N. V. Petrova

Perinucleolar relocalization and nucleolin as crucial events in the transcriptional activation of key genes in mantle cell lymphoma

In mantle cell lymphoma (MCL), one allele of the cyclin D1 (Ccnd1) gene is translocated from its normal localization on chromosome 11 to chromosome 14. This is considered as the crucial event in the transformation process of a normal naive B-cell; however, the actual molecular mechanism leading to Ccnd1 activation remains to be deciphered. Using a combination of three-dimensional and immuno-fluorescence in situ hybridization experiments, the radial position of the 2 Ccnd1 alleles was investigated in MCL-derived cell lines and malignant cells from affected patients. The translocated Ccnd1 allele was observed significantly more distant from the nuclear membrane than its nontranslocated counterpart, with a very high proportion of IgH-Ccnd1 chromosomal segments localized next to a nucleolus. These perinucleolar areas were found to contain active RNA polymerase II (PolII) clusters. Nucleoli are rich in nucleolin, a potent transcription factor that we found to bind sites within the Ccnd1 gene specifically in MCL cells and to activate Ccnd1 transcription. We propose that the Ccnd1 transcriptional activation in MCL cells relates to the repositioning of the rearranged IgH-Ccnd1-carrying chromosomal segment in a nuclear territory with abundant nucleolin and active PolII molecules. Similar transforming events could occur in Burkitt and other B-cell lymphomas.

Specific radial positions of centromeres of human chromosomes X, 1, and 19 remain unchanged in chromatin-depleted nuclei of primary human fibroblasts: Evidence for the organizing role of the nuclear matrix

Radial positions of centromeres of human chromosomes X, 1, and 19 were determined in the nuclei of primary fibroblasts before and after removal of 60%–80% of chromatin. It has been demonstrated that the specific radial positions of these centromeres (more central for the chromosome 19 centromere and more peripheral for the centromeres of chromosomes 1 and X) remain unchanged in chromatin‐depleted nuclei. Additional digestion of nuclear RNA did not influence this specific distribution. These results strongly suggest that the characteristic organization of interphase chromosomes is supported by the proteinous nuclear matrix and is not maintained by simple repulsing of negatively charged chromosomes.

Changes in chromosome positioning may contribute to the development of diseases related to X-chromosome aneuploidy

The radial positions of the centromeric regions of chromosomes 1 and X were determined in normal male fibroblasts (XY) and in fibroblasts from a patient with a rare case of XXXXY polysomy. The centromeric regions and presumably the whole territories of active X chromosomes were demonstrated to occupy similar, although not identical, positions in XY and XXXXY cells. The centromeres of inactive X chromosomes (Barr bodies) were located closer to the nuclear periphery as compared with the centromeres of active X chromosomes. In addition, it was established that the nuclear radial position of gene‐rich chromosome 1 was changed in XXXXY cells as compared to normal XY cells. The data are discussed in the context of the hypothesis postulating that changes in nuclear positioning of chromosomal territories induced by the presence of extra copies of individual chromosomes may contribute to the development of diseases related to different polysomies. J. Cell. Physiol. 213: 278–283, 2007.

Transcription factor RUNX1

Transcription factor RUNX1 is one of the key regulatory proteins in vertebrates. RUNX1 controls hematopoiesis and angiogenesis and is indispensable for the emergence of sites of definitive hematopoiesis during embryogenesis and for blood stem cells differentiation in adult bone marrow. The RUNX1 gene is a frequent target of chromosomal translocations causing acute leukemias. Many human leukemias are some-how associated with RUNX1 mutations. Nevertheless, the precise mechanism guiding the tissue-specific manner of RUNX1 expression remains unknown. The review summarizes the experimental data accumulated over the past twenty years, beginning from the date of the first annotation of the RUNX1 cDNA sequence. The structure, isoforms, covalent modifications, and role in various regulatory cascades are considered for the RUNX1 transcription factor, as well as the RUNX1 expression regulation, mutations, and the involvement in chromosomal translocations.

Genetic and Epigenetic Mechanisms of β-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 β-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 β-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 β-globin genes, leads to development of severe diseases–hemoglobinopathies. In most cases, reactivation of the fetal hemoglobin gene in patients with β-thalassemia and sickle cell disease can reduce negative consequences of irreversible alterations of expression of the β-globin genes. This review focuses on the current state of research on genetic and epigenetic mechanisms underlying stage-specific switching of β-globin genes.

