Cohesin Complex: Structure and Principles of Interaction with DNA.

Accurate duplication and separation of long linear genomic DNA molecules is associated with a number of purely mechanical problems. SMC complexes are key components of the cellular machinery that ensures decatenation of sister chromosomes and compaction of genomic DNA during division. Cohesin, one of the essential eukaryotic SMC complexes, has a typical ring structure with intersubunit pore through which DNA molecules can be threaded. Capacity of cohesin for such topological entrapment of DNA is crucial for the phenomenon of post-replicative association of sister chromatids better known as cohesion. Recently, it became apparent that cohesin and other SMC complexes are, in fact, motor proteins with a very peculiar movement pattern leading to formation of DNA loops. This specific process has been called loop extrusion. Extrusion underlies multiple functions of cohesin beyond cohesion, but molecular mechanism of the process remains a mystery. In this review, we summarized the data on molecular architecture of cohesin, effect of ATP hydrolysis cycle on this architecture, and known modes of cohesin-DNA interactions. Many of the seemingly disparate facts presented here will probably be incorporated in a unified mechanistic model of loop extrusion in the not-so-distant future.

Cohesin-Dependent Loop Extrusion: Molecular Mechanics and Role in Cell Physiology.

The most prominent representatives of multisubunit SMC complexes, cohesin and condensin, are best known as structural components of mitotic chromosomes. It turned out that these complexes, as well as their bacterial homologues, are molecular motors, the ATP-dependent movement of these complexes along DNA threads leads to the formation of DNA loops. In recent years, we have witnessed an avalanche-like accumulation of data on the process of SMC dependent DNA looping, also known as loop extrusion. This review briefly summarizes the current understanding of the place and role of cohesin-dependent extrusion in cell physiology and presents a number of models describing the potential molecular mechanism of extrusion in a most compelling way. We conclude the review with a discussion of how the capacity of cohesin to extrude DNA loops may be mechanistically linked to its involvement in sister chromatid cohesion.

Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells.

Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

Studying RNA-DNA interactome by Red-C identifies noncoding RNAs associated with various chromatin types and reveals transcription dynamics.

Non-coding RNAs (ncRNAs) participate in various biological processes, including regulating transcription and sustaining genome 3D organization. Here, we present a method termed Red-C that exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncovered the RNA-DNA interactome of human K562 cells and identified hundreds of ncRNAs enriched in active or repressed chromatin, including previously undescribed RNAs. Analysis of the RNA-DNA interactome also allowed us to trace the kinetics of messenger RNA production. Our data support the model of co-transcriptional intron splicing, but not the hypothesis of the circularization of actively transcribed genes.

C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries.

Studies performed using Hi-C and other high-throughput whole-genome C-methods have demonstrated that 3D organization of eukaryotic genomes is functionally relevant. Unfortunately, ultra-deep sequencing of Hi-C libraries necessary to detect loop structures in large vertebrate genomes remains rather expensive. However, many studies are in fact aimed at determining the fine-scale 3D structure of comparatively small genomic regions up to several Mb in length. Such studies typically focus on the spatial structure of domains of coregulated genes, molecular mechanisms of loop formation, and interrogation of functional significance of GWAS-revealed polymorphisms. Therefore, a handful of molecular techniques based on Hi-C have been developed to address such issues. These techniques commonly rely on in-solution hybridization of Hi-C/3C-seq libraries with pools of biotinylated baits covering the region of interest, followed by deep sequencing of the enriched library. Here, we describe a new protocol of this kind, C-TALE (Chromatin TArget Ligation Enrichment). Preparation of hybridization probes from bacterial artificial chromosomes and an additional round of enrichment make C-TALE a cost-effective alternative to existing many-versus-all C-methods.

Single-cell genome-wide studies give new insight into nongenetic cell-to-cell variability in animals.

