Euryarchaeal genomes are folded into SMC-dependent loops and domains, but lack transcription-mediated compartmentalization.

Hi-C has become a routine method for probing the 3D organization of genomes. However, when applied to prokaryotes and archaea, the current protocols are expensive and limited in their resolution. We develop a cost-effective Hi-C protocol to explore chromosome conformations of these two kingdoms at the gene or operon level. We first validate it on E. coli and V. cholera, generating sub-kilobase-resolution contact maps, and then apply it to the euryarchaeota H. volcanii, Hbt. salinarum, and T. kodakaraensis. With a resolution of up to 1 kb, we explore the diversity of chromosome folding in this phylum. In contrast to crenarchaeota, these euryarchaeota lack (active/inactive) compartment-like structures. Instead, their genomes are composed of self-interacting domains and chromatin loops. In H. volcanii, these structures are regulated by transcription and the archaeal structural maintenance of chromosomes (SMC) protein, further supporting the ubiquitous role of these processes in shaping the higher-order organization of genomes.

From Loops to Looks: Transcription Factors and Chromatin Organization Shaping Terminal B Cell Differentiation.

B lymphopoiesis is tightly regulated at the level of gene transcription. In recent years, investigators have shed light on the transcription factor networks and the epigenetic machinery involved at all differentiation steps of mammalian B cell development. During terminal differentiation, B cells undergo dramatic changes in gene transcriptional programs to generate germinal center B cells, plasma cells and memory B cells. Recent evidence indicates that mature B cell formation involves an essential contribution from 3D chromatin conformations through its interplay with transcription factors and epigenetic machinery. Here, we provide an up-to-date overview of the coordination between transcription factors, epigenetic changes, and chromatin architecture during terminal B cell differentiation, focusing on recent discoveries and technical advances for studying 3D chromatin structures.

Principles of Chromosome Architecture Revealed by Hi-C.

Chromosomes are folded and compacted in interphase nuclei, but the molecular basis of this folding is poorly understood. Chromosome conformation capture methods, such as Hi-C, combine chemical crosslinking of chromatin with fragmentation, DNA ligation, and high-throughput DNA sequencing to detect neighboring loci genome-wide. Hi-C has revealed the segregation of chromatin into active and inactive compartments and the folding of DNA into self-associating domains and loops. Depletion of CTCF, cohesin, or cohesin-associated proteins was recently shown to affect the majority of domains and loops in a manner that is consistent with a model of DNA folding through extrusion of chromatin loops. Compartmentation was not dependent on CTCF or cohesin. Hi-C contact maps represent the superimposition of CTCF/cohesin-dependent and -independent folding states.

Domain Model of Eukaryotic Genome Organization: From DNA Loops Fixed on the Nuclear Matrix to TADs.

The article reviews the development of ideas on the domain organization of eukaryotic genome, with special attention on the studies of DNA loops anchored to the nuclear matrix and their role in the emergence of the modern model of eukaryotic genome spatial organization. Critical analysis of results demonstrating that topologically associated chromatin domains are structural-functional blocks of the genome supports the notion that these blocks are fundamentally different from domains whose existence was proposed by the domain hypothesis of eukaryotic genome organization formulated in the 1980s. Based on the discussed evidence, it is concluded that the model postulating that eukaryotic genome is built from uniformly organized structural-functional blocks has proven to be untenable.

The regulation of chromosome segregation via centromere loops.

Biophysical studies of the yeast centromere have shown that the organization of the centromeric chromatin plays a crucial role in maintaining proper tension between sister kinetochores during mitosis. While centromeric chromatin has traditionally been considered a simple spring, recent work reveals the centromere as a multifaceted, tunable shock absorber. Centromeres can differ from other regions of the genome in their heterochromatin state, supercoiling state, and enrichment of structural maintenance of chromosomes (SMC) protein complexes. Each of these differences can be utilized to alter the effective stiffness of centromeric chromatin. In budding yeast, the SMC protein complexes condensin and cohesin stiffen chromatin by forming and cross-linking chromatin loops, respectively, into a fibrous structure resembling a bottlebrush. The high density of the loops compacts chromatin while spatially isolating the tension from spindle pulling forces to a subset of the chromatin. Paradoxically, the molecular crowding of chromatin via cohesin and condensin also causes an outward/poleward force. The structure allows the centromere to act as a shock absorber that buffers the variable forces generated by dynamic spindle microtubules. Based on the distribution of SMCs from bacteria to human and the conserved distance between sister kinetochores in a wide variety of organisms (0.4 to 1 micron), we propose that the bottlebrush mechanism is the foundational principle for centromere function in eukaryotes.

Polycomb-mediated chromatin loops revealed by a subkilobase-resolution chromatin interaction map.

