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).

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.

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.