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Proper expression of genes requires communication with their regulatory elements that can be located elsewhere along the chromosome. The physics of chromatin fibers imposes a range of constraints on such communication. The molecular and biophysical mechanisms by which chromosomal communication is established, or prevented, have become a topic of intense study, and important roles for the spatial organization of chromosomes are being discovered. Here we present a view of the interphase 3D genome characterized by extensive physical compartmentalization and insulation on the one hand and facilitated long-range interactions on the other. We propose the existence of topological machines dedicated to set up and to exploit a 3D genome organization to both promote and censor communication along and between chromosomes., National Human Genome Research Institute (U.S.) (Grant R01 HG003143), National Human Genome Research Institute (U.S.) (Grant U54 HG007010), National Human Genome Research Institute (U.S.) (Grant U01 HG007910), National Cancer Institute (U.S.) (Grant U54 CA193419), National Institutes of Health (U.S.) (Grant U54 DK107980), National Institutes of Health (U.S.) (Grant U01 DA 040588), National Institute of General Medical Sciences (U.S.) (Grant R01 GM 112720), National Institute of Allergy and Infectious Diseases (U.S.) (Grant U01 R01 AI 117839)

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization., National Institutes of Health (U.S.) (Grant R01 GM114190), National Human Genome Research Institute (U.S.) (Grant R01 HG003143)

Predicting and constraining RNA virus evolution require understanding the molecular factors that define the mutational landscape accessible to these pathogens. RNA viruses typically have high mutation rates, resulting in frequent production of protein variants with compromised biophysical properties. Their evolution is necessarily constrained by the consequent challenge to protein folding and function. We hypothesized that host proteostasis mechanisms may be significant determinants of the fitness of viral protein variants, serving as a critical force shaping viral evolution. Here, we test that hypothesis by propagating influenza in host cells displaying chemically-controlled, divergent proteostasis environments. We find that both the nature of selection on the influenza genome and the accessibility of specific mutational trajectories are significantly impacted by host proteostasis. These findings provide new insights into features of host-pathogen interactions that shape viral evolution, and into the potential design of host proteostasis-targeted antiviral therapeutics that are refractory to resistance., National Institutes of Health (U.S.) (Award 1DP2GM119162), National Institutes of Health (U.S.) (Grant P30-ES002109)

Topologically associating domains (TADs) are fundamental structural and functional building blocks of human interphase chromosomes, yet the mechanisms of TAD formation remain unclear. Here, we propose that loop extrusion underlies TAD formation. In this process, cis-acting loop-extruding factors, likely cohesins, form progressively larger loops but stall at TAD boundaries due to interactions with boundary proteins, including CTCF. Using polymer simulations, we show that this model produces TADs and finer-scale features of Hi-C data. Each TAD emerges from multiple loops dynamically formed through extrusion, contrary to typical illustrations of single static loops. Loop extrusion both explains diverse experimental observations—including the preferential orientation of CTCF motifs, enrichments of architectural proteins at TAD boundaries, and boundary deletion experiments—and makes specific predictions for the depletion of CTCF versus cohesin. Finally, loop extrusion has potentially far-ranging consequences for processes such as enhancer-promoter interactions, orientation-specific chromosomal looping, and compaction of mitotic chromosomes., National Institutes of Health (U.S.) (grant R01 GM114190), National Institutes of Health (U.S.) (grant U54 DK107980), National Institutes of Health (U.S.) (grant R01 G003143), National Science Foundation (U.S.) (1504942)