The locations of chromatin loops in Drosophila were determined by Hi-C (chemical cross-linking, restriction digestion, ligation, and high-throughput DNA sequencing). Whereas most loop boundaries or "anchors" are associated with CTCF protein in mammals, loop anchors in Drosophila were found most often in association with the polycomb group (PcG) protein Polycomb (Pc), a subunit of polycomb repressive complex 1 (PRC1). Loops were frequently located within domains of PcG-repressed chromatin. Promoters located at PRC1 loop anchors regulate some of the most important developmental genes and are less likely to be expressed than those not at PRC1 loop anchors. Although DNA looping has most commonly been associated with enhancer-promoter communication, our results indicate that loops are also associated with gene repression.

Lessons we can learn from neurons to make cancer cells quiescent.

Cancer is a major concern for contemporary societies. However, the incidence of cancer is unevenly distributed among tissues and cell types. In particular, the evidence indicates that neurons are absolutely resistant to cancer and this is commonly explained on the basis of the known postmitotic state of neurons. The dominant paradigm on cancer understands this problem as a disease caused by mutations in cellular genes that result in unrestrained cell proliferation and eventually in tissue invasion and metastasis. However, the evidence also shows that mutations and gross chromosomal anomalies are common in functional neurons that nevertheless do not become neoplastic. This fact suggests that in the real nonexperimental setting mutations per se are not enough for inducing carcinogenesis but also that the postmitotic state of neurons is not genetically controlled or determined, otherwise there should be reports of spontaneously transformed neurons. Here we discuss the evidence that the postmitotic state of neurons has a structural basis on the high stability of their nuclear higher order structure that performs like an absolute tumor suppressor. We also discuss evidence that it is possible to induce a similar structural postmitotic state in nonneural cell types as a practical strategy for stopping or reducing the progression of cancer.

DNA loop domain organization in nucleoids from cells of different types.

The loop domain organization of chromatin plays an important role in transcription regulation and thus may be assumed to vary in cells of different types. We investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay) for nucleoids obtained from human lymphocytes, lymphoblasts and glioblastoma T98G cells. The results confirm our previous observation that there are three parts of DNA in nucleoids: DNA on the nucleoid surface, loops up to ∼150 kb inside the nucleoid, and larger loops that cannot migrate. However, the relative amounts of the three parts were found to be very different for different cell types. The distributions of the loop length up to 150 kb were shown to be exponential, with the distribution parameter, the loop density, to be dependent on the cell type.

The interphase mammalian chromosome as a structural system based on tensegrity.

Each mammalian chromosome is constituted by a DNA fiber of macroscopic length that needs to be fitted in a microscopic nucleus. The DNA fiber is subjected at physiological temperature to random thermal bending and looping that must be constrained so as achieve structural stability thus avoiding spontaneous rupturing of the fiber. Standard textbooks assume that chromatin proteins are primarily responsible for the packaging of DNA and so of its protection against spontaneous breakage. Yet the dynamic nature of the interactions between chromatin proteins and DNA is unlikely to provide the necessary long-term structural stability for the chromosomal DNA. On the other hand, longstanding evidence indicates that stable interactions between DNA and constituents of a nuclear compartment commonly known as the nuclear matrix organize the chromosomal DNA as a series of topologically constrained, supercoiled loops during interphase. This results in a primary level of DNA condensation and packaging within the nucleus, as well as in protection against spontaneous DNA breakage, independently of chromatin proteins which nevertheless increase and dynamically modulate the degree of DNA packaging and its role in the regulation of DNA function. Thus current evidence, presented hereunder, supports a model for the organization of the interphase chromosome as resilient system that satisfies the principles of structural tensegrity.

DNA Brushing Shoulders: Targeted Looping and Scanning of Large DNA Strands.

We present a nanofluidic device for targeted manipulations in the quarternary structure of single DNA molecules. We demonstrate the folding and unfolding of hairpin-shaped regions, similar to chromatin loops. These loops are stable for minutes at nanochannel junctions. We demonstrate continuous scanning of two DNA segments that occupy a common nanovolume. We present a model governing the stability of loop folds and discuss how the system achieves specific DNA configurations without operator intervention.

Polymer Modeling Reveals Interplay between Physical Properties of Chromosomal DNA and the Size and Distribution of Condensin-Based Chromatin Loops.

