Induction of the Erythroid Differentiation of K562 Cells Is Coupled with Changes in the Inter-Chromosomal Contacts of rDNA Clusters.

The expression of clusters of rDNA genes influences pluripotency; however, the underlying mechanisms are not yet known. These clusters shape inter-chromosomal contacts with numerous genes controlling differentiation in human and Drosophila cells. This suggests a possible role of these contacts in the formation of 3D chromosomal structures and the regulation of gene expression in development. However, it has not yet been demonstrated whether inter-chromosomal rDNA contacts are changed during differentiation. In this study, we used human leukemia K562 cells and induced their erythroid differentiation in order to study both the changes in rDNA contacts and the expression of genes. We observed that approximately 200 sets of rDNA-contacting genes are co-expressed in different combinations in both untreated and differentiated K562 cells. rDNA contacts are changed during differentiation and coupled with the upregulation of genes whose products are mainly located in the nucleus and are highly associated with DNA- and RNA-binding, along with the downregulation of genes whose products mainly reside in the cytoplasm or intra- or extracellular vesicles. The most downregulated gene is ID3, which is known as an inhibitor of differentiation, and thus should be switched off to allow for differentiation. Our data suggest that the differentiation of K562 cells leads to alterations in the inter-chromosomal contacts of rDNA clusters and 3D structures in particular chromosomal regions as well as to changes in the expression of genes located in the corresponding chromosomal domains. We conclude that approximately half of the rDNA-contacting genes are co-expressed in human cells and that rDNA clusters are involved in the global regulation of gene expression.

Genome-Wide Study of Colocalization between Genomic Stretches: A Method and Applications to the Regulation of Gene Expression.

In this paper, we describe a method for the study of colocalization effects between stretch-stretch and stretch-point genome tracks based on a set of indices varying within the (-1, +1) interval. The indices combine the distances between the centers of neighboring stretches and their lengths. The extreme boundaries of the interval correspond to the complete colocalization of the genome tracks or its complete absence. We also obtained the relevant criteria of statistical significance for such indices using the complete permutation test. The method is robust with respect to strongly inhomogeneous positioning and length distribution of the genome tracks. On the basis of this approach, we created command-line software, the Genome Track Colocalization Analyzer. The software was tested, compared with other available packages, and applied to particular problems related to gene expression. The package, Genome Track Colocalization Analyzer (GTCA), is freely available to the users. GTCA complements our previous software, the Genome Track Analyzer, intended for the search for pairwise correlations between point-like genome tracks (also freely available). The corresponding details are provided in Data Availability Statement at the end of the text.

Genes Possessing the Most Frequent DNA DSBs Are Highly Associated with Development and Cancers, and Essentially Overlap with the rDNA-Contacting Genes.

Double-strand DNA breakes (DSBs) are the most deleterious and widespread examples of DNA damage. They inevitably originate from endogenous mechanisms in the course of transcription, replication, and recombination, as well as from different exogenous factors. If not properly repaired, DSBs result in cell death or diseases. Genome-wide analysis of DSBs has revealed the numerous endogenous DSBs in human chromosomes. However, until now, it has not been clear what kind of genes are preferentially subjected to breakage. We performed a genetic and epigenetic analysis of the most frequent DSBs in HEK293T cells. Here, we show that they predominantly occur in the active genes controlling differentiation, development, and morphogenesis. These genes are highly associated with cancers and other diseases. About one-third of the genes possessing frequent DSBs correspond to rDNA-contacting genes. Our data suggest that a specific set of active genes controlling morphogenesis are the main targets of DNA breakage in human cells, although there is a specific set of silent genes controlling metabolism that also are enriched in DSBs. We detected this enrichment by different activators and repressors of transcription at DSB target sites, as well breakage at promoters. We propose that both active transcription and silencing of genes give a propensity for DNA breakage. These results have implications for medicine and gene therapy.

Fragments of rDNA Genes Scattered over the Human Genome Are Targets of Small RNAs.

