Multiscale modelling of chromatin 4D organization in SARS-CoV-2 infected cells.

SARS-CoV-2 can re-structure chromatin organization and alter the epigenomic landscape of the host genome, but the mechanisms that produce such changes remain unclear. Here, we use polymer physics to investigate how the chromatin of the host genome is re-organized upon infection with SARS-CoV-2. We show that re-structuring of A/B compartments can be explained by a re-modulation of intra-compartment homo-typic affinities, which leads to the weakening of A-A interactions and the enhancement of A-B mixing. At the TAD level, re-arrangements are physically described by a reduction in the loop extrusion activity coupled with an alteration of chromatin phase-separation properties, resulting in more intermingling between different TADs and a spread in space of the TADs themselves. In addition, the architecture of loci relevant to the antiviral interferon response, such as DDX58 or IFIT, becomes more variable within the 3D single-molecule population of the infected model, suggesting that viral infection leads to a loss of chromatin structural specificity. Analysing the time trajectories of pairwise gene-enhancer and higher-order contacts reveals that this variability derives from increased fluctuations in the chromatin dynamics of infected cells. This suggests that SARS-CoV-2 alters gene regulation by impacting the stability of the contact network in time.

Multiscale modelling of chromatin 4D organization in SARS-CoV-2 infected cells.

SARS-CoV-2 is able to re-structure chromatin organization and alters the epigenomic landscape of the host genome, though the mechanisms that produce such changes are still poorly understood. Here, we investigate with polymer physics chromatin re-organization of the host genome, in space and time upon SARS-CoV-2 viral infection. We show that re-structuring of A/B compartments is well explained by a re-modulation of intra-compartment homotypic affinities, which leads to the weakening of A-A interactions and enhances A-B mixing. At TAD level, re-arrangements are physically described by a general reduction of the loop extrusion activity coupled with an alteration of chromatin phase-separation properties, resulting in more intermingling between different TADs and spread in space of TADs themselves. In addition, the architecture of loci relevant to the antiviral interferon (IFN) response, such as DDX58 or IFIT, results more variable within the 3D single-molecule population of the infected model, suggesting that viral infection leads to a loss of chromatin structural specificity. Analysis of time trajectories of pairwise gene-enhancer and higher-order contacts reveals that such variability derives from a more fluctuating dynamics in infected case, suggesting that SARS-CoV-2 alters gene regulation by impacting the stability of the contact network in time. Overall, our study provides the first polymer-physics based 4D reconstruction of SARS-CoV-2 infected genome with mechanistic insights on the consequent gene mis-regulation.

Phase separation of ecDNA aggregates establishes in-trans contact domains boosting selective MYC regulatory interactions.

Extrachromosomal DNAs (ecDNAs) are found in the nucleus of an array of human cancer cells where they can form clusters that were associated to oncogene overexpression, as they carry genes and cis-regulatory elements. Yet, the mechanisms of aggregation and gene amplification beyond copy-number effects remain mostly unclear. Here, we investigate, at the single molecule level, MYC-harboring ecDNAs of COLO320-DM colorectal cancer cells by use of a minimal polymer model of the interactions of ecDNA BRD4 binding sites and BRD4 molecules. We find that BRD4 induces ecDNAs phase separation, resulting in the self-assembly of clusters whose predicted structure is validated against HiChIP data (Hung et al., 2021). Clusters establish in-trans associated contact domains (I-TADs) enriched, beyond copy number, in regulatory contacts among specific ecDNA regions, encompassing its PVT1-MYC fusions but not its other canonical MYC copy. That explains why the fusions originate most of ecDNA MYC transcripts (Hung et al., 2021), and shows that ecDNA clustering per se is important but not sufficient to amplify oncogene expression beyond copy-number, reconciling opposite views on the role of clusters (Hung et al., 2021; Zhu et al., 2021; Purshouse et al. 2022). Regulatory contacts become strongly enriched as soon as half a dozen ecDNAs aggregate, then saturate because of steric hindrance, highlighting that even cells with few ecDNAs can experience pathogenic MYC upregulations. To help drug design and therapeutic applications, with the model we dissect the effects of JQ1, a BET inhibitor. We find that JQ1 reverses ecDNA phase separation hence abolishing I-TADs and extra regulatory contacts, explaining how in COLO320-DM cells it reduces MYC transcription (Hung et al., 2021).

Physics-Based Polymer Models to Probe Chromosome Structure in Single Molecules.

