The ctf13-30/CTF13 genomic haploinsufficiency modifier screen identifies the yeast chromatin remodeling complex RSC, which is required for the establishment of sister chromatid cohesion

Abstract

The budding yeast centromere-kinetochore complex ensures high-fidelity chromosome segregation in mitosis and meiosis by mediating the attachment and movement of chromosomes along spindle microtubules. To identify new genes and pathways whose function impinges on chromosome transmission, we developed a genomic haploinsufficiency modifier screen and used ctf13-30, encoding a mutant core kinetochore protein, as the reference point. We demonstrate through a series of secondary screens that the genomic modifier screen is a successful method for identifying genes that encode nonessential proteins required for the fidelity of chromosome segregation. One gene isolated in our screen was RSC2, a nonessential subunit of the RSC chromatin remodeling complex. rsc2 mutants have defects in both chromosome segregation and cohesion, but the localization of kinetochore proteins to centromeres is not affected. We determined that, in the absence of RSC2, cohesin could still associate with chromosomes but fails to achieve proper cohesion between sister chromatids, indicating that RSC has a role in the establishment of cohesion. In addition, numerous subunits of RSC were affinity purified and a new component of RSC, Rtt102, was identified. Our work indicates that only a subset of the nonessential RSC subunits function in maintaining chromosome transmission fidelity.

FIG. 1.

Schematic outline of the yeast genomic modifier screen. In our screen we use growth as a testable phenotype. ++, wild-type growth; +, mild slow growth, −, no growth or slow growth. X₁, deletion of gene 1, X₂, deletion of gene 2, etc. Heterozygous ctf13-30/CTF13 causes a mild slow-growth phenotype compared to that of wild-type (X/X) strains. Heterozygous deletions that modified the phenotype produced by heterozygous ctf13-30/CTF13 were sought. Most unlinked heterozygous deletions do not affect the mild growth defect of heterozygous ctf13-30/CTF13 mutants (row 2). Enhancers are genes whose heterozygous deletion in combination with ctf13-30/CTF13 causes a more severe slow-growth phenotype of ctf13-30/CTF13 mutants (row 3). Suppressors are genes whose heterozygous deletion in combination with ctf13-30/CTF13 causes a rescue of the slow-growth phenotype of ctf13-30/CTF13 mutants (row 4). In the ctf13-30/CTF13 example described in the text the mild slow-growth phenotype of ctf13-30/CTF13 mutants compared to that of wild-type cells did not allow efficient detection of traditional suppressors. Numerous single heterozygous deletions produce haploinsufficiency (rows 5 and 6). False positives are detected in cases where the single heterozygous deletion itself causes a severe growth defect (row 5). In some cases the haploinsufficiency phenotype displayed by a single heterozygous diploid was suppressed by the addition of heterozygous ctf13-30/CTF13 (row 6). These represent genetic interactions in the opposite direction, and we have called these gene suppressors.

FIG. 2.

Ndc10, Ctf3, and Cse4 coimmunoprecipitation with CEN DNA is not dependent on Rsc2. Anti-Myc or anti-HA ChIP assays were performed with chromatin extracts of wild-type (RSC2) or rsc2Δ mutant cells expressing either Ndc10-Myc (YVM499 and YKB121), Ctf3-Myc (YVM 218 and YKB127), or Cse4-HA (YVM1141 and YKB124), as well as with cells expressing no tag (YPH499). Cells were grown to mid-log phase in YPD medium at 25°C, and then the culture was shifted to 37°C for 3 h before chromatin was isolated for IP. Multiplex PCR was performed to amplify the centromeric DNA of CEN1, CEN3, and a non-CEN locus, PGK1, for both whole-cell extracts (WCE) and immunoprecipitates.

FIG. 3.

rsc2Δ mutant cells exhibit defects in sister chromatid cohesion. The numbers of GFP signals were scored in wild-type cells (YPH1477), three independent isolates of rsc2Δ cells (YKB235a to -c), and three independent isolates of rsc1Δ cells (YKB177) arrested for 3 h at 37°C in G₁ with α-factor or in G₂ with nocodazole. The data shown represent the averages of three independent experiments. One hundred cells were counted for each sample.

FIG. 4.

rsc2Δ mutant cells exhibit defects in establishment of sister chromatid cohesion. (A) Wild-type cells (Wt; RSC2) and rsc2Δ mutants expressing either Smc3-HA (YKB353 or YKB355) or Scc1-HA (YKB342 or YKB341) were blocked in G₁ by α-factor treatment and released at 37°C. Chromosome spreads shown are of cells were taken prior to release or at 100 min postrelease. DNA was stained with DAPI (4′,6′-diamidino-2-phenylindole). Chromatin-associated Scc1-HA and Smc3-HA were detected by indirect immunofluorescence. (B) Wild-type (YPH1477; circle), scc1-73 (YKB426; triangle), and rsc2Δ (YKB235; square) cells were arrested with α-factor and released into nocodazole media at 25°C. Cultures were shifted to 37°C after >90% of cells had budded (time zero), samples were taken every 20 min for 180 min, and the percentages of sister chromatid separation were scored. One hundred cells were scored per sample; two separate experiments were performed with nearly identical results.

FIG. 5.

Purification of RSC protein complexes and associated proteins. TAPs of the RSC complex were carried out on strains containing either no tagged proteins or TAP-tagged versions of Rsc2 (NJK208), Rsc3 (NJK203), Rsc4 (NJK210), Rsc6 (NJK213), Np16 (NJK212), Rsc8 (NJK211), Rsc58 (NJK192), and Rtt102 (NJK182). (A) Silver-stained sodium dodecyl sulfate gels of some of the RSC affinity purifications. Proteins copurifying with the TAP-tagged proteins were identified by either MALDI-ToFMS or LC/MS/MS. (B) Clustered summary of the proteins identified copurifying with the TAP-tagged versions of RSC proteins. Red boxes, proteins identified by MALDI-ToF MS; blue boxes, proteins identified by LC/MS/MS; black boxes, proteins identified by both methods. Proteins were listed only if they were identified as copurifying with at least two TAP-tagged baits.