PgmNr Y501: The RSC complex functions to maintain ploidy in Saccharomyces cerevisiae.

Authors:
T. L. Sing 1,2 ; M. P. Hung 1,2 ; S. Ohnuki 3 ; J. Ou 1,2 ; B. J. San Luis 2,4 ; M. Costanzo 2,4 ; C. Boone 2,4 ; Y. Ohya 3 ; G. W. Brown 1,2


Institutes
1) Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada; 2) Donnelly Centre for Cellular and Biomolecular Research, Toronto, Ontario, Canada; 3) Department of Integrated Biosciences, University of Tokyo, Tokyo, Japan; 4) Department of Molecular Genetics, University of Toronto, Ontario, Canada.


Keyword: Cell Cycle/Growth Control/Metabolism

Abstract:

Ploidy is tightly regulated in eukaryotic cells, and is critical for cell function and survival. Cells must coordinate multiple pathways to ensure replicated DNA is segregated both accurately and in a timely fashion to prevent changes in chromosome number. Several cellular processes have been implicated in ploidy maintenance, including spindle pole body (SPB) duplication, mitotic spindle formation and kinetochore attachment.

We have discovered an unanticipated role for 6 of 7 non-essential subunits of the RSC (Remodels the Structure of Chromatin) complex in ploidy maintenance. Using flow cytometry, we demonstrated that deletion of RSC1, RSC2, RSC30, LDB7, NPL6, or HTL1, but not RTT102, causes a rapid transition from haploid to diploid DNA content following germination of haploid mutant spores. Unlike normal diploids that arise from mating, rsc∆ mutant diploids retained the ability to mate, indicating that diploidization is the result of autopolyploidization rather than mating-type switching. Interestingly, morphological analysis revealed that diploidized rsc∆ mutants have an elongated bud phenotype, which makes them distinct from wild type diploids.

Microscopic analysis revealed that rsc∆ mutants do indeed have defects in SPB duplication and maturation that explain the increase-in-ploidy phenotype. Using RNA sequencing, we showed that these defects were not due to obvious changes in transcript levels in the rsc∆ mutants. Therefore, we performed synthetic genetic array (SGA) analysis, which implicated the RSC complex in regulating proteins important for inserting newly synthesized SPBs into the nuclear envelope.  Surprisingly, we were unable to detect physical interactions between the RSC complex and SPBs by yeast 2-hybrid (Y2H) or bimolecular fluorescence complementation (BiFC). Instead, evidence has lead us to a model where the RSC complex facilitates nuclear pore complex (NPC) stability and/or localization, which is necessary for proper SPB insertion. 

Understanding how the RSC complex regulates ploidy will contribute to an integrated view of how multiple pathways converge to maintain the haploid state and propagate it from one generation to the next.



Yeast Database Genetic Index
1. gene symbol: RTT102; systematic name: YGR275W
2. gene symbol: RSC1; systematic name: YGR056W
3. gene symbol: RSC2; systematic name: YLR357W
4. gene symbol: RSC30; systematic name: YHR056C
5. gene symbol: LDB7; systematic name: YBL006C
6. gene symbol: NPL6; systematic name: YMR091C
7. gene symbol: HTL1; systematic name: YCR020W-B