Dynamics of Whole-Genome Contacts of Nucleoli in Cells Suggests a Role for rDNA Genes in Global Epigenetic Regulation.

Chromosomes are organized into 3D structures that are important for the regulation of gene expression and differentiation. Important role in formation of inter-chromosome contacts play rDNA clusters that make up nucleoli. In the course of differentiation, heterochromatization of rDNA units in mouse cells is coupled with the repression or activation of different genes.

[Drosophila rDNA Genes Shape the Stable Contacts with the Tlk Gene at the Expression Area of Small RNAs and Affect on Looped Domains inside the Gene].

In experiments on mouse and human cells it was demonstrated that rDNA plays an important role in epigenetic regulation of many genes. To identify and study rDNA-contacting genes in Drosophila we used the 4С (circular chromosome conformation capture) approach. We detected very stable contacts of rDNA genes within a 5-kb region inside the Tlk gene residing in X chromosome.

[Contact Sites of rDNA Clusters with FANK1 Gene Correspond to Repressed Chromatin].

rDNA genes play an important role in epigenetic regulation and in differentiation of eukaryotic cells. Using the 4C (circular chromosome conformation capture) approach and model HEK293T cells, we analyzed the rDNA-contacting gene FANK1, using anchor located inside rDNA genes. At the 5' end of the FANK1 gene we detected frequent contacts with rDNA clusters.

Interchromosomal Contacts of rDNA Clusters with DUX Genes in Human Chromosome 4 Are Very Sensitive to Heat Shock Treatment.

In order to study the effects of heat shock treatment on the distribution of rDNA contacts at the region possessing DUX genes inside chromosome 4 we used 4C approach. Our data indicate that the treatment removes the frequent rDNA contacts in this region. The recent data on involvement of superenhancers that are decorated by broad H3K27ac marks in the phase separation mechanisms and the previous data demonstrating that these broad marks are the favorite sites of rDNA contacts taken together with our data on sensitivity of the contacts to the heat shock treatment suggest that the phase separation mechanisms are involved in the reversible rDNA-mediated regulation of gene expression via the contacts.

rDNA Clusters Make Contact with Genes that Are Involved in Differentiation and Cancer and Change Contacts after Heat Shock Treatment.

Human rDNA clusters form numerous contacts with different chromosomal regions as evidenced by chromosome conformation capture data. Heterochromatization of rDNA genes leads to heterochromatization in different chromosomal regions coupled with the activation of the transcription of genes related to differentiation. These data suggest a role for rDNA clusters in the regulation of many human genes.

[Homeotic DUX4 Genes that Control Human Embryonic Development at the Two-Cell Stage Are Surrounded by Regions Contacting with rDNA Gene Clusters].

Many human genes that control human embryonic development and differentiation of human cells form chromosomal contact with rRNA gene clusters, which are involved in the epigenetic regulation of many genes. The sites of rRNA gene contact often fall on extended (up to 50 kb) regions containing a chromatin mark, H3K27ac histone, typical for superenhancers, as well as on pericentromeric and subtelomeric regions of chromosomes. We found that the DUX4 genes located in the subtelomeric region of human chromosome 4 are surrounded by regions that are often in contact with the rRNA genes.

[Mutation Frequencies in RNAi Targets in HIV-1 Genomes Obtained from Blood Plasma of Patients Receiving Anti-Retroviral Therapy].

Gene therapy for AIDS based on RNA interference (RNAi) is currently looked upon as a promising alternative to conventional antiretroviral chemotherapy. The high variability of HIV-1 is the main challenge in developing new approaches to AIDS therapy. To date, about 18 million HIV-1 infected individuals receive antiretroviral therapy worldwide.

[Mutation Frequencies in HIV-1 Genome in Regions Containing Efficient RNAi Targets As Calculated from Ultra-Deep Sequencing Data].

HIV-1 is one of the most variable viruses. The development of gene therapy technology using RNAi for AIDS/HIV-1 treatment is a potential alternative for traditional anti-retroviral therapy. Anti-HIV-1 siRNA should aim to exploit the most conserved viral targets.

Link Between Double-Strand DNA Break Hotspots and Transcription Regulation: Forum Domains - 50-250 kb Chromosome Regions Containing Coordinately Expressed Genes.

The data on forum domains formed by DNA double-strand break (DSB) hotspots are reviewed including forum domain identification by pulsed-field gel electrophoresis, whole genome mapping of these domains using deep sequencing strategies, analysis of gene expression in forum domains, and binding of nuclear proteins to their boundaries. Earlier unpublished data by the authors are presented. The "piano playing" hypothesis is suggested based on coordinated active transcription in some of the forum domains and coordinated silencing in the majority of them.

A Bioinformatic Pipeline for Monitoring of the Mutational Stability of Viral Drug Targets with Deep-Sequencing Technology.

The efficient development of antiviral drugs, including efficient antiviral small interfering RNAs (siRNAs), requires continuous monitoring of the strict correspondence between a drug and the related highly variable viral DNA/RNA target(s). Deep sequencing is able to provide an assessment of both the general target conservation and the frequency of particular mutations in the different target sites. The aim of this study was to develop a reliable bioinformatic pipeline for the analysis of millions of short, deep sequencing reads corresponding to selected highly variable viral sequences that are drug target(s).