The mechanism by which chromatids and chromosomes are segregated during mitosis and meiosis is a major puzzle of biology and biophysics. Using polymer simulations of chromosome dynamics, we show that a single mechanism of loop extrusion by condensins can robustly compact, segregate and disentangle chromosomes, arriving at individualized chromatids with morphology observed in vivo. Our model resolves the paradox of topological simplification concomitant with chromosome 'condensation', and explains how enzymes a few nanometers in size are able to control chromosome geometry and topology at micron length scales. We suggest that loop extrusion is a universal mechanism of genome folding that mediates functional interactions during interphase and compacts chromosomes during mitosis., National Science Foundation (U.S.) (Grant DMR-1206868), National Science Foundation (U.S.) (Grant MCB-1022117), National Institutes of Health (U.S.) (NIH Grant GM105847), National Institutes of Health (U.S.) (NIH Grant CA193419), National Institutes of Health (U.S.) (NIH grant GM114190), National Institutes of Health (U.S.) (NIH grant R01HG003143), National Institutes of Health (U.S.) (NIH grant DK107980)

Peptide-recognition modules (PRMs) are used throughout biology to mediate protein–protein interactions, and many PRMs are members of large protein domain families. Recent genome-wide measurements describe networks of peptide–PRM interactions. In these networks, very similar PRMs recognize distinct sets of peptides, raising the question of how peptide-recognition specificity is achieved using similar protein domains. The analysis of individual protein complex structures often gives answers that are not easily applicable to other members of the same PRM family. Bioinformatics-based approaches, one the other hand, may be difficult to interpret physically. Here we integrate structural information with a large, quantitative data set of SH2 domain–peptide interactions to study the physical origin of domain–peptide specificity. We develop an energy model, inspired by protein folding, based on interactions between the amino-acid positions in the domain and peptide. We use this model to successfully predict which SH2 domains and peptides interact and uncover the positions in each that are important for specificity. The energy model is general enough that it can be applied to other members of the SH2 family or to new peptides, and the cross-validation results suggest that these energy calculations will be useful for predicting binding interactions. It can also be adapted to study other PRM families, predict optimal peptides for a given SH2 domain, or study other biological interactions, e.g. protein–DNA interactions., National Institutes of Health. National Centers for Biomedical Computing (Informatics for Integrating Biology and the Bedside), National Institutes of Health (U.S.) (grant U54LM008748)

Metazoan genomes are spatially organized at multiple scales, from packaging of DNA around individual nucleosomes to segregation of whole chromosomes into distinct territories1–5. At the intermediate scale of kilobases to megabases, which encompasses the sizes of genes, gene clusters and regulatory domains, the three-dimensional (3D) organization of DNA is implicated in multiple gene regulatory mechanisms2–4,6–8, but understanding this organization remains a challenge. At this scale, the genome is partitioned into domains of different epigenetic states that are essential for regulating gene expression9–11. Here, we investigate the 3D organization of chromatin in different epigenetic states using super-resolution imaging. We classified genomic domains in Drosophila cells into transcriptionally active, inactive, or Polycomb-repressed states and observed distinct chromatin organizations for each state. Remarkably, all three types of chromatin domains exhibit power-law scaling between their physical sizes in 3D and their domain lengths, but each type has a distinct scaling exponent. Polycomb-repressed chromatin shows the densest packing and most intriguing folding behaviour in which packing density increases with domain length. Distinct from the self-similar organization displayed by transcriptionally active and inactive chromatin, the Polycomb-repressed domains are characterized by a high degree of chromatin intermixing within the domain. Moreover, compared to inactive domains, Polycomb-repressed domains spatially exclude neighbouring active chromatin to a much stronger degree. Computational modelling and knockdown experiments suggest that reversible chromatin interactions mediated by Polycomb-group proteins plays an important role in these unique packaging properties of the repressed chromatin. Taken together, our super-resolution images reveal distinct chromatin packaging for different epigenetic states at the kilobase-to-megabase scale, a length scale that is directly relevant to genome regulation.

