The role of transcript regions and amino acid choice in nucleosome positioning.

Eukaryotic DNA is organized and compacted in a string of nucleosomes, DNA-wrapped protein cylinders. The positions of nucleosomes along DNA are not random but show well-known base pair sequence preferences that result from the sequence-dependent elastic and geometric properties of the DNA double helix. Here, we focus on DNA around transcription start sites, which are known to typically attract nucleosomes in multicellular life forms through their high GC content. We aim to understand how these GC signals, as observed in genome-wide averages, are produced and encoded through different genomic regions (mainly 5' UTRs, coding exons, and introns). Our study uses a bioinformatics approach to decompose the genome-wide GC signal into between-region and within-region signals. We find large differences in GC signal contributions between vertebrates and plants and, remarkably, even between closely related species. Introns contribute most to the GC signal in vertebrates, while in plants the exons dominate. Further, we find signal strengths stronger on DNA than on mRNA, suggesting a biological function of GC signals along the DNA itself, as is the case for nucleosome positioning. Finally, we make the surprising discovery that both the choice of synonymous codons and amino acids contribute to the nucleosome positioning signal.

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.

Markedly Different Effects of Monovalent Cations on the Efficiency of Gene Expression.

The effect of monovalent cations on a cell-free transcription-translation (TX-TL) system is examined using a luciferase assay. It is found that the potency for all ions analyzed here is in the order Rb+  > K+  > Cs+  > Na+  ≈ Li+  > (CH3 )4 N+ , where Rb+ is most efficient at promoting TX-TL and the ions of Li+ , Na+ , and (CH3 )4 N+ exhibit an inhibitory effect. Similar promotion/inhibition effects are observed for cell-free TL alone with an mRNA template.

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.

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.

Circuit topology analysis of cellular genome reveals signature motifs, conformational heterogeneity, and scaling.

Reciprocal regulation of genome topology and function is a fundamental and enduring puzzle in biology. The wealth of data provided by Hi-C libraries offers the opportunity to unravel this relationship. However, there is a need for a comprehensive theoretical framework in order to extract topological information for genome characterization and comparison. Here, we develop a toolbox for topological analysis based on Circuit Topology, allowing for the quantification of inter- and intracellular genomic heterogeneity, at various levels of fold complexity: pairwise contact arrangement, higher-order contact arrangement, and topological fractal dimension. Single-cell Hi-C data were analyzed and characterized based on topological content, revealing not only a strong multiscale heterogeneity but also highly conserved features such as a characteristic topological length scale and topological signature motifs in the genome. We propose that these motifs inform on the topological state of the nucleus and indicate the presence of active loop extrusion.

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.

Circuit Topology Analysis of Polymer Folding Reactions.

Circuit topology is emerging as a versatile measure to classify the internal structures of folded linear polymers such as proteins and nucleic acids. The topology framework can be applied to a wide range of problems, most notably molecular folding reactions that are central to biology and molecular engineering. In this Outlook, we discuss the state-of-the art of the technology and elaborate on the opportunities and challenges that lie ahead.

Pioneer transcription factors in chromatin remodeling: The kinetic proofreading view.

Pioneer transcription factors are a recently defined class of transcription factors which can bind directly to nucleosomal DNA; they play a key role in gene activation in certain pathways. Here we quantify their role in the initiation of nucleosome displacement within the kinetic proofreading scenario of chromatin remodeling. The model allows one to perform remodeling efficiency comparisons for scenarios involving different types of transcription factors and remodelers as a function of their binding and unbinding rates and concentrations. Our results demonstrate a way to fine-tune the specificity of processes that modify the chromatin structure in transcriptional initiation.

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.

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.

Histone mark recognition controls nucleosome translocation via a kinetic proofreading mechanism: Confronting theory and high-throughput experiments.

Chromatin remodelers are multidomain enzymatic motor complexes that displace nucleosomes along DNA and hence "remodel chromatin structure," i.e., they dynamically reorganize nucleosome positions in both gene activation and gene repression. Recently, experimental insights from structural biology methods and remodeling assays have substantially advanced the understanding of these key chromatin components. Here we confront the kinetic proofreading scenario of chromatin remodeling, which proposes a mechanical link between histone residue modifications and the ATP-dependent action of remodelers, with recent experiments. We show that recent high-throughput data on nucleosome libraries assayed with remodelers from the Imitation Switch family are in accord with our earlier predictions of the kinetic proofreading scenario. We make suggestions for experimentally verifiable predictions of the kinetic proofreading scenarios for remodelers from other families.

Shortest paths through synonymous genomes.

The elasticity of the DNA double helix varies with the underlying base pair sequence. This allows one to put mechanical cues into sequences that in turn influence the packaging of DNA into nucleosomes, DNA-wrapped protein cylinders. Nucleosomes dictate a broad range of biological processes, ranging from gene regulation, recombination, and replication to chromosome condensation. Here we map base pair sequences onto graphs and use shortest paths algorithms to determine which DNA stretches are easiest or hardest to bend inside a nucleosome. We further demonstrate how genetic and mechanical information can be multiplexed by studying paths through graphs of synonymous codons. Using this method we find that nucleosomes can be placed by mechanical cues nearly everywhere on the genome of baker's yeast (Saccharomyces cerevisiae).

The nucleosome: from structure to function through physics.

Eukaryotic cells must fit meters of DNA into micron-sized cell nuclei and, at the same time, control and modulate the access to the genetic material. The necessary amount of DNA compaction is achieved via multiple levels of structural organization, the first being the nucleosome-a unique complex of histone proteins with ∼150 base pairs of DNA. Here we use specific examples to demonstrate that many aspects of the structure and function of nucleosomes can be understood using principles of basic physics, physics-based tools and models. For instance, the stability of a single nucleosome and the accessibility to its DNA depend sensitively on the charges in the histone core, which can be changed by post-translational modifications. The positions of nucleosomes along DNA molecules depend on the sequence-dependent shape and elasticity of the DNA double helix that has to be wrapped into the nucleosome complex. Larger-scale structures composed of multiple nucleosomes, that is nucleosome arrays, depend in turn on the interactions between its constituents that result from delicately tuned electrostatics.

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.

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.

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.