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

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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. Some molecules responsible for altering the 3-D organization of chromosomes work as complexes of more than ten different proteins, and many are conserved in fungi, plants, and animals. Two such complexes are called “remodels the structure of chromatin” (RSC) in yeast and “switching deficient/sucrose non-fermenting” (SWI/SNF) in man. SWI/SNF is known to inhibit the advent of multiple types of human cancers. Since cancer is a disease whereby cells unduly divide, we sought to define when in the yeast cell division cycle RSC executes essential functions. Using a generic method to induce inactivation of essential proteins in otherwise healthy yeast cells, we found that the RSC complex is important before chromosome replication as well as before chromosome segregation. Interestingly, combining two of the mutations we had generated caused doubling of the entire chromosome complement of yeast. As it is known that such multiplication of the cellular chromosome complements results in an increased malleability of the genetic patrimony, which itself is known to underlie some of the aggressive traits of human cancers, our discovery suggests new models as to why SWI/SNF is such a potent tumor suppressor, and this may in turn provide valuable new inroads for cancer treatment.