Preferential Co-Expression and Colocalization of rDNA-Contacting Genes with LincRNAs Suggest Their Involvement in Shaping Inter-Chromosomal Interactions with Nucleoli.

Different developmental genes shape frequent dynamic inter-chromosomal contacts with rDNA units in human and Drosophila cells. In the course of differentiation, changes in these contacts occur, coupled with changes in the expression of hundreds of rDNA-contacting genes. The data suggest a possible role of nucleoli in the global regulation of gene expression. However, the mechanism behind the specificity of these inter-chromosomal contacts, which are rebuilt in every cell cycle, is not yet known. Here, we describe the strong association of rDNA-contacting genes with numerous long intergenic non-coding RNAs (lincRNAs) in HEK293T cells and in initial and differentiated K562 cells. We observed that up to 600 different lincRNAs were preferentially co-expressed with multiple overlapping sets of rDNA-contacting developmental genes, and there was a strong correlation between the genomic positions of rDNA-contacting genes and lincRNA mappings. These two findings suggest that lincRNAs might guide the corresponding developmental genes toward rDNA clusters. We conclude that the inter-chromosomal interactions of rDNA-contacting genes with nucleoli might be guided by lincRNAs, which might physically link particular genomic regions with rDNA clusters.

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.

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.

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.

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/.

Structural attributes of nucleotide sequences in promoter regions of supercoiling-sensitive genes: how to relate microarray expression data with genomic sequences.

The level of supercoiling in the chromosome can affect gene expression. To clarify the basis of supercoiling sensitivity, we analyzed the structural features of nucleotide sequences in the vicinity of promoters for the genes with expression enhanced and decreased in response to loss of chromosomal supercoiling in Escherichia coli. Fourier analysis of promoter sequences for supercoiling-sensitive genes reveals the tendency in selection of sequences with helical periodicities close to 10nt for relaxation-induced genes and to 11nt for relaxation-repressed genes. The helical periodicities in the subsets of promoters recognized by RNA polymerase with different sigma factors were also studied. A special procedure was developed for the study of correlations between the intensities of periodicities in promoter sequences and the expression levels of corresponding genes. Significant correlations of expression with the AT content and with AT periodicities about 10, 11, and 50nt indicate their role in regulation of supercoiling-sensitive genes.