[DNA Damage Response in Nucleoli].

Nucleoli, the largest subnuclear compartments, are formed around arrays of ribosomal gene repeats transcribed by RNA polymerase I. The primary function of nucleoli is ribosome biogenesis. Specific DNA damage response mechanisms exist to maintain the genomic stability of ribosomal repeats. Here, we provide a snapshot of our current understanding of processes involved in nucleolar DNA damage response. We discuss structure and function of ribosomal repeats, techniques developed for studying DNA damage response in nucleoli, as well as molecular mechanisms of DNA damage-induced repression of nucleolar transcription and nucleoli reorganization.

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.

[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 Organization of the Eukaryotic Cell Nucleus and Transcription Regulation: Introduction to This Special Issue of Biochemistry (Moscow).

This issue of Biochemistry (Moscow) is devoted to the cell nucleus and mechanisms of transcription regulation. Over the years, biochemical processes in the cell nucleus have been studied in isolation, outside the context of their spatial organization. Now it is clear that segregation of functional processes within a compartmentalized cell nucleus is very important for the implementation of basic genetic processes. The functional compartmentalization of the cell nucleus is closely related to the spatial organization of the genome, which in turn plays a key role in the operation of epigenetic mechanisms. In this issue of Biochemistry (Moscow), we present a selection of review articles covering the functional architecture of the eukaryotic cell nucleus, the mechanisms of genome folding, the role of stochastic processes in establishing 3D architecture of the genome, and the impact of genome spatial organization on transcription regulation.

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.

Genetic and Epigenetic Mechanisms of ß-Globin Gene Switching.

Vertebrates have multiple forms of hemoglobin that differ in the composition of their polypeptide chains. During ontogenesis, the composition of these subunits changes. Genes encoding different α- and ß-polypeptide chains are located in two multigene clusters on different chromosomes. Each cluster contains several genes that are expressed at different stages of ontogenesis. The phenomenon of stage-specific transcription of globin genes is referred to as globin gene switching. Mechanisms of expression switching, stage-specific activation, and repression of transcription of α- and ß-globin genes are of interest from both theoretical and practical points of view. Alteration of balanced expression of globin genes, which usually occurs due to damage to adult ß-globin genes, leads to development of severe diseases - hemoglobinopathies. In most cases, reactivation of the fetal hemoglobin gene in patients with ß-thalassemia and sickle cell disease can reduce negative consequences of irreversible alterations of expression of the ß-globin genes. This review focuses on the current state of research on genetic and epigenetic mechanisms underlying stage-specific switching of ß-globin genes.

[Intersubunit Mobility of the Ribosome].

Ribosomes are ribonucleoprotein nanoparticles synthesizing all proteins in living cells. The function of the ribosome is to translate the genetic information encoded in a nucleotide sequence of mRNA into the amino acid sequence of a protein. Each translation step (occurring after the codon-dependent binding of the aminoacyl-tRNA with the ribosome and mRNA) includes (i) the transpeptidation reaction and (ii) the translocation that unidirectionally drives the mRNA chain and mRNA-bound tRNA molecules through the ribosomal intersubunit space; the latter process is driven by the free energy of the chemical reaction of transpeptidation. Thus, the translating ribosome can be considered a conveying protein-synthesizing molecular machine. In this review we analyze the role of ribosomal intersubunit mobility in the process of translocation.

[Role of the Nucleolus in Rearrangements of the IGH Locus].

The review summarizes the results from a series of studies focusing on the role that the nucleolus plays in maturation of the IGH locus and the choice of its partner genes in leukemia-associated translocations. The role of nuclear compartmentalization and nuclear localization of translocated oncogenes in ectopic activation of their transcription is discussed.

[Comparative analysis of the synchronization methods of normal and transformed human cells].

Reactions of genetically identical cells to various exogenous and endogenous stimuli can vary significantly. One of the main factors of this non-genetic cellular heterogeneity is the cell cycle. The most convenient way to study the subcellular processes depending on the cell cycle stage is the synchronization of the cells. Toxic inhibitors of DNA replication and/or mitotic spindle assembly are typically used to synchronize cells. It is important to accurately select the synchronization method for a particular experiment. In this study, we performed a comparative analysis of the synchronization methods of normal and transformed human cells, paying special attention to the accuracy of synchronization and toxicity of the methods used.

[Main regulatory element (MRE) of the Danio rerio α/ß-globin gene domain exerts enhancer activity toward the promoters of the embryonic-larval and adult globin genes].

In warm-blooded vertebrates, the α- and ß-globin genes are organized in domains of different types and are regulated in different fashion. In cold-blooded vertebrates and, in particular, the tropical fish Danio rerio, the α- and ß-globin genes form two gene clusters. A major D. rerio globin gene cluster is in chromosome 3 and includes the α- and ß-globin genes of embryonic-larval and adult types. The region upstream of the cluster contains c16orf35, harbors the main regulatory element (MRE) of the α-globin gene domain in warm-blooded vertebrates. In this study, transient transfection of erythroid cells with genetic constructs containing a reporter gene under the control of potential regulatory elements of the domain was performed to characterize the promoters of the embryonic-larval and adult α- and ß-globin genes of the major cluster. Also, in the 5th intron of c16orf35 in Danio reriowas detected a functional analog of the warm-blooded vertebrate MRE. This enhancer stimulated activity of the promoters of both adult and embryonic-larval α- and ß-globin genes.

