Multiscale Bayesian simulations reveal functional chromatin condensation of gene loci.

Chromatin, the complex assembly of DNA and associated proteins, plays a pivotal role in orchestrating various genomic functions. To aid our understanding of the principles underlying chromatin organization, we introduce Hi-C metainference, a Bayesian approach that integrates Hi-C contact frequencies into multiscale prior models of chromatin. This approach combines both bottom-up (the physics-based prior) and top-down (the data-driven posterior) strategies to characterize the 3D organization of a target genomic locus. We first demonstrate the capability of this method to accurately reconstruct the structural ensemble and the dynamics of a system from contact information. We then apply the approach to investigate the Sox2, Pou5f1, and Nanog loci of mouse embryonic stem cells using a bottom-up chromatin model at 1 kb resolution. We observe that the studied loci are conformationally heterogeneous and organized as crumpled globules, favoring contacts between distant enhancers and promoters. Using nucleosome-resolution simulations, we then reveal how the Nanog gene is functionally organized across the multiple scales of chromatin. At the local level, we identify diverse tetranucleosome folding motifs with a characteristic distribution along the genome, predominantly open at cis-regulatory elements and compact in between. At the larger scale, we find that enhancer-promoter contacts are driven by the transient condensation of chromatin into compact domains stabilized by extensive internucleosome interactions. Overall, this work highlights the condensed, but dynamic nature of chromatin in vivo, contributing to a deeper understanding of gene structure-function relationships.

Lateral interactions govern self-assembly of the bacterial biofilm matrix protein BslA.

The soil bacterium Bacillus subtilis is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give the B. subtilis biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in bslA deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods.

Bi-directional nucleosome sliding by the Chd1 chromatin remodeler integrates intrinsic sequence-dependent and ATP-dependent nucleosome positioning.

Chromatin remodelers use a helicase-type ATPase motor to shift DNA around the histone core. Although not directly reading out the DNA sequence, some chromatin remodelers exhibit a sequence-dependent bias in nucleosome positioning, which presumably reflects properties of the DNA duplex. Here, we show how nucleosome positioning by the Chd1 remodeler is influenced by local DNA perturbations throughout the nucleosome footprint. Using site-specific DNA cleavage coupled with next-generation sequencing, we show that nucleosomes shifted by Chd1 can preferentially localize DNA perturbations - poly(dA:dT) tracts, DNA mismatches, and single-nucleotide insertions - about a helical turn outside the Chd1 motor domain binding site, super helix location 2 (SHL2). This phenomenon occurs with both the Widom 601 positioning sequence and the natural +1 nucleosome sequence from the Saccharomyces cerevisiae SWH1 gene. Our modeling indicates that localization of DNA perturbations about a helical turn outward from SHL2 results from back-and-forth sliding due to remodeler action on both sides of the nucleosome. Our results also show that barrier effects from DNA perturbations can be extended by the strong phasing of nucleosome positioning sequences.

Molecular dynamics simulations for the study of chromatin biology.

The organization of Eukaryotic DNA into chromatin has profound implications for the processing of genetic information. In the past years, molecular dynamics (MD) simulations proved to be a powerful tool to investigate the mechanistic basis of chromatin biology. We review recent all-atom and coarse-grained MD studies revealing how the structure and dynamics of chromatin underlie its biological functions. We describe the latest method developments; the structural fluctuations of nucleosomes and the various factors affecting them; the organization of chromatin fibers, with particular emphasis on its liquid-like character; the interactions and dynamics of transcription factors on chromatin; and how chromatin organization is modulated by molecular motors acting on DNA.

The lane-switch mechanism for nucleosome repositioning by DNA translocase.

