The Role of Liquid-Liquid Phase Separation in the Compartmentalization of Cell Nucleus and Spatial Genome Organization.

Functional compartmentalization of the cell nucleus plays an important role in the regulation of genome activity by providing accumulation of enzymes and auxiliary factors in the reaction centers, such as transcription factories, Cajal bodies, speckles, etc. The mechanisms behind the nucleus functional compartmentalization are still poorly understood. There are reasons to believe that the key role in the nucleus compartmentalization belongs to the process of liquid-liquid phase separation. In this brief review, we analyze results of experimental studies demonstrating that liquid-liquid phase separation not only governs functional compartmentalization of the cell nucleus but also contributes to the formation of the 3D genomic architecture.

Modification of Nuclear Compartments and the 3D Genome in the Course of a Viral Infection.

The review addresses the question of how the structural and functional compartmentalization of the cell nucleus and the 3D organization of the cellular genome are modified during the infection of cells with various viruses. Particular attention is paid to the role of the introduced changes in the implementation of the viral strategy to evade the antiviral defense systems and provide conditions for viral replication. The discussion focuses on viruses replicating in the cell nucleus. Cytoplasmic viruses are mentioned in cases when a significant reorganization of the nuclear compartments or the 3D genome structure occurs during an infection with these viruses.

[3D Genomics].

The development of new research methods significantly changed our views on the role that the 3D organization of the genome plays in its functional activity. It was found that the genome is subdivided into structural-functional units that restrict the area of enhancer action at the level of spatial organization. Spatial reconfiguration of an extended genomic fragment was identified as a potential mechanism that activates or represses various genes. Accordingly, a distorted spatial organization of the genome often causes various diseases, including cancer. All these observations contributed to the emergence of 3D genomics as a new avenue of research. The review summarizes the most important discoveries in the field of 3D genomics and discusses the directions of its further development.

Structural-Functional Domains of the Eukaryotic Genome.

It is well known that DNA folding in the eukaryotic cell nucleus is tightly coupled with the operation of epigenetic mechanisms defining the repertoires of the genes expressed in different types of cells. To understand these mechanisms, it is important to know how DNA is packaged in chromatin. About 30 years ago a hypothesis was formulated, according to which epigenetic mechanisms operate not at the level of individual genes, but rather groups of genes localized in structurally and functionally isolated genomic segments that were called structural and functional domains. The question of what exactly these domains constitute has been re-examined multiple times as our knowledge of principles of chromatin folding has changed. In this review, we discuss structural and functional genomic domains in light of the current model of interphase chromosome organization based on the results of analysis of spatial proximity between remote genomic elements.

Dissipative particle dynamics for systems with high density of charges: Implementation of electrostatic interactions.

In this work, we study the question of how to introduce electrostatic interactions in dissipative particle dynamics (DPD) method in order to correctly reproduce the properties of systems with high density of charges, including those with inhomogeneous charge distribution. To this end, we formulate general requirements for the electrostatic force in DPD and propose a new functional form of the force which suits better for satisfying these requirements than the previously used ones. In order to verify the proposed model, we study the problem of a single polyelectrolyte chain collapse and compare the results with molecular dynamics (MD) simulations in which the exact Coulomb force is used. We show that an excellent quantitative agreement between MD and DPD models is observed if the length parameter D of the proposed electrostatic force is chosen properly; the recommendations concerning the choice of this parameter value are given based on the analysis of a polyelectrolyte chain collapse behavior. Finally, we demonstrate the applicability of DPD with the proposed electrostatic force to studying microphase separation phenomenon in polyelectrolyte melts and show that the same values of D as in the case of single chain collapse should be used, thus indicating universality of the model. Due to the charge correlation attraction, a long-range order in such melts can be observed even at zero Flory-Huggins parameter.

[Regulatory elements of the eukaryotic genome controlling transcription].