Transient DNA loops occur throughout the genome due to thermal fluctuations of DNA and the function of SMC complex proteins such as condensin and cohesin. Transient crosslinking within and between chromosomes and loop extrusion by SMCs have profound effects on high-order chromatin organization and exhibit specificity in cell type, cell cycle stage, and cellular environment. SMC complexes anchor one end to DNA with the other extending some distance and retracting to form a loop. How cells regulate loop sizes and how loops distribute along chromatin are emerging questions. To understand loop size regulation, we employed bead-spring polymer chain models of chromatin and the activity of an SMC complex on chromatin. Our study shows that (1) the stiffness of the chromatin polymer chain, (2) the tensile stiffness of chromatin crosslinking complexes such as condensin, and (3) the strength of the internal or external tethering of chromatin chains cooperatively dictate the loop size distribution and compaction volume of induced chromatin domains. When strong DNA tethers are invoked, loop size distributions are tuned by condensin stiffness. When DNA tethers are released, loop size distributions are tuned by chromatin stiffness. In this three-way interaction, the presence and strength of tethering unexpectedly dictates chromatin conformation within a topological domain.

Loop detection using Hi-C data with HiCExplorer.

BACKGROUND: Chromatin loops are an essential factor in the structural organization of the genome; however, their detection in Hi-C interaction matrices is a challenging and compute-intensive task. The approach presented here, integrated into the HiCExplorer software, shows a chromatin loop detection algorithm that applies a strict candidate selection based on continuous negative binomial distributions and performs a Wilcoxon rank-sum test to detect enriched Hi-C interactions. RESULTS: HiCExplorer's loop detection has a high detection rate and accuracy. It is the fastest available CPU implementation and utilizes all threads offered by modern multicore platforms. CONCLUSIONS: HiCExplorer's method to detect loops by using a continuous negative binomial function combined with the donut approach from HiCCUPS leads to reliable and fast computation of loops. All the loop-calling algorithms investigated provide differing results, which intersect by $\sim 50\%$ at most. The tested in situ Hi-C data contain a large amount of noise; achieving better agreement between loop calling algorithms will require cleaner Hi-C data and therefore future improvements to the experimental methods that generate the data.

emDNA - A Tool for Modeling Protein-decorated DNA Loops and Minicircles at the Base-pair Step Level.

Computational modeling of nucleic acids plays an important role in molecular biology, enhancing our general understanding of the relationship between structure and function. Biophysical studies have provided a wealth of information on how double-helical DNA responds to proteins and other molecules in its local environment but far less understanding of the larger scale structural responses found in protein-decorated loops and minicircles. Current computational models of DNA range from detailed all-atom molecular dynamics studies, which produce rich time and spatially dependent depictions of small DNA fragments, to coarse-grained simulations, which sacrifice detailed physical and chemical information to treat larger-scale systems. The treatment of DNA used here, at the base-pair step level with rigid-body parameters, allows one to develop models hundreds of base pairs long from local, sequence-specific features found from experiment. The emDNA software takes advantage of this framework, producing optimized structures of DNA at thermal equilibrium with built-in or user-generated elastic models. The program, in combination with the case studies included in this article, allows users of any skill level to develop and investigate mesoscale models of their own design. The functionality of emDNA includes a tool to incorporate experiment-specific configurations, e.g., protein-bound and/or melted DNA from known high-resolution structures, within higher-order 3D models by fixing the orientation and position of user-specified base pairs. The software provides a new avenue into multiscale genetic modeling, giving a wide range of users a deeper understanding of DNA mesoscale organization and the opportunity to pose new questions in genetic research. The publicly available emDNA software, including build instructions and usage information, is available on GitHub (https://nicocvn.github.io/emDNA/).

Detection of Allele-Specific 3D Chromatin Interactions Using High-Resolution In-Nucleus 4C-seq.

Chromosome conformation capture techniques are a set of methods used to determine 3D genome organization through the capture and identification of physical contacts between pairs of genomic loci. Among them, 4C-seq (circular chromosome conformation capture coupled to high-throughput sequencing) allows for the identification and quantification of the sequences interacting with a preselected locus of interest. 4C-seq has been widely used in the literature, mainly to study chromatin loops between enhancers and promoters or between CTCF binding sites and to identify chromatin domain boundaries. As 3D-contacts may be established in an allele-specific manner, we describe an up-to-date allele-specific 4C-seq protocol, starting from the selection of allele-specific viewpoints to Illumina sequencing. This protocol has mainly been optimized for cultured mammalian cells, but can be adapted for other cell types with relatively minor changes in initial steps.

Cohesin: behind dynamic genome topology and gene expression reprogramming.

Beyond its originally discovered role tethering replicated sister chromatids, cohesin has emerged as a master regulator of gene expression. Recent advances in chromatin topology resolution and single-cell studies have revealed that cohesin has a pivotal role regulating highly dynamic chromatin interactions linked to transcription control. The dynamic association of cohesin with chromatin and its capacity to perform loop extrusion contribute to the heterogeneity of chromatin contacts. Additionally, different cohesin subcomplexes, with specific properties and regulation, control gene expression across the cell cycle and during developmental cell commitment. Here, we discuss the most recent literature in the field to highlight the role of cohesin in gene expression regulation during transcriptional shifts and its relationship with human diseases.

