DNA double-strand breaks (DSBs) are involved in many cellular mechanisms, including replication, transcription, and genome rearrangements. The recent observation that hot spots of DSBs in human chromosomes delimit DNA domains that possess coordinately expressed genes suggests a strong relationship between the organization of transcription patterns and hot spots of DSBs. In this study, we performed mapping of hot spots of DSBs in a human 43-kb ribosomal DNA (rDNA) repeated unit. We observed that rDNA units corresponded to the most fragile sites in human chromosomes and that these units possessed at least nine specific regions containing clusters of extremely frequently occurring DSBs, which were located exclusively in non-coding intergenic spacer (IGS) regions. The hot spots of DSBs corresponded to only a specific subset of DNase-hypersensitive sites, and coincided with CTCF, PARP1, and HNRNPA2B1 binding sites, and H3K4me3 marks. Our rDNA-4C data indicate that the regions of IGS containing the hot spots of DSBs often form contacts with specific regions in different chromosomes, including the pericentromeric regions, as well as regions that are characterized by H3K27ac and H3K4me3 marks, CTCF binding sites, ChIA-PET and RIP signals, and high levels of DSBs. The data suggest a strong link between chromosome breakage and several different mechanisms of epigenetic regulation of gene expression.

Gene-specific silencing refers to a phenomenon in which expression of an individual gene can be specifically repressed by different mechanisms on the levels of transcription, RNA splicing, transport, degradation in nuclei or cytoplasm, or blocking of translation. In different species gene-specific silencing was observed by expression or injections of antiparallel double-stranded RNA formed by a fragment of mRNA and antisense RNA. Here we show a potent and specific gene silencing in bacteria by expression of RNA, that is complementary in a parallel orientation to Escherichia coli lon mRNA. Moreover, the expression of parallel RNA is more effective at producing interference than expression of antisense RNA corresponding to the same mRNA region. Both effects of interference mediated either by parallel RNA or antiparallel RNA gradually decrease up to the 40th generation. Together with in vitro nuclease protection studies these results indicate that a parallel RNA duplex might be formed in vivo and both types of duplexes, antiparallel or parallel, can induce gene-specific silencing by similar mechanisms.There has been dramatic recent progress in uncovering the gene-specific silencing in a number of organisms (1-3). Several lines of evidence suggest that dsRNA 1 is the effector molecule responsible for RNA-mediated silencing or co-suppression. dsRNA is formed by mRNA and antisense RNA, that corresponds to the non-coding strand of the same gene. However, in experiments on Caenorhabditis elegans it was demonstrated that injections of gel-purified antisense RNA corresponding to an abundant transcript is less active at producing interference than in vitro annealed dsRNA samples (1). The purification was performed to remove the traces of dsRNA from in vitro synthesized RNA preparations because of the nonspecific activity of RNA polymerases.It was concluded that the observation of co-suppression and RNA interference uncovered the existence of a novel cellular mechanisms for regulation of gene expression (2, 4). The phenomenon has been described in fungi, protozoa, plants, invertebrates, and vertebrates (5-10). This suggests a evolutionary conservation of the physiological mechanisms involved.The mechanisms of RNAi remain largely unknown. It was concluded that RNAi and co-suppression work by an equivalent core mechanism produces decrease or elimination of a target mRNA transcript (2,3,4,6). In experiments on gene silencing ("quelling") in Neurospora crassa it was shown that in the mutant defective in quelling the gene specifying RNA-dependent RNA polymerase was affected (5). The genes involved in RNA degradation also could be connected with RNAi (12). It was considered that RNAi mechanisms might operate at the level of transcription and involve proteins of the polycomb complex (13). But recently it was clearly demonstrated that this transcriptional cosuppression mechanism is distinct from RNAi and involve homology recognition at the DNA level (14). The fact demonstrates that although the phenomenology of gene-specific silenc...

Genome instability plays a key role in multiple biological processes and diseases, including cancer. Genome-wide mapping of DNA double-strand breaks (DSBs) is important for understanding both chromosomal architecture and specific chromosomal regions at DSBs. We developed a method for precise genome-wide mapping of blunt-ended DSBs in human chromosomes, and observed non-random fragmentation and DSB hot spots. These hot spots are scattered along chromosomes and delimit protected 50–250 kb DNA domains. We found that about 30% of the domains (denoted forum domains) possess coordinately expressed genes and that PARP1 and HNRNPA2B1 specifically bind DNA sequences at the forum domain termini. Thus, our data suggest a novel type of gene regulation: a coordinated transcription or silencing of gene clusters delimited by DSB hot spots as well as PARP1 and HNRNPa2B1 binding sites.