In this review, we discuss regulatory elements of the eukaryotic genome that control transcription. We describe the functional anatomy of tissue specific promoters and promoters of house-keeping genes; current methodologies for prediction and identification of enhancers; the role of insulators in the enhancer-promoter communication and maintenance of the epigenetic status of genomic domains; and briefly discuss the relationship between the interphase chromatin topology and regulation of transcription. Considering these questions, we pay particular attention to the new data obtained in works on the high-throughput sequencing of primary transcripts and the genome-wide analysis of histone modifications.

Multiblock copolymers prepared by patterned modification: Analytical theory and computer simulations.

We describe a special type of multiblock copolymers which are synthesized by a hypothetic procedure of the modification of monomer units in a polymer melt according to a certain geometrical criterion. In particular, we explore the case of lamellar-like structures: the sequence statistics of the resulting multiblock copolymers is described and their ability to self-assemble is studied. It is found that the block-size distribution P(k) for such random copolymers contains a large fraction of short blocks with the asymptotic dependence ∼k(-3/2), where k is the block size. A characteristic feature of such multiblock copolymers is their extremely high block-size polydispersity with the polydispersity index being proportional to the space period of the modification. The morphological behavior of such copolymers is simulated by means of dissipative particle dynamics. A stable self-assembled lamellar structure is observed, but the domain size appears to be sufficiently larger than the initial pattern period.

Anomalous diffusion in fractal globules.

The fractal globule state is a popular model for describing chromatin packing in eukaryotic nuclei. Here we provide a scaling theory and dissipative particle dynamics computer simulation for the thermal motion of monomers in the fractal globule state. Simulations starting from different entanglement-free initial states show good convergence which provides evidence supporting the existence of a unique metastable fractal globule state. We show monomer motion in this state to be subdiffusive described by ⟨X(2)(t)⟩∼t(αF) with αF close to 0.4. This result is in good agreement with existing experimental data on the chromatin dynamics, which makes an additional argument in support of the fractal globule model of chromatin packing.

[Compartmentalization of the cell nucleus and spatial organization of the genome].

The eukaryotic cell nucleus is one of the most complex cell organelles. Despite the absence of membranes, the nuclear space is divided into numerous compartments where different processes in- volved in the genome activity take place. The most important nuclear compartments include nucleoli, nuclear speckles, PML bodies, Cajal bodies, histone locus bodies, Polycomb bodies, insulator bodies, transcription and replication factories. The structural basis for the nuclear compartmentalization is provided by genomic DNA that occupies most of the nuclear volume. Nuclear compartments, in turn, guide the chromosome folding by providing a platform for the spatial interaction of individual genomic loci. In this review, we discuss fundamental principles of higher order genome organization with a focus on chromosome territories and chromosome domains, as well as consider the structure and function of the key nuclear compartments. We show that the func- tional compartmentalization of the cell nucleus and genome spatial organization are tightly interconnected, and that this form of organization is highly dynamic and is based on stochastic processes.

[Spatial organization of interphase chromosomes and the role of chromatin fiber dynamycs in the positioning of genome elements].

Many studies are devoted to the analysis ofinterphase chromosome architecture due to the evidence of functional-dependent spatial organization of the genome. These studies are based on classical cytological methods as well as on biochemical approaches (3C, 4C, 5C, Hi-C) allowing for the detection of long-range interactions between fragments of chromatin fibers, including the genome-wide interactions. In this review, we discuss the results of these researches which make it possible to explain functional-dependent multilevel compartmentalization of the nucleous and unravel the principals of high-level chromatin organization. Special attention is paid to the enchancer-promoter interactions important for the regulation of gene expression. Accordingly, we consider the model of an active chromatin hub and the alternative model of an active chromatin compartment, which was proposed based on reconsideration of some steps of the 3C procedure.

[Spatial organization of house-keeping genes in interphase nuclei].

