PLoS Genet. 2007 Jun;3(6):e92. Epub 2007 Apr 20.

Reverse genetic analysis of the yeast RSC chromatin remodeler reveals a role for RSC3 and SNF5 homolog 1 in ploidy maintenance.

Campsteijn C, Wijnands-Collin AM, Logie C.

Source

Molecular Biology Department, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, Netherlands.

Abstract

The yeast "remodels the structure of chromatin" (RSC) complex is a multi-subunit "switching deficient/sucrose non-fermenting" type ATP-dependent nucleosome remodeler, with human counterparts that are well-established tumor suppressors. Using temperature-inducible degron fusions of all the essential RSC subunits, we set out to map RSC requirement as a function of the mitotic cell cycle. We found that RSC executes essential functions during G1, G2, and mitosis. Remarkably, we observed a doubling of chromosome complements when degron alleles of the RSC subunit SFH1, the yeast hSNF5 tumor suppressor ortholog, and RSC3 were combined. The requirement for simultaneous deregulation of SFH1 and RSC3 to induce these ploidy shifts was eliminated by knockout of the S-phase cyclin CLB5 and by transient depletion of replication origin licensing factor Cdc6p. Further, combination of the degron alleles of SFH1 and RSC3, with deletion alleles of each of the nine Cdc28/Cdk1-associated cyclins, revealed a strong and specific genetic interaction between the S-phase cyclin genes CLB5 and RSC3, indicating a role for Rsc3p in proper S-phase regulation. Taken together, our results implicate RSC in regulation of the G1/S-phase transition and establish a hitherto unanticipated role for RSC-mediated chromatin remodeling in ploidy maintenance.

PMID:: 17542652; [PubMed - indexed for MEDLINE]
PMCID:: PMC1885278

Free PMC Article

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Figure 1

Conditional Depletion of RSC Subunits Terminally Arrests Cells at Multiple Stages of the Cell Cycle

(A) Characterization of yeast strains bearing degron alleles of RSC subunits. Strains were cultured in galactose media at 25 °C (left panel, top lanes) or 37 °C (left panel, bottom lanes) to deplete degron fusions of RSC subunits for 3, 6, and 9 h (only 9 h shown), and a 10-fold serial dilution was spotted onto rich medium and incubated for 3 d at 25 °C (left panel; see Materials and Methods). In parallel, aliquots were taken following depletion and analyzed for DNA content by flow cytometry (right panel). For a more complete set of strains, see Figure S2.
(B) Growth curves under nonpermissive conditions of several strains used in (A) □, Wild type;

, sth1^td;

, sfh1^td;⧫, rsc3^td; •, rsc4^td; █ , rsc6^td; ▴ , rsc9^td;▴ , rsc58^td; ⧫, rsc8^td.
(C) Cells were shifted from YEP-glucose medium supplemented with 0.1 mM CuSO₄ at 25 °C to YEP-galactose supplemented with 0.1 mM CuSO₄ at 25 °C, and 10-fold dilutions were spotted after 0 or 16 h onto glucose containing plates with 0.1 mM CuSO₄ and incubated at 25 °C. All strains displayed in this figure contain the P_gal::UBR1 allele.

Reverse Genetic Analysis of the Yeast RSC Chromatin Remodeler Reveals a Role for RSC3 and SNF5 Homolog 1 in Ploidy Maintenance