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

Sir2 is a highly conserved NAD+-dependent histone deacetylase that functions in heterochromatin formation and promotes replicative lifespan (RLS) in the budding yeast, Saccharomyces cerevisiae. Within the yeast rDNA locus, Sir2 is required for efficient cohesin recruitment and maintaining stability of the tandem array. In addition to the previously reported depletion of Sir2 in replicatively aged cells, we discovered that subunits of the Sir2 containing complexes, SIR and RENT, were depleted. Several other rDNA structural protein complexes also exhibited age-related depletion, most notably the cohesin complex. We hypothesized that mitotic chromosome instability (CIN) due to cohesin depletion could be a driver of replicative aging. ChIP assays of the residual cohesin (Mcd1-13xMyc) in moderately aged cells showed strong depletion from the rDNA and initial redistribution to the point centromeres, which was then lost in older cells. Despite the shift in cohesin distribution, sister chromatid cohesion was partially attenuated in aged cells and the frequency of chromosome loss was increased. This age-induced CIN was exacerbated in strains lacking Sir2 and its paralog, Hst1, but suppressed in strains that stabilize the rDNA array due to deletion of FOB1 or through caloric restriction (CR). Furthermore, ectopic expression of MCD1 from a doxycycline-inducible promoter was sufficient to suppress rDNA instability in aged cells and to extend RLS. Taken together we conclude that age-induced depletion of cohesin and multiple other nucleolar chromatin factors destabilize the rDNA locus, which then results in general CIN and aneuploidy that shortens RLS.

Sir2是一种高度保守的烟酰胺腺嘌呤二核苷酸（NAD+）依赖的组蛋白去乙酰化酶，在出芽酵母（酿酒酵母，Saccharomyces cerevisiae）中参与异染色质形成，并可延长其复制寿命（RLS）。在酵母核糖体DNA（rDNA）位点内，Sir2对于高效募集黏连蛋白（cohesin）以及维持串联阵列的稳定性是必需的。除此前已报道的复制衰老细胞中Sir2的表达耗竭外，本研究还发现包含Sir2的SIR复合物与RENT复合物的亚基同样出现了耗竭。另有多种rDNA结构蛋白复合物也表现出衰老相关的表达耗竭，其中尤以黏连蛋白复合物最为显著。我们提出假说：黏连蛋白耗竭引发的有丝分裂染色体不稳定性（CIN）可能是复制衰老的驱动因素。对中度衰老细胞中残留的黏连蛋白（Mcd1-13xMyc）进行的染色质免疫沉淀（ChIP）实验显示，该蛋白从rDNA位点发生显著耗竭，并最初重新分布至着丝粒区域，但在更老龄的细胞中这种重新分布现象也随之消失。尽管黏连蛋白的分布发生了上述改变，衰老细胞中的姐妹染色单体黏连仍出现了部分减弱，且染色体丢失的频率有所升高。这种衰老诱导的CIN在缺失Sir2及其旁系同源基因HST1的菌株中会进一步加剧，但在因敲除FOB1或通过热量限制（CR）稳定rDNA串联阵列的菌株中则受到抑制。此外，通过多西环素诱导型启动子异位表达MCD1，足以抑制衰老细胞中的rDNA不稳定现象，并延长复制寿命（RLS）。综合以上实验结果，我们得出结论：衰老诱导的黏连蛋白及多种其他核仁染色质因子的耗竭会使rDNA位点失稳，进而引发全身性的染色体不稳定性与非整倍性（aneuploidy），最终缩短复制寿命（RLS）。