Segregation of α- and ß-Globin Gene Cluster in Vertebrate Evolution: Chance or Necessity?

The review is devoted to the patterns of evolution of α- and ß-globin gene domains. A hypothesis is presented according to which segregation of the ancestral cluster of α/ß-globin genes in Amniota occurred due to the performance by α-globins and ß-globins of non-canonical functions not related to oxygen transport.

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 ß-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 ß-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.

Distinct Patterns of Colocalization of the CCND1 and CMYC Genes With Their Potential Translocation Partner IGH at Successive Stages of B-Cell Differentiation.

The immunoglobulin heavy chain (IGH) locus is submitted to intra-chromosomal DNA breakages and rearrangements during normal B cell differentiation that create a risk for illegitimate inter-chromosomal translocations leading to a variety of B-cell malignancies. In most Burkitt's and Mantle Cell lymphomas, specific chromosomal translocations juxtapose the IGH locus with a CMYC or Cyclin D1 (CCND1) gene, respectively. 3D-fluorescence in situ hybridization was performed on normal peripheral B lymphocytes induced to mature in vitro from a naive state to the stage where they undergo somatic hypermutation (SHM) and class switch recombination (CSR). The CCND1 genes were found very close to the IGH locus in naive B cells and further away after maturation. In contrast, the CMYC alleles became localized closer to an IGH locus at the stage of SHM/CSR. The colocalization observed between the two oncogenes and the IGH locus at successive stages of B-cell differentiation occurred in the immediate vicinity of the nucleolus, consistent with the known localization of the RAGs and AID enzymes whose function has been demonstrated in IGH physiological rearrangements. We propose that the chromosomal events leading to Mantle Cell lymphoma and Burkitt's lymphoma are favored by the colocalization of CCND1 and CMYC with IGH at the time the concerned B cells undergo VDJ recombination or SHM/CSR, respectively. J. Cell. Biochem. 117: 1506-1510, 2016. © 2016 Wiley Periodicals, Inc.

Characterization of the enhancer element of the Danio rerio minor globin gene locus.

In Danio rerio, the alpha- and beta-globin genes are present in two clusters: a major cluster located on chromosome 3 and a minor cluster located on chromosome 12. In contrast to the segregated alpha- and beta-globin gene domains of warm-blooded animals, in Danio rerio, each cluster contains both alpha- and beta-globin genes. Expression of globin genes present in the major cluster is controlled by an erythroid-specific enhancer similar to the major regulatory element of mammalian and avian alpha-globin gene domains. The enhancer controlling expression of the globin genes present in the minor locus has not been identified yet. Based on the distribution of epigenetic marks, we have selected two genomic regions that might harbor an enhancer of the minor locus. Using transient transfection of constructs with a reporter gene, we have demonstrated that a ~500-bp DNA fragment located ~1.7 Kb upstream of the αe4 gene possesses an erythroid-specific enhancer active with respect to promoters present in both the major and the minor globin gene loci of Danio rerio. The identified enhancer element harbors clustered binding sites for GATA-1, NF-E2, and EKLF similar to the enhancer of the major globin locus on chromosome 3. Both enhancers appear to have emerged as a result of independent evolution of a duplicated regulatory element present in an ancestral single alpha-/beta-globin locus that existed before teleost-specific genome duplication.

Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub.

The current progress in the study of the spatial organization of interphase chromosomes became possible owing to the development of the chromosome conformation capture (3C) protocol. The crucial step of this protocol is the proximity ligation-preferential ligation of DNA fragments assumed to be joined within nuclei by protein bridges and solubilized as a common complex after formaldehyde cross-linking and DNA cleavage. Here, we show that a substantial, and in some cases the major, part of DNA is not solubilized from cross-linked nuclei treated with restriction endonuclease(s) and sodium dodecyl sulphate and that this treatment neither causes lysis of the nucleus nor drastically affects its internal organization. Analysis of the ligation frequencies of the mouse ß-globin gene domain DNA fragments demonstrated that the previously reported 3C signals were generated predominantly, if not exclusively, in the insoluble portion of the 3C material. The proximity ligation thus occurs within the cross-linked chromatin cage in non-lysed nuclei. The finding does not compromise the 3C protocol but allows the consideration of an active chromatin hub as a folded chromatin domain or a nuclear compartment rather than a rigid complex of regulatory elements.

Dynamics of double strand breaks and chromosomal translocations.

Chromosomal translocations are a major cause of cancer. At the same time, the mechanisms that lead to specific chromosomal translocations that associate different gene regions remain largely unknown. Translocations are induced by double strand breaks (DSBs) in DNA. Here we review recent data on the mechanisms of generation, mobility and repair of DSBs and stress the importance of the nuclear organization in this process.

