Session S39: Physics of Genome Organization: from DNA to Chromatin II

Inferring the locations of DNA bound proteins from Hi-C data

Eukaryotic DNA can be found in either a tightly packed state (heterochromatin) or an open conformation (euchromatin). Certain proteins that bind to the DNA are responsible for setting up these two types of states. They interact with each other, and generate spatially separated compartments in the DNA through the formation of loops. In this talk I will present a combination of analytic and simulation results for the effects of protein-protein interactions on the large-scale 3D structure of chromatin. Using these findings we have developed a maximum-likelihood method for inferring the distribution of DNA bound factors that can help refine and make new predictions for the locations of proteins responsible of structuring the chromosome.   

Coalescence Model for Crumpled Globules Formed in Polymer Collapse

The rapid collapse of a polymer, due to external forces or changes in solvent, yields a long-lived "crumpled globule." The conjectured fractal structure shaped by hierarchical collapse dynamics has proved difficult to establish, even with large simulations. To unravel this puzzle, we study a coarse-grained model of in-falling spherical blobs that coalesce upon contact. Distances between pairs of monomers are assigned upon their initial coalescence, and do not "equilibrate" subsequently. Surprisingly, the model reproduces quantitatively the dependence of distance on segment length, suggesting that the slow approach to scaling is related to the wide distribution of blob sizes.   

Probing nuclear dynamics and architecture using single-walled carbon nanotubes

Chromatin is a multiscale dynamic architecture that acts as a template for many biochemical processes such as transcription and DNA replication. Recent developments such as Hi-C technology enable an identification of chromatin interactions across an entire genome. However, a single cell dynamic view of chromatin organization is far from understood. We discuss a new live cell imaging technique to probe the dynamics of the nucleus at a single cell level using single-walled carbon nanotubes (SWNTs). SWNTs are non-perturbing rigid rods (diameter of 1 nm and length of roughly 100 nm) that fluoresce in the near infrared region. Due to their high aspect ratio, they can diffuse in tight spaces and report on the architecture and dynamics of the nucleoplasm. We develop 3D imaging and tracking of SWNTs in the volume of the nucleus using double helix point spread function microscopy (DH-PSF) and discuss the capabilities of the DH-PSF for inferring the 3D orientation of nanotubes based on vectorial diffraction theory.   

Fractionation of Exosomes and DNA using Size-Based Separation at the Nanoscale

Exosomes, a key target of “liquid biopsies”, are nano-vesicles found in nearly all biological fluids. Exosomes are secreted by eukaryotic and prokaryotic cells alike, and contain information about their originating cells, including surface proteins, cytoplasmic proteins, and nucleic acids. One challenge in studying exosome morphology is the difficulty of sorting exosomes by size and surface markers. Common separation techniques for exosomes include ultracentrifugation and ultrafiltration, for preparation of large volume samples, but these techniques often show contamination and significant heterogeneity between preparations. To date, deterministic lateral displacement (DLD) pillar arrays in silicon have proven an efficient technology to sort, separate, and enrich micron-scale particles including human parasites, eukaryotic cells, blood cells, and circulating tumor cells in blood; however, the DLD technology has never been translated to the true nanoscale, where it could function on bio-colloids such as exosomes. We have fabricated nanoscale DLD (nanoDLD) arrays capable of rapidly sorting colloids down to 20 nm in continuous flow, and demonstrated size sorting of individual exosome vesicles and dsDNA polymers, opening the potential for on-chip biomolecule separation and diagnosti   

The impact of non-uniform capsid charge density on virus assembly

Many spherical viruses efficiently encapsulate their genome into shells (capsids) with icosahedral symmetry. Under many circumstances, this process is spontaneous and is primarily driven by the electrostatic interaction between positively charged capsid proteins and negatively charged genome. Through the free energy minimization of a generic potential, we calculate the optimal encapsulated genome length. In this talk, I will present our results due to a non-uniform charge distribution on the shell and its impact on the optimal size of encapsulated genome.   

\textbf{Single molecule fluorescence studies of transition paths in DNA hairpin folding }

DNA hairpins are the simplest structures for investigating fundamental aspects of nucleic acid folding mechanisms. For two-state hairpins, all of the mechanistic information on how the hairpin folds is contained in the transition path (TP), the rare event in single molecule trajectories when the free energy barrier between folded and unfolded states is actually crossed. The only previous experimental study of TPs in nucleic acids used optical tweezer measurements and Szabo's analytical theory for diffusive barrier crossing to reconstruct the free energy surface for an indirect determination of average TP times (Neupane \textit{et al}. \textit{PRL} 2012). We used confocal single molecule FRET and maximum likelihood analysis of photon trajectories to determine an upper bound of 2.5 $\mu $s for the average TP time of a DNA hairpin (Truex \textit{et al}., \textit{PRL} 2015), compared to the value of 4 $\mu $s predicted by Neupane \textit{et al}., providing an important test of energy landscape theory. Current experiments are aimed at eventually characterizing structural changes during TPs, which will provide a very demanding test of mechanisms predicted by both theoretical models and simulations.   

