Condensin is required for condensation of rDNA array during starvation. (A) Loss of condensin blocks rDNA condensation during starvation. Early log-phase WT and ycs4-2 cells in synthetic complete (SC) medium (+N) were shifted from 23 to 37°C or were allowed to remain at 23°C for 2 h and then changed to the nutrient starvation (−N) medium or fresh SC medium (+N) and incubated for 30 min. rDNA structure was analyzed by FISH with a 25S rDNA probe (bright white). The boxed cell is enlarged on the right. Nuclear DNA was stained with DAPI (light gray). A color version of this figure is available at The EMBO Journal Online. (B) Condensin is required for rDNA condensation during rapamycin treatment. Early log-phase cultures of WT, brn1-9, ycs4-2 and ycs5-1 cells in YPD were shifted from 23 to 37°C or were allowed to remain at 23°C for 2 h, which was followed by incubation without or with 200 nM rapamycin for 30 min. rDNA and nuclei were analyzed by FISH (bright white) and DAPI (light gray), respectively. A color version of this figure is available at The EMBO Journal Online. (C) Condensin is required for rapamycin-induced nucleolar contraction. The same as Figure 1B except nucleolar structure was analyzed by IF with a Nop1 antibody (bright white). Nuclear DNA was stained with DAPI (light gray). A color version of this figure is available at The EMBO Journal Online. (D) Condensin is required for maintaining the condensed nucleolar structure in the presence of rapamycin. Early log-phase WT and ycs4-2 cells were treated with 200 nM rapamycin for 1 h at 23°C to induce nucleolar contraction and then shifted to 37°C. The condensed nucleolus is quantitated (N=100 for each data point) and plotted against Ycs4-2 level. (E) Rapid loss of Ycs4-2 at the restrictive temperature. Ycs4-2 with chromosomally tagged Myc12 in the ycs4-2 strain is analyzed by Western blot at different conditions.

Rpd3 is required for rapamycin-induced nucleolar contraction and condensin loading to rDNA. (A) Rpd3 is required for rapamycin induction of condensin relocation to the nucleolus and nucleolar contraction. WT and rpd3Δ cells expressing Ycs4-Myc9 were treated with 200 nM rapamycin for 1 h. Nucleolar structure and Ycs4-Myc9 localization were analyzed by IF staining with Nop1 (bright white) and Myc antibodies (bright white), respectively. Nuclear DNA was stained with DAPI (light gray). A color version of this figure is available at The EMBO Journal Online. (B) The samples in (A) were analyzed for Ycs4 loading to the promoter of 35S rDNA by ChIP. CUP1 was used as a control.

Transcriptional repression does not cause nucleolar contraction and condensin loading. (A) Loss of Rrn3 is insufficient to cause nucleolar contraction. Early log-phase WT and rrn3-1 cells carrying chromosomally tagged YCS4-Myc9 in YPD were shifted from 23 to 38°C and incubated for 3 h. Merged pictures of nuclear (bright white) and nucleolar (light gray) staining are shown. (B) Loss of Rrn3 is insufficient to cause condensin loading to rDNA array. Samples in (A) were examined for Ycs4-Myc9 association with 35S rDNA promoter and CUP1 by ChIP with Myc antibody (9E10). –Ab, no antibody was used as a negative control. (C) Loss of Pol I is insufficient to cause nucleolar contraction. Early log-phase WT (RPA190), rpa190-1 and rpa190-3 cells in YPD were shifted from 23 to 37°C and incubated for 3 h. Merged staining of Nop1 and DAPI is shown. (D) Loss of Pol I is insufficient to cause H4 deacetylation. Samples in (C) were examined for H4 acetylation level at 35S rDNA promoter and 25S transcribing regions by ChIP with anti-hyperacetylated histone H4 antibody. CAb, rabbit pre-serum was used as a negative control.

Condensin maintains rDNA stability during transcriptional repression. (A) rpa190-1/3 mutations cause fragmented nucleolus phenotype. Early log-phase WT and rpa190-1/3 cells were shifted from 23 to 37°C in YPD for different times. Merged images of the nucleolus with the nucleus at 5 h are shown. Images of other time points and a color version of this figure are available at The EMBO Journal Online. (B) rpa190-1/3 mutations cause accumulation of cells with rDNA distributed throughout the entire nucleus. rpa190-1/3 cells were incubated at 37°C for 5 h and then analyzed for the nucleolar and rDNA structures by IF and FISH, respectively. Typical images of cells containing fragmented nucleolus or high levels of ERCs are shown. A color version of this figure is available at The EMBO Journal Online. (C) Rapamycin treatment suppresses the ERC formation in Pol I mutants. Early log-phase WT and rpa190-1/3 cells were shifted from 23 to 37°C for 2.5 h to inactivate Pol I, followed by incubation with or without rapamycin for different times. rDNA was analyzed by FISH. The upper panel shows the experimental strategy. Boxed cells were enlarged for better visualization. Images of other time points and a color version of this figure are available at The EMBO Journal Online. (D) Condensin is required for suppression of ERC formation in Pol I mutant in the presence of rapamycin. Single mutant (rpa190-1 YCS4) and double mutant (rpa190-1 ycs4-2) were shifted from 23 to 37°C for 2.5 h to inactivate Pol I and condensin, followed by incubation with or without rapamycin for 3 h. Merged images of the nucleolus with the nucleus are shown. A color version of this figure is available at The EMBO Journal Online.