How DNA is organized in three dimensions inside the cell nucleus and how this affects the ways in which cells access, read and interpret genetic information are among the longest standing questions in cell biology. Using newly developed molecular, genomic and computational approaches based on the chromosome conformation capture technology (such as 3C, 4C, 5C and Hi-C), the spatial organization of genomes is being explored at unprecedented resolution. Interpreting the increasingly large chromatin interaction data sets is now posing novel challenges. Here we describe several types of statistical and computational approaches that have recently been developed to analyse chromatin interaction data., National Institutes of Health (U.S.), National Human Genome Research Institute (U.S.) (HG003143), National Human Genome Research Institute (U.S.) (HG003143-06S1), W. M. Keck Foundation, Spain. Ministerio de Ciencia e Innovación (BFU2010-19310/BMC), Human Frontier Science Program (Strasbourg, France) (RGP0044/2011), European Union (BLUEPRINT project (EU FP7 grant agreement 282510)), National Cancer Institute (U.S.) (Physical Sciences Oncology Center at MIT, U54CA143874)

Chromosomal translocations are frequent features of cancer genomes that contribute to disease progression. These rearrangements result from formation and illegitimate repair of DNA double-strand breaks (DSBs), a process that requires spatial colocalization of chromosomal breakpoints. The “contact first” hypothesis suggests that translocation partners colocalize in the nuclei of normal cells, prior to rearrangement. It is unclear, however, the extent to which spatial interactions based on three-dimensional genome architecture contribute to chromosomal rearrangements in human disease. Here we intersect Hi-C maps of three-dimensional chromosome conformation with collections of 1,533 chromosomal translocations from cancer and germline genomes. We show that many translocation-prone pairs of regions genome-wide, including the cancer translocation partners BCR-ABL and MYC-IGH, display elevated Hi-C contact frequencies in normal human cells. Considering tissue specificity, we find that translocation breakpoints reported in human hematologic malignancies have higher Hi-C contact frequencies in lymphoid cells than those reported in sarcomas and epithelial tumors. However, translocations from multiple tissue types show significant correlation with Hi-C contact frequencies, suggesting that both tissue-specific and universal features of chromatin structure contribute to chromosomal alterations. Our results demonstrate that three-dimensional genome architecture shapes the landscape of rearrangements directly observed in human disease and establish Hi-C as a key method for dissecting these effects., National Human Genome Research Institute (U.S.) (grant T32 HG002295), United States. Dept. of Defense (National Defense Science and Engineering Graduate Fellowship Program), Fannie and John Hertz Foundation, National Institute of General Medical Sciences (U.S.) (grant 5T32 GM008313), National Institute of General Medical Sciences (U.S.) (Medical Scientist Training Program)

Three-dimensional (3D) organization of genomes affects critical cellular processes such as transcription, replication, and deoxyribo nucleic acid (DNA) repair. While previous studies have investigated the natural role, the 3D organization plays in limiting a possible set of genomic rearrangements following DNA repair, the influence of specific organizational principles on this process, particularly over longer evolutionary time scales, remains relatively unexplored. In budding yeast S.cerevisiae, chromosomes are organized into a Rabl-like configuration, with clustered centromeres and telomeres tethered to the nuclear periphery. Hi-C data for S.cerevisiae show that a consequence of this Rabl-like organization is that regions equally distant from centromeres are more frequently in contact with each other, between arms of both the same and different chromosomes. Here, we detect rearrangement events in Saccharomyces species using an automatic approach, and observe increased rearrangement frequency between regions with higher contact frequencies. Together, our results underscore how specific principles of 3D chromosomal organization can influence evolutionary events., National Institutes of Health (U.S.) (Grant GM114190)

Advances in microscopy and genomic techniques have provided new insight into spatial chromatin organization inside of the nucleus. In particular, chromosome conformation capture data has highlighted the relevance of polymer physics for high-order chromatin organization. In this context, we review basic polymer states, discuss how an appropriate polymer model can be determined from experimental data, and examine the success and limitations of various polymer models of higher-order interphase chromatin organization. By taking into account topological constraints acting on the chromatin fiber, recently developed polymer models of interphase chromatin can reproduce the observed scaling of distances between genomic loci, chromosomal territories, and probabilities of contacts between loci measured by chromosome conformation capture methods. Polymer models provide a framework for the interpretation of experimental data as ensembles of conformations rather than collections of loops, and will be crucial for untangling functional implications of chromosomal organization., National Cancer Institute (U.S.). Physical Sciences-Oncology Center (MIT, (U54CA143874))

