Temperature-dependent regulation of rDNA condensation inSaccharomyces cerevisiae

Abstract

Chromatin condensation during mitosis produces detangled and discrete DNA entities required for high fidelity sister chromatid segregation during mitosis and positions DNA away from the cleavage furrow during cytokinesis. Regional condensation during G1 also establishes a nuclear architecture through which gene transcription is regulated but remains plastic so that cells can respond to changes in nutrient levels, temperature and signaling molecules. To date, however, the potential impact of this plasticity on mitotic chromosome condensation remains unknown. Here, we report results obtained from a new condensation assay that wildtype budding yeast cells exhibit dramatic changes in rDNA conformation in response to temperature. rDNA hypercondenses in wildtype cells maintained at 37°C, compared with cells maintained at 23°C. This hypercondensation machinery can be activated during preanaphase but readily inactivated upon exposure to lower temperatures. Extended mitotic arrest at 23°C does not result in hypercondensation, negating a kinetic-based argument in which condensation that typically proceeds slowly is accelerated when cells are placed at 37°C. Neither elevated recombination nor reduced transcription appear to promote this hypercondensation. This heretofore undetected temperature-dependent hypercondensation pathway impacts current views of chromatin structure based on conditional mutant gene analyses and significantly extends our understanding of physiologic changes in chromatin architecture in response to hypothermia.

Promotion of hyperthermic-induced rDNA hypercondensation inSaccharomyces cerevisiae

“…Cell cycle progression from log phase into mitosis was confirmed by flow cytometry (Figure 1A). As expected, mitotic wildtype cells maintained at 23°C contained long rDNA loops while the rDNA of mitotic cells shifted to 37°C during the final hour of incubation hypercondensed into very short loops (Figure 1B), consistent with prior findings (Shen and Skibbens, 2017a; Matos-Perdomo and Machín, 2018a). Prior analyses of these cells revealed that only a fraction of scc2-4 mutant cells contain condensed rDNA loci at 23°C, a level which is retained after shifting to 37°C during the final hour of incubation (Shen and Skibbens, 2017b).…”

“…Cohesins play a critical role in chromosome condensation, including across the rDNA locus, such that mutation in either cohesin subunits (Mcd1/Scc1, Pds5 or Scc3) or regulators (Eco1 or Scc2) all result in severe rDNA condensation defects (Guacci et al, 1997; D’Ambrosio et al, 2008; Tong and Skibbens, 2015; Skibbens et al, 1999; Hartman et al, 2000; Woodman et al, 2015; Orgil et al, 2015). These observations formally suggest that de novo cohesin deposition during mitosis may play a critical role in hyperthermic-induced rDNA hypercondensation, in contrast to the decondensation of rDNA into ‘puffs’ that occurs upon either cohesin inactivation or dissociation (Guacci et al, 1997; Ciosk et al, 2000; Shen and Skibbens, 2017a). Here, we test whether de novo cohesin deposition promotes hyperthermic-induced rDNA hypercondensation by inactivating the Scc2,4 heterocomplex that is required for cohesin deposition onto DNA (Ciosk et al, 2000; Watrin et al, 2006).…”

“…Wildtype cells shifted to an elevated temperature during mitosis exhibit rDNA hypercondensation (Shen and Skibbens, 2017a; Matos-Perdomo and Machín, 2018a), but the structural basis for this dramatic change in chromatin structure remains unknown. Cohesins play a critical role in chromosome condensation, including across the rDNA locus, such that mutation in either cohesin subunits (Mcd1/Scc1, Pds5 or Scc3) or regulators (Eco1 or Scc2) all result in severe rDNA condensation defects (Guacci et al, 1997; D’Ambrosio et al, 2008; Tong and Skibbens, 2015; Skibbens et al, 1999; Hartman et al, 2000; Woodman et al, 2015; Orgil et al, 2015).…”

“…In budding yeast, rDNA forms a diffuse puff-like structure during G1 phase that coalesces into a tight loop-like structure during mitosis (Guacci et al, 1994; Guacci et al, 1997). The importance of these architectural changes is highlighted by the development of numerous strategies that include FISH, GFP-tagged rDNA binding proteins, and a streamlined intercalating-dye method which now provides for rapid and efficient quantification of rDNA condensation (Guacci et al, 1994; Guacci et al, 1997; D’Ambrosio et al, 2008; Lopez-Serra et al, 2013; Tong and Skibbens, 2015; Lavoie et al, 2002; Lavoie et al, 2004; Shen and Skibbens, 2017a). To date, these condensation assays have helped elucidate the role of highly conserved cohesin and condensin complexes in regulating rDNA architecture.…”

“…Despite the intense focus on yeast rDNA architecture over the last two decades (Xu et al, 2017; Guacci et al, 1994; Lavoie et al, 2004; Freeman et al, 2000; Castaño et al, 1996; Sullivan et al, 2004; Gard et al, 2009; Hong and Seong, 2014), an additional rDNA state was only recently discovered in which mitotic cells induce a hyper-condensed rDNA state in response to elevated temperature (Shen and Skibbens, 2017a; Matos-Perdomo and Machín, 2018a). This hyperthermic-induced rDNA hypercondensation is both rapidly induced and reversible.…”