Mutation of YCS4, a budding yeast condensin subunit, affects mitotic and nonmitotic chromosome behavior

Abstract

The budding yeast YCS4 gene encodes a conserved regulatory subunit of the condensin complex. We isolated an allele of this gene in a screen for mutants defective in sister chromatid separation or segregation. The phenotype of the ycs4-1 mutant is similar to topoisomerase II mutants and distinct from the esp1-1 mutant: the topological resolution of sister chromatids is compromised in ycs4-1 despite normal removal of cohesins from mitotic chromosomes. Consistent with a role in sister separation, YCS4 function is required to localize DNA topoisomerase I and II to chromosomes. Unlike its homologs in Xenopus and fission yeast, Ycs4p is associated with chromatin throughout the cell cycle; the only change in localization occurs during anaphase when the protein is enriched at the nucleolus. This relocalization may reveal the specific challenge that segregation of the transcriptionally hyperactive, repetitive array of rDNA genes can present during mitosis. Indeed, segregation of the nucleolus is abnormal in ycs4-1 at the nonpermissive temperature. Interrepeat recombination in the rDNA array is specifically elevated in ycs4-1 at the permissive temperature, suggesting that the Ycs4p plays a role at the array aside from its segregation. Furthermore, ycs4-1 is defective in silencing at the mating type loci at the permissive temperature. Taken together, our data suggest that there are mitotic as well as nonmitotic chromosomal abnormalities associated with loss of condensin function in budding yeast.

Figure 1

YCS4 is required for sister chromatid separation. (A) Sister chromatid separation phenotypes of ycs4-1 and top2-4 are similar. Wild-type, ycs4-1, and top2-4 cells released from α-factor arrest (t = 0) into media at the nonpermissive temperature (37^oC) were scored for sister separation by microscopy at three loci along chromosome IV over time. After 1 h, α-factor was added back to the media to prevent cells from entering the next cell cycle. Strains contained Lac operator repeats integrated at the TRP1 locus near the centromere (cen-proximal) (wild-type, SBY215, ycs4-1, NBY241, top2-4, NBY92), on the arm (wild-type, NBY291, ycs4-1, NBY327, top2-4, NBY259) and at the telomere (wild-type, NBY292, ycs4-1, NBY323, top2-4, NBY455) of the chromosome. Although sister chromatid separation is normal at all three loci in wild type, the ycs4-1 mutants, like the top2-4 mutants, show differential ability to separate loci based on the proximity to the centromere. (B) In ycs4-1, sister chromatid separation is compromised in the ab-sence of spindle forces. Wild-type (SBY215), ycs4-1 (NBY241), top2-4 (NBY92), mad2Δ (NBY113), ycs4-1mad2Δ (NBY275), and top2-4mad2Δ (NBY258) strains were released from α-factor arrest (t = 0) into media with benomyl and nocodazole at the nonpermissive temperature (37^oC); all strains contained the Lac operator array at the TRP1 locus of chromosome IV. After 1 h, α-factor was added back to the media to prevent cells from entering the next cell cycle. Sister chromatid separation was scored over time. In the absence of microtubules, sister chromatid separation is delayed in ycs4-1.

Figure 2

Cohesin binding and displacement is normal in ycs4-1. (A) Cohesin subunit Mcd1p/Scc1p was localized by indirect immunofluorescence on chromosome spreads in wild-type (SBY376) and ycs4-1 (NBY284) strains containing 3XHA-epitope tagged Mcd1p/Scc1p. Samples were fixed and stained at the indicated times during a synchronous cell cycle at the nonpermissive temperature (37^oC). DNA staining (DAPI) is shown in the left panels, anti-LacI antibody staining is shown in the middle panels, and anti-HA antibody staining is shown in the right panels. G1, S phase, and an-aphase spreads prepared from wild-type (top) and ycs4-1 (bottom) cells are shown. Mcd1p is absent from G1 and anaphase spreads but is present on spreads from cells in S phase in both wild-type and ycs4-1. Bar, 10 μm. (B) Quantified results of Mcd1p localization to chromosomes. The percentage of chromosomes with Mcd1p localized to chromosome spreads is represented versus time for wild type (SBY376) and ycs4-1 (NBY284).