Small noncoding RNAs of different origins and classes play several roles in the regulation of gene expression. Here, we show that diverged and rearranged fragments of rDNA units are scattered throughout the human genome and that endogenous small noncoding RNAs are processed by the Microprocessor complex from specific regions of ribosomal RNAs shaping hairpins. These small RNAs correspond to particular sites inside the fragments of rDNA that mostly reside in intergenic regions or the introns of about 1500 genes. The targets of these small ribosomal RNAs (srRNAs) are characterized by a set of epigenetic marks, binding sites of Pol II, RAD21, CBP, and P300, DNase I hypersensitive sites, and by enrichment or depletion of active histone marks. In HEK293T cells, genes that are targeted by srRNAs (srRNA target genes) are involved in differentiation and development. srRNA target genes are enriched with more actively transcribed genes. Our data suggest that remnants of rDNA sequences and srRNAs may be involved in the upregulation or downregulation of a specific set of genes in human cells. These results have implications for diverse fields, including epigenetics and gene therapy.

The Role of rDNA Clusters in Global Epigenetic Gene Regulation.

The regulation of gene expression has been studied for decades, but the underlying mechanisms are still not fully understood. As well as local and distant regulation, there are specific mechanisms of regulation during development and physiological modulation of gene activity in differentiated cells. Current research strongly supports a role for the 3D chromosomal structure in the regulation of gene expression. However, it is not known whether the genome structure reflects the formation of active or repressed chromosomal domains or if these structures play a primary role in the regulation of gene expression. During early development, heterochromatinization of ribosomal DNA (rDNA) is coupled with silencing or activation of the expression of different sets of genes. Although the mechanisms behind this type of regulation are not known, rDNA clusters shape frequent inter-chromosomal contacts with a large group of genes controlling development. This review aims to shed light on the involvement of clusters of ribosomal genes in the global regulation of gene expression. We also discuss the possible role of RNA-mediated and phase-separation mechanisms in the global regulation of gene expression by nucleoli.

Chromosomal Translocations in NK-Cell Lymphomas Originate from Inter-Chromosomal Contacts of Active rDNA Clusters Possessing Hot Spots of DSBs.

Endogenous hot spots of DNA double-strand breaks (DSBs) are tightly linked with transcription patterns and cancer. There are nine hot spots of DSBs (denoted Pleiades) in human rDNA units that are located exclusively inside the intergenic spacer (IGS). Profiles of Pleiades coincide with the profiles of γ-H2AX, 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. Circular chromosome conformation capture (4C) data indicate that the rDNA units often make contact with a specific set of chromosomal regions containing genes that are involved in differentiation and cancer. Interestingly, these regions also often possess hot spots of DSBs that provide the potential for Robertsonian and oncogenic translocations. In this study, we searched for translocations in which rDNA clusters are involved. The whole genome sequence (WGS) data of normal T cells and NK-cell lymphomas from the same individuals revealed numerous translocations in which Pleiades were involved. The sites of these translocations in normal T cells and in the lymphomas were mostly different, although there were also some common sites. The genes at translocations in normal cells and in lymphomas are associated with predominantly non-overlapping lists of genes that are depleted with silenced genes. Our data indicate that rDNA-mediated translocations occur at about the same frequency in the normal T cells and NK-lymphoma cells but differ at particular sites that correspond to open chromatin. We conclude that oncogenic translocations lead to dysregulation of a specific set of genes controlling development. In normal T cells and in NK cells, there are hot spots of translocations at sites possessing strong H3K27ac marks. The data indicate that Pleiades are involved in rDNA-mediated translocation.

Structural coordinates: A novel approach to predict protein backbone conformation.

MOTIVATION: Local protein structure is usually described via classifying each peptide to a unique class from a set of pre-defined structures. These classifications may differ in the number of structural classes, the length of peptides, or class attribution criteria. Most methods that predict the local structure of a protein from its sequence first rely on some classification and only then proceed to the 3D conformation assessment. However, most classification methods rely on homologous proteins' existence, unavoidably lose information by attributing a peptide to a single class or suffer from a suboptimal choice of the representative classes. RESULTS: To alleviate the above challenges, we propose a method that constructs a peptide's structural representation from the sequence, reflecting its similarity to several basic representative structures. For 5-mer peptides and 16 representative structures, we achieved the Q16 classification accuracy of 67.9%, which is higher than what is currently reported in the literature. Our prediction method does not utilize information about protein homologues but relies only on the amino acids' physicochemical properties and the resolved structures' statistics. We also show that the 3D coordinates of a peptide can be uniquely recovered from its structural coordinates, and show the required conditions under various geometric constraints.

