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. 2023 Apr 12;80(5):121.
doi: 10.1007/s00018-023-04770-3.

Actively transcribed rDNA and distal junction (DJ) sequence are involved in association of NORs with nucleoli

Mikhail Liskovykh #  1 , Nikolai S Petrov #  2 , Vladimir N Noskov  2 , Hiroshi Masumoto  3 , William C Earnshaw  4 , David Schlessinger  5 , Svetlana A Shabalina  6 , Vladimir Larionov  7 , Natalay Kouprina  8
Affiliations

Actively transcribed rDNA and distal junction (DJ) sequence are involved in association of NORs with nucleoli

Mikhail Liskovykh et al. Cell Mol Life Sci. .

Abstract

Although they are organelles without a limiting membrane, nucleoli have an exclusive structure, built upon the rDNA-rich acrocentric short arms of five human chromosomes (nucleolar organizer regions or NORs). This has raised the question: what are the structural features of a chromosome required for its inclusion in a nucleolus? Previous work has suggested that sequences adjacent to the tandemly repeated rDNA repeat units (DJ, distal junction sequence) may be involved, and we have extended such studies by addressing several issues related to the requirements for the association of NORs with nucleoli. We exploited both a set of somatic cell hybrids containing individual human acrocentric chromosomes and a set of Human Artificial Chromosomes (HACs) carrying different parts of a NOR, including an rDNA unit or DJ or PJ (proximal junction) sequence. Association of NORs with nucleoli was increased when constituent rDNA was transcribed and may be also affected by the status of heterochromatin blocks formed next to the rDNA arrays. Furthermore, our data suggest that a relatively small size DJ region, highly conserved in evolution, is also involved, along with the rDNA repeats, in the localization of p-arms of acrocentric chromosomes in nucleoli. Thus, we infer a cooperative action of rDNA sequence-stimulated by its activity-and sequences distal to rDNA contributing to incorporation into nucleoli. Analysis of NOR sequences also identified LncRNA_038958 in the DJ, a candidate transcript with the region of the suggested promoter that is located close to the DJ/rDNA boundary and contains CTCF binding sites. This LncRNA may affect RNA Polymerase I and/or nucleolar activity. Our findings provide the basis for future studies to determine which RNAs and proteins interact critically with NOR sequences to organize the higher-order structure of nucleoli and their function in normal cells and pathological states.