Spatial organization of the eukaryotic genome is tightly connected to its functioning. In particular, the interaction of gene promoters with remote enhancer elements in active chromatin hubs, as well as the recruitment of genes to the common transcription factories plays an important role in regulation of gene transcription. Most of works related to the analysis of spatial interaction of genome regulatory elements relies on models of tissue-specific genes. Meanwhile, it remains unclear to which extent the spatial organization of chromosomes is guided by house-keeping genes that are transcribed in most of cell types and outnumber the transcribed tissue-specific genes. To address this question, we used the 4C technique to characterize genome-wide the spatial contacts of the chicken house-keeping genes CARHSP1 and TRAP1 situated on chromosome 14. The promoters of these genes had an increased frequency of interaction with chromosome regions enriched in CpG islands and binding motifs for the ubiquitous transcription factor Sp1, both of which mark promoters of house-keeping genes, and overall with transcriptionally active regions. By contrast, the analysis of interaction of a gene poor region of chromosome 14 revealed no such preferences. The evidence for the interaction of house-keeping gene promoters were also obtained in independent cytological experiments aimed at visualization of non-methylated CpG islands in individual nuclei of human cells, which showed clustering of CpG islands in the nuclear space. Altogether, the results of our work suggest that the interaction of house-keeping genes constitutes an important factor that determines the spatial organization of interphase chromosomes.

[The chicken beta-globin genes: a model system for studying transcription regulation at the level of genomic domains].

In the last quarter of the XX century, as a result of studies performed on a number of model systems, a hypothesis was formulated according to which the genome of higher eukaryotes consists of functionally isolated areas named genomic domains. Each domain includes one or more genes and a regulatory system that is normally active only in respect of this domain and allows it to achieve the regulatory autonomy of the neighboring chromosome regions. A genomic domain is characterized by the spectra of covalent histone modifications which define the boundaries of the domain and the degree of chromatin condensation within it, and so, the probability of transcription activation of genes within the domain. Development of the domain hypothesis of genome organization became possible to a large extent through the study of mechanisms of transcriptional regulation of the globin genes in vertebrates. One of the most popular models in this field of molecular biology is the chicken beta-globin gene domain. Based on this model system, the fundamental principles of complex enhancer action in higher eukaryotes have been described, the properties of insulators and functional units of enhancers and promoters have been studied, the influence of covalent histone modifications on the level of chromatin condensation and their role in the regulation of transcription within the domain have been investigated. In this review we summarize the data on the study of the chicken beta-globin gene domain, as well as consider the domain hypothesis of eukaryotic genome organization.

Domains of α- and ß-globin genes in the context of the structural-functional organization of the eukaryotic genome.

The eukaryotic cell genome has a multilevel regulatory system of gene expression that includes stages of preliminary activation of genes or of extended genomic regions (switching them to potentially active states) and stages of final activation of promoters and maintaining their active status in cells of a certain lineage. Current views on the regulatory systems of transcription in eukaryotes have been formed based on results of systematic studies on a limited number of model systems, in particular, on the α- and ß-globin gene domains of vertebrates. Unexpectedly, these genomic domains harboring genes responsible for the synthesis of different subunits of the same protein were found to have a fundamentally different organization inside chromatin. In this review, we analyze specific features of the organization of the α- and ß-globin gene domains in vertebrates, as well as principles of activities of the regulatory systems in these domains. In the final part of the review, we attempt to answer the question how the evolution of α- and ß-globin genes has led to segregation of these genes into two distinct types of chromatin domains situated on different chromosomes.

Transcription factories in the context of the nuclear and genome organization.

In the eukaryotic nucleus, genes are transcribed in transcription factories. In the present review, we re-evaluate the models of transcription factories in the light of recent and older data. Based on this analysis, we propose that transcription factories result from the aggregation of RNA polymerase II-containing pre-initiation complexes assembled next to each other in the nuclear space. Such an aggregation can be triggered by the phosphorylation of the C-terminal domain of RNA polymerase II molecules and their interaction with various transcription factors. Individual transcription factories would thus incorporate tissue-specific, co-regulated as well as housekeeping genes based only on their initial proximity to each other in the nuclear space. Targeting genes to be transcribed to protein-dense factories that contain all factors necessary for transcription initiation and elongation through chromatin templates clearly favors a more economical utilization and better recycling of the transcription machinery.

[Patterns of the histone modifications across the chicken alpha-globin genes domain].