Enhancer Function in the 3D Genome.

In this review, we consider various aspects of enhancer functioning in the context of the 3D genome. Particular attention is paid to the mechanisms of enhancer-promoter communication and the significance of the spatial juxtaposition of enhancers and promoters in 3D nuclear space. A model of an activator chromatin compartment is substantiated, which provides the possibility of transferring activating factors from an enhancer to a promoter without establishing direct contact between these elements. The mechanisms of selective activation of individual promoters or promoter classes by enhancers are also discussed.

Mapping of the nuclear matrix-bound chromatin hubs by a new M3C experimental procedure.

We have developed an experimental procedure to analyze the spatial proximity of nuclear matrix-bound DNA fragments. This protocol, referred to as Matrix 3C (M3C), includes a high salt extraction of nuclei, the removal of distal parts of unfolded DNA loops using restriction enzyme treatment, ligation of the nuclear matrix-bound DNA fragments and a subsequent analysis of ligation frequencies. Using the M3C procedure, we have demonstrated that CpG islands of at least three housekeeping genes that surround the chicken α-globin gene domain are assembled into a complex (presumably, a transcription factory) that is stabilized by the nuclear matrix in both erythroid and non-erythroid cells. In erythroid cells, the regulatory elements of the α-globin genes are attracted to this complex to form a new assembly: an active chromatin hub that is linked to the pre-existing transcription factory. The erythroid-specific part of the assembly is removed by high salt extraction. Based on these observations, we propose that mixed transcription factories that mediate the transcription of both housekeeping and tissue-specific genes are composed of a permanent compartment containing integrated into the nuclear matrix promoters of housekeeping genes and a 'guest' compartment where promoters and regulatory elements of tissue-specific genes can be temporarily recruited.

TMEM8 - a non-globin gene entrapped in the globin web.

For more than 30 years it was believed that globin gene domains included only genes encoding globin chains. Here we show that in chickens, the domain of alpha-globin genes also harbor the non-globin gene TMEM8. It was relocated to the vicinity of the alpha-globin cluster due to inversion of an approximately 170-kb genomic fragment. Although in humans TMEM8 is preferentially expressed in resting T-lymphocytes, in chickens it acquired an erythroid-specific expression profile and is upregulated upon terminal differentiation of erythroblasts. This correlates with the presence of erythroid-specific regulatory elements in the body of chicken TMEM8, which interact with regulatory elements of the alpha-globin genes. Surprisingly, TMEM8 is not simply recruited to the alpha-globin gene domain active chromatin hub. An alternative chromatin hub is assembled, which includes some of the regulatory elements essential for the activation of globin gene expression. These regulatory elements should thus shuttle between two different chromatin hubs.

Co-Regulated Genes and Gene Clusters.

There are many co-regulated genes in eukaryotic cells. The coordinated activation or repression of such genes occurs at specific stages of differentiation, or under the influence of external stimuli. As a rule, co-regulated genes are dispersed in the genome. However, there are also gene clusters, which contain paralogous genes that encode proteins with similar functions. In this aspect, they differ significantly from bacterial operons containing functionally linked genes that are not paralogs. In this review, we discuss the reasons for the existence of gene clusters in vertebrate cells and propose that clustering is necessary to ensure the possibility of selective activation of one of several similar genes.

Manipulation of Cellular Processes via Nucleolus Hijaking in the Course of Viral Infection in Mammals.

Due to their exceptional simplicity of organization, viruses rely on the resources, molecular mechanisms, macromolecular complexes, regulatory pathways, and functional compartments of the host cell for an effective infection process. The nucleolus plays an important role in the process of interaction between the virus and the infected cell. The interactions of viral proteins and nucleic acids with the nucleolus during the infection process are universal phenomena and have been described for almost all taxonomic groups. During infection, proteins of the nucleolus in association with viral components can be directly used for the processes of replication and transcription of viral nucleic acids and the assembly and transport of viral particles. In the course of a viral infection, the usurpation of the nucleolus functions occurs and the usurpation is accompanied by profound changes in ribosome biogenesis. Recent studies have demonstrated that the nucleolus is a multifunctional and dynamic compartment. In addition to the biogenesis of ribosomes, it is involved in regulating the cell cycle and apoptosis, responding to cellular stress, repairing DNA, and transcribing RNA polymerase II-dependent genes. A viral infection can be accompanied by targeted transport of viral proteins to the nucleolus, massive release of resident proteins of the nucleolus into the nucleoplasm and cytoplasm, the movement of non-nucleolar proteins into the nucleolar compartment, and the temporary localization of viral nucleic acids in the nucleolus. The interaction of viral and nucleolar proteins interferes with canonical and non-canonical functions of the nucleolus and results in a change in the physiology of the host cell: cell cycle arrest, intensification or arrest of ribosome biogenesis, induction or inhibition of apoptosis, and the modification of signaling cascades involved in the stress response. The nucleolus is, therefore, an important target during viral infection. In this review, we discuss the functional impact of viral proteins and nucleic acid interaction with the nucleolus during infection.