A requiem to the nuclear matrix: from a controversial concept to 3D organization of the nucleus.

The first papers coining the term "nuclear matrix" were published 40 years ago. Here, we review the data obtained during the nuclear matrix studies and discuss the contribution of this controversial concept to our current understanding of nuclear architecture and three-dimensional organization of genome.

Heat Stress-Induced Transcriptional Repression.

Heat stress is one of the most popular models for studying the regulation of gene expression. For decades, researchers' attention was focused on the study of the mechanisms of transcriptional activation of stress-induced genes. Although the phenomenon of heat stress-induced global transcriptional repression is known for a long time, the exact molecular mechanisms of such a repression are poorly explored. In this mini-review, we attempt to summarize the existing experimental data on heat stress-induced transcriptional repression.

[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.

[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.

[Detection of complementary transcripts in intergenic region of the chicken α-globin gene domain].

Using strand-specific reverse transcription followed by Real Time PCR analysis we have characterized the transcription profile of the segment of chicken α-globin gene domain harboring embryonic gene π, adult gene αD and spacer region separating these genes. It has been demonstrated that in erythroid cells of adult lineage the spacer region is transcribed in both directions. These results suggest a possibility that switching of α-globin genes expression is controlled by RNA-interference mechanism.

[Joint locus of a/b-globin genes in Danio rerio is segregated into structural subdomains active at different stages of development].

In the domain model of eukaryotic genome organization, the functional unit of the genome, along with the relevant regulatory elements, is considered to be a gene or a gene family. In hot-blooded vertebrate animals, the domains of a- and b-globin genes are positioned at different chromosomes and are organized and regulated in different fashion. In cold-blooded animals, in particular in tropical fish Danio rerio, a- and b globin genes are located in a common gene cluster. However, the joint a/b-globin gene cluster is subdivided into two development stage-specific subdomains, the adult one and the embryonic-larval one. In an attempt to find out whether this functional segregation correlates with structural segregation of the domain we compared the DNase I sensitivity and profiles of histone modifications of adult and embryonic-larval segments of the domain in cultured D. rerio fibroblasts. We have demonstrated that, in these nonerythroid cells, adult and embryonic- larval subdomains possess different DNase I sensitivities and different profiles of H3K27me3, a histone modification introduced by PRC2 complex. These observations suggest that joint a/b globin gene domain of Danio rerio is segregated into two structural subdomain harboring adult and embryonic-larval globin genes.

Heat stress induces formation of cytoplasmic granules containing HSC70 protein.

Using indirect immunofluorescence, in this study we showed that the constitutive heat shock protein HSC70 forms granule-like structures in the cytoplasm of human cells several days after the exposure to heat stress. It was shown that this effect is not the result of HSC70 overexpression under heat stress and is not due to the formation of hyperthermia-induced translational stress granules in the cytoplasm.

Nuclear localization of translocation partners in differentiating B-cells.

We studied the nuclear localization and relative position in the nuclear space of malignant translocation partner genes c-Myc, CCND1, and IGH locus in naive and differentiating B cells. We have shown that, during B-cell maturation, c-Myc and IGH loci become closer to each other. In differentiating lymphocytes, those alleles of c-Myc and IGH that are in close spatial proximity to each other are closer to the nucleolus. For the CCND1 locus, no correlation between the proximity of loci and nuclear localization was found. These data suggest that the close spatial proximity of c-Myc and IGH loci during B-cell maturation increase the probability of malignant translocation.

Nuclear matrix and structural and functional compartmentalization of the eucaryotic cell nucleus.

Becoming popular at the end of the 20th century, the concept of the nuclear matrix implies the existence of a nuclear skeleton that organizes functional elements in the cell nucleus. This review presents a critical analysis of the results obtained in the study of nuclear matrix in the light of current views on the organization of the cell nucleus. Numerous studies of nuclear matrix have failed to provide evidence of the existence of such a structure. Moreover, the existence of a filamentous structure that supports the nuclear compartmentalization appears to be unnecessary, since this function is performed by the folded genome itself.

Evolution of α- and ß-globin genes and their regulatory systems in light of the hypothesis of domain organization of the genome.

The α- and ß-globin gene domains are a traditional model for study of the domain organization of the eucaryotic genome because these genes encode hemoglobin, a physiologically important protein. The α-globin and ß-globin gene domains are organized in completely different ways, while the expression of globin genes is tightly coordinated, which makes it extremely interesting to study the origin of these genes and the evolution of their regulatory systems. In this review, the organization of the α- and ß-globin gene domains and their genomic environment in different taxonomic groups are comparatively analyzed. A new hypothesis of possible evolutionary pathways for segregated α- and ß-globin gene domains of warm-blooded animals is proposed.