Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure.

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.

Detecting radio- and chemoresistant cells in 3D cancer co-cultures using chromatin biomarkers.

The heterogenous treatment response of tumor cells limits the effectiveness of cancer therapy. While this heterogeneity has been linked to cell-to-cell variability within the complex tumor microenvironment, a quantitative biomarker that identifies and characterizes treatment-resistant cell populations is still missing. Herein, we use chromatin organization as a cost-efficient readout of the cells' states to identify subpopulations that exhibit distinct responses to radiotherapy. To this end, we developed a 3D co-culture model of cancer spheroids and patient-derived fibroblasts treated with radiotherapy. Using the model we identified treatment-resistant cells that bypassed DNA damage checkpoints and exhibited an aggressive growth phenotype. Importantly, these cells featured more condensed chromatin which primed them for treatment evasion, as inhibiting chromatin condensation and DNA damage repair mechanisms improved the efficacy of not only radio- but also chemotherapy. Collectively, our work shows the potential of using chromatin organization to cost-effectively study the heterogeneous treatment susceptibility of cells and guide therapeutic design.

Chromatin as self-returning walks: From population to single cell and back.

With a growing understanding of the chromatin structure, many efforts remain focused on bridging the gap between what is suggested by population-averaged data and what is visualized for single cells. A popular approach to traversing these scales is to fit a polymer model to Hi-C contact data. However, Hi-C is an average of millions to billions of cells, and each cell may not contain all population-averaged contacts. Therefore, we employ a novel approach of summing individual chromosome trajectories-determined by our Self-Returning Random Walk model-to create populations of cells. We allow single cells to consist of disparate structures and reproduce a variety of experimentally relevant contact maps. We show that the amount of shared topology between cells, and their mechanism of formation, changes the population-averaged structure. Therefore, we present a modeling technique that, with few constraints and little oversight, can be used to understand which single-cell chromatin structures underlie population-averaged behavior.

Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function.

Extending across multiple length scales, dynamic chromatin structure is linked to transcription through the regulation of genome organization. However, no individual technique can fully elucidate this structure and its relation to molecular function at all length and time scales at both a single-cell level and a population level. Here, we present a multitechnique nanoscale chromatin imaging and analysis (nano-ChIA) platform that consolidates electron tomography of the primary chromatin fiber, optical super-resolution imaging of transcription processes, and label-free nano-sensing of chromatin packing and its dynamics in live cells. Using nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nanometers, sub-megabase genomic size, and an internal fractal structure. The chromatin packing behavior of these domains exhibits a complex bidirectional relationship with active gene transcription. Furthermore, we found that properties of PDs are correlated among progenitor and progeny cells across cell division.

Physical and data structure of 3D genome.

With the textbook view of chromatin folding based on the 30-nm fiber being challenged, it has been proposed that interphase DNA has an irregular 10-nm nucleosome polymer structure whose folding philosophy is unknown. Nevertheless, experimental advances suggest that this irregular packing is associated with many nontrivial physical properties that are puzzling from a polymer physics point of view. Here, we show that the reconciliation of these exotic properties necessitates modularizing three-dimensional genome into tree data structures on top of, and in striking contrast to, the linear topology of DNA double helix. These functional modules need to be connected and isolated by an open backbone that results in porous and heterogeneous packing in a quasi-self-similar manner, as revealed by our electron and optical imaging. Our multiscale theoretical and experimental results suggest the existence of higher-order universal folding principles for a disordered chromatin fiber to avoid entanglement and fulfill its biological functions.

Dynamic Crowding Regulates Transcription.

The nuclear environment is highly crowded by biological macromolecules, including chromatin and mobile proteins, which alter the kinetics and efficiency of transcriptional machinery. These alterations have been described, both theoretically and experimentally, for steady-state crowding densities; however, temporal changes in crowding density ("dynamic crowding") have yet to be integrated with gene expression. Dynamic crowding is pertinent to nuclear biology because processes such as chromatin translocation and protein diffusion lend to highly mobile biological crowders. Therefore, to capture such dynamic crowding and investigate its influence on transcription, we employ a three-pronged, systems-molecular approach. A system of chemical reactions represents the transcription pathway, the rates of which are determined by molecular-scale simulations; Brownian dynamics and Monte Carlo simulations quantify protein diffusion and DNA-protein binding affinity, dependent on macromolecular density. Altogether, this approach shows that transcription depends critically on dynamic crowding as the gene expression resultant from dynamic crowding can be profoundly different than that of steady-state crowding. In fact, expression levels can display both amplification and suppression and are notably different for genes or gene populations with different chemical and structural properties. These properties can be exploited to impose circadian expression, which is asymmetric and varies in strength, or to explain expression in cells under biomechanical stress. Therefore, this work demonstrates that dynamic crowding nontrivially alters transcription kinetics and presents dynamic crowding within the bulk nuclear nanoenvironment as a novel regulatory framework for gene expression.

Encapsulation of Nanoparticles During Polymer Micelle Formation: A Dissipative Particle Dynamics Study.

The formation of block copolymer micelles with and without hydrophobic nanoparticles is simulated using dissipative particle dynamics. We use the model developed by Spaeth et al. [ Spaeth , J. R. , Kevrekidis , I. G. , and Panagiotopoulos , A. Z. J. Chem. Phys. 2011 , 134 ( (16) ) 164902 ], and drive micelle formation by adjusting the interaction parameters linearly over time to represent a rapid change from organic solvent to water. For different concentrations of added nanoparticles, we determine characteristic times for micelle formation and coagulation, and characterize micelles with respect to size, polydispersity, and nanoparticle loading. Four block copolymers with different numbers of hydrophobic and hydrophilic polymer beads, are examined. We find that increasing the number of hydrophobic beads on the polymer decreases the micelle formation time and lowers polydispersity in the final micelle distribution. Adding more nanoparticles to the simulation has a negligible effect on micelle formation and coagulation times, and monotonically increases the polydispersity of the micelles for a given polymer system. The presence of relatively stable free polymer in one system decreases the amount of polymer encapsulating the nanoparticles, and results in an increase in polydispersity and the number of nanoparticles per micelle for that system, especially at high nanoparticle concentration. Longer polymers lead to micelles with a more uniform nanoparticle loading.