Over the last decade, and especially after the advent of fluorescent in situ hybridization imaging and chromosome conformation capture methods, the availability of experimental data on genome three-dimensional organization has dramatically increased. We now have access to unprecedented details of how genomes organize within the interphase nucleus. Development of new computational approaches to leverage this data has already resulted in the first three-dimensional structures of genomic domains and genomes. Such approaches expand our knowledge of the chromatin folding principles, which has been classically studied using polymer physics and molecular simulations. Our outlook describes computational approaches for integrating experimental data with polymer physics, thereby bridging the resolution gap for structural determination of genomes and genomic domains., Spain. Ministerio de Ciencia e Innovación (BFU2010-19310), National Cancer Institute (U.S.), David H. Koch Institute for Integrative Cancer Research at MIT

The fractal globule is a compact polymer state that emerges during polymer condensation as a result of topological constraints which prevent one region of the chain from passing across another one. This long-lived intermediate state was introduced in 1988 (Grosberg et al. 1988) and has not been observed in experiments or simulations until recently (Lieberman-Aiden et al. 2009). Recent characterization of human chromatin using a novel chromosome conformational capture technique brought the fractal globule into the spotlight as a structural model of human chromosome on the scale of up to 10 Mb (Lieberman-Aiden et al. 2009). Here, we present the concept of the fractal globule, comparing it to other states of a polymer and focusing on its properties relevant for the biophysics of chromatin. We then discuss properties of the fractal globule that make it an attractive model for chromatin organization inside a cell. Next, we connect the fractal globule to recent studies that emphasize topological constraints as a primary factor driving formation of chromosomal territories. We discuss how theoretical predictions, made on the basis of the fractal globule model, can be tested experimentally. Finally, we discuss whether fractal globule architecture can be relevant for chromatin packing in other organisms such as yeast and bacteria., National Cancer Institute (U.S.)

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity (2-6). Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible (7-10). We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments. Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

Protein knots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Seven distinctly knotted folds have already been identified. It is by and large unclear how these exceptional structures actually fold, and only recently, experiments and simulations have begun to shed some light on this issue. In checking the new protein structures submitted to the Protein Data Bank, we encountered the most complex and the smallest knots to date: A recently uncovered α-haloacid dehalogenase structure contains a knot with six crossings, a so-called Stevedore knot, in a projection onto a plane. The smallest protein knot is present in an as yet unclassified protein fragment that consists of only 92 amino acids. The topological complexity of the Stevedore knot presents a puzzle as to how it could possibly fold. To unravel this enigma, we performed folding simulations with a structure-based coarse-grained model and uncovered a possible mechanism by which the knot forms in a single loop flip., Author Summary Knots are ubiquitous in many aspects of our life, but remain elusive in proteins. The multitude of protein structures archived in the Protein Data Bank can be grouped into several hundred patterns, but only a handful are folded into knots. Combing through the recently added structures we found several novel knotted proteins. A microbial enzyme that catalyzes the breakdown of pollutants is the most complex protein knot encountered so far (similar to a knot used by stevedores for lifting cargo). The smallest knotted protein on the other hand consists of only 92 amino acids. The existence of these complex motifs demonstrates that the ability of self assembly goes far beyond normal expectations. Aided by computer simulations we present evidence which suggests that the Stevedore protein knot, despite its topological complexity, may actually form in a single flipping movement.

