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.

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.

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.

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.

Gene expression patterns are very sensitive to external influences and are reflected in phenotypic changes. It was previously described that transferring melanoma cells from a plastic surface to Matrigel led to formation of de novo vascular networks—vasculogenic mimicry—that are characteristic to a stemness phenotype in aggressive tumors. Up to now there was no detailed data about the gene signature accompanying this process. Here, we show that this transfer shortly led to extremely strong epigenetic changes in gene expression in the melanoma cells. We observed that on Matrigel numerous genes controlling ribosome biogenesis were upregulated. However, most of the activated genes were inhibitors of the differentiation genes (ID1, ID2, and ID3). At the same time, the genes that control differentiation were downregulated. Both the upregulated and the downregulated genes are simultaneously targeted by different transcription factors shaping sets of co-expressed genes. The specific group of downregulated genes shaping contacts with rDNA genes are also associated with the H3K27me3 mark and with numerous lincRNAs and miRNAs. We conclude that the stemness phenotype of melanoma cells is due to the downregulation of developmental genes and formation of dedifferentiated cells.

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.