Mechanisms mediating suppression of globin gene transcription in Danio rerio nonerythroid cells.

We studied the repression of adult and embryo-larval genes of the major globin gene locus in D. rerio fibroblasts. The results obtained suggest that at least some of the globin genes are repressed by Polycomb, similarly to human α-globin genes. Furthermore, within two α/ß globin gene pairs, repression of α-type and ß-type genes appears to be mediated by different mechanisms, as increasing the level of histone acetylation can activate transcription of only ß-type genes.

Nucleolus: A Central Hub for Nuclear Functions.

The nucleolus is the largest and most studied nuclear body, but its role in nuclear function is far from being comprehensively understood. Much work on the nucleolus has focused on its role in regulating RNA polymerase I (RNA Pol I) transcription and ribosome biogenesis; however, emerging evidence points to the nucleolus as an organizing hub for many nuclear functions, accomplished via the shuttling of proteins and nucleic acids between the nucleolus and nucleoplasm. Here, we discuss the cellular mechanisms affected by shuttling of nucleolar components, including the 3D organization of the genome, stress response, DNA repair and recombination, transcription regulation, telomere maintenance, and other essential cellular functions.

Unusual compartmentalization of CTCF and other transcription factors in the course of terminal erythroid differentiation.

It is demonstrated that in chicken embryonic and mature erythrocyte nuclei the distribution of a versatile transcription factor CTCF differs drastically from its distribution in nuclei of proliferating erythroid and non-erythroid cells. In the latter case CTCF was distributed throughout the whole nucleus volume, being concentrated in many small compartments (punctuate nuclear staining). In contrast, in embryonic and mature erythrocytes CTCF was concentrated in a limited number of large compartments. These large CTCF-containing compartments were not observed in other cells. Occasionally, but not in all cells, some of these compartments were localized close to nucleoli but did not colocalize with them. In mature erythrocytes a clear exclusion of CTCF-containing compartments from the chromatin domain was observed. This exclusion correlated with a tight association of CTCF with the nuclear matrix. Concentration in relatively large compartments and exclusion from the chromatin domain in nuclei of mature erythrocytes were also observed for RNA polymerase II and several transcription factors. The data are discussed in the context of a hypothesis postulating that relocalization of different components of the transcriptional machinery from the chromatin domain into the interchromatin compartment is an important step of the terminal inactivation of chicken erythrocyte nuclei.

Repositioning of ETO gene in cells treated with VP-16, an inhibitor of DNA-topoisomerase II.

The translocation t(8;21)(q22;q22) affecting AML1 and ETO genes is known to be one of the frequent chromosome translocations in acute myeloid leukemia. But no data have been available up to date concerning mutual positioning of these particular genes in the nucleus of a living cell as well as the mechanism of their rapprochement and realignment. Here we show that there is no proximity between these two genes in the primary nuclei of normal human male fibroblasts and moreover that these genes are located in different nuclear layers. But we further show that treatment of cells with VP-16 (etoposide), an inhibitor of DNA topoisomerase II widely used in anticancer chemotherapy, causes the ETO gene repositioning which allows AML1 and ETO genes to be localized in the same nuclear layer. Inhibitor studies demonstrate that such an effect is likely to be connected with the formation of stalled cleavable complexes on DNA. Finally, inhibition of ETO gene repositioning by 2,3-butanedione monoxime (BDM) suggests that this process depends on nuclear myosin. Together, our data corroborate the so called "breakage first" model of the origins of recurrent reciprocal translocation.

Chromatin domains and regulation of transcription.