Human chromosomes have a complex 3D spatial organization in the cell nucleus, which comprises a hierarchy of physical interactions across genomic scales. Such an architecture serves important functional roles, as genes and their regulators have to physically interact to control gene regulation. However, the molecular mechanisms underlying the formation of those contacts remain poorly understood. Here, we describe a polymer-physics-based approach to investigate the machinery shaping genome folding and function. In silico model predictions on DNA single-molecule 3D structures are validated against independent super-resolution single-cell microscopy data, supporting a scenario whereby chromosome architecture is controlled by thermodynamics mechanisms of phase separation. Finally, as an application of our methods, the validated single-polymer conformations of the theory are used to benchmark powerful technologies to probe genome structure, such as Hi-C, SPRITE, and GAM.

Unveiling the Machinery behind Chromosome Folding by Polymer Physics Modeling.

Understanding the mechanisms underlying the complex 3D architecture of mammalian genomes poses, at a more fundamental level, the problem of how two or multiple genomic sites can establish physical contacts in the nucleus of the cells. Beyond stochastic and fleeting encounters related to the polymeric nature of chromatin, experiments have revealed specific, privileged patterns of interactions that suggest the existence of basic organizing principles of folding. In this review, we focus on two major and recently proposed physical processes of chromatin organization: loop-extrusion and polymer phase-separation, both supported by increasing experimental evidence. We discuss their implementation into polymer physics models, which we test against available single-cell super-resolution imaging data, showing that both mechanisms can cooperate to shape chromatin structure at the single-molecule level. Next, by exploiting the comprehension of the underlying molecular mechanisms, we illustrate how such polymer models can be used as powerful tools to make predictions in silico that can complement experiments in understanding genome folding. To this aim, we focus on recent key applications, such as the prediction of chromatin structure rearrangements upon disease-associated mutations and the identification of the putative chromatin organizing factors that orchestrate the specificity of DNA regulatory contacts genome-wide.

Loop-extrusion and polymer phase-separation can co-exist at the single-molecule level to shape chromatin folding.

Loop-extrusion and phase-separation have been proposed as mechanisms that shape chromosome spatial organization. It is unclear, however, how they perform relative to each other in explaining chromatin architecture data and whether they compete or co-exist at the single-molecule level. Here, we compare models of polymer physics based on loop-extrusion and phase-separation, as well as models where both mechanisms act simultaneously in a single molecule, against multiplexed FISH data available in human loci in IMR90 and HCT116 cells. We find that the different models recapitulate bulk Hi-C and average multiplexed microscopy data. Single-molecule chromatin conformations are also well captured, especially by phase-separation based models that better reflect the experimentally reported segregation in globules of the considered genomic loci and their cell-to-cell structural variability. Such a variability is consistent with two main concurrent causes: single-cell epigenetic heterogeneity and an intrinsic thermodynamic conformational degeneracy of folding. Overall, the model combining loop-extrusion and polymer phase-separation provides a very good description of the data, particularly higher-order contacts, showing that the two mechanisms can co-exist in shaping chromatin architecture in single cells.

The Physics of DNA Folding: Polymer Models and Phase-Separation.

Within cell nuclei, several biophysical processes occur in order to allow the correct activities of the genome such as transcription and gene regulation. To quantitatively investigate such processes, polymer physics models have been developed to unveil the molecular mechanisms underlying genome functions. Among these, phase-separation plays a key role since it controls gene activity and shapes chromatin spatial structure. In this paper, we review some recent experimental and theoretical progress in the field and show that polymer physics in synergy with numerical simulations can be helpful for several purposes, including the study of molecular condensates, gene-enhancer dynamics, and the three-dimensional reconstruction of real genomic regions.

Polymer physics reveals a combinatorial code linking 3D chromatin architecture to 1D chromatin states.

The mammalian genome has a complex, functional 3D organization. However, it remains largely unknown how DNA contacts are orchestrated by chromatin organizers. Here, we infer from only Hi-C the cell-type-specific arrangement of DNA binding sites sufficient to recapitulate, through polymer physics, contact patterns genome wide. Our model is validated by its predictions in a set of duplications at Sox9 against available independent data. The binding site types fall in classes that well match chromatin states from segmentation studies, yet they have an overlapping, combinatorial organization along chromosomes necessary to accurately explain contact specificity. The chromatin signatures of the binding site types return a code linking chromatin states to 3D architecture. The code is validated by extensive de novo predictions of Hi-C maps in an independent set of chromosomes. Overall, our results shed light on how 3D information is encrypted in 1D chromatin via the specific combinatorial arrangement of binding sites.

A Polymer Physics Model to Dissect Genome Organization in Healthy and Pathological Phenotypes.

