[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.

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.

[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.

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.

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.

Mechanisms controlling activation of the alpha-globin gene domain in chicken erythroid cells.

In this review we consider the organization of the chicken alpha-globin gene domain and mechanisms regulating the activity of this tissue-specific gene domain located in a potentially active (characterized by an increased sensitivity to nucleases) chromatin configuration in cells of all lineages. Both regulatory mechanisms ensuring repression of alpha-globin genes in non-erythroid cells and mechanisms responsible for activation of transcription of these genes during erythroid cell differentiation are discussed. The analysis of the structure-function organization of the chicken alpha-globin gene domain presented in this review is based mainly on the authors' own results obtained over the last 20 years. On discussing the hypotheses explaining the mechanisms controlling the functional activity of chicken alpha-globin gene domain, data obtained in studies of alpha-globin gene domains of other vertebrates are also analyzed.

Non-clonability correlates with genomic instability: a case study of a unique DNA region.

Instability of eukaryotic DNA in constructs propagated in prokaryotic hosts is a frequently observed phenomenon. With the exception of a very high A+T-content and the presence of multiple repetitions, no general rule at the basis of this phenomenon is actually known. The intergenic spacer located between the pi and alpha(D) chicken alpha-type globin genes is frequently deleted from recombinant phages and plasmids. Here we have cloned this DNA fragment using a specially designed bacterial strain (SURE competent cells, Stratogene). Comparative analysis of DNA of recombinant clones bearing deletions and clones containing the intact genomic DNA fragment has revealed two important DNA sequence motifs that contribute to the unclonability of eukaryotic DNA in prokaryotic cells. First, the similarity to bacterial transposons (i.e. the presence of repeats flanking a several kilobase DNA fragment) may cause the loss of the fragment during propagation of the recombinant DNA in E. coli. Second, a high content of rotationally correlated kinkable elements (TG*CA steps) may result in non-clonability of the DNA sequence. Interestingly, the latter type of "unclonable" DNA sequence motifs identified in the globin gene domain is unstable (frequently rearranged) also in the eukaryotic chromosome resulting in a local polymorphism. In the chicken domain of alpha globin genes this unstable DNA sequence seems to be partially protected by interaction with nuclear matrix proteins.

Nonlymphoid cultured cells possess a system controlling cellular compatibility.

We show that various nonlymphoid cultured cells can activate the production of cytotoxic factors in response to direct contact with cells of a different kind. Accumulation of cytotoxic factors in the medium was detected 1 h after contact of K562 and L929 cells or after contact of L929 cells with purified membranes of K562 cells. TNF-alpha or immunologically related proteins, or both, but not Fas-ligand or lymphotoxin, were also accumulated in membranes of K562 and L929 cells shortly after these cells had been allowed to contact each other. The cytotoxic factors expressed by nonlymphoid cells trigger apoptosis of target cells. These observations strongly suggest that nonlymphoid cells possess molecular mechanisms controlling cellular compatibility.

Extensive methylation of a part of the CpG island located 3.0-4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells.

Here, we show that in the chicken genome, the domain of alpha-globin genes is preceded by a CpG island of which the downstream part ( approximately 0.65 kbp) is heavily methylated in lymphoid cells; it is either non-methylated or undermethylated in erythroid cells. Recombinant plasmids were constructed with the corresponding DNA fragment (called "uCpG") placed upstream to a reporter CAT gene expressed from the promoter of the alpha(D) chicken globin gene. Selective methylation of CpG dinucleotides within the uCpG fragment suppressed fivefold the expression of the CAT gene, when neither this gene itself nor the alpha(D) promoter were methylated. Methylation of CpG dinucleotides within the alpha(D) gene promoter did not modify the suppression effect exerted by methylated uCpG. We interpret these results within the frame of the hypothesis postulating, that methylation of the upstream CpG island of the chicken alpha-globin gene domain may play an essential role in silencing the alpha-globin genes in non-erythroid cells.

Functional analysis of DNA sequences located within a cluster of DNase I hypersensitive sites colocalizing with a MAR element at the upstream border of the chicken alpha-globin gene domain.

We have cloned and sequenced a genomic DNA fragment of chicken containing a cluster of DNase I hypersensitive sites (DHS) located 11-15 kb upstream from the first gene of the alpha-globin gene domain and including a constitutive DHS flanked by two erythroid-specific ones. A 1.2-kb subfragment of the DNA fragment under study located upstream to the constitutive DHS and colocalizing roughly with one of the erythroid-specific DHS was shown to possess the properties of a matrix association region (MAR). The cloned DNA sequences were tested for their ability to serve as promoters and/or influence transcription from the promoter of the alphaD globin gene. In the region studied, we did not find any promoters or enhancers that were active in erythroid cells. The whole DNase I hypersensitive region and some of its subfragments showed a silencing effect when placed downstream from the reporter gene. The expression of the reporter gene was completely abolished, however, when these DNA fragments were placed between the alphaD promoter and the reporter gene. Thus, they seem to act as transcription "terminators." Numerous polyadenylation signals (AATAAA) and an AT-rich palindrome were found within the sequenced DNA fragment. These observations are discussed within the frame of the hypothesis postulating that continuous transcription is essential for maintaining the active status of genomic domains. Furthermore, it is suggested that the DNA fragment studied contains a negative control element that keeps globin genes silent within the chromatin domain permanently open in nonerythroid cells.

Interbands of Drosophila melanogaster polytene chromosomes contain matrix association regions.

The DNA of three previously cloned interband regions (85D9/D10, 86B4/B6, and 61C7/C8) of Drosophila melanogaster polytene chromosomes has been tested for the presence of matrix association regions (MAR), using the in vitro matrix-binding assay of Cockerill and Garrard. MARs were found in all three interband regions under study. These results are discussed in frames of a model postulating that interband regions of polytene chromosomes correspond to the chromosomal DNA loop borders, which can be identified in interphase nuclei using biochemical approaches.