Evolution of the Genome 3D Organization: Comparison of Fused and Segregated Globin Gene Clusters

The genomes are folded in a complex three-dimensional (3D) structure. Some features of this organization are common for all eukaryotes, but little is known about its evolution. Here, we have studied the 3D organization and regulation of zebrafish globin gene domain and compared its organization and regulation with those of other vertebrate species. In birds and mammals, the α- and β-globin genes are segregated into separate clusters located on different chromosomes and organized into chromatin domains of different types, whereas in cold-blooded vertebrates, including Danio rerio, α- and β-globin genes are organized into common clusters. The major globin gene locus of Danio rerio is of particular interest as it is located in a genomic area that is syntenic in vertebrates and is controlled by a conserved enhancer. We have found that the major globin gene locus of Danio rerio is structurally and functionally segregated into two spatially distinct subloci harboring either adult or embryo-larval globin genes. These subloci demonstrate different organization at the level of chromatin domains and different modes of spatial organization, which appears to be due to selective interaction of the upstream enhancer with the sublocus harboring globin genes of the adult type. These data are discussed in terms of evolution of linear and 3D organization of gene clusters in vertebrates.

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/β-globin gene domain exerts enhancer activity toward the promoters of the embryonic-larval and adult globin genes].

In warm-blooded vertebrates, the α- and β-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 β-globin genes form two gene clusters. A major D. rerio globin gene cluster is in chromosome 3 and includes the α- and β-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 β-globin genes of the major cluster. Also, in the 5th intron of c16orf35 in Danio rerio was 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 β-globin genes.

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

The α- and β-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 β-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 β-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 β-globin gene domains of warm-blooded animals is proposed.

Chromatin structure of the joint α/β-globin gene locus of Danio rerio.

59 Although the domains of α and β globin genes of homoiothermal vertebrates are evolutionarily and functionally closely related, they are organized in a fundamentally different manner and are located on different chromosomes. In poikilothermal vertebrates, α and β globin genes are located in close proximity to each other; this configuration is considered ancestral. The purpose of this study was to investigate the domain organization of the joint α/β globin gene locus of Danio rerio. The chromatin status of the joint domain is characterized. It is shown that, judging by its properties, the domain of the joint α/β globin genes of D. rerio is an open domain similar to the domain of α globin genes of homoiothermal animals. Within the concept of domain organization of the genome, a functional unit of the eukaryotic genome is domain—a long chromosomal region containing one or several functionally related genes and their cis reg ulatory elements [1]. A conventional model in the studies of the domain organization of the genome is tissue specific genes. It is known that, in chickens and placental mammals, the genes encoding α and β globin subunits of hemoglobin are located on different chromosomes and arranged in the chromatin domains of fundamentally different types [2]. The α globin gene domain is classified with the open type domains. In the open type domains, chromatin is present in a potentially active (sensitive to DNase I) configuration in both erythroid and nonerythroid cells and replicates in the early S phase of the cell cycle [3]. α Globin genes are tightly integrated with their genomic envi ronment (the main regulatory element of α globin genes in all homoiothermal animals is located in the intron of the neighboring gene) [4]. The β globin gene domain of homoiothermal ani mals is a classic example of a closed type domain. In erythroid cells, chromatin in the β globin domain is sensitive to DNase and undergoes an early replication. In the nonerythroid cells, the β globin gene domain is relatively resistant to the treatment with DNase, is replicated at the end of the S phase [5], and is isolated from the genomic environment by insulators. Signifi cant differences in the packing pattern of the domains of α and β globin genes are correlated with the differ ences in the mechanisms of regulation of their expres sion [6]. As already mentioned, in the genomes of homoio thermal animals, α and β globin genes are located on different chromosomes. However, in the common ancestor of vertebrates, α and β globin genes were apparently located on the same chromosome [7]. Cer tain information about the organization of the ances tral domain can be obtained by studying the organiza tion of the fusion domain of α/β globin genes of mod ern teleost fish, in which adult α and β globin genes (hbaa and hbba) are duplicated and located on the same chromosome in close proximity to each other [8]. In this paper, we attempted to determine the type of genomic domains to which the joint cluster of adult α/β globin genes belongs. The conclusions as to whether this fusion domain belongs to the open or closed type domains can be made on the basis of studying the chromatin sensitivity in the domain to DNase and analyzing the acetylation profile of histones in chromatin for α globin gene, β globin gene, and their common promoter in erythroid and nonerythroid cells. Experiments were performed with erythrocytes of adult fish, in which the adult globin genes are expressed, and cultured fibroblasts of Danio rerio (ATCC no. CRL 2298), in which the globin genes are not expressed. If the fusion domain belongs to the open type domains, a high sensitivity of chromatin to DNase due to retaining of acetylation of histones in chromatin should be retained in fibroblasts, where the globin genes are inactive. The comparison of the kinetics of DNase cleavage of the studied genomic fragment and fragments containing an known tran scriptionally active gene (β actin) and a known repressed gene (crystallin) provides insights on the Chromatin Structure of the Joint α/β Globin Gene Locus of Danio rerio