Translocases such as DNA/RNA polymerases, replicative helicases, and exonucleases are involved in eukaryotic DNA transcription, replication, and repair. Since eukaryotic genomic DNA wraps around histone octamers and forms nucleosomes, translocases inevitably encounter nucleosomes. A previous study has shown that a nucleosome repositions downstream when a translocase collides with the nucleosome. However, the molecular mechanism of the downstream repositioning remains unclear. In this study, we identified the lane-switch mechanism for downstream repositioning with molecular dynamics simulations and validated it with restriction enzyme digestion assays and deep sequencing assays. In this mechanism, after a translocase unwraps nucleosomal DNA up to the site proximal to the dyad, the remaining wrapped DNA switches its binding lane to that vacated by the unwrapping, and the downstream DNA rewraps, completing downstream repositioning. This mechanism may have broad implications for transcription through nucleosomes, histone recycling, and nucleosome remodeling.

The kinetic landscape of nucleosome assembly: A coarse-grained molecular dynamics study.

The organization of nucleosomes along the Eukaryotic genome is maintained over time despite disruptive events such as replication. During this complex process, histones and DNA can form a variety of non-canonical nucleosome conformations, but their precise molecular details and roles during nucleosome assembly remain unclear. In this study, employing coarse-grained molecular dynamics simulations and Markov state modeling, we characterized the complete kinetics of nucleosome assembly. On the nucleosome-positioning 601 DNA sequence, we observe a rich transition network among various canonical and non-canonical tetrasome, hexasome, and nucleosome conformations. A low salt environment makes nucleosomes stable, but the kinetic landscape becomes more rugged, so that the system is more likely to be trapped in off-pathway partially assembled intermediates. Finally, we find that the co-operativity between DNA bending and histone association enables positioning sequence motifs to direct the assembly process, with potential implications for the dynamic organization of nucleosomes on real genomic sequences.

Nucleosomes as allosteric scaffolds for genetic regulation.

Nucleosomes are stable yet highly dynamic complexes exhibiting diverse types of motions, such as sliding, DNA unwrapping, and disassembly, encoding a landscape with a large number of metastable states. In this review, describing recent studies on these nucleosome structure changes, we propose that the nucleosome can be viewed as an ideal allosteric scaffold: regulated by effector molecules such as transcription factors and chromatin remodelers, the nucleosome controls the downstream gene activity. Binding of transcription factors to the nucleosome can enhance DNA unwrapping or slide the DNA, altering either the binding or the unbinding of other transcription factors to nearby sites. ATP-dependent chromatin remodelers induce a series of DNA deformations, which allosterically propagate throughout the nucleosome to induce DNA sliding or histone exchange.

Langmuir-Blodgett technique for anisotropic colloids: Young investigator perspective.

Langmuir-Blodgett (LB) technique, which involves the formation of interfacial layers and the subsequent transfer onto a solid substrate, provides one of the most versatile means to characterize the adsorption, self-assembly, and rheological properties of interfacial colloids. In this review, we summarized the relevant studies on anisotropic colloids, which exhibit a richer self-assembly potential than their spherical counterparts. Hard particles with different shape and aspect ratio induce interfacial distortions when trapped at a liquid interface; the ensuing capillary interactions can drive the formation of complex two-dimensional (2D) structures with interesting properties. Soft colloids such as proteins, on the other hand, possess intrinsic anisotropy in both their shape and interactions. Exploring the shape/orientation dependent interactions and the created structures are important for diverse applications. Here, we present the comprehensive state of anisotropic colloids at liquid interfaces, and try to develop a deeper understanding of their self-assembly dynamics.

Chromatin remodelers couple inchworm motion with twist-defect formation to slide nucleosomal DNA.

ATP-dependent chromatin remodelers are molecular machines that control genome organization by repositioning, ejecting, or editing nucleosomes, activities that confer them essential regulatory roles on gene expression and DNA replication. Here, we investigate the molecular mechanism of active nucleosome sliding by means of molecular dynamics simulations of the Snf2 remodeler translocase in complex with a nucleosome. During its inchworm motion driven by ATP consumption, the translocase overwrites the original nucleosome energy landscape via steric and electrostatic interactions to induce sliding of nucleosomal DNA unidirectionally. The sliding is initiated at the remodeler binding location via the generation of a pair of twist defects, which then spontaneously propagate to complete sliding throughout the entire nucleosome. We also reveal how remodeler mutations and DNA sequence control active nucleosome repositioning, explaining several past experimental observations. These results offer a detailed mechanistic picture of remodeling important for the complete understanding of these key biological processes.