Huge numbers of cells form an adult animal body, ranging from several thousands in Placozoa and small nematodes to many billions in mammals. Cells are classified into separate groups known as cell types by their morphological and biochemical features. Six to several hundreds of spatially ordered cell types are recognized in different animals. This complex organization develops from one cell, a zygote, during ontogeny, and its dynamic equilibrium is often maintained in the adult body. One of the key challenges in biology is to understand the mechanisms that sustain the reproducible development of a complex ordered cell ensemble such as the animal body from a single cell. How cells with identical genomes stably maintain one of the numerous possible phenotypes? How the cell differentiation lineage is selected during development? What genes play a key role in maintaining cell identity, and how do they determine expression of other genes characteristic of the relevant cell type? How does the basically stochastic nature of transcription in an isolated cell affect the stability of cell identity, the selection of a cell lineage, and the variability of cell responses to external stimuli? Better-grounded answers to these questions have become possible with recent progress in single-cell genome-wide analysis techniques, which combine the high throughput of biochemical methods and the differential nature of microscopy. The techniques are still in their infancy, and their further development will certainly revolutionize many fields of life sciences and, in particular, developmental biology. Here, we summarize the main results that have been obtained in single-cell genome-wide analyses and describe the nongenetic cell-to-cell variability in animals.

Bench Research Informed by GWAS Results.

Scientifically interesting as well as practically important phenotypes often belong to the realm of complex traits. To the extent that these traits are hereditary, they are usually 'highly polygenic'. The study of such traits presents a challenge for researchers, as the complex genetic architecture of such traits makes it nearly impossible to utilise many of the usual methods of reverse genetics, which often focus on specific genes. In recent years, thousands of genome-wide association studies (GWAS) were undertaken to explore the relationships between complex traits and a large number of genetic factors, most of which are characterised by tiny effects. In this review, we aim to familiarise 'wet biologists' with approaches for the interpretation of GWAS results, to clarify some issues that may seem counterintuitive and to assess the possibility of using GWAS results in experiments on various complex traits.

Sensitivity of cohesin-chromatin association to high-salt treatment corroborates non-topological mode of loop extrusion.

Cohesin is a key organizer of chromatin folding in eukaryotic cells. The two main activities of this ring-shaped protein complex are the maintenance of sister chromatid cohesion and the establishment of long-range DNA-DNA interactions through the process of loop extrusion. Although the basic principles of both cohesion and loop extrusion have been described, we still do not understand several crucial mechanistic details. One of such unresolved issues is the question of whether a cohesin ring topologically embraces DNA string(s) during loop extrusion. Here, we show that cohesin complexes residing on CTCF-occupied genomic sites in mammalian cells do not interact with DNA topologically. We assessed the stability of cohesin-dependent loops and cohesin association with chromatin in high-ionic-strength conditions in G1-synchronized HeLa cells. We found that increased salt concentration completely displaces cohesin from those genomic regions that correspond to CTCF-defined loop anchors. Unsurprisingly, CTCF-anchored cohesin loops also dissipate in these conditions. Because topologically engaged cohesin is considered to be salt resistant, our data corroborate a non-topological model of loop extrusion. We also propose a model of cohesin activity throughout the interphase, which essentially equates the termination of non-topological loop extrusion with topological loading of cohesin. This theoretical framework enables a parsimonious explanation of various seemingly contradictory experimental findings.

LASCA: loop and significant contact annotation pipeline.

Chromatin loops represent one of the major levels of hierarchical folding of the genome. Although the situation is evolving, current methods have various difficulties with the accurate mapping of loops even in mammalian Hi-C data, and most of them fail to identify chromatin loops in animal species with substantially different genome architecture. This paper presents the loop and significant contact annotation (LASCA) pipeline, which uses Weibull distribution-based modeling to effectively identify loops and enhancer-promoter interactions in Hi-C data from evolutionarily distant species: from yeast and worms to mammals. Available at: https://github.com/ArtemLuzhin/LASCA_pipeline .

Novel Approaches for Identifying the Molecular Background of Schizophrenia.

