Slow chromatin dynamics enhances promoter accessibility to transcriptional condensates.

Enhancers are DNA sequences at a long genomic distance from target genes. Recent experiments suggest that enhancers are anchored to the surfaces of condensates of transcription machinery and that the loop extrusion process enhances the transcription level of their target genes. Here, we theoretically study the polymer dynamics driven by the loop extrusion of the linker DNA between an enhancer and the promoter of its target gene to calculate the contact probability of the promoter to the transcription machinery in the condensate. Our theory predicts that when the loop extrusion process is active, the contact probability increases with increasing linker DNA length. This finding reflects the fact that the relaxation time, with which the promoter stays in proximity to the surface of the transcriptional condensate, increases as the length of the linker DNA increases. This contrasts the equilibrium case for which the contact probability between the promoter and the transcription machineries is smaller for longer linker DNA lengths.

The Anticancer Drug Daunomycin Directly Affects Gene Expression and DNA Structure.

Daunomycin (DM), an anthracycline antibiotic, is frequently used to treat various cancers, but the direct effects of DM on gene expression and DNA structure are unclear. We used an in vitro cell-free system, optimized with spermine (SP), to study the effect of DM on gene expression. A bimodal effect of DM on gene expression, weak promotion followed by inhibition, was observed with increasing concentration of DM. We also performed atomic force microscopy observation to measure how DM affects the higher-order structure of DNA induced with SP. DM destroyed SP-induced flower-like conformations of DNA by generating double-strand breaks, and this destructive conformational change of DNA corresponded to the inhibitory effect on gene expression. Interestingly, the weakly enhanced cell-free gene expression occurred as DNA conformations were elongated or relaxed at lower DM concentrations. We expect these newly unveiled DM effects on gene expression and the higher-order structure of DNA will contribute further to the development and refinement of useful anticancer therapy chemicals.

Computer simulations of chromatin phase separation.

Several simulation studies have recently appeared in Biophysical Journal that investigate the formation of biomolecular condensates in the nucleus. These structures explain a large variety of biological phenomena, from epigenetic inheritance, to enhancer-promoter interactions, to the spatial organization of the entire cell nucleus.

Loop extrusion driven volume phase transition of entangled chromosomes.

Experiments on reconstituted chromosomes have revealed that mitotic chromosomes are assembled even without nucleosomes. When topoisomerase II (topo II) is depleted from such reconstituted chromosomes, these chromosomes are not disentangled and form "sparklers," where DNA and linker histone are condensed in the core and condensin is localized at the periphery. To understand the mechanism of the assembly of sparklers, we here take into account the loop extrusion by condensin in an extension of the theory of entangled polymer gels. The loop extrusion stiffens an entangled DNA network because DNA segments in the elastically effective chains are translocated to loops, which are elastically ineffective. Our theory predicts that the loop extrusion by condensin drives the volume phase transition that collapses a swollen entangled DNA gel because the stiffening of the network destabilizes the swollen phase. This may be an important piece to understand the mechanism of the assembly of mitotic chromosomes.

Multiplexing mechanical and translational cues on genes.

The genetic code gives precise instructions on how to translate codons into amino acids. Due to the degeneracy of the genetic code-18 out of 20 amino acids are encoded for by more than one codon-more information can be stored in a basepair sequence. Indeed, various types of additional information have been discussed in the literature, e.g., the positioning of nucleosomes along eukaryotic genomes and the modulation of the translating efficiency in ribosomes to influence cotranslational protein folding. The purpose of this study is to show that it is indeed possible to carry more than one additional layer of information on top of a gene. In particular, we show how much translation efficiency and nucleosome positioning can be adjusted simultaneously without changing the encoded protein. We achieve this by mapping genes on weighted graphs that contain all synonymous genes, and then finding shortest paths through these graphs. This enables us, for example, to readjust the disrupted translational efficiency profile after a gene has been introduced from one organism (e.g., human) into another (e.g., yeast) without greatly changing the nucleosome landscape intrinsically encoded by the DNA molecule.