Novel technologies revealed a nontrivial spatial organization of the chromosomes within the cell nucleus, which includes different levels of compartmentalization and architectural patterns. Notably, such complex three-dimensional structure plays a crucial role in vital biological functions and its alterations can produce extensive rewiring of genomic regulatory regions, thus leading to gene misexpression and disease. Here, we show that theoretical and computational approaches, based on polymer physics, can be employed to dissect chromatin contacts in three-dimensional space and to predict the effects of pathogenic structural variants on the genome architecture. In particular, we discuss the folding of the human EPHA4 and the murine Pitx1 loci as case studies.

Physical mechanisms of chromatin spatial organization.

In higher eukaryotes, chromosomes have a complex three-dimensional (3D) conformation in the cell nucleus serving vital functional purposes, yet their folding principles remain poorly understood at the single-molecule level. Here, we summarize recent approaches from polymer physics to comprehend the physical mechanisms underlying chromatin architecture. In particular, we focus on two models that have been supported by recent, growing experimental evidence, the Loop Extrusion model and the Strings&Binders phase separation model. We discuss their key ingredients, how they compare to experimental data and some insight they provide on chromatin architecture and gene regulation. Progress in that research field are opening the possibility to predict how genomic mutations alter the network of contacts between genes and their regulators and how that is linked to genetic diseases, such as congenital disorders and cancer.

Dynamic and equilibrium properties of finite-size polymer models of chromosome folding.

Novel technologies are revealing that chromosomes have a complex three-dimensional organization within the cell nucleus that serves functional purposes. Models from polymer physics have been developed to quantitively understand the molecular principles controlling their structure and folding mechanisms. Here, by using massive molecular-dynamics simulations we show that classical scaling laws combined with finite-size effects of a simple polymer model can effectively explain the scaling behavior that chromatin exhibits at the topologically associating domains level, as revealed by experimental observations. Model results are then validated against recently published high-resolution in situ Hi-C data.

Polymer models are a versatile tool to study chromatin 3D organization.

The development of new experimental technologies is opening the way to a deeper investigation of the three-dimensional organization of chromosomes inside the cell nucleus. Genome architecture is linked to vital functional purposes, yet a full comprehension of the mechanisms behind DNA folding is still far from being accomplished. Theoretical approaches based on polymer physics have been employed to understand the complexity of chromatin architecture data and to unveil the basic mechanisms shaping its structure. Here, we review some recent advances in the field to discuss how Polymer Physics, combined with numerical Molecular Dynamics simulation and Machine Learning based inference, can capture important aspects of genome organization, including the description of tissue-specific structural rearrangements, the detection of novel, regulatory-linked architectural elements and the structural variability of chromatin at the single-cell level.

Comparison of the Hi-C, GAM and SPRITE methods using polymer models of chromatin.

Hi-C, split-pool recognition of interactions by tag extension (SPRITE) and genome architecture mapping (GAM) are powerful technologies utilized to probe chromatin interactions genome wide, but how faithfully they capture three-dimensional (3D) contacts and how they perform relative to each other is unclear, as no benchmark exists. Here, we compare these methods in silico in a simplified, yet controlled, framework against known 3D structures of polymer models of murine and human loci, which can recapitulate Hi-C, GAM and SPRITE experiments and multiplexed fluorescence in situ hybridization (FISH) single-molecule conformations. We find that in silico Hi-C, GAM and SPRITE bulk data are faithful to the reference 3D structures whereas single-cell data reflect strong variability among single molecules. The minimal number of cells required in replicate experiments to return statistically similar contacts is different across the technologies, being lowest in SPRITE and highest in GAM under the same conditions. Noise-to-signal levels follow an inverse power law with detection efficiency and grow with genomic distance differently among the three methods, being lowest in GAM for genomic separations >1 Mb.

CTCF mediates dosage- and sequence-context-dependent transcriptional insulation by forming local chromatin domains.

Insulators play a critical role in spatiotemporal gene regulation in animals. The evolutionarily conserved CCCTC-binding factor (CTCF) is required for insulator function in mammals, but not all of its binding sites act as insulators. Here we explore the sequence requirements of CTCF-mediated transcriptional insulation using a sensitive insulator reporter in mouse embryonic stem cells. We find that insulation potency depends on the number of CTCF-binding sites in tandem. Furthermore, CTCF-mediated insulation is dependent on upstream flanking sequences at its binding sites. CTCF-binding sites at topologically associating domain boundaries are more likely to function as insulators than those outside topologically associating domain boundaries, independently of binding strength. We demonstrate that insulators form local chromatin domain boundaries and weaken enhancer-promoter contacts. Taken together, our results provide genetic, molecular and structural evidence connecting chromatin topology to the action of insulators in the mammalian genome.

Promoter-proximal CTCF binding promotes distal enhancer-dependent gene activation.

