Depletion of limiting rDNA structural complexes triggers chromosomal instability and replicative aging ofSaccharomyces cerevisiae

Abstract

20Sir2 is a highly conserved NAD + -dependent histone deacetylase that functions in 21 heterochromatin formation and promotes replicative lifespan (RLS) in the budding yeast, 22Saccharomyces cerevisiae. Within the yeast rDNA locus, Sir2 is required for efficient cohesin 23 recruitment and maintaining stability of the tandem array. In addition to the rDNA, ChIP-seq of 24 an epitope-tagged cohesin subunit (Mcd1-13xMyc) in a sir2∆ mutant revealed subtle reductions 25 of cohesin binding at all 16 centromeres. Coupled with the previously reported chromosome 26 instability in sir2∆ cells and depletion of Sir2 in aged cells, we hypothesized that mitotic 27 chromosome instability (CIN) due to Sir2 depletion could be a driver of replicative aging. In 28 addition to Sir2, we discovered that other subunits of the Sir2-containing SIR and RENT 29 complexes were depleted in aged cells, as were subunits of the cohesin and monopolin/cohibin 30 complexes, implying the possibility of CIN. ChIP assays of the residual Mcd1-13xMyc in aged 31 cells showed strong depletion from the rDNA and possible redistribution to centromeres, most 32 likely in an attempt to maintain chromosome stability. Despite the shift in cohesin distribution, 33 sister chromatid cohesion was partially attenuated in old cells and the frequency of chromosome 34 loss was increased. This age-induced CIN was exacerbated in strains lacking Sir2 and its paralog, 35Hst1, but suppressed in strains that stabilize the rDNA array due to the deletion of FOB1 or 36 through caloric restriction (CR). Furthermore, ectopic expression of MCD1 from a doxycycline-37 inducible promoter was sufficient to suppress to rDNA instability in aged cells and to extend 38 RLS. Taken together we conclude that age-induced depletion of cohesin and multiple other 39 nucleolar chromatin factors destabilize the rDNA locus, which then results in general CIN and 40 aneuploidy that shortens RLS. 41

Fob1-dependent condensin recruitment and loop extrusion on yeast chromosome III

“…A differential chromatin interaction (DCI) score was calculated for each 5kb segment across the genome to measure the difference in normalized contact between each segment and its +/-200kb flanking regions (Fig 5E-F). Segments with the strongest negative DCI scores, representing the most decreased interactions in the lrs4 Δ mutant compared to WT (Fig S3C), were predominantly from a region on the right arm of chrXII between the centromere and rDNA array (Fig 6E and S3E), corresponding to a large condensin-dependent chromatin loop (Fine et al, 2019; Lazar-Stefanita et al, 2017; Wang et al , 2021). Furthermore, the RDT1 -containing chrIII segment (coordinates 30000 to 35000) showed the 2 nd strongest negative DCI score (Fig 5E and S3E).…”

“…Despite such limitations, there are still multiple biochemically generated architectural features within the yeast genome, including abundant self-associating chromatin domains detected by Micro-C that only span across one to five genes and are dependent on chromatin remodeling factors (Hsieh et al, 2015; Hsieh et al , 2016). There are also biochemically generated large-scale features, such as the rDNA array and the condensin-dependent TAD formed between the centromere and rDNA on chrXII (Fig 7; (Duan et al , 2010; Fine et al , 2019; Lazar-Stefanita et al , 2017)). The MAT a-and MAT α-specific conformations of chrIII comprise the other major large-scale architectural features of the budding yeast genome (Belton et al , 2015; Miele et al , 2009).…”

Contribution of spontaneous mutations to quantitative and molecular variation at the highly repetitive rDNA locus in yeast

“…2014), genome integrity (Ide et al . 2010; Kobayashi 2011, 2014; Fine et al . 2019), stress response (Salim et al .…”