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. 2023 Feb 28;120(9):e2219126120.
doi: 10.1073/pnas.2219126120. Epub 2023 Feb 23.

Two differentially stable rDNA loci coexist on the same chromosome and form a single nucleolus

Luciana Lazar-Stefanita  1   2 , Jingchuan Luo  1   2 , Max A B Haase  1   2   3 , Weimin Zhang  1   2 , Jef D Boeke  1   2   4
Affiliations

Two differentially stable rDNA loci coexist on the same chromosome and form a single nucleolus

Luciana Lazar-Stefanita et al. Proc Natl Acad Sci U S A. .

Abstract

The nucleolus is the most prominent membraneless compartment within the nucleus-dedicated to the metabolism of ribosomal RNA. Nucleoli are composed of hundreds of ribosomal DNA (rDNA) repeated genes that form large chromosomal clusters, whose high recombination rates can cause nucleolar dysfunction and promote genome instability. Intriguingly, the evolving architecture of eukaryotic genomes appears to have favored two strategic rDNA locations-where a single locus per chromosome is situated either near the centromere (CEN) or the telomere. Here, we deployed an innovative genome engineering approach to cut and paste to an ectopic chromosomal location-the ~1.5 mega-base rDNA locus in a single step using CRISPR technology. This "megablock" rDNA engineering was performed in a fused-karyotype strain of Saccharomyces cerevisiae. The strategic repositioning of this locus within the megachromosome allowed experimentally mimicking and monitoring the outcome of an rDNA migratory event, in which twin rDNA loci coexist on the same chromosomal arm. We showed that the twin-rDNA yeast readily adapts, exhibiting wild-type growth and maintaining rRNA homeostasis, and that the twin loci form a single nucleolus throughout the cell cycle. Unexpectedly, the size of each rDNA array appears to depend on its position relative to the CEN, in that the locus that is CEN-distal undergoes size reduction at a higher frequency compared to the CEN-proximal counterpart. Finally, we provided molecular evidence supporting a mechanism called paralogous cis-rDNA interference, which potentially explains why placing two identical repeated arrays on the same chromosome may negatively affect their function and structural stability.

Keywords: Hi-C maps; cis duplicated rDNA loci; megablock chromosome engineering; nucleolus.

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Conflict of interest statement

The authors have organizational affiliations to disclose: J.D.B. is a Founder and Director of CDI Labs, Inc., a Founder of Neochromosome, Inc, a Founder of and Consultant to ReOpen Diagnostics, and serves or served on the Scientific Advisory Board of the following: Logomix, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Sangamo, Inc., Tessera Therapeutics, inc., and the Wyss Institute. The remaining authors declare no competing interests. The authors have stock ownership to disclose: J.D.B. has substantive interest in CDI Labs, Neochromosome, and ReOpen Diagnostics.