The CCCTC-binding factor (CTCF) works together with the cohesin complex to drive the formation of chromatin loops and topologically associating domains, but its role in gene regulation has not been fully defined. Here, we investigated the effects of acute CTCF loss on chromatin architecture and transcriptional programs in mouse embryonic stem cells undergoing differentiation to neural precursor cells. We identified CTCF-dependent enhancer-promoter contacts genome-wide and found that they disproportionately affect genes that are bound by CTCF at the promoter and are dependent on long-distance enhancers. Disruption of promoter-proximal CTCF binding reduced both long-range enhancer-promoter contacts and transcription, which were restored by artificial tethering of CTCF to the promoter. Promoter-proximal CTCF binding is correlated with the transcription of over 2,000 genes across a diverse set of adult tissues. Taken together, the results of our study show that CTCF binding to promoters may promote long-distance enhancer-dependent transcription at specific genes in diverse cell types.

Divergent Transcription of the Nkx2-5 Locus Generates Two Enhancer RNAs with Opposing Functions.

Enhancer RNAs (eRNAs) are a subset of long noncoding RNA generated from genomic enhancers: they are thought to act as potent promoters of the expression of nearby genes through interaction with the transcriptional and epigenomic machineries. In the present work, we describe two eRNAs transcribed from the enhancer of Nkx2-5-a gene specifying a master cardiomyogenic lineage transcription factor (TF)-which we call Intergenic Regulatory Element Nkx2-5 Enhancers (IRENEs). The IRENEs are encoded, respectively, on the same strand (SS) and in the divergent direction (div) respect to the nearby gene. Of note, these two eRNAs have opposing roles in the regulation of Nkx2-5: IRENE-SS acts as a canonical promoter of transcription, whereas IRENE-div represses the activity of the enhancer through recruitment of the histone deacetylase sirtuin 1. Thus, we have identified an autoregulatory loop controlling expression of the master cardiac TF NKX2-5, in which one eRNA represses transcription.

Polymer physics indicates chromatin folding variability across single-cells results from state degeneracy in phase separation.

The spatial organization of chromosomes has key functional roles, yet how chromosomes fold remains poorly understood at the single-molecule level. Here, we employ models of polymer physics to investigate DNA loci in human HCT116 and IMR90 wild-type and cohesin depleted cells. Model predictions on single-molecule structures are validated against single-cell imaging data, providing evidence that chromosomal architecture is controlled by a thermodynamics mechanism of polymer phase separation whereby chromatin self-assembles in segregated globules by combinatorial interactions of chromatin factors that include CTCF and cohesin. The thermodynamics degeneracy of single-molecule conformations results in broad structural and temporal variability of TAD-like contact patterns. Globules establish stable environments where specific contacts are highly favored over stochastic encounters. Cohesin depletion reverses phase separation into randomly folded states, erasing average interaction patterns. Overall, globule phase separation appears to be a robust yet reversible mechanism of chromatin organization where stochasticity and specificity coexist.

A Dynamic Folded Hairpin Conformation Is Associated with α-Globin Activation in Erythroid Cells.

We investigate the three-dimensional (3D) conformations of the α-globin locus at the single-allele level in murine embryonic stem cells (ESCs) and erythroid cells, combining polymer physics models and high-resolution Capture-C data. Model predictions are validated against independent fluorescence in situ hybridization (FISH) data measuring pairwise distances, and Tri-C data identifying three-way contacts. The architecture is rearranged during the transition from ESCs to erythroid cells, associated with the activation of the globin genes. We find that in ESCs, the spatial organization conforms to a highly intermingled 3D structure involving non-specific contacts, whereas in erythroid cells the α-globin genes and their enhancers form a self-contained domain, arranged in a folded hairpin conformation, separated from intermingling flanking regions by a thermodynamic mechanism of micro-phase separation. The flanking regions are rich in convergent CTCF sites, which only marginally participate in the erythroid-specific gene-enhancer contacts, suggesting that beyond the interaction of CTCF sites, multiple molecular mechanisms cooperate to form an interacting domain.

Computational approaches from polymer physics to investigate chromatin folding.

Microscopy and sequencing-based technologies are providing increasing insights into chromatin architecture. Nevertheless, a full comprehension of chromosome folding and its link with vital cell functions is far from accomplished at the molecular level. Recent theoretical and computational approaches are providing important support to experiments to dissect the three-dimensional structure of chromosomes and its organizational mechanisms. Here, we review, in particular, the String&Binders polymer model of chromatin that describes the textbook scenario where contacts between distal DNA sites are established by cognate binders. It has been shown to recapitulate key features of chromosome folding and to be able at predicting how phenotypes causing structural variants rewire the interactions between genes and regulators.