Recent advances in psychiatric genetics have led to the discovery of dozens of genomic loci associated with schizophrenia. However, a gap exists between the detection of genetic associations and understanding the underlying molecular mechanisms. This review describes the basic approaches used in the so-called post-GWAS studies to generate biological interpretation of the existing population genetic data, including both molecular (creation and analysis of knockout animals, exploration of the transcriptional effects of common variants in human brain cells) and computational (fine-mapping of causal variability, gene set enrichment analysis, partitioned heritability analysis) methods. The results of the crucial studies, in which these approaches were used to uncover the molecular and neurobiological basis of the disease, are also reported.

A modified protocol of Capture-C allows affordable and flexible high-resolution promoter interactome analysis.

Large-scale epigenomic projects have mapped hundreds of thousands of potential regulatory sites in the human genome, but only a small proportion of these elements are proximal to transcription start sites. It is believed that the majority of these sequences are remote promoter-activating genomic sites scattered within several hundreds of kilobases from their cognate promoters and referred to as enhancers. It is still unclear what principles, aside from relative closeness in the linear genome, determine which promoter(s) is controlled by a given enhancer; however, this understanding is of great fundamental and clinical relevance. In recent years, C-methods (chromosome conformation capture-based methods) have become a powerful tool for the identification of enhancer-promoter spatial contacts that, in most cases, reflect their functional link. Here, we describe a new hybridisation-based promoter Capture-C protocol that makes use of biotinylated dsDNA probes generated by PCR from a custom pool of long oligonucleotides. The described protocol allows high-resolution promoter interactome description, providing a flexible and cost-effective alternative to the existing promoter Capture-C modifications. Based on the obtained data, we propose several tips on probe design that could potentially improve the results of future experiments.

The anti-cancer drugs curaxins target spatial genome organization.

Recently we characterized a class of anti-cancer agents (curaxins) that disturbs DNA/histone interactions within nucleosomes. Here, using a combination of genomic and in vitro approaches, we demonstrate that curaxins strongly affect spatial genome organization and compromise enhancer-promoter communication, which is necessary for the expression of several oncogenes, including MYC. We further show that curaxins selectively inhibit enhancer-regulated transcription of chromatinized templates in cell-free conditions. Genomic studies also suggest that curaxins induce partial depletion of CTCF from its binding sites, which contributes to the observed changes in genome topology. Thus, curaxins can be classified as epigenetic drugs that target the 3D genome organization.

Activation of the alpha-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure.

BACKGROUND: In homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation. RESULTS: We found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation. CONCLUSIONS: Our findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing topologically associating domain.

The Role of Crowding Forces in Juxtaposing ß-Globin Gene Domain Remote Regulatory Elements in Mouse Erythroid Cells.

The extremely high concentration of macromolecules in a eukaryotic cell nucleus indicates that the nucleoplasm is a crowded macromolecular solution in which large objects tend to gather together due to crowding forces. It has been shown experimentally that crowding forces support the integrity of various nuclear compartments. However, little is known about their role in control of chromatin dynamics in vivo. Here, we experimentally addressed the possible role of crowding forces in spatial organization of the eukaryotic genome. Using the mouse ß-globin domain as a model, we demonstrated that spatial juxtaposition of the remote regulatory elements of this domain in globin-expressing cells may be lost and restored by manipulation of the level of macromolecular crowding. In addition to proving the role of crowding forces in shaping interphase chromatin, our results suggest that the folding of the chromatin fiber is a major determinant in juxtaposing remote genomic elements.

Actual ligation frequencies in the chromosome conformation capture procedure.

Chromosome conformation capture (3C) and derivative experimental procedures are used to estimate the spatial proximity between different genomic elements, thus providing information about the 3D organization of genomic domains and whole genomes within the nucleus. All C-methods are based on the proximity ligation-the preferential ligation of joined DNA fragments obtained upon restriction enzyme digestion of in vivo cross-linked chromatin. Here, using the mouse beta-globin genes in erythroid cells as a model, we estimated the actual frequencies of ligation between the fragments bearing the promoter of the major beta-globin gene and its distant enhancers and showed that the number of ligation products produced does not exceed 1% of all fragments subjected to the ligation. Although this low yield of 3C ligation products may be explained entirely by technical issues, it may as well reflect a low frequency of interaction between DNA regulatory elements in vivo.