DNA sliding in nucleosomes via twist defect propagation revealed by molecular simulations.

While nucleosomes are highly stable structures as fundamental units of chromatin, they also slide along the DNA, either spontaneously or by active remodelers. Here, we investigate the microscopic mechanisms of nucleosome sliding by multiscale molecular simulations, characterizing how the screw-like motion of DNA proceeds via the formation and propagation of twist defects. Firstly, coarse-grained molecular simulations reveal that the sliding dynamics is highly dependent on DNA sequence. Depending on the sequence and the nucleosome super-helical location, we find two distinct types of twist defects: a locally under-twisted DNA region, previously observed in crystal structures, and a locally over-twisted DNA, an unprecedented feature. The stability of the over-twist defect was confirmed via all-atom simulations. Analysis of our trajectories via Markov state modeling highlights how the sequence-dependence of the sliding dynamics is due to the different twist defect energy costs, and in particular how nucleosome regions where defects cannot easily form introduce the kinetic bottlenecks slowing down repositioning. Twist defects can also mediate sliding of nucleosomes made with strong positioning sequences, albeit at a much lower diffusion coefficient, due to a high-energy intermediate state. Finally, we discuss how chromatin remodelers may exploit these spontaneous fluctuations to induce unidirectional sliding of nucleosomes.

Sequence-dependent nucleosome sliding in rotation-coupled and uncoupled modes revealed by molecular simulations.

While nucleosome positioning on eukaryotic genome play important roles for genetic regulation, molecular mechanisms of nucleosome positioning and sliding along DNA are not well understood. Here we investigated thermally-activated spontaneous nucleosome sliding mechanisms developing and applying a coarse-grained molecular simulation method that incorporates both long-range electrostatic and short-range hydrogen-bond interactions between histone octamer and DNA. The simulations revealed two distinct sliding modes depending on the nucleosomal DNA sequence. A uniform DNA sequence showed frequent sliding with one base pair step in a rotation-coupled manner, akin to screw-like motions. On the contrary, a strong positioning sequence, the so-called 601 sequence, exhibits rare, abrupt transitions of five and ten base pair steps without rotation. Moreover, we evaluated the importance of hydrogen bond interactions on the sliding mode, finding that strong and weak bonds favor respectively the rotation-coupled and -uncoupled sliding movements.

Adsorption of the natural protein surfactant Rsn-2 onto liquid interfaces.

To stabilize foams, droplets and films at liquid interfaces a range of protein biosurfactants have evolved in nature. Compared to synthetic surfactants, these combine surface activity with biocompatibility and low solution aggregation. One recently studied example is Rsn-2, a component of the foam nest of the frog Engystomops pustulosus, which has been predicted to undergo a clamshell-like opening transition at the air-water interface. Using atomistic molecular dynamics simulations and surface tension measurements we study the adsorption of Rsn-2 onto air-water and cyclohexane-water interfaces. The protein adsorbs readily at both interfaces, with adsorption mediated by the hydrophobic N-terminus. At the cyclohexane-water interface the clamshell opens, due to the favourable interaction between hydrophobic residues and cyclohexane molecules and the penetration of cyclohexane molecules into the protein core. Simulations of deletion mutants showed that removal of the N-terminus inhibits interfacial adsorption, which is consistent with the surface tension measurements. Deletion of the hydrophilic C-terminus also affects adsorption, suggesting that this plays a role in orienting the protein at the interface. The characterisation of the interfacial behaviour gives insight into the factors that control the interfacial adsorption of proteins, which may inform new applications of this and similar proteins in areas including drug delivery and food technology and may also be used in the design of synthetic molecules showing similar changes in conformation at interfaces.