The Chromosome Conformation Capture (3C) in Drosophila melanogaster.

The discovery of the DNA double helix by Watson and Crick in 1953 was the first report showing that the genomic information is not contained in a stretched linear molecule. After that, a huge advance in the knowledge of the structure of the eukaryotic genome in the nuclear space has been made over the last decades, bringing us to the widely accepted concept that the genome is packaged into hierarchical levels of higher-order three-dimensional structures. The spatial organization of the eukaryotic genome has direct influence on fundamental nuclear processes that include transcription, replication, and DNA repair. The idea that structural alterations of chromosomes may cause disease goes back to the early nineteenth century. Big effort has been devoted to the study of the three-dimensional architecture of the genome and its functional implications. In this chapter, I will describe the chromosome conformation capture (3C), one of the first techniques used to detect and measure the frequency of interactions between genomic sequences that are kept in spatial proximity in the nucleus.

Wheat chromatin architecture is organized in genome territories and transcription factories.

BACKGROUND: Polyploidy is ubiquitous in eukaryotic plant and fungal lineages, and it leads to the co-existence of several copies of similar or related genomes in one nucleus. In plants, polyploidy is considered a major factor in successful domestication. However, polyploidy challenges chromosome folding architecture in the nucleus to establish functional structures. RESULTS: We examine the hexaploid wheat nuclear architecture by integrating RNA-seq, ChIP-seq, ATAC-seq, Hi-C, and Hi-ChIP data. Our results highlight the presence of three levels of large-scale spatial organization: the arrangement into genome territories, the diametrical separation between facultative and constitutive heterochromatin, and the organization of RNA polymerase II around transcription factories. We demonstrate the micro-compartmentalization of transcriptionally active genes determined by physical interactions between genes with specific euchromatic histone modifications. Both intra- and interchromosomal RNA polymerase-associated contacts involve multiple genes displaying similar expression levels. CONCLUSIONS: Our results provide new insights into the physical chromosome organization of a polyploid genome, as well as on the relationship between epigenetic marks and chromosome conformation to determine a 3D spatial organization of gene expression, a key factor governing gene transcription in polyploids.

The Genome in a Three-Dimensional Context: Deciphering the Contribution of Noncoding Mutations at Enhancers to Blood Cancer.

Associations between blood cancer and genetic predisposition, including both inherited variants and acquired mutations and epimutations, have been well characterized. However, the majority of these variants affect noncoding regions, making their mechanisms difficult to hypothesize and hindering the translation of these insights into patient benefits. Fueled by unprecedented progress in next-generation sequencing and computational integrative analysis, studies have started applying combinations of epigenetic, genome architecture, and functional assays to bridge the gap between noncoding variants and blood cancer. These complementary tools have not only allowed us to understand the potential malignant role of these variants but also to differentiate key variants, cell-types, and conditions from misleading ones. Here, we briefly review recent studies that have provided fundamental insights into our understanding of how noncoding mutations at enhancers predispose and promote blood malignancies in the context of spatial genome architecture.

Physical principles and new applications of comet assay.

The comet assay is a sensitive method to assess DNA damages in single cells. The approach consists of an analysis of electrophoretic migration of DNA from nucleoids obtained after cell lysis in a thin layer of agarose. Although the method is widely used the physical mechanisms of DNA track formation remained to be rather elusive for a long time. This review is devoted to our recent results pertaining to this subject, using an original approach based on the kinetic measurements of the comet formation. We argue that linear DNA fragments give an essential contribution into the tail formation in the alkaline conditions and, at neutral pH, when the level of DNA damages is very high. On the other hand, in the neutral comet assay at low levels of DNA damages (and also in the case of undamaged cells) the tail is formed by extended DNA loops. These loops are about the same as chromatin loops in the cell nuclei. Kinetic measurements in the comet assay give an opportunity to investigate the topology of the loops and large-scale features of the loop domain organization (and re-organization) in nucleoids obtained from different cell types.

Extra views on structure and dynamics of DNA loops on nucleosomes studied with molecular simulations.

It has been shown experimentally that the action of the RSC chromatin remodeler leads to the formation of an irregular, partially remodeled nucleosome, termed a remosome. The remosome contains an extra 30-40 base pairs of DNA compared to a canonical nucleosome. Large-scale molecular simulations have provided information on the probable structure of remosomes and have explained why they remain stable in the absence of RSC. Here we explain how these simulations were carried out and what the resulting remosome models imply in terms of the mechanism of action of RSC. We notably show that local kinks within DNA are key in explaining how extra DNA can be in added to nucleosomes without unduly disturbing DNA-histone binding.