Individual effects of the copia and gypsy enhancer and insulator on chromatin marks, eRNA synthesis, and binding of insulator proteins in transfected genetic constructs.

Enhancers and insulators are involved in the regulation of gene expression, but the basic underlying mechanisms of action of these elements are unknown. We analyzed the individual effects of the enhancer and the insulator from Drosophila mobile elements copia [enh(copia)] and gypsy using transfected genetic constructs in S2 cells. This system excludes the influence of genomic cis regulatory elements.

Six Highly Conserved Targets of RNAi Revealed in HIV-1-Infected Patients from Russia Are Also Present in Many HIV-1 Strains Worldwide.

RNAi has been suggested for use in gene therapy of HIV/AIDS, but the main problem is that HIV-1 is highly variable and could escape attack from the small interfering RNAs (siRNAs) due to even single nucleotide substitutions in the potential targets. To exhaustively check the variability in selected RNA targets of HIV-1, we used ultra-deep sequencing of six regions of HIV-1 from the plasma of two independent cohorts of patients from Russia. Six RNAi targets were found that are invariable in 82%-97% of viruses in both cohorts and are located inside the domains specifying reverse transcriptase (RT), integrase, vpu, gp120, and p17.

Analysis of Variability in HIV-1 Subtype A Strains in Russia Suggests a Combination of Deep Sequencing and Multitarget RNA Interference for Silencing of the Virus.

Any method for silencing the activity of the HIV-1 retrovirus should tackle the extremely high variability of HIV-1 sequences and mutational escape. We studied sequence variability in the vicinity of selected RNA interference (RNAi) targets from isolates of HIV-1 subtype A in Russia, and we propose that using artificial RNAi is a potential alternative to traditional antiretroviral therapy. We prove that using multiple RNAi targets overcomes the variability in HIV-1 isolates.

[Mutation frequencies in HIV-1 subtype-A genome in regions containing efficient RNAi targets].

The development of gene-therapy technology using RNAi for AIDS/HIV-1 treatment is a prospective alternative to traditional anti-retroviral therapy. RNAi targets could be selected in HIV-1 transcripts and in CCR5 mRNA. Previously, we experimentally selected a number of efficient siRNAs that target HIV-1 RNAs.

Hot spots of DNA double-strand breaks in human rDNA units are produced in vivo.

Endogenous hot spots of DNA double-strand breaks (DSBs) are tightly linked with transcription patterns and cancer genomics(1,2). There are nine hot spots of DSBs located in human rDNA units(3-6). Here we describe that the profiles of these hot spots coincide with the profiles of γ-H2AX or H2AX, strongly suggesting a high level of in vivo breakage inside rDNA genes.

Conserved sequences in the current strains of HIV-1 subtype A in Russia are effectively targeted by artificial RNAi in vitro.

Highly active antiretroviral therapy has greatly reduced the morbidity and mortality of AIDS. However, many of the antiretroviral drugs are toxic with long-term use, and all currently used anti-HIV agents generate drug-resistant mutants. Therefore, there is a great need for new approaches to AIDS therapy.

Genome-wide mapping of hot spots of DNA double-strand breaks in human cells as a tool for epigenetic studies and cancer genomics.

Hot spots of DNA double-strand breaks (DSBs) are associated with coordinated expression of genes in chromosomal domains (Tchurikov et al., 2011 [1]; 2013). These 50-150-kb DNA domains (denoted "forum domains") can be visualized by separation of undigested chromosomal DNA in pulsed-field agarose gels (Tchurikov et al.

Hot spots of DNA double-strand breaks and genomic contacts of human rDNA units are involved in epigenetic regulation.

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.

Molecular analysis of transcription start sites of separate copies of the suffix short retroelement in genome of Drosophila.

In order to study TSS in the suffix element, we used total RNA isolated from ovaries of Drosophila melanogaster. Using a 5'-RACE System (Invitrogen) and 454 sequencing, we found the full-length suffix sense transcripts. However, most 5'-RACE reads (>70%) correspond to 5'-truncated transcripts lacking the first 33-39 nucleotides.

DNA double-strand breaks coupled with PARP1 and HNRNPA2B1 binding sites flank coordinately expressed domains in human chromosomes.

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.

Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells.

Cytotoxic exogenous RNases triggering apoptotic response in malignant cells have potential as anticancer drugs; surprisingly, detailed characterization of the RNase-induced apoptosis has not been conducted so far. Here we show that a cytotoxic RNase from Bacillus intermedius (binase) induces extrinsic and intrinsic apoptotic pathways in leukemic Kasumi-1 cells. The experiments were performed using TaqMan Array Human Apoptosis 96-well Plate for gene expression analysis, and flow cytometry.

Generation of genetic constructs that simultaneously express several shRNAs.

RNAi has potential as an antiviral gene therapy strategy. Cassette constructs simultaneously expressing several siRNAs could prove to be the most efficient technique in developing gene therapy approaches for highly mutable viruses such as HIV-1. Here we describe a rapid and cost-saving protocol to generate cassettes that simultaneously express three siRNAs for repression of HIV-1 and CCR5 transcripts.