Compartmentalization and compaction of DNA in the nucleus is the characteristic feature of eukaryotic cells. A fully extended DNA molecule has to be compacted 100,000 times to fit within the nucleus. At the same time it is critical that various DNA regions remain accessible for interaction with regulatory factors and transcription/replication factories. This puzzle is solved at the level of DNA packaging in chromatin that occurs in several steps: rolling of DNA onto nucleosomes, compaction of nucleosome fiber with formation of the so-called 30 nm fiber, and folding of the latter into the giant (50-200 kbp) loops, fixed onto the protein skeleton, the nuclear matrix. The general assumption is that DNA folding in the cell nucleus cannot be uniform. It has been known for a long time that a transcriptionally active chromatin fraction is more sensitive to nucleases; this was interpreted as evidence for the less tight compaction of this fraction. In this review we summarize the latest results on structure of transcriptionally active chromatin and the mechanisms of transcriptional regulation in the context of chromatin dynamics. In particular the significance of histone modifications and the mechanisms controlling dynamics of chromatin domains are discussed as well as the significance of spatial organization of the genome for functioning of distant regulatory elements.

Induction of transcription within chromosomal DNA loops flanked by MAR elements causes an association of loop DNA with the nuclear matrix.

The spatial organization of an approximately 170 kb region of human chromosome 19, including CD22 and GPR40-GPR43 genes, was studied using in situ hybridization of a set of cosmid and PAC probes with nuclear halos prepared from proliferating and differentiated HL60 cells. The whole region under study was found to be looped out into the nuclear halo in proliferating cells. It is likely that the loop observed was attached to the nuclear matrix via MAR elements present at the flanks of the area under study. Upon dimethyl sulfoxide-induced differentiation of the cells the looped fragment became associated with the nuclear matrix. This change in the spatial organization correlated with the activation of transcription of at least two (CD22 and GPR43) genes present within the loop. The data obtained are discussed in the framework of the hypothesis postulating that the spatial organization of chromosomal DNA is maintained via constitutive (basic) and facultative (transcription-related) interactions of the latter with the nuclear matrix.

The IGH locus relocalizes to a "recombination compartment" in the perinucleolar region of differentiating B-lymphocytes.

The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events during B-cell differentiation including somatic hypermutation and class switch recombination which mark the end of immunoglobulin gene maturation in germinal centers of secondary lymph nodes. These two events rely on the activity of activation-induced cytidine deaminase (AID) which requires DNA double strand breaks be created, a potential danger to the cell. Applying 3D-fluorescence in situ hybridization coupled with immunofluorescence staining to a previously described experimental system recapitulating normal B-cell differentiation ex vivo, we have kinetically analyzed the radial positioning of the two IGH gene loci as well as their proximity with the nucleolus, heterochromatin and γH2AX foci. Our observations are consistent with the proposal that these IGH gene rearrangements take place in a specific perinucleolar "recombination compartment" where AID could be sequestered thus limiting the extent of its potentially deleterious off-target effects.

Visualization of individual DNA loops and a map of loop domains in the human dystrophin gene.

The organization of the human dystrophin gene into loop domains has been studied using two different experimental approaches: excision of DNA loops mediated by nuclear matrix-bound topoisomerase II and in situ hybridization of different probes with histone-depleted nuclei (nuclear halos). Our objective was to examine if the DNA loops mapped by this biochemical approach coincide with loops visualized by microscopy. The results obtained using both approaches were in good agreement. Eight loops separated by attachment regions of different length were mapped in the upstream part (up to exon 54) of the gene by topoisomerase II-mediated excision. One of these loops was then directly visualized by in situ hybridization of the corresponding bacmid clone with nuclear halos. This is the first direct demonstration that a DNA domain mapped as a loop using a biochemical approach corresponds to a loop visible on cytological preparations. The validity of this result and of the whole map of loop domains was confirmed by in situ hybridization using probes derived from other attachment regions or loops mapped by topoisomerase II-mediated cleavage; these probes hybridized on the core or halo region, respectively, of nuclear halos. Our results demonstrate that a single transcription unit may be organized into several loops and that DNA loop attachment regions may be fairly long. Three out of four replication origins mapped in this gene co-localize with loop attachment regions, and the major deletion hot spot is harbored in an attachment region. These results strongly suggest that partitioning of genomic DNA into specific loops attached to a skeletal structure is a characteristic feature of eukaryotic chromosome organization in interphase.

The upstream area of the chicken alpha-globin gene domain is transcribed in both directions in the same cells.

It was demonstrated previously that in erythroid chicken cells an extended upstream area of the alpha-globin gene domain is transcribed in both directions as a part of ggPRX gene and a part of a full domain transcript of the alpha-globin gene domain. Here, we show that both DNA chains of the above-mentioned region are transcribed in the same cells and that the corresponding transcripts coexist in nuclei. The data obtained suggest that cells possess a molecular mechanism which in some cases prevents the formation of dsRNA and subsequent destruction of both transcripts in spite of the presence of complementary RNA chains in the cell nucleus.