The binding of a transcription factor (TF) to a DNA operator site can initiate or repress the expression of a gene. Computational prediction of sites recognized by a TF has traditionally relied upon knowledge of several cognate sites, rather than an ab initio approach. Here, we examine the possibility of using structure-based energy calculations that require no knowledge of bound sites but rather start with the structure of a protein–DNA complex. We study the PurR Escherichia coli TF, and explore to which extent atomistic models of protein–DNA complexes can be used to distinguish between cognate and noncognate DNA sites. Particular emphasis is placed on systematic evaluation of this approach by comparing its performance with bioinformatic methods, by testing it against random decoys and sites of homologous TFs. We also examine a set of experimental mutations in both DNA and the protein. Using our explicit estimates of energy, we show that the specificity for PurR is dominated by direct protein–DNA interactions, and weakly influenced by bending of DNA., National Science Foundation (U.S.) (Grant DMR-08- 03315), Deutsche Forschungsgemeinschaft (DFG) (Grant VI237/1), NEC Research Support Fund, National Institutes of Health. National Centers for Biomedical Computing (Informatics for Integrating Biology and the Bedside), National Institutes of Health (U.S.) (3U54LM008748-04S1)

KNOTS (http://knots.mit.edu) is a web server that detects knots in protein structures. Several protein structures have been reported to contain intricate knots. The physiological role of knots and their effect on folding and evolution is an area of active research. The user submits a PDB id or uploads a 3D protein structure in PDB or mmCIF format. The current implementation of the server uses the Alexander polynomial to detect knots. The results of the analysis that are presented to the user are the location of the knot in the structure, the type of the knot and an interactive visualization of the knot. The results can also be downloaded and viewed offline. The server also maintains a regularly updated list of known knots in protein structures., National Science Foundation (U.S.) (grant DMR-04-26677), Deutsche Forschungsgemeinschaft (DFG) (grant VI237/1), Alfred P. Sloan Foundation (Research Fellowship)

Strong experimental and theoretical evidence shows that transcription factors and other specific DNA-binding proteins find their sites using a two-mode search: alternating between 3D diffusion through the cell and 1D sliding along the DNA. We consider the role spatial effects in the mechanism on two different scales. First, we reconcile recent experimental findings by showing that the 3D diffusion of the transcription factor is often local, i.e. the transcription factor lands quite near its dissociation site. Second, we discriminate between two types of searches: global searches and local searches. We show that these searches differ significantly in average search time and the variability of search time. Using experimentally measured parameter values, we also show that 1D and 3D search is not optimally balanced, leading to much larger estimates of search time. Together, these results lead to a number of biological implications including suggestions of how prokaryotes and eukaryotes achieve rapid gene regulation and the relationship between the search mechanism and noise in gene expression., Comment: 16 pages, 4 figures

The classic model of eukaryotic gene expression requires direct spatial contact between a distal enhancer and a proximal promoter. Recent Chromosome Conformation Capture (3C) studies show that enhancers and promoters are embedded in a complex network of looping interactions. Here we use a polymer model of chromatin fiber to investigate whether, and to what extent, looping interactions between elements in the vicinity of an enhancer-promoter pair can influence their contact frequency. Our equilibrium polymer simulations show that a chromatin loop, formed by elements flanking either an enhancer or a promoter, suppresses enhancer-promoter interactions, working as an insulator. A loop formed by elements located in the region between an enhancer and a promoter, on the contrary, facilitates their interactions. We find that different mechanisms underlie insulation and facilitation; insulation occurs due to steric exclusion by the loop, and is a global effect, while facilitation occurs due to an effective shortening of the enhancer-promoter genomic distance, and is a local effect. Consistently, we find that these effects manifest quite differently for in silico 3C and microscopy. Our results show that looping interactions that do not directly involve an enhancer-promoter pair can nevertheless significantly modulate their interactions. This phenomenon is analogous to allosteric regulation in proteins, where a conformational change triggered by binding of a regulatory molecule to one site affects the state of another site., National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54-CA143874-04), Massachusetts Institute of Technology. Undergraduate Research Opportunities Program, National Science Foundation (U.S.). Massachusetts Institute of Technology. Program for Research in Mathematics, Engineering and Science for High School Students (PRIMES)