Figures

Fig. 1.
Fig. 1.
Transplantation of the rDNA megablock to an ectopic genome location. (A) Diagram showing chromosome XII of wild-type yeast (n = 16, BY4741) that contains the rDNA locus (green), and below a simplified representation of nuclear organization in yeast, where examples of chromosome arms (gray lines) are anchored at the nuclear membrane through CEN and TEL, and the crescent-shaped nucleolus (green) consisting of rDNA genes. (B) Representative microscopy images of yeast nuclei with one or two rDNA loci (black arrowheads in the schematic indicate rDNA-containing chromosomes in WZ499 strain). (C) Schema illustrating the relocation (arrowheads) of the rDNA megablock (black box) on the fused megachromosome C (chr C: color code corresponds to the design of chromosome fusion) in n = 3 yeast strains (JL411 has a CEN distal rDNA locus, whereas, rDNA is CEN proximal in JL665). Colored blocks indicate sequence homology of the repair donors at CRISPR-Cas9 cutting sites. (D) Hi-C contact maps of chr C shown in gray on the x and y axes of each map. The rDNA locus is CEN distal on the Left, whereas, after transplantation, it appears CEN proximal on the Right. rDNA distances from the CENs (dark gray) are indicated atop the maps.
Fig. 2.
Fig. 2.
Effects of rDNA duplication on cell fitness and transcriptomics. (A) Estimate of total length of the rDNA array by deep sequencing: % of rDNA aligned reads normalized by genome size (12.3 Mb). Schematics of chromosome XII (n = 16, BY4741) and megachromosome C (n = 3) are shown on the bottom of the plot (gray) and the position of the rDNA locus is highlighted in green. The parentheses indicate 1xrDNA locus per chromosome either CEN proximal or CEN distal (strains: JL411, JL665, and LS88); whereas, the configuration with 2xrDNA loci on chr C is shown without parentheses (independent isolates of LS71: C1 to C3). (B) Serial dilution assay on different growth conditions at 30 °C of n = 16 (BY4741) and n = 3 isolates with either single (JL411 and JL665) or duplicated rDNA (LS71). (C) Schematics of RNA-based assays in n = 3 isolates with 1xrDNA (JL411 and JL665) vs. 2xrDNA (LS71). RT-qPCR bar plot used to estimate changes in the synthesis of the ribosomal RNA precursor (ETS1) relative to the control messenger RNA, UBC6. (D) Volcano plot showing RNA-seq data comparing transcriptomes of 1xrDNA (JL411 and JL665) and 2xrDNA (LS71) strains in triplicates. Red and blue dots indicate differentially expressed genes in 2xrDNA vs. 1xrDNA: in blue down-regulated, in red up-regulated (P < 10 to 5, |fold change| > 2).
Fig. 3.
Fig. 3.
Stability of the rDNA locus is a function of chromosome position. (A) Hi-C contact maps of megachromosome C (chr C in gray on the x and y axes of each contact map) in n = 3 strains with either one or two rDNA loci. Bottom Left triangle map: average representation of two merged contact maps with 1xrDNA on chr C, either CEN proximal (JL665) or CEN distal (JL411). Top Right map: average representation of three merged contact maps with 2xrDNA on chr C CEN proximal and distal (LS71 C1 to C3). rDNA distances from the CEN (dark gray) are indicated at the top of the map. (B) Quantifications of Hi-C contacts between the rDNA-flanking regions on chr C (500 kb long), highlighted in blue rectangles in panel A (rectangles 1 & 3 represent sequences upstream of the CEN-proximal rDNA boundary; whereas, 2 & 4 represent sequences downstream of the CEN-distal boundary), and the 1.9 Mb rDNA intervening sequence. Mean, median of absolute contact values, and their P values, calculated using the Kolmogorov–Smirnov test (K-S test), are indicated for each strain with one or two rDNA loci. (C and D) Estimate of rDNA array sizes. PFGE of BamHI-digested chromosomes (C) and the corresponding Southern blot with unique probes designed for each rDNA position (D). Each lane represents an independent isolate of n = 3 strains with 1xrDNA, either CEN proximal or CEN distal, or 2xrDNA in cis CEN proximal and distal. Schematic indicates the position of rDNA arrays and probes (cyan or fuchsia) on chr C. PFGE run specifications: S. pombe program for megasize chromosome separation.
Fig. 4.
Fig. 4.
Twin rDNA loci in cis form a single nucleolus during cell division. (A) Representative microscopy images of n = 3 strains with one rDNA locus (CEN proximal) or two loci in cis (CEN proximal and distal). Cells were synchronized in G1 (LS88 and LS90 strains grown in alpha factor at 25 °C) and late anaphase (LS95 and LS103 strains with cdc15-2 ts mutation grown at 37 °C) before imaging. The insertion of white lines indicates that cells originated from different locations in the field of view. (B, Top) Hi-C maps of megachromosomes in n = 3, with two rDNA loci in cis on chr C (dashed outline), from cells synchronized in G1 (Top Right triangle map) and late anaphase (Bottom Left triangle map). Bottom: contact variation maps (log2-ratio) of chr C with 1xrDNA vs. 2xrDNA in G1 (Top Right triangle map) vs. late anaphase (Bottom Left triangle map). Black arrowheads point at contact variations adjacent to the rDNA loci. Blue to red color scale indicates contact increase on chr C with 2xrDNA relative to 1xrDNA. The linear distance between the two rDNA loci in cis is indicated on the schematic of chr C. (C) Quantifications of Hi-C contacts between rDNA-flanking regions on chr C (dashed outline in the top triangle maps in panel B), as described in Fig. 3B. Relative contact reduction in anaphase (gray) compared to G1 (blue) is indicated for each strain with: one, two, and non-rDNA–containing proximal or distal locus.
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