The Molecular Atlas Project

The promise of super-resolution microscopy is a technology to discover new biological mechanisms that occur at smaller length scales then previously observable. However, with higher-resolution, we generally lose the larger spatial context of the image itself. The \textit{Molecular Atlas Project} (MAP) directly asks how these competing interests between super-resolution imaging and broader spatially contextualized information can be reconciled. MAP enables us to acquire, visualize, explore, and annotate proteomic image data representing 7 orders of magnitude in length ranging from molecular (nm) to tissue (cm) scales. This multi-scale understanding is made possible by combining multiplexed DNA-PAINT, a DNA nanotechnology approach to super-resolution imaging, with ``big-data'' strategies for information management and image visualization. With these innovations combined, MAP enables us to explore cell-specific heterogeneity in ductal carcinoma for \textit{every cell }in a cm-sized tissue section, analyze organoid growth for advances in high-throughput tissue-on-a-chip technology, and examine individual synapses for connectome mapping over extremely wide areas. Ultimately, MAP is a fundamentally new way to interact with multiscale biophysical data.   

simulation of the DNA force-extension curve

A molecular dynamics simulation study of the force-extension curve of double-stranded DNA is presented. Extended simulations of the DNA at multiple points along the force-extension curve are conducted with DNA end-to-end length constrained at each point. The calculated force-extension curve qualitatively reproduces the experimental one. The DNA conformational ensemble at each extension shows that the famous plateau of the force-extension curve results from B-DNA melting, whereas the formation of the earlier-predicted novel DNA conformation called 'zip-DNA' takes place at extensions past the plateau. An extensive analysis of the DNA conformational ensemble in terms of base configuration, backbone configuration, solvent interaction energy, etc., is conducted in order to elucidate the physical origin of DNA elasticity and the main interactions responsible for the shape of the force-extension curve.   

Characterization of the full base pairing probability distribution in RNA secondary structure folding

Below the denaturation temperature of RNA, its secondary structures can exist in one of two phases: a strongly disordered, low-temperature glass phase and a weakly disordered, high-temperature molten phase. The probability of two bases pairing in these phases have been shown to scale with the distance between the two bases as -3/2 and -4/3 in the molten and glass phases, respectively. In this study, we characterize the full probability distributions of pair binding both near and far from the critical point rather than just the behavior of their means studied before. We anticipate that this approach allows one to more closely probe the nature of the phase transition and better measure the system's critical exponents close to and at its critical point.   

Gene dosage imbalance during DNA replication controls bacterial cell-fate decision

Genes encoding proteins in a common regulatory network are frequently located close to one another on the chromosome to facilitate co-regulation or couple gene expression to growth rate. Contrasting with these observations, here we demonstrate a functional role for the arrangement of \textit{Bacillus subtilis} sporulation network genes on opposite sides of the chromosome. We show that the arrangement of two sporulation network genes, one located close to the origin, the other close to the terminus leads to a transient gene dosage imbalance during chromosome replication. This imbalance is detected by the sporulation network to produce cell-cycle coordinated pulses of the sporulation master regulator Spo0A\textasciitilde P. This pulsed response allows cells to decide between sporulation and continued vegetative growth during each cell-cycle spent in starvation. Furthermore, changes in DNA replication and cell-cycle parameters with decreased growth rate in starvation conditions enable cells to indirectly detect starvation without the need for evaluating specific metabolites. The simplicity of the uncovered coordination mechanism and starvation sensing suggests that it may be widely applicable in a variety of gene regulatory and stress-response settings.   

Torque-induced buckling behavior in stretched intertwined DNAs

Two intertwined DNA molecules (a DNA 'braid') is a common occurrence in the cell and is a relevant substrate for the study of topoisomerase and recombination enzymes. Single molecule experiments have observed the signature of a buckling transition in braids under tensile and torsional stress. We present a free energy model for braided DNA to investigate the mechanical properties of these structures. Our model is based on the semi-flexible polymer model for double helix DNA and is in quantitative accord with the experiments. We identify coexistence of a force-extended state with a plectonemically buckled state, which is reminiscent of single supercoiled DNA behavior. However, the absence of an intrinsic twist modulus in braided DNA results in unique mechanical properties such as non-linear torque in the extended state. At the buckling transition, we predict a jump in the braid extension due to the plectoneme end loop which acts as a nucleation barrier. We investigate the effect of salt concentration on the mechanical response of braids, e.g. we find that buckling starts at a lower linking number for lower salt concentration, the opposite of what is seen for single supercoiled DNAs. Also, concentrations less than 20 mM monovalent salt favor formation of multiple plectoneme domains.   

Sequence Heterogeneity Accelerates Protein Search for Targets on DNA

The process of protein search for specific binding sites on DNA is fundamentally important since it marks the beginning of all major biological processes. We present a theoretical investigation that probes the role of DNA sequence symmetry, heterogeneity and chemical composition in the protein search dynamics. Using a discrete-state stochastic approach with a first-passage events analysis, which takes into account the most relevant physical-chemical processes, a full analytical description of the search dynamics is obtained. It is found that, contrary to existing views, the protein search is generally faster on DNA with more heterogeneous sequences. In addition, the search dynamics might be affected by the chemical composition near the target site. The physical origins of these phenomena are discussed. Our results suggest that biological processes might be effectively regulated by modifying chemical composition, symmetry and heterogeneity of a genome.   

SA1 and TRF1 synergistically bind to telomeric DNA and promote DNA-DNA pairing

Impaired telomere cohesion leads to increased aneuploidy and early onset of tumorigenesis. Cohesion is thought to occur through the entrapment of two DNA strands within tripartite cohesin ring(s), along with a fourth subunit (SA1/SA2). Surprisingly, cohesion rings are not essential for telomere cohesion, which instead requires SA1 and shelterin proteins including TRF1. However, neither this unique cohesion mechanism at telomeres or DNA-binding properties of SA1 is understood. Here, using single-molecule fluorescence imaging of quantum dot-labeled proteins on DNA we discover that while SA1 diffuses across multiple telomeric and non-telomeric regions, the diffusion mediated through its N-terminal domain is slower at telomeric regions. However, addition of TRF1 traps SA1 within telomeric regions, which form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy. Together, these experimental results and coarse-grained molecular dynamics simulations suggest that TRF1 and SA1 synergistically interact with DNA to support telomere cohesion without cohesin rings.