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.
Chromosomes communicate in the 3D genome to ensure proper gene expression. Molecular mechanisms of chromosome interaction and biophysical properties of topological machines are key for our understanding of this communication.
Analysis of the spatial organization of chromosomes reveals complex three-dimensional networks of chromosomal interactions. These interactions affect gene expression at multiple levels, including ...long-range control by distant enhancers and repressors, coordinated expression of genes, and modification of epigenetic states. Major challenges now include deciphering the mechanisms by which loci come together and understanding the functional consequences of these often transient associations.
The higher order arrangement of nucleosomes and the level of compaction of the chromatin fiber play important roles in the control of gene expression and other genomic activities. Analysis of ...chromatin in vitro has suggested that under near physiological conditions chromatin fibers can become highly compact and that the level of compaction can be modulated by histone modifications. However, less is known about the organization of chromatin fibers in living cells. Here, we combine chromosome conformation capture (3C) data with distance measurements and polymer modeling to determine the in vivo mass density of a transcriptionally active 95-kb GC-rich domain on chromosome III of the yeast Saccharomyces cerevisiae. In contrast to previous reports, we find that yeast does not form a compact fiber but that chromatin is extended with a mass per unit length that is consistent with a rather loose arrangement of nucleosomes. Analysis of 3C data from a neighboring AT-rich chromosomal domain indicates that chromatin in this domain is more compact, but that mass density is still well below that of a canonical 30 nm fiber. Our approach should be widely applicable to scale 3C data to real spatial dimensions, which will facilitate the quantification of the effects of chromatin modifications and transcription on chromatin fiber organization.
Recent studies have shown that chromosomes in a range of organisms are compartmentalized in different types of chromatin domains. In mammals, chromosomes form compartments that are composed of ...smaller Topologically Associating Domains (TADs). TADs are thought to represent functional domains of gene regulation but much is still unknown about the mechanisms of their formation and how they exert their regulatory effect on embedded genes. Further, similar domains have been detected in other organisms, including flies, worms, fungi and bacteria. Although in all these cases these domains appear similar as detected by 3C-based methods, their biology appears to be quite distinct with differences in the protein complexes involved in their formation and differences in their internal organization. Here we outline our current understanding of such domains in different organisms and their roles in gene regulation.
Active and inactive chromatin are spatially separated in the nucleus. In Hi-C data, this is reflected by the formation of compartments, whose interactions form a characteristic checkerboard pattern ...in chromatin interaction maps. Only recently have the mechanisms that drive this separation come into view. Here, we discuss new insights into these mechanisms and possible functions in genome regulation. Compartmentalization can be understood as a microphase-segregated block co-polymer. Microphase separation can be facilitated by chromatin factors that associate with compartment domains, and that can engage in liquid–liquid phase separation to form subnuclear bodies, as well as by acting as bridging factors between polymer sections. We then discuss how a spatially segregated state of the genome can contribute to gene regulation, and highlight experimental challenges for testing these structure–function relationships.
Recent studies have shown that chromosome and nuclear compartmentalization may be driven by a process of phase separation.Subnuclear positioning of compartments can be achieved by tethering to the lamina, nucleoli, and other subnuclear bodies.Compartmentalization can be counteracted by loop extrusion.Phase-separated compartments may function in transcriptional control by in- and excluding specific cofactors required for gene regulation.Recently developed tools will allow researchers to interrogate the function of long-range compartment interactions and phase-separated regions of the nucleus.
During interphase, the eukaryotic genome is organized into chromosome territories that are spatially segregated into compartment domains. The extent to which interacting domains or chromosomes are ...entangled is not known. We analyze series of co-occurring chromatin interactions using multi-contact 3C (MC-3C) in human cells to provide insights into the topological entanglement of chromatin. Multi-contact interactions represent percolation paths (C-walks) through three-dimensional (3D) chromatin space. We find that the order of interactions within C-walks that occur across interfaces where chromosomes or compartment domains interact is not random. Polymer simulations show that such C-walks are consistent with distal domains being topologically insulated, that is, not catenated. Simulations show that even low levels of random strand passage, for example by topoisomerase II, would result in entanglements, increased mixing at domain interfaces and an order of interactions within C-walks not consistent with experimental MC-3C data. Our results indicate that, during interphase, entanglements between chromosomes and chromosomal domains are rare.
Despite advances in DNA sequencing technology, assembly of complex genomes remains a major challenge, particularly for genomes sequenced using short reads, which yield highly fragmented assemblies. ...Here we show that genome-wide in vivo chromatin interaction frequency data, which are measurable with chromosome conformation capture-based experiments, can be used as genomic distance proxies to accurately position individual contigs without requiring any sequence overlap. We also use these data to construct approximate genome scaffolds de novo. Applying our approach to incomplete regions of the human genome, we predict the positions of 65 previously unplaced contigs, in agreement with alternative methods in 26/31 cases attempted in common. Our approach can theoretically bridge any gap size and should be applicable to any species for which global chromatin interaction data can be generated.
Mitotic chromosomes fold as compact arrays of chromatin loops. To identify the pathway of mitotic chromosome formation, we combined imaging and Hi-C analysis of synchronous DT40 cell cultures with ...polymer simulations. Here we show that in prophase, the interphase organization is rapidly lost in a condensin-dependent manner, and arrays of consecutive 60-kilobase (kb) loops are formed. During prometaphase, ~80-kb inner loops are nested within ~400-kb outer loops. The loop array acquires a helical arrangement with consecutive loops emanating from a central "spiral staircase" condensin scaffold. The size of helical turns progressively increases to ~12 megabases during prometaphase. Acute depletion of condensin I or II shows that nested loops form by differential action of the two condensins, whereas condensin II is required for helical winding.
•Recent innovations in Hi-C experimentation are combined in a single protocol.•The rationale for each step in the procedure is provided.•A detailed step-by-step protocol is described for performing ...Hi-C 2.0.
Chromosome conformation capture-based methods such as Hi-C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair-wise interactions). Here, we describe an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (kb) resolution. It also includes steps to reduce random ligation and the generation of uninformative molecules, such as unligated ends, to improve the amount of valid intra-chromosomal read pairs. This protocol allows for obtaining information on conformational structures such as compartment and topologically associating domains, as well as high-resolution conformational features such as DNA loops.
The spatial organization of chromosomes inside the cell nucleus is still poorly understood. This organization is guided by intra- and interchromosomal contacts and by interactions of specific ...chromosomal loci with relatively fixed nuclear 'landmarks' such as the nuclear envelope and the nucleolus. Researchers have begun to use new molecular genome-wide mapping techniques to uncover both types of molecular interactions, providing insights into the fundamental principles of interphase chromosome folding.
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