Using native chromatin immunoprecipitation (N-ChIP) followed by TaqMan RT-PCR quantitative analysis we have determined the profiles of histone acetylation and histone methylation within the alpha-globin gene domain before and after switching of embryonic globin genes expression. The results obtained do not support a supposition that the inactivation of the embryonic alpha-type globin gene pi in erythroid cells of the adult lineage is mediated via formation of an inactive chromatin domain. On the other hand we have demonstrated that suppression of the gene pi activity in erythroid cells of adult lineage correlates with the decrease of the histone acetylation level within the embryonic subdomain of the alpha-globin gene domain.

[Expansion of the functional domain of chicken alpha-globin genes].

The transcriptional domain of chicken alpha-globin genes was shown to contain the non-globin coding erythroid-specific TMEM8 gene inducible upon terminal differentiation of erythroblasts. Acquirement by the chicken TMEM8 gene of the erythroid-specific expression correlates with its approachment to the cluster of alpha-globin genes as a result of inversion of a 170-kb chromosomal segment. The human TMEM8 gene is located far from the globin genes and is not expressed in erythroblasts. Transcription of the TMEM8 gene and adult globins in differentiated chicken erythroid cells is controlled by alternative activatory hubs sharing two regulatory elements (including the erythroid enhancer). A conclusion is made that in mature erythroblasts these regulatory elements shuttle between two different activatory hubs.

Transcription factories and spatial organization of eukaryotic genomes.

The phenomenon of association of transcribed genes into so-called transcription factories and also the role of these associations in spatial organization of the eukaryotic genome are actively discussed in the modern literature. Some authors think that the association of transcribed genes into transcription factories constitutes a major factor supporting the function-dependent three-dimensional organization of the interphase genome. In spite of the obvious interest in the problem of spatial organization of transcription in the eukaryotic cell nucleus, the number of experimental studies of transcriptional factories remains rather limited and the results of these studies are often contradictory. In the current review we have tried to critically re-evaluate the published experimental results that constitute the basis for current models and also the models themselves. We have especially analyzed the existing contradictions and attempted to explain them whenever possible. We also discuss new models that can explain the biological significance of clustering of transcribed genes and show possible mechanisms of the origin of transcription factories in the course of evolution.

The organization in micro-loops of an extended fragment of chicken chromosome 14, including the alpha globin gene cluster in the erythroid cells.

It has been shown that the activation of tissue-specific gene transcription during the course of cell differentiation is associated with a spatial reorganization of the genomic domains harboring those specific genes. This reorganization consists of the relocation to the nuclear matrix of the whole genomic domain containing one or more of the genes being transcribed. However, it remains unclear whether, during this process, extended areas of the genome also become attached to the nuclear matrix. We studied the genome´s pattern of interaction with the nuclear matrix in both erythroid and non-erythroid cells of chickens, using a 220Kb region of chromosome #14, which contains the alpha-globin gene cluster and some surrounding house-keeping genes. The results show that in erythroid cells, the fragment of the genome containing the alpha-globin gene domain became spatially arranged into micro-loops which could not be detected by mapping experiments.

Study of spatial organization of chicken alpha-globin gene domain by 3C technique.

This work deals with 3C (Chromosome Conformation Capture) analysis of the chicken alpha-globin gene domain in embryonic erythrocytes and lymphoid cells. Ligation products were quantitatively analyzed by real-time PCR with TaqMan probes. It was found that in lymphoid cells, where alpha-globin gene is not active, the domain has a relatively extended configuration. In embryonic erythrocytes that transcribe alpha(D) and alpha(A) genes, simultaneous interaction of several domain elements was revealed including the major regulatory element, the erythroid-specific DNase I hypersensitive site at a distance of 9 kb upstream from the alpha-globin gene cluster (-9 DHS), promoter of the housekeeping gene CGTHBA, the alpha(D)-globin gene promoter, and the erythroid-specific enhancer located after the alpha-globin gene cluster. We suppose that such interaction is necessary to provide for the active transcription status of the chicken alpha-globin gene domains in erythroid cells.