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.

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.

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.

Self assembled linear polymeric chains with tuneable semiflexibility using isotropic interactions.

We propose a two-body spherically symmetric (isotropic) potential such that particles interacting by the potential self-assemble into linear semiflexible polymeric chains without branching. By suitable control of the potential parameters, we can control the persistence length of the polymer and can even introduce a controlled number of branches. Thus we show how to achieve effective directional interactions starting from spherically symmetric potentials. The self-assembled polymers have an exponential distribution of chain lengths akin to what is observed for worm-like micellar systems. On increasing particle density, the polymeric chains self-organize to an ordered line-hexagonal phase where every chain is surrounded by six parallel chains, the transition is first order. On further increase in monomer density, the order is destroyed and we get a branched gel-like phase. This potential can be used to model semi-flexible equilibrium polymers with tunable semiflexibility and excluded volume. The use of the potential is computationally cheap and hence can be used to simulate and probe equilibrium polymer dynamics with long chains. The potential also gives a plausible method of tuning colloidal interactions in experiments such that one can obtain self-assembling polymeric chains made up of colloids and probe polymer dynamics using an optical microscope. Furthermore, we show how a modified potential leads to the observation of an intermediate nematic phase of self-assembled chains in between the low density disordered phase and the line-ordered hexagonal phase.

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.

Polymer physics models reveal structural folding features of single-molecule gene chromatin conformations.

Here, we employ polymer physics models of chromatin to investigate the 3D folding of a 2Mb wide genomic region encompassing the human LTN1 gene, a crucial DNA locus involved in key cellular functions. Through extensive Molecular Dynamics simulations, we reconstruct in-silico the ensemble of single-molecule LTN1 3D structures, which we benchmark against recent in-situ Hi-C 2.0 data. The model-derived single molecules are then used to predict structural folding features at the single-cell level, providing testable predictions for super-resolution microscopy experiments.

Attention to Detail: The Effect of Fluoroscopic Parallax on Limb Alignment Assessment During Corrective Osteotomy.

BACKGROUND: The accuracy of a corrective osteotomy is dependent on many factors. One error rarely considered is using noncentered fluoroscopic imaging to assess intraoperative alignment. This study quantified the coronal alignment error produced by visual parallax per interval changes in vertical and horizontal positioning of the C-arm and alignment rod during intraoperative evaluation. METHODS: Unilateral hip, ankle, and knee fluoroscopic images were obtained from a single intact cadaveric specimen. A center-center fluoroscopic image was obtained by moving the C-arm appeared in the center square of the nine-box grid. With the base of the C-arm stationary, the radiograph generator/intensifier portion of the C-arm was translated medially until the target bone appeared on the edge of the fluoroscopic image. RESULTS: One hundred eight images were obtained. Measurement error increased by an average of 14% per 10 mm of horizontal C-arm offset. Minimal effect was seen if the obtained image was within 5 mm of the true center; however, once 55 mm of offset was reached, all experimental conditions resulted in at least 10 mm of parallax error. CONCLUSION: Our results demonstrate that small variations in C-arm positioning can create statistically significant inaccuracies when assessing limb alignment using intraoperative fluoroscopy.

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.

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.

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.

Microarray analysis of cellular thermotolerance.

BACKGROUND AND OBJECTIVES: Previously, we have shown that a 43°C pretreatment can provide thermotolerance to a following, more severe, thermal stress at 45°C. Using cells that lack the Hsp70 gene, we have also shown that there is still some thermotolerance in the absence of HSP70 protein. The purpose of this study was to determine which genes play a role in thermotolerance by measuring viability and proliferation of the cells at 2 days after heating. Specifically, we wanted to understand which pathways may be responsible for protecting cells in the absence of HSP70. STUDY DESIGN/MATERIALS AND METHODS: Murine embryonic fibroblast cells with and without Hsp70 (MEF(+/+) and MEF(-/-), respectively) were exposed to a mild heat shock of 43°C for 30 minutes in a constant temperature water bath. After 3 hours of recovery, RNA was harvested from three heated samples alongside three untreated controls using a MicroRNeasy kit with DNAse treatment. RNA quality was verified by an Agilent Bioanalyzer. The RNA was then converted to cDNA and hybridized to Affymetrix gene expression DNA microarrays. The genes that showed a twofold change (up or down) relative to unheated controls were filtered by t-test for significance at a threshold of P < 0.05 using Genespring software. Data were verified by qRT-PCR. Genes were then categorized based upon their ontology. RESULTS: While many genes were similarly upregulated, the main difference between cell types was an increase in transcription factors and nucleic acid binding proteins. Several genes known to be involved in the heat response were upregulated more than twofold (Hsp70, Hsp40, Hsp110, Hsp25, Atf3), however, another well studied heat responsive gene Hsp90 only increased by 1.5-fold under these conditions despite its role in thermotolerance. CONCLUSIONS: The data herein presents genetic pathways which are candidates for further study of pretreatment protocols in laser irradiation.