Keywords: Human artificial chromosome (HAC); Nucleolar organizer regions (NORs); Ribosomal DNA; Transformation-associated recombination (TAR); rDNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
a Schematic representation of the acrocentric human chromosomes. Scheme of an acrocentric chromosome with the rDNA portion and distal (DJ) and proximal (PJ) junction sequences. rDNA repeats are located on q Arm of the chromosome. The number of rDNA repeats varies on the chromosomes. Each ~ 13.3 kb rDNA repeat is composed of the transcribed region encoding 45S rRNA (5′-ETS, 18S, ITS1, 5.8S, ITS2, 28S and 3′-ETS) and a ~ 30.7 kb IGS (intergenic spacer sequence). The 13.3 kb transcribed human 45S rDNA region is shown in blue. Telo-telomere regions. CEN-centromere region. b Southern blot analysis of six human/mouse hybrid cell lines containing human chromosomes 13, 14, 15, 21 and 22 [A9 (#13) 89-2 (chr13), A9 hygro14 10F (chr14), A9 (Neo15)-3 (chr15), A9 #21-16 (chr21), A9#22 (γ2) (chr22γ2) and A9HyTK-22 (chr22TK)]. Genomic DNA possessing one of the acrocentric chromosomes was isolated from each hybrid cell line, digested by EcoRV and separated by CHEF gel electrophoresis (range 50 kb–1.1 Mb). The rDNA repeats were detected with a radioactively labeled probe specific to the rDNA intergenic spacer (IGS). Red arrowheads indicate the fragments that contain rDNA repeats. M1 a marker [CHEF DNA Size Lambda Ladder (BIO-RAD)]. c FISH analysis of human/mouse hybrid cell line A9#22 (γ2) containing chromosome 22 with the human IGS FISH probe specific to the intergenic spacer of the rDNA unit (see “Materials and methods”). Chromosome 22 (green) is indicated by a white arrowhead. d A copy number of the rDNA repeats in a human acrocentric chromosome in each hybrid cell line was estimated by quantitative real-time PCR (qPCR). e 3D immuno-FISH was performed on human/mouse hybrid cells. Anti-RRP1 antibodies (Nop52) (red) used to visualize nucleoli were combined with a BAC FISH probe containing the IGS sequence (green). Nuclei were stained with DAPI (blue). Nucleolar- and non-nucleolar-associated NORs are indicated by white arrowheads. f Quantification of 3D immuno-FISH showing the percentage of cells in which the NORs from human acrocentric chromosomes and human non-acrocentric chromosomes 4 and 18 associated with mouse nucleoli. For each cell line, the experiment was replicated three times with the total number of the nucleolar localized and non-localized chromosomes ranged from 141 to 317 (Table S3)
Fig. 2
Fig. 2
Transcription reactivation of the silent rDNA cluster from the human chromosome 13 in a mouse background. a Scheme of the experiment. On day 0, A9#13 89-2 human/mouse hybrid cells carrying the human chromosome 13 were infected with four lentiviruses coding transcription factors TAF1A, TAF1B, TAF1C, and TAF1D with an efficiency close to 100%. The cells were incubated for 7 days and then transferred to the gelatin-covered microscopy glass slide. After that, the 3D immuno-FISH analysis was performed. b 3D immuno-FISH was performed on A9#13 89-2 human/mouse hybrid cells. Anti-RRP1 antibodies (Nop52) (red) used to visualize nucleoli were combined with a BAC FISH probe containing the IGS sequence (green). Nuclei were stained with DAPI (blue). Nucleolar- and non-nucleolar-associated NORs are indicated by white arrowheads. c, d Quantification of 3D immuno-FISH analysis showing the percentage of cells in which the human chromosome 13 is associated with mouse nucleoli before and after reactivation of transcription of the human ribosomal rDNA genes. Transcription is activated twofold compared to the basic level of transcription which is considered as 1 (b). As a control #1, untreated A9#13 89-2 human/mouse hybrid cells by were used. As a control #2, infection of A9#13 89-2 cells by LVTHM-EGFP lentiviruses were used (b). The total number of the nucleolar localized and non-localized chromosomes ranges from 331 to 405 (Table S3). Percentages were calculated for replicate cultures. Significant differences were calculated using a two-tailed nonparametric Mann–Whitney U test. Statistically significant difference is indicated with a red asterisk (c, d)
Fig. 3
Fig. 3
a TAR isolation of the rDNA repeat (rDNA; TAR1 construct), transcribed (45S; TAR2 construct) and non-transcribed (IGS; TAR3 construct) parts of the rDNA repeat, proximal junction (PJ; TAR4 construct) and distal junction (PJ; TAR5 construct) regions, and the IGS-Δ construct in which the 3′ end of the IGS sequence is deleted (see “Materials and methods” for detail). b Schemes of the HAC vector carrying either the rDNA repeat (HAC/rDNA), transcribed (HAC/45S) and non-transcribed (HAC/IGS) parts of the rDNA repeat, the PJ fragment (HAC/PJ), the DJ fragment (HAC/DJ) or the IGS-Δ BAC construct (HAC/IGS-Δ). A HAC carrying the GFP transgene (HAC/GFP) was used as a control. c 3D immuno-FISH in human HT1080 cells. Anti-RRP1 antibodies (Nop52) (red) used to visualize nucleoli were combined with the PNA probe for the alphoidtetO array of the HAC (green). Nuclei were stained with DAPI (blue). Nucleolar- and non-nucleolar-associated NORs are indicated by white arrowheads. d Quantification of 3D immuno-FISH showing the percentage of cells in which the HACs carrying different constructs are associated with mouse nucleoli. For each construct, the experiment was replicated three times (Table S5). Significant differences were calculated using a two-tailed nonparametric Mann–Whitney U test. Statistically significant differences are marked with red asterisks
Fig. 4
Fig. 4
Effect of chromatin status on HAC localization in the nucleolus in human HT1080 cells. a Scheme of the experiment. On day 0, the cells with HAC/GFP, HAC/rDNA and HAC/DJ were transfected with tetR-tTAVP64 with an efficiency close to 100%. Cells were incubated for 7 days in the presence or absence of doxycycline and then transferred onto the gelatin-covered microscopy glass slide. On day 10, 3D immuno-FISH was performed. b 3D immuno-FISH with anti-RRP1 antibodies (Nop52) (red) and the PNA probe for the alphoidtetO array of the HAC (green). Nuclei were stained with DAPI (blue). Nucleolar- and non-nucleolar-associated HACs are indicated by white arrowheads. c Relative changes of HACs colocalization with the nucleoli in dox-plus versus dox-minus medium after transfection by the tetR-tTAVP64 fusion protein genes. Transfection by tetR-tTAVP64 in a dox-minus medium leads to its binding to tetO sequences on the HAC kinetochore and reduction of HAC/rDNA and HAC/DJ association with the nucleoli (1.5-fold and 1.5-fold decrease, respectively). Significant differences were calculated using a two-tailed nonparametric Mann–Whitney U test. Statistically significant differences are indicated with red asterisks
Fig. 5
Fig. 5
Comparative analysis of the proximal 100 kb sequence of the human DJ region with primate genomes. Schematic representation of the proximal end of the human DJ region (contigs IDs: GL000220 and AL592188.60) and its comparative analysis are based on UCSC genomic data (https://genome.ucsc.edu/). The top 15 primate genomes with unambiguous levels of similarity/identity are presented, based on multiple alignments of 30 mammalian genomes. The 15 pairwise alignments of each specie with the human genome (green boxes) are shown below the NCBI RefSeq annotation of the genes as a wiggle track (“Multiz Alignments of 30 mammals”), where height indicates an alignment quality. NCBI annotations of seven exons of LncRNA NR_038958 located in the proximal region of DJ and other noncoding RNAs, including ribosomal RNAs, are shown as blue boxes
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