Resolution of human ribosomal DNA occurs in anaphase, dependent on tankyrase 1, condensin II, and topoisomerase IIα

Abstract

Formation of individualized sister chromatids is essential for their accurate segregation. In budding yeast, while most of the genome segregates at the metaphase to anaphase transition, resolution of the ribosomal DNA (rDNA) repeats is delayed. The timing and mechanism in human cells is unknown. Here we show that resolution of human rDNA occurs in anaphase after the bulk of the genome, dependent on tankyrase 1, condensin II, and topoisomerase IIα. Defective resolution leads to rDNA bridges, rDNA damage, and aneuploidy of an rDNA-containing acrocentric chromosome. Thus, temporal regulation of rDNA segregation is conserved between yeast and man and is essential for genome integrity.

Keywords: condensin; rDNA; tankyrase; topoisomerase IIa.

Figure 1.

rDNA resolution is delayed until anaphase and depends on tankyrase 1. (A) Schematic diagram of the rDNA arrays on the p-arms of the five acrocentric human chromosomes. (PJ) Proximal junction; (DJ) distal junction. The repeating unit is a 43-kb gene comprised of pre-rRNA and untranscribed IGS (intergenic spacer). (B–G) rDNA is not resolved in prophase. FISH analysis of mitotic cells isolated by shake-off from asynchronously growing HeLa cells and probed for rDNA (red) (B,D,F) and 15 cen (green) (B), DJ (green) (D), or PJ (green) (F). (C,E,G) Quantification of results in B, D, and F, respectively. Mitotic cells were scored as having rDNA, DJ, or PJ unresolved if ≥50% of their loci appeared as singlets. Average of two independent experiments ± SEM. (C) n = 54–83 cells each. (E) n = 32 cells each. (G) n = 50–51 cells each. (*) P ≤ 0.05, (**) P ≤ 0.01, Student's unpaired t-test. (H–J) rDNA is resolved late in anaphase, dependent on tankyrase 1. Following a 48-h transfection with GFP or TNKS1 siRNA, HeLa cells were arrested in prometaphase by a 16-h nocodazole treatment (H) or in anaphase by a 4.5-h nocodazole treatment followed by a 45-min release (I), isolated by mitotic shake-off, and analyzed by FISH using rDNA (red) and 13/21 cen (green) probes. (J) Quantification of results in H and I. (pro) Prometaphase; (ana) anaphase. Mitotic cells with three or more stretched rDNA lines were scored as having stretched rDNA. For anaphase, only cells with separated centromeres were scored. n = 31–51 cells. (K–M) FISH analysis of rDNA stretching in mitotic cells isolated by shake-off from asynchronously growing HeLa cells following a 48-h transfection with GFP or TNKS1 siRNA and probed for rDNA (red) (K–M) and 15 cen (green) (M). Deconvolved images are shown. (L) Quantification of results in K. Mitotic cells with three or more stretched rDNA lines were scored as having stretched rDNA. Average of two independent experiments ± SEM. n = 300–596 cells each. (**) P ≤ 0.01, Student's unpaired t-test. (B,D,F,H,I,K,M) DNA was stained with DAPI (blue). Bar, 2 μm.

Figure 2.

rDNA resolution requires CAP-D3/condensin II. (A) Schematic diagram of condensins I and II. Horizontal lines indicate CAP-D2 and D3. TNKS-binding sites at positions 5 and 519 are indicted in CAP-D3. (B–D) Depletion of SMC2 leads to rDNA stretching. Following a 48-h transfection with GFP or SMC2 siRNA, HeLa cells were analyzed by immunoblot (B) or isolated by mitotic shake-off and analyzed by FISH using rDNA (red) and 13/21 cen (green) probes (C) and quantified (D). Mitotic cells with three or more stretched rDNA lines were scored as having stretched rDNA. Average of two independent experiments (n = 63–70 cells each) ± SEM. (**) P ≤ 0.01, Student's unpaired t-test. (E–G) CAP-D3, not D2, is required for rDNA resolution in anaphase. HeLa cell lines stably expressing CAP-D3 or CAP-D2 shRNAs were analyzed by immunoblot (E) or isolated by mitotic shake-off following a 4.5-h incubation in nocodazole and 45-min release into anaphase and analyzed by FISH using rDNA (red) and 13/21 cen (green) probes (F) and quantified (G). Anaphase cells (separated centromeres) with ≥3 stretched rDNA lines were scored as having stretched rDNA. Average of two independent experiments ± SEM. n = 56–112 cells each. (*) P ≤ 0.05, Student's unpaired t-test. (H) CAP-D3, but not D2, coimmunoprecipitates with TNKS1. Immunoblot analysis of HeLa cells immunoprecipitated with control or TNKS1 antibodies and probed with the indicated antibodies. (I–K) The TNKS-binding site in CAP-D3 at amino acid 519 is required for resolution of cohesion. CAP-D3 shRNA#2 cells were transfected with GFP-Vec, GFP-CAP-D3 wild type, or GFP-CAP-D3 single G10A or G524, or double GAA mutant plasmids for 18 h and analyzed by immunoblot (I) or isolated by mitotic shake-off following a 4.5 h incubation in nocodazole and 45-min release into anaphase (J) and analyzed by FISH using rDNA (red) probes and quantified (K). Anaphase cells (separated centromeres) with three or more stretched rDNA lines were scored as having stretched rDNA. Average of two independent experiments ± SEM. n = 56–112 cells each. (**) P ≤ 0.01, Student's unpaired t-test. (L) CAP-D3 GAA mutant does not bind tankyrase 1 but retains its association with SMC2. Immunoblot analysis of U2OS cells immunoprecipitated with anti-GFP antibody following transfection with GFP-Vec, GFP-CAP-D3 wild type, or GFP-CAP-D3 GAA and probed with the indicated antibodies. (C,F,J) DNA was stained with DAPI (blue). Bar, 2 μm.