Rapamycin causes rapid relocation of condensin into the nucleolus and loading of condensin to rDNA array. (A) Rapamycin causes Ycs4 to rapidly relocate into the nucleolus. Early log-phase cultures of untagged (BLY03) and Ycs4-Myc12 chromosomally tagged (ZW206) cells in YPD were treated with or without 200 nM rapamycin for 1 h at 23°C. Nucleolar structure and distribution of Ycs4-Myc12 were examined by IF with a monoclonal Nop1 antibody (bright white) and the A14 rabbit polyclonal anti-Myc antibody (bright white), respectively. The nuclei were stained with DAPI (light gray). A color version of this figure is available at The EMBO Journal Online. (B) Rapamycin causes enrichment of Ycs4 on rDNA array. Early log-phase untagged (BLY03) and Ycs4-Myc12 (ZW206) cells were treated with 200 nM rapamycin for 30 min. Ycs4-Myc12 protein associated with rDNA chromatin was determined by ChIP with an anti-Myc antibody (9E10). Polymerase chain reactions (PCR) were performed with rDNA primer pairs as indicated in Supplementary Figure 2D. –Ab, no antibody as a negative control. The right-hand panel shows quantification of the level of Ycs4-Myc12 bound to rDNA chromatin. (C) Rapamycin causes dissociation of Ycs4 from other chromosomal regions. Samples from (B) were analyzed for Ycs4-Myc12 associated with CUP1 and ACT1. The right-hand panel shows quantification of the level of Ycs4-Myc12 bound to the CUP1 and ACT1 loci. (D) Rapamycin causes dissociation of Ycs4 from ribosomal protein (RP) genes. Samples from (B) were analyzed for Ycs4-Myc12 associated with RPL30 and RPS26A. The right-hand panel shows quantification of the level of Ycs4-Myc12 bound to the RPL30 and RPS26A loci.

Histone deacetylation at H4 K5,12 is necessary and sufficient to cause condensin enrichment to the nucleolus and rDNA array. (A) Deacetylation at H4 K5,12 is necessary and sufficient to relocalize Ycs4-Myc9 to the nucleolus. WT, H4 K5,12G and H4 K5,12R strains carrying Ycs4-Myc9 were treated without or with rapamycin for 1 h and analyzed for Ycs4-Myc9 localization by IF with a Myc antibody. The nucleolus and nucleus are marked by IF with a Nop1 antibody and DAPI staining, respectively. A color version of this figure is available at The EMBO Journal Online. (B) Deacetylation at H4 K5,12 is necessary and sufficient to cause condensin loading to rDNA. The samples in Figure 4A were analyzed for Ycs4 loading to 35S rDNA promoter by ChIP.

Rapamycin treatment in the absence of condensin causes the fragmented nucleolus phenotype as a result of ERC formation. (A) Rapamycin treatment of condensin mutant causes fragmented nucleolus phenotype. Early log-phase WT and ycs4-2 cells in YPD were shifted from 23 to 37°C for 2 h, followed by incubation with 200 nM rapamycin (+Rap) or a drug carrier (−Rap) for 0, 1, 3 and 6 h. Nucleolar structure was visualized by IF with a Nop1 antibody (bright white). Merged images of representative cells with DAPI staining (light gray) are shown. A color version of this figure is available at The EMBO Journal Online. (B) Quantification of results from (A). N=100. (C) top1Δtop2-4 cells show similar fragmented nucleolus phenotype and distribution of rDNA throughout the entire nucleus. top1Δtop2-4 cells cultured at 23°C were analyzed for nucleolar and rDNA structures by IF and FISH, respectively. Merged images with DAPI staining are shown. A color version of this figure is available at The EMBO Journal Online. (D) Rapamycin treatment in the absence of condensin causes rDNA to distribute throughout the entire nucleus. Early log-phase ycs4-2 cells in YPD were shifted from 23 to 37°C for 2 h, followed by incubation with 200 nM rapamycin (+Rap) or drug carrier (−Rap) for 6 h. rDNA structure was visualized by FISH with a 25S rDNA probe (bright white). Merged images with DAPI staining are shown. The arrowhead points to a cell containing rDNA distributed throughout the entire nucleus. A color version of this figure is available at The EMBO Journal Online. (E) Loss of condensin causes accumulation of extrachromosomal rDNA circles (ERCs). Early log-phase WT and ycs4-2 (y-4) cells in YPD were shifted from 23 to 37°C for 0 and 6 h. Total DNA was isolated, separated by agarose DNA gel electrophoresis and analyzed by Southern blot with a ³²P-labeled 25S rDNA probe. top1Δtop2-4 cells cultured at 37°C for 2 h were used as a control. Gen rDNA, genomic rDNA.