Bacterial RNases are promising tools for the development of anticancer drugs. Neoplastic transformation leads to enhanced accumulation of rRNA and tRNA, and altered expression of regulatory noncoding RNAs. Cleavage of RNA in cancer cells is the main reason for the cytotoxic effects of exogenic RNases. We have shown that binase, a cytotoxic ribonuclease from Bacillus intermedius, affects the total amount of intracellular RNA and the expression of proapoptotic and antiapoptotic mRNAs. For four cell lines, we visualized cellular RNA by fluorescence microscopy, and determined RNA levels, viability and apoptosis by flow cytometry. We found that the level of cellular RNA was decreased in cells that were sensitive to the cytotoxic effects of binase. The RNA level was lowered by 44% in HEK cells transfected with the hSK4 gene of the Ca2+‐activated potassium channels (HEKhSK4) and by 20% in kit‐transformed myeloid progenitor FDC‐P1iR1171 cells. The most significant decrease in RNA levels was registered in the subpopulations of apoptotic cells. However, the binase‐induced RNA decrease did not correlate with apoptosis. Kit‐transformed cells with binase‐induced RNA decrease retained viability if the interleukin‐dependent proliferation pathway was activated. Using quantitative RT‐PCR with RNA samples isolated from the binase‐treated HEKhSK4 cells, we found that the amount of mRNA of the antiapoptotic bcl‐2 gene in vivo was reduced about two‐fold. In contrast, expression of the proapoptotic genes p53 and hSK4 was increased 1.5‐fold and 4.3‐fold, respectively. These results show that binase is a regulator of RNA‐dependent processes of cell proliferation and apoptosis.

The main epigenetic mechanisms in regulation of gene expression are discussed. The definition of epigenetics and its specific mechanisms including DNA methylation and gene imprinting, modifications of nucleosomal histones associated with silencing or activation of gene transcription, RNA interference, chromosomal silencing, and the role of mobile elements are discussed.

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Some RNases selectively attack malignant cells, triggering an apoptotic response, and therefore are considered as alternative chemotherapeutic drugs. Here we studied the effects of Bacillus intermedius RNase (binase) on murine myeloid progenitor cells FDC-P1; transduced FDC-P1 cells ectopically expressing mutated human KIT N822K oncogene and/or human AML1-ETO oncogene; and human leukemia Kasumi-1 cells expressing both of these oncogenes. Expression of both KIT and AML1-ETO oncogenes makes FDC-P1 cells sensitive to the toxic effects of binase. Kasumi-1 cells were the most responsive to the toxic actions of binase among the cell lines used in this work with an IC50 value of 0.56 µM. Either blocking the functional activity of the KIT protein with imatinib or knocking-down oncogene expression using lentiviral vectors producing shRNA against AML1-ETO or KIT eliminated the sensitivity of Kasumi-1 cells to binase toxic action and promoted their survival, even in the absence of KIT-dependent proliferation and antiapoptotic pathways. Here we provide evidence that the cooperative effect of the expression of mutated KIT and AML1-ETO oncogenes is crucial for selective toxic action of binase on malignant cells. These findings can facilitate clinical applications of binase providing a useful screen based on the presence of the corresponding target oncogenes in malignant cells.

We have observed the existence of a parallel complementary region between two Drosophila DNA sequences, fragments of the suffix [(1986) EMBO J, 5, 2341-23471 and a 5'-non-coding sequence of the alcohol dehydrogenase gene [(1983[( ) Cell 33, 125-1331. The region includes -40 bp, 76% of which are complementary in the same polarity. Synthetic complementary 16 bp oligonucleotides corresponding to this region which were bound by the 5'-ends through a 1.6-hexanediol bridge form a duplex which displays both melting and annealing as judged by UV absorbance. Anti parallel complementary 16 bp long oligonucleotides bound by the 5'-3' ends through the same bridge and a single-strand sequence were used as controls. The Hoechst 33 258 drug binds to this parallel duplex of DNA; however, the properties of such a complex testify against the B-form of the duplex.