotypic epigenetic marks. Biochemical data and population assays suggest that these chromatin marks are required to maintain transcription condensates in specific locations in the nucleus. Conversely, nuclear condensates have been shown to displace chromatin as they grow. We investigate how functional chromatin elements co-organize spatially with respect to transcription condensates using super-resolution microscopy. Our single cell assays reveal cell-to-cell variability obscured by ensemble data. Our work provides new insights into the complex interplay between biomolecular condensates and chromatin topologies.   

Single molecule tracking reveals distinct mobility states that contribute to the dynamics and function of nuclear receptors

Transcription factors (TFs) regulate gene expression by binding to specific consensus motifs within the local chromatin context. The mechanisms by which TFs navigate the nuclear environment as they search for binding sites remains unclear. Single molecule tracking (SMT) has emerged as a powerful approach to explore mechanisms of TF movement and chromatin interactions in living cells. We used SMT in concert with a machine-learning based classification algorithm to directly measure the intranuclear dynamics of nuclear receptors. We apply this framework to study the dynamics of the peroxisome proliferator-activated receptor (PPAR)-γ2, which is an important regulator of glucose metabolism and insulin sensitivity in adipocytes. We show that PPARγ2 exhibits two distinct low mobility states, similar to other nuclear hormone receptors. Through mutagenesis and transient expression studies, we identify PPARγ2 domains that contribute to the dynamics of these two states. Our analyses constrain the physical mechanisms that underlie the origins of these two states and reveal their implications for transcriptional control. Our data provide the first dynamic characterization of PPARγ2 — a TF that is the focus of several anti-diabetes therapeutics.   

Compressing cell nuclei: From nonlinear mechanics to nontrivial shapes

Abnormal nuclear shapes are a feature of many human pathologies, such as metastatic cancer, Hutchinson-Gilford progeria syndrome (rapid aging), and mandibulofacial dysplasia disorder. Understanding these nuclear aberrations from a mechanical point of view may reveal physical mechanisms of cell nuclear function and suggest new approaches to clinical therapies. Recent experimental studies show that the nucleus takes irregular shapes called blebs and mechanically stiffens under compression. We have developed a Brownian-dynamics-based model of a nucleus subjected to uniaxial compression. Our model consists of a polymeric protein shell called the lamina, with a chain of monomeric subunits representing chromatin inside the shell. Chromatin binds to itself randomly via crosslinkers and to the lamina via linkages. Extensile and contractile motors remodeling the chromatin represent the ATP-driven activity in the nucleus. We find that with fast compression in the low-strain regime, the nucleus demonstrates compression stiffening behavior. We also observe localized bulges on the nuclear surface, indicating the possible formation of blebs. For the same strains, with slow compression, the nucleus shows a more ellipsoidal shape, with fewer bulges as compared to fast compression. Our results suggest that during fast compression cycles, compressive forces do not propagate far into the nucleus. However, with slow compression, forces can propagate further which leads chromatin to rearrange itself to accommodate the high strains. Our model captures nuclear compression stiffening and shows how different nuclear morphologies arise in response to applied forces.   

Phase separation in sequence and space: Coupling folding landscape of chromatin with epigenetics

Chromosomal regions adopt heritable stable states resulting in bistable gene expression without changes to the underlying DNA sequence. Such epigenetic control is due to covalent modifications of histones, which are inextricably linked to chromatin structure.  An associated positive feedback mechanism has also been posited wherein enzymes catalyse reactions to similarly modify nucleosomes in close proximity. Theorists have long sought analytically tractable mathematical models that capture the interplay between chromatin structure and the spread/maintenance of epigenetic marks. We propose a model wherein the polymer is modelled as a matrix of contacts between sites (nucleosomes); additionally, each site carries a binary variable denoting its epigenetic state. Following our previous work, we then treat both the addition/removal of epigenetic marks, and making/breaking of contacts in the polymer chain as a stochastic reaction network, and employ field theoretic methods to study the coupled system. We are interested in interrogating the effects of dynamical asymmetry, modulating cooperativity of polymer folding on the coupled landscape, and the formation and maintenance of spatiotemporal domains, and biological consequences thereof to processes such as gene regulation and cellular differentiation.   

Nonlinear mechanics of human mitotic chromosomes

In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized X-shaped metaphase chromosomes. This dramatic metamorphosis has been observed using microscopy for over a century, and yet remarkably little is known about the structural organization of a metaphase chromosome. Here, we probe chromosome organization via force-extension experiments in a novel optical trap set-up. We find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behavior, distinct from classical polymer models. To explain this anomalous stiffening, we introduce a Hierarchical Worm-like Chain (HWLC) model that describes the chromosome as a heterogeneous assembly of worm-like chains (WLCs).  In this framework, the collective stiffening behaviour of a HWLC is attributed to the broad distribution of the mechanical properties of the assembly components. Finally, we propose that studying the mechanical response of protein degraded or disease-associated chromosomes could lead to new insight on chromosomal organization.   

Enzymatic activity in chromatin organization

Spatial organization of chromatin is critical for genome regulation. In literature, various types of affinity mediators and enzymes have been attributed for spatial organization of chromatin from thermodynamics perspective. However, at the mechanistic level, enzymes act in their unique ways. Here, we construct a polymer physics model following the mechanistic scheme of Topoisomerase-II, an enzyme resolving topological constraints of chromatin, and investigate its role on interphase chromatin organization. Our computer simulations demonstrate Topoisomerase-II's calibre in phase separating chromatin into eu- and heterochromatic regions with a characteristic wall-like organization of the euchromatic regions. We argue that transient phantomness of the euchromatin due to enzymatic activity of Topoisomerase-II induces this phase separation. Motivated from a recent experimental report, we further extend our model to a bidisperse setting and show that the characteristic features of the enzymatic activity driven phase separation survives there. The existence of these characteristic features, even under the non-localized action of the enzyme, highlights the critical role of enzymatic activity in chromatin organization, and points out the importance of further experiments along this line.