Figure 3.

Topo IIα is required for rDNA resolution. (A,B) Inhibition of Topo IIα leads to rDNA stretching. HeLa cells were isolated by mitotic shake-off following a 2-h treatment with (vehicle) DMSO or the Topo IIα inhibitors etoposide or ICRF-193 and analyzed by FISH using rDNA (red) and 13/21 cen (green) probes (A) and quantified (B). Mitotic cells with three or more stretched rDNA lines were scored as having stretched rDNA. Average of two independent experiments (n = 50–95 cells each) ± SEM. (*) P ≤ 0.05, (**) P ≤ 0.01, Student's unpaired t-test. (C–F) Topo IIα overexpression rescues stretched rDNA in TNKS1- or CAP-D3-depleted cells. TNKS1 (C,D) or CAP-D3 (E,F) shRNA HeLa cell lines were transfected with vector, Flag-TNKS1, Flag-Topo IIα, or GFP-CAP-D3 plasmids for 18 h; incubated for an additional 4.5 h in nocodazole; released for 45 min into anaphase; isolated by mitotic shake-off; and analyzed by FISH using rDNA (red) and 13/21 cen (green) probes (C,E) and quantified (D,F). Anaphase cells (separated centromeres) with three or more stretched rDNA lines were scored as having stretched rDNA. Average of two independent experiments ± SEM. (D) n = 51–111 cells each. (F) n = 76–127 cells each. (*) P ≤ 0.05, (**) P ≤ 0.01, Student's unpaired t-test. (G–K) TNKS1 or CAP-D3 depletion leads to rDNA segregation defects that are exacerbated by topo IIα inhibition. HeLa cell lines stably expressing GFP, TNKS1, CAP-D3, or CAP-D2 shRNA were treated with nocodazole for 1 h and released in DMSO or 50 μm etoposide for 1 h and analyzed by FISH using an rDNA probe (red) (G) and quantified (H). Arrows indicate DAPI-staining bridges. Late anaphase cells with rDNA laggards and bridges were scored. Average of three independent experiments ± SEM. n = 45–112 cells each. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001, Student's unpaired t-test. (I,J) Double depletion of TNKS1 and CAP-D3 does not exacerbate the phenotype. (I) CAP-D3 shRNA cells treated with GFP or TNKS1 siRNA for 48 h were analyzed by immunoblot (I) or FISH (J) following treatment with nocodazole for 1 h and release in DMSO for 1 h and quantified. Late anaphase cells with rDNA laggards and bridges were scored. Average of three independent experiments ± SEM. n = 41–58 cells each. (K) Graphical representation of the frequency of rDNA-positive DAPI bridges versus total DAPI bridges from H. (A,C,E,G) DNA was stained with DAPI (blue). Bar, 2 μm.

Figure 4.

Impaired rDNA segregation leads to genomic instability. (A,B) TNKS1-depleted cells have rDNA bridges in late anaphase. HeLa cells were isolated by mitotic shake-off from asynchronously growing cultures following 48-h transfection with GFP or TNKS1 siRNA and analyzed by FISH using an rDNA probe (red; deconvolved images are shown) (A) and quantified (B). Arrow indicates DAPI-staining bridge. Late anaphase cells with rDNA laggards and bridges were scored. Average of two independent experiments ± SEM. n = 25–34 late anaphase cells each. (C–G) Increased aneuploidy of the acrocentric chromosome 15 in TNKS1-depleted cells. FISH analysis of interphase cells from (C) HTC75 or (F) BJ-hTERT cells stably expressing GFP or TNKS1 shRNA using a 15 cen (green) or 10 cen (red) probe. (D,G) Quantification of the deviation from the modal copy number for C and F, respectively. (D) Average of two independent experiments ± SEM. n = 201–222 cells each. (G) Average of three independent experiments ± SEM. n = 108–140 cells each. (*) P ≤ 0.05; (**) P ≤ 0.01, Student's unpaired t-test. (E) Immunoblot analysis of BJ-hTERT cell lines stably expressing GFP or TNKS1 shRNA. (H,I) Increased rDNA-containing micronuclei in TNKS1-depleted cells. FISH analysis of HeLa cells following 72-h transfection with GFP or TNKS1 siRNA with an rDNA probe (red). (I) Quantification of interphase cells containing one or more micronuclei without (−) or with (+) rDNA. Average of two independent experiments ± SEM. n = 301–388 cells each. (*) P ≤ 0.05, Student's unpaired t-test. (J,K) Increased rDNA associated DNA damage foci in TNKS1-depleted cells. (J) Asynchronous HeLa cells were fixed in 3.7% paraformaldehyde and analyzed by immunofluorescence by dual staining with anti-UBF (red) and γH2AX (green) antibodies. (K) Quantification of mitotic cells containing two or more colocalizing foci. Average of two independent experiments ± SEM. n = 36–65 cells each. (*) P ≤ 0.05, Student's unpaired t-test.