Figure 3

DNA topoisomerases I and II are absent from chromosomes in ycs4-1. (A) Top2p was localized by indirect immunofluorescence on chromosome spreads in wild-type (NBY474) and ycs4-1 (NBY322) strains containing 3XHA-epitope tagged Top2p during a synchronous cell cycle at the nonpermissive temperature (37^oC). Wild-type chromosome spreads are on the right and ycs4-1 spreads are on the left; 0- and 30-min time points are shown. For each, DNA staining (DAPI) is shown on the left and anti-HA antibody staining is shown on the right. Wild-type spreads maintain the punctate Top2p. There is a dramatic loss of Top2p from chromosomes in ycs4-1 spreads within 30 min of the shift to 37^oC. Bar, 10 μm. (B) Quan-tified results of Top2p localization. The percentage of chromosomes with Top2p versus time is represented for wild type (NBY474) and ycs4-1 (NBY322). (C) Quantified results of Top1p localization. Top1p was localized by indirect immunofluorescence on chromosome spreads in wild-type (NBY496) and ycs4-1 (NBY498) strains containing 3XHA-epitope tagged Top1p during a synchronous cell cycle at the nonpermissive temperature (37^oC).

Figure 4

Ycs4p is nuclear protein throughout the cell cycle and becomes enriched at the nucleolus at anaphase. Indirect immunofluorescence was performed on Ycs4p-13Xmyc in cells with the chromosomal rDNA array (NBY333) and without, rdnΔ (NBY508). DNA staining (DAPI) is shown in the panels in the first set of vertical panels, anti-myc antibody staining that recognized Ycs4p-13Xmyc in the second set, anti-Nop1p staining in the third set, and a merge of the Ycs4p and Nop1p staining in the final set of panels. Bar, 10 μm.

Figure 5

YCS4 is required for mitotic condensation, stability, and segregation of rDNA. (A) The rDNA is decondensed in ycs4-1 mutants. Fluorescent in situ hybridization with probes against the rDNA was performed on wild type (NBY8) and ycs4-1 (NBY319) arrested in metaphase at the nonpermissive temperature (37^oC). Compared with the loops present in wild-type rDNA, the rDNA in ycs4-1 cells appears collapsed into an amorphous mass at the periphery of the DNA staining. Bar, 10 μm. (B) Results of in situ hybridization were quantified. In 69% of ycs4-1 cells arrested in metaphase, the rDNA appears decondensed, as compared with 4.5% of wild-type cells. (C) Segregation of the nucleolus is defective in ycs4-1. Indirect immunofluorescence for the nucleolar marker Nop1p was performed on wild-type (NBY8) and ycs4-1 (NBY319) cells in anaphase (120 min after release from G1). The percentage of cells with segregated or unsegregated nucleoli was scored and graphed. Only 10% of wild-type cells in anaphase have not segregated their nucleolus; 45% of ycs4-1 cells with elongated spindles still contain the nucleolus in the mother bud (as identified by the persistence of the α-factor induced shmoo). (D) Loss of nucleolar structure may inhibit its segregation. A wild-type (NBY8) cell is shown on the left and a ycs4-1 mutant (NBY319) cell on the right. DNA staining (DAPI) is shown in the top pair of panels, anti-tubulin antibody staining in the middle, and anti-Nop1p antibody staining on the bottom. The wild-type cell has segregated its crescent-shaped nucleolus into each cell body. The nucleolus in the ycs4-1 cell has lost its structure and is found in the mother cell body; the amorphous Nop1p-containing mass is typical of the 45% of cells with an unsegregated nucleolus. Bar, 10 μm.

Figure 6

ycs4-1 is defective in silencing at the silent mating type loci but not at the telomeres. (A) Deletion of the HMLα locus suppresses ycs4-1's growth on media containing α-factor. Serial dilutions of wild-type (NBY8), wild-type with the HMLα silent mating type locus deleted (hmlΔ) (NBY585), ycs4-1 (NBY316), ycs4-1 with the HMLα silent mating type locus deleted (hmlΔ) (NBY319) on YPD with and without 1 μg/ml α-factor (all strains are bar1Δ). Deletion of the HMLα locus suppresses ycs4-1′s growth on media containing α-factor. (B) Silencing at the telomeres is unaffected by the ycs4-1 mutation at the permissive temperature. Serial dilutions of wild type (NBY374) and ycs4-1 (NBY377), both containing a URA3 reporter construct at the telomere to assay silencing.