Daria M. Fedoseeva

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. 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.

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. 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.

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. RNAi is a powerful means of inhibiting HIV-1 production in human cells. We propose to use RNAi for gene therapy of HIV/AIDS. Previously we identified a number of new biologically active siRNAs targeting several moderately conserved regions in HIV-1 transcripts. Here we analyze the heterogeneity of nucleotide sequences in three RNAi targets in sequences encoding the reverse transcriptase and integrase domains of current isolates of HIV-1 subtype A in Russia. These data were used to generate genetic constructs expressing short hairpin RNAs 28-30-bp in length that could be processed in cells into siRNAs. After transfection of the constructs we observed siRNAs that efficiently attacked the selected targets. We expect that targeting several viral genes important for HIV-1 reproduction will help overcome the problem of viral adaptation and will prevent the appearance of RNAi escape mutants in current virus strains, an important feature of gene therapy of HIV/AIDS.

Mapping of genomic double-strand breaks by ligation of biotinylated oligonucleotides to forum domains: Analysis of the data obtained for human rDNA units

DNA double-strand breaks (DSBs) are associated with different physiological and pathological processes in different organisms. To understand the role of DSBs in multiple cellular mechanisms, a robust method for genome-wide mapping of chromosomal breaks at one-nucleotide resolution is required. Many years ago, we detected large DNA fragments migrating from DNA-agarose plugs in pulsed-field gels, which we named ‘forum domains’ [1,2]. Recently, we developed a method for genome-wide mapping of DSBs that produces these 50–150 kb DNA domains using microarrays or 454 sequencing (Tchurikov et al., 2011; 2013). Now we have used Illumina sequencing to map DSBs in repetitive rDNA units in human HEK293T cells. Here we describe in detail the experimental design and bioinformatics analysis of the data deposited in the Gene Expression Omnibus with accession number GSE49302 and associated with the study published in the Journal of Molecular Cell Biology (Tchurikov et al., 2014).

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 genomics1,2. There are nine hot spots of DSBs located in human rDNA units3–6. 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. The data were confirmed by microscopic observation of the largest γ-H2AX foci inside nucleoli in interphase chromosomes. In metaphase chromosomes, we observed that only some portion of rDNA clusters possess γ-H2AX foci and that all γ-H2AX foci co-localize with UBF-1 binding sites, which strongly suggests that only active rDNA units possess the hot spots of DSBs. Both γ-H2AX and UBF-1 are epigenetically inherited and thus indicate the rDNA units that were active in the previous cell cycle. These results have implications for diverse fields, including epigenetics and cancer genomics.

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.

Abstract 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. The optimal number of targets critically depends on the conservation of the target sequences. The total number of targets that are conserved with a probability of 0.7–0.8 should exceed at least 2. Combining deep sequencing and multitarget RNAi may provide an efficient approach to cure HIV/AIDS.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. The optimal number of targets critically depends on the conservation of the target sequences. The total number of targets that are conserved with a probability of 0.7-0.8 should exceed at least 2. Combining deep sequencing and multitarget RNAi may provide an efficient approach to cure HIV/AIDS.

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). The suggested bioinformatic pipeline combines the available programs and the ad hoc scripts based on an original algorithm of the search for the conserved targets in the deep sequencing data. We also present the statistical criteria for the threshold of reliable mutation detection and for the assessment of variations between corresponding data sets. These criteria are robust against the possible sequencing errors in the reads. As an example, the bioinformatic pipeline is applied to the study of the conservation of RNA interference (RNAi) targets in human immunodeficiency virus 1 (HIV-1) subtype A. The developed pipeline is freely available to download at the website http://virmut.eimb.ru/. Brief comments and comparisons between VirMut and other pipelines are also presented.

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. The analysis of mutation frequencies and their characteristics inside the targets suggests a likely role for APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, A3G) in G-to-A mutations and a predominant effect of RT biases in the detected variability of the virus. The lowest frequency of mutations was detected in the central part of all six targets. We also discovered that the identical RNAi targets are present in many HIV-1 strains from many countries and from all continents. The data are important for both the understanding of the patterns of HIV-1 mutability and properties of RT and for the development of gene therapy approaches using RNAi for the treatment of HIV/AIDS.

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., 1988; 1992) and used for genome-wide mapping of the DSBs that produce them. Recently, we described nine hot spots of DSBs in human rDNA genes and observed that, in rDNA units, the hot spots coincide with CTCF binding sites and H3K4me3 marks (Tchurikov et al., 2014), suggesting a role for DSBs in active transcription. Here we have used Illumina sequencing to map DSBs in chromosomes of human HEK293T cells, and describe in detail the experimental design and bioinformatics analysis of the data deposited in the Gene Expression Omnibus with accession number GSE53811 and associated with the study published in DNA Research (Kravatsky et al., 2015). Our data indicate that H3K4me3 marks often coincide with hot spots of DSBs in HEK293T cells and that the mapping of these hot spots is important for cancer genomic studies.

Molecular analysis of enhancer RNAs and chromatin modifications in the region of their synthesis in Drosophila cells possessing genetic constructs.

7 The mechanisms of action of enhancers and insuu lators remain unclear, although ample data have been accumulated as a result of studying them by genetic and molecular methods. It is known that enhancers induce the synthesis of noncoding, soocalled enhancer RNA (eRNA) on DNA sequences located between the enhancer and promoter [1, 2]. This synthesis proceeds both downstream and upstream of enhancers, and its level in both directions is comparable to the level of transcription of the gene controlled by the enhancer [2]. Recently, using the ChIPPSeq approach, a number of histone modifications, which are assumed to be caused by enhancers, have been identified in the Drosophila chromatin [3, 4]. However, it cannot be ruled out that these modifications could also be caused by other elements of the Drosophila genome, because a possible effect of other elements in this approach was not controlled and prevented. Recently, we have devell oped a model system for analysis of individual regulaa tory elements in Drosophila, which is based on the use of transfected genetic constructs that contain specific regulatory elements and the luciferase reporter gene [2, 5–7]. This system makes it possible to rule out the cisseffect of other possible genomic regulators on the distribution of chromatin modifications in genetic constructs. In this work, to study the mechanisms of action of enhancers and insulators, we have used this system containing the enhancer of the copia transposs able element (enh(copia)) and the gypsy insulator. The objective of this study was to determine the size of eRNA, to identify those histone modifications that can be caused by the enhancer when it is isolated from other possible regulatory elements, and to find out how the insulator located in different positions in the genetic constructs affects the synthesis of eRNA and relevant histone modifications. As a result, we were the first to obtain data showing that the length of eRNA in Drosophila varies from 300 to 1050 bp and that the syn thesis of these eRNAs is inhibited by the insulator. Using this model system, we also revealed that only some of the previously described histone modificaa tions are caused directly by the enhancer. We assume that the enhancer through enhancerrassociated proo teins and protein complexes causes a number of chroo matin modifications. The latter can serve as signals for the synthesis of eRNA. The results of this study conn firmed our earlier assumptions that enhancers …