The Conformation of Interfacially Adsorbed Ranaspumin-2 Is an Arrested State on the Unfolding Pathway.

Ranaspumin-2 (Rsn-2) is a surfactant protein found in the foam nests of the túngara frog. Previous experimental work has led to a proposed model of adsorption that involves an unusual clam-shell-like unhinging of the protein at an interface. Interestingly, there is no concomitant denaturation of the secondary structural elements of Rsn-2 with the large-scale transformation of its tertiary structure. In this work we use both experiment and simulation to better understand the driving forces underpinning this unusual process. We develop a modified Go-model approach where we have included explicit representation of the side chains to realistically model the interaction between the secondary structure elements of the protein and the interface. Doing so allows for the study of the underlying energy landscape that governs the mechanism of Rsn-2 interfacial adsorption. Experimentally, we study targeted mutants of Rsn-2, using the Langmuir trough, pendant drop tensiometry, and circular dichroism, to demonstrate that the clam-shell model is correct. We find that Rsn-2 adsorption is in fact a two-step process: the hydrophobic N-terminal tail recruits the protein to the interface after which Rsn-2 undergoes an unfolding transition that maintains its secondary structure. Intriguingly, our simulations show that the conformation Rsn-2 adopts at an interface is an arrested state along the denaturation pathway. More generally, our computational model should prove a useful, and computationally efficient, tool in studying the dynamics and energetics of protein-interface interactions.

The Bacterial Hydrophobin BslA is a Switchable Ellipsoidal Janus Nanocolloid.

BslA is an amphiphilic protein that forms a highly hydrophobic coat around Bacillus subtilis biofilms, shielding the bacterial community from external aqueous solution. It has a unique structure featuring a distinct partition between hydrophilic and hydrophobic surfaces. This surface property is reminiscent of synthesized Janus colloids. By investigating the behavior of BslA variants at water-cyclohexane interfaces through a set of multiscale simulations informed by experimental data, we show that BslA indeed represents a biological example of an ellipsoidal Janus nanoparticle, whose surface interactions are, moreover, readily switchable. BslA contains a local conformational toggle, which controls its global affinity for, and orientation at, water-oil interfaces. This adaptability, together with single-point mutations, enables the fine-tuning of its solvent and interfacial interactions, and suggests that BslA could be a basis for biotechnological applications.

Interfacial self-assembly of a bacterial hydrophobin.

The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-positive bacterium Bacillus subtilis forms biofilms that exhibit a characteristic wrinkled morphology and a highly hydrophobic surface. A critical component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. We recently reported the structure of BslA, and noted the presence of a large surface-exposed hydrophobic patch. Such surface patches are also observed in the class of surface-active proteins known as hydrophobins, and are thought to mediate their interfacial activity. However, although functionally related to the hydrophobins, BslA shares no sequence nor structural similarity, and here we show that the mechanism of action is also distinct. Specifically, our results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aqueous solution by adopting a random coil conformation, enabling the protein to be soluble and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side chains into the air or oil phase and forming a three-stranded ß-sheet. This form then self-assembles into a well-ordered 2D rectangular lattice that stabilizes the interface. By replacing a hydrophobic leucine in the center of the cap with a positively charged lysine, we changed the energetics of adsorption and disrupted the formation of the 2D lattice. This limited structural metamorphosis represents a previously unidentified environmentally responsive mechanism for interfacial stabilization by proteins.

Quantifying disorder through conditional entropy: an application to fluid mixing.

In this paper, we present a method to quantify the extent of disorder in a system by using conditional entropies. Our approach is especially useful when other global, or mean field, measures of disorder fail. The method is equally suited for both continuum and lattice models, and it can be made rigorous for the latter. We apply it to mixing and demixing in multicomponent fluid membranes, and show that it has advantages over previous measures based on Shannon entropies, such as a much diminished dependence on binning and the ability to capture local correlations. Further potential applications are very diverse, and could include the study of local and global order in fluid mixtures, liquid crystals, magnetic materials, and particularly biomolecular systems.