Dynamics of Whole-Genome Contacts of Nucleoli in Drosophila 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. Furthermore, the nucleoli of human cells shape the direct contacts with genes that are involved in differentiation and cancer. Here, we identified and categorized the genes located in the regions where rDNA clusters make frequent contacts. Using a 4C approach, we demonstrate that in Drosophila S2 cells, rDNA clusters form contacts with genes that are involved in chromosome organization and differentiation. Heat shock treatment induces changes in the contacts between nucleoli and hundreds of genes controlling morphogenesis. We show that nucleoli form contacts with regions that are enriched with active or repressive histone marks and where small non-coding RNAs are mapped. These data indicate that rDNA contacts are involved in the repression and activation of gene expression and that rDNA clusters orchestrate large groups of Drosophila genes involved in differentiation.

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. However, the genes that reside within the rDNA-contacting regions have not been identified. The purpose of this study was to detect and characterize the regions where rDNA clusters make frequent contacts and to identify and categorize genes located in these regions. We analyzed the regions that contact rDNA using 4C data and show that these regions are enriched with genes specifying transcription factors and non-coding RNAs involved in differentiation and development. The rDNA-contacting genes are involved in neuronal development and are associated with different cancers. Heat shock treatment led to dramatic changes in the pattern of rDNA-contacting sites, especially in the regions possessing long stretches of H3K27ac marks. Whole-genome analysis of rDNA-contacting sites revealed specific epigenetic marks and the transcription sites of 20-100 nt non-coding RNAs in these regions. The rDNA-contacting genes jointly regulate many genes that are involved in the control of transcription by RNA polymerase II and the development of neurons. Our data suggest a role for rDNA clusters in the differentiation of human cells and carcinogenesis.

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.

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. 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.

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. 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.

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.

Genome-wide study of correlations between genomic features and their relationship with the regulation of gene expression.

The broad class of tasks in genetics and epigenetics can be reduced to the study of various features that are distributed over the genome (genome tracks). The rapid and efficient processing of the huge amount of data stored in the genome-scale databases cannot be achieved without the software packages based on the analytical criteria. However, strong inhomogeneity of genome tracks hampers the development of relevant statistics. We developed the criteria for the assessment of genome track inhomogeneity and correlations between two genome tracks. We also developed a software package, Genome Track Analyzer, based on this theory. The theory and software were tested on simulated data and were applied to the study of correlations between CpG islands and transcription start sites in the Homo sapiens genome, between profiles of protein-binding sites in chromosomes of Drosophila melanogaster, and between DNA double-strand breaks and histone marks in the H. sapiens genome. Significant correlations between transcription start sites on the forward and the reverse strands were observed in genomes of D. melanogaster, Caenorhabditis elegans, Mus musculus, H. sapiens, and Danio rerio. The observed correlations may be related to the regulation of gene expression in eukaryotes. Genome Track Analyzer is freely available at http://ancorr.eimb.ru/.

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.

Genome-wide profiling of forum domains in Drosophila melanogaster.

Forum domains are stretches of chromosomal DNA that are excised from eukaryotic chromosomes during their spontaneous non-random fragmentation. Most forum domains are 50-200 kb in length. We mapped forum domain termini using FISH on polytene chromosomes and we performed genome-wide mapping using a Drosophila melanogaster genomic tiling microarray consisting of overlapping 3 kb fragments. We found that forum termini very often correspond to regions of intercalary heterochromatin and regions of late replication in polytene chromosomes. We found that forum domains contain clusters of several or many genes. The largest forum domains correspond to the main clusters of homeotic genes inside BX-C and ANTP-C, cluster of histone genes and clusters of piRNAs. PRE/TRE and transcription factor binding sites often reside inside domains and do not overlap with forum domain termini. We also found that about 20% of forum domain termini correspond to small chromosomal regions where Ago1, Ago2, small RNAs and repressive chromatin structures are detected. Our results indicate that forum domains correspond to big multi-gene chromosomal units, some of which could be coordinately expressed. The data on the global mapping of forum domains revealed a strong correlation between fragmentation sites in chromosomes, particular sets of mobile elements and regions of intercalary heterochromatin.