S. V. Razin

Dynamics of unfolded nucleosomal fiber.

The rigidity of chromatin fiber solenoidal structure in different states of condensation was evaluated with the help of gel-electrophoresis. A new property of the unfolded nucleosomal fiber-the capacity to condense with temperature-was demonstrated. These results together with our previously obtained data (W.A. Krajewski et al., Mol. Gen. Genet. 230, pp. 442-448, 1991; W.A. Krajewski et al., Ibid. 231, pp. 17-22, 1991) testify that changes in DNA linking number of transcriptionally active minichromosomes arise in vivo from alteration of nucleosomal solenoid parameters (i.e. from supernucleosomal level of chromatin organization), rather than from core histone modifications only or from increased flexibility of DNA within nucleosomes.

Flexibility of DNA Within Transcriptionally Active Nucleosomes: Analysis by Circular Dichroism Measurements

The conformational flexibility of DNA in transcriptionally active chromatin fractions has been estimated by circular dichroism spectroscopy analysis and was found to be restricted in the same fashion as in bulk chromatin. The observation is discussed in the context of different models of active chromatin organization.

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.

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

Heat-shock induced γH2AX foci are associated with the nuclear matrix only in S-phase cells

130 The phosphorylated form of histone H2AX (γH2AX) is a marker of double stranded DNA breaks and damaged replicative forks. Its phosphorylation occurs not only in response to various genotoxic agents but also as a result of thermal stress. The nature of the inducer that triggers the formation of γH2AX foci determines the extent of their interaction with the nuclear matrix structure. In this work we analyzed whether the γH2AX foci formed during hyperthermia were associated with the nuclear matrix. It was found that this association depended on the cell cycle phase and was specific for S phase cells.

Inhibition of DNA topoisomerase II with etoposide induces association of DNA topoisomerase IIα, DNA topoisomerase IIβ, and nucleolin with BCR 2 of the ETO gene

Chromosomal translocation t(8;21)(q22;q22), which affects AML1 and ETO genes, is often detected in patients with acute myeloid leukemia. The mechanisms that ensure preferential rearrangement at topoisomerase breakpoints remain obscure. It is known that the preferential breakpoints in DNA strands are clustered within the narrow breakpoint-clustering regions (BCRs). We have earlier shown that, in the nuclei of human primary fibroblasts, these two genes are not located close to one another; furthermore, they are located in different chromosome layers. We discovered that the treatment of cells with etoposide (VP-16), an inhibitor of DNA topoisomerase II, changed the preferential nuclear location of the ETO gene so that the AML1 and ETO genes became located in the same chromosome layer. Inhibitor analysis showed that this effect was most likely due to formation of specific stalled cleavable complexes on DNA in the presence of etoposide and that nuclear myosin is apparently involved in the translocation. In this study, we showed that doublestrand breaks are clustered in the immediate vicinity of the nucleolus, which directly indicates that the latter may be involved in illegitimate recombination leading to the occurrence of translocations. We continued studies using chromatin precipitation and real-time TaqMan PCR and found that the BCR2 region of the ETO gene was enriched not only with the DNA topoisomerase II α but also with the nucleolus-specific proteins DNA topoisomerase II β and nucleolin.

Genes surrounding the cluster of tissue-specific α-globin genes in chicken genome are expressed in both erythroid and lymphoid cells

224 The cluster of chicken α -globin genes comprises three genes— α A, α D, and π —that are transcribed only in erythroid cells, although they are located in the open chromatin region of all types of cells [1]. These tissuespecific genes are surrounded by the housekeeping genes; the level of expression of the latter remains unknown. In this study, we have assessed the transcriptional state of the genes surrounding the domain of chicken α -globin genes as well as relative levels of their expression in erythroid and lymphoid cells. The genes flanking the α -globin gene cluster on the telomere side (five genes located in a 130-kb genomic region) and on the centromere side (four genes located in a 85-kb genome region) proved to be expressed in both erythroid and lymphoid cells. Note that the level of expression of two genes ( TMEM6 and TMEM8 ) that are immediately adjacent to the cluster of α -globin genes in the direction of the centromere was significantly higher than the level of expression of the same gene in the lymphoid cells. The remaining genes displayed similar levels of expression in the erythroid and lymphoid cells. In the chicken genome, the cluster of α -globin genes is located in the pretelomeric region of chromosome 14. The genes flanking this cluster are shown in Fig. 1, whereas their functions can be seen from Table 1. Note that the functions of these genes are known only for humans [2]. In all vertebrate animals, a rather extended region of synteny was found, which includes the cluster of α -globin genes and some flanking genes [3–5]. In hens, this region is the shortest and it includes genes located in the region flanking the α -globin gene cluster from the side of centromere. The relative positions of some unrelated genes and regulatory elements are evolutionarily conserved, suggesting the possibility of interaction between these genes and regulatory elements. Indeed, in mice, the promoters of these genes moved to the transcriptional centers responsible for transcription of the housekeeping gene around the globin-gene cluster [6]. We assume that a similar mechanism underlies the regulation of expression of chicken α -globin genes. This assumption was indirectly confirmed by our previous data on different spatial organization of chicken α -globin domain and the surrounding genes in erythroid and nonerythroid cells. To further confirm the above assumption, it was necessary to determine which genes surrounding the α -globin domain in the chicken genome are constantly expressed (i.e., are the housekeeping genes).The goal of this study was to determine the transcriptional state of the genes nearest to chicken α -globin genes was determined in cells that differentiated in either the erythroid or nonerythroid directions.