Aging-induced nucleolar decline and chromosomal instability

项目摘要。 异染色质由大量重复的DNA结构域组成，如着丝粒、端粒、rDNA、 以及在整个间期保持组织成紧密染色质结构的反转录转座子， 当染色体的其他部分(常染色质)通常解聚时。异染色质是 通常抑制转录，在维持基因组稳定性方面发挥重要作用，无论是 它通过促进着丝粒功能、端粒保护或抑制重组 在潜在的DNA重复之间，以帮助保持基因组的完整性。情况就是这样。 RDNA基因座，是一种研究相对较少和不寻常的异染色质形式，由线粒体组成 重复的rRNA基因。矛盾的是，rDNA被RNA聚合酶I(Pol I)大量转录到 合成核糖体RNA，但保留几个关键的异染色质特征，如抑制 依赖于RNA聚合酶II(POL II)的转录的重组和“沉默”。萌芽中 酵母rDNA，来自基因间隔区的非编码RNA的转录必须被沉默 保守的NAD+依赖的组蛋白去乙酰基酶Sir2，以维持rDNA的稳定性并支持复制 寿命。值得注意的是，Sir2对这些非编码RNA的沉默实际上需要依赖Pol I 转录大的rRNA编码基因。因此，核仁具有相当复杂的和 动态染色质环境旨在优化rRNA合成，同时保持 串联阵列。在发芽酵母的复制老化过程中，Sir2蛋白和粘附素一起 复合体和其他核蛋白随着细胞的老化而逐渐耗尽。这将导致 RDNA异染色质的劣化和阵列的不稳定性。有趣的是，复制的 衰老细胞也具有由rDNA不稳定性驱动的染色体不稳定(CIN)表型。 粘附素Mcd1亚基的过表达抑制AGE诱导的rDNA和CIN 表型，并有力地延长寿命。该项目中的实验旨在识别 确定由Sir2和粘附素稳定的rDNA阵列如何提高保真度 染色体分离。我们假设rDNA与基因组的相互作用包括 着丝粒在年龄诱导的CIN表型中起重要作用。因此，我们将开发一种 定义全基因组rDNA接触的方法，并测试这些接触如何随年龄或何时变化 RRNA的合成受到了影响。机械实验也将解决为什么某些原子核 随着年龄的增长，蛋白质被耗尽或不稳定，并确定了额外的剂量依赖寿命 各种因素。我们期待这些对酵母的研究将为未来的结构功能提供一个范例 哺乳动物细胞衰老过程中rdna异染色质的研究。

Project Summary. Heterochromatin consists of large domains of repetitive DNA such as centromeres, telomeres, rDNA, and retrotransposons that remain organized into compact chromatin structures throughout interphase, when other portions of the chromosomes (euchromatin) usually decondense. Heterochromatin is generally repressive to transcription and plays important roles in maintaining genome stability, whether it is through facilitating centromere function, telomere protection, or suppressing recombination between the underlying DNA repeats to help maintain genomic integrity. Such is the case with the rDNA locus, a relatively understudied and unusual form of heterochromatin consisting of tandemly repeated rRNA genes. Paradoxically, the rDNA is heavily transcribed by RNA polymerase I (Pol I) to synthesize ribosomal RNA, yet retains several key heterochromatin characteristics such as suppression of recombination and “silencing” of RNA polymerase II (Pol II)-dependent transcription. In budding yeast rDNA, transcription of non-coding RNAs from the intergenic spacers must be silenced by the conserved NAD+-dependent histone deacetylase Sir2 to maintain rDNA stability and support replicative lifespan. Remarkably, silencing of these non-coding RNAs by Sir2 actually requires Pol I-dependent transcription of the large rRNA coding genes. Therefore, the nucleolus has a rather complex and dynamic chromatin environment designed to optimize rRNA synthesis while maintaining integrity of the tandem array. During replicative aging of budding yeast, the Sir2 protein, along with the cohesin complex, and other nuclear proteins, are progressively depleted as the cells get older. This results in deterioration of the rDNA heterochromatin and instability of the array. Interestingly, the replicatively aging cells also have a chromosome instability (CIN) phenotype that is driven by the rDNA instability. Overexpression of the Mcd1 subunit of cohesin suppresses the age-induced rDNA and CIN phenotypes, and strongly extends lifespan. The experiments in this