Ensembles of Breathing Nucleosomes: A Computational Study.

About three-fourths of the human DNA molecules are wrapped into nucleosomes, protein spools with DNA. Nucleosomes are highly dynamic, transiently exposing their DNA through spontaneous unspooling. Recent experiments allowed to observe the DNA of an ensemble of such breathing nucleosomes through x-ray diffraction with contrast matching between the solvent and the protein core. In this study, we calculate such an ensemble through a Monte Carlo simulation of a coarse-grained nucleosome model with sequence-dependent DNA mechanics. Our analysis gives detailed insights into the sequence dependence of nucleosome breathing observed in the experiment and allows us to determine the adsorption energy of the DNA bound to the protein core as a function of the ionic strength. Moreover, we predict the breathing behavior of other potentially interesting sequences and compare the findings to earlier related experiments.

The biology and polymer physics underlying large-scale chromosome organization.

Chromosome large-scale organization is a beautiful example of the interplay between physics and biology. DNA molecules are polymers and thus belong to the class of molecules for which physicists have developed models and formulated testable hypotheses to understand their arrangement and dynamic properties in solution, based on the principles of polymer physics. Biologists documented and discovered the biochemical basis for the structure, function and dynamic spatial organization of chromosomes in cells. The underlying principles of chromosome organization have recently been revealed in unprecedented detail using high-resolution chromosome capture technology that can simultaneously detect chromosome contact sites throughout the genome. These independent lines of investigation have now converged on a model in which DNA loops, generated by the loop extrusion mechanism, are the basic organizational and functional units of the chromosome.

Dilution of contact frequency between superenhancers by loop extrusion at interfaces.

The loop extrusion theory predicts that cohesin acts as a molecular motor that extrudes chromatin fibers to produce loops. Hi-C experiments have detected relatively high contact frequencies between superenhancers. These probably result from the fact that superenhancers are localized at condensates of transcriptional activators and coactivators. The contact frequency between superenhancers is enhanced by auxin treatment that removes cohesin from chromatin. Motivated by these experimental results, we here treat chromatin at the surface of a condensate as a loop extruding polymer brush. Our theory predicts that the lateral pressure generated by the brush decreases with decreasing the loading rate of cohesin. This is because loop extrusion actively transfers chain segments at the vicinity of the interface. Our theory thus predicts that the increase of contact frequency by auxin treatment results from the fact that suppressing the loop extrusion process induces the dissolution of molecular components to the nucleoplasm, decreasing the average distance between superenhancers.

The Latest Twists in Chromatin Remodeling.

In its most restrictive interpretation, the notion of chromatin remodeling refers to the action of chromatin-remodeling enzymes on nucleosomes with the aim of displacing and removing them from the chromatin fiber (the effective polymer formed by a DNA molecule and proteins). This local modification of the fiber structure can have consequences for the initiation and repression of the transcription process, and when the remodeling process spreads along the fiber, it also results in long-range effects essential for fiber condensation. There are three regulatory levels of relevance that can be distinguished for this process: the intrinsic sequence preference of the histone octamer, which rules the positioning of the nucleosome along the DNA, notably in relation to the genetic information coded in DNA; the recognition or selection of nucleosomal substrates by remodeling complexes; and, finally, the motor action on the nucleosome exerted by the chromatin remodeler. Recent work has been able to provide crucial insights at each of these three levels that add new twists to this exciting and unfinished story, which we highlight in this perspective.

Genomes of Multicellular Organisms Have Evolved to Attract Nucleosomes to Promoter Regions.

Sequences that influence nucleosome positioning in promoter regions, and their relation to gene regulation, have been the topic of much research over the last decade. In yeast, significant nucleosome-depleted regions are found, which facilitate transcription. With the arrival of nucleosome positioning maps for the human genome, it was discovered that in our genome, unlike in that of yeast, promoters encode for high nucleosome occupancy. In this work, we look at the genomes of a range of different organisms, to provide a catalog of nucleosome positioning signals in promoters across the tree of life. We utilize a computational model of the nucleosome, based on crystallographic analyses of the structure and elasticity of the nucleosome, to predict the nucleosome positioning signals in promoter regions. To be able to apply our model to large genomic datasets, we introduce an approximative scheme that makes use of the limited range of correlations in nucleosomal sequence preferences to create a computationally efficient approximation of the full biophysical model. Our predictions show that a clear distinction between unicellular and multicellular life is visible in the intrinsically encoded nucleosome affinity. Furthermore, the strength of the nucleosome positioning signals correlates with the complexity of the organism. We conclude that encoding for high nucleosome occupancy, as in the human genome, is in fact a universal feature of multicellular life.

Benchmarking and refining probability-based models for nucleosome-DNA interaction.

BACKGROUND: In investigations of nucleosome positioning preferences, a model that assigns an affinity to a given sequence is necessary to make predictions. One important class of models, which treats a nucleosome sequence as a Markov chain, has been applied with success when informed with experimentally measured nucleosomal sequence preferences. RESULTS: We find that we can also use such models as a fast approximative scheme for computationally expensive biophysical models, vastly increasing their reach. Employing these models in this way also allows us to benchmark them for the first time. Doing so for the approximative in silico models indirectly tells us about the accuracy we can expect of them when applied to real data. CONCLUSION: We find that models presented in the literature should perform well, but this performance depends on factors such as the order of the Markov model, the preprocessing of the probability distributions on which the model is based, and the size and quality of the sequence ensemble from which those distributions are calculated.

Transcription dynamics stabilizes nucleus-like layer structure in chromatin brush.

We use a brush of DNA in a solution of transcriptional machinery and histone proteins to theoretically predict that this brush shows phase separation due to the instability arising from the disassembly of nucleosomes during transcription. In the two-phase coexistent state, collapsed chains (with relatively large nucleosome occupancy) lie at the grafting surface and swollen chains (with relatively small nucleosome occupancy) are distributed at the space above the collapsed chains, analogous to the structure of chromatin in differentiated cells. This layer structure is stabilized by the lateral osmotic pressure of swollen chains. For a relatively small grafting density, DNA brushes show tricritical points because the entropic elasticity with respect to the lateral excursion of swollen chains balances with the lateral osmotic pressure of these chains. At the tricritical points, DNA brushes show large fluctuations of local nucleosome concentration, which may be reminiscent of the fluctuations observed in embryonic stem cells.

The role of DNA sequence in nucleosome breathing.

Roughly 3/4 of human genomes are sequestered by nucleosomes, DNA spools with a protein core, dictating a broad range of biological processes, ranging from gene regulation, recombination, and replication, to chromosome condensation. Nucleosomes are dynamical structures and temporarily expose wrapped DNA through spontaneous unspooling from either end, a process called site exposure or nucleosome breathing. Here we ask how this process is influenced by the mechanical properties of the wrapped DNA, which is known to depend on the underlying base pair sequence. Using a coarse-grained nucleosome model we calculate the accessibility profiles for site exposure. We find that the process is very sensitive to sequence effects, so that evolution could potentially tune the accessibility of nucleosomal DNA and would only need a small number of mutations to do so.

Transcription Driven Phase Separation in Chromatin Brush.

We theoretically predict the local density of nucleosomes on DNA brushes in a solution of molecules, which are necessary for transcription and the assembly of nucleosomes. Our theory predicts that in a confined space, DNA brushes show phase separation, where a region of relatively large nucleosomal occupancy coexists with a region of smaller nucleosomal occupancy. This phase separation is driven by an instability arising from the fact that the rate of transcription increases as the nucleosomal occupancy decreases due to the excluded volume interactions between nucleosomes and RNA polymerase during thermal diffusion and, in turn, nucleosomes are (in some cases) desorbed from DNA when RNA polymerase collides with nucleosomes during transcription. The miscibility phase diagram shows critical points, which are sensitive to the rate constants involved in transcription, the changes of interactions of DNA chain segments by assembling nucleosomes, and pressures that are applied to the brushes.

Computational study of remodeling in a nucleosomal array.

Chromatin remodeling complexes utilize the energy of ATP hydrolysis to change the packing state of chromatin, e.g. by catalysing the sliding of nucleosomes along DNA. Here we present simple models to describe experimental data of changes in DNA accessibility along a synthetic, repetitive array of nucleosomes during remodeling by the ACF enzyme or its isolated ATPase subunit, ISWI. We find substantial qualitative differences between the remodeling activities of ISWI and ACF. To understand better the observed behavior for the ACF remodeler, we study more microscopic models of nucleosomal arrays.

Crenarchaeal chromatin proteins Cren7 and Sul7 compact DNA by inducing rigid bends.

Archaeal chromatin proteins share molecular and functional similarities with both bacterial and eukaryotic chromatin proteins. These proteins play an important role in functionally organizing the genomic DNA into a compact nucleoid. Cren7 and Sul7 are two crenarchaeal nucleoid-associated proteins, which are structurally homologous, but not conserved at the sequence level. Co-crystal structures have shown that these two proteins induce a sharp bend on binding to DNA. In this study, we have investigated the architectural properties of these proteins using atomic force microscopy, molecular dynamics simulations and magnetic tweezers. We demonstrate that Cren7 and Sul7 both compact DNA molecules to a similar extent. Using a theoretical model, we quantify the number of individual proteins bound to the DNA as a function of protein concentration and show that forces up to 3.5 pN do not affect this binding. Moreover, we investigate the flexibility of the bending angle induced by Cren7 and Sul7 and show that the protein-DNA complexes differ in flexibility from analogous bacterial and eukaryotic DNA-bending proteins.

Rigid-body molecular dynamics of DNA inside a nucleosome.

The majority of eukaryotic DNA, about three quarter, is wrapped around histone proteins forming so-called nucleosomes. To study nucleosomal DNA we introduce a coarse-grained molecular dynamics model based on sequence-dependent harmonic rigid base pair step parameters of DNA and nucleosomal binding sites. Mixed parametrization based on all-atom molecular dynamics and crystallographic data of protein-DNA structures is used for the base pair step parameters. The binding site parameters are adjusted by experimental B-factor values of the nucleosome crystal structure. The model is then used to determine the energy cost for placing a twist defect into the nucleosomal DNA which allows us to use Kramers theory to calculate nucleosome sliding caused by such defects. It is shown that the twist defect scenario together with the sequence-dependent elasticity of DNA can explain the slow time scales observed for nucleosome mobility along DNA. With this method we also show how the twist defect mechanism leads to a higher mobility of DNA in the presence of sin mutations near the dyad axis. Finally, by performing simulations on 5s rDNA, 601, and telomeric base pair sequences, it is demonstrated that the current model is a powerful tool to predict nucleosome positioning.

On the stability of fractal globules.

The fractal globule, a self-similar compact polymer conformation where the chain is spatially segregated on all length scales, has been proposed to result from a sudden polymer collapse. This state has gained renewed interest as one of the prime candidates for the non-entangled states of DNA molecules inside cell nuclei. Here, we present Monte Carlo simulations of collapsing polymers. We find through studying polymers of lengths between 500 and 8000 that a chain collapses into a globule, which is neither fractal, nor as entangled as an equilibrium globule. To demonstrate that the non-fractalness of the conformation is not just the result of the collapse dynamics, we study in addition the dynamics of polymers that start from fractal globule configurations. Also in this case the chain moves quickly to the weakly entangled globule where the polymer is well mixed. After a much longer time the chain entangles reach its equilibrium conformation, the molten globule. We find that the fractal globule is a highly unstable conformation that only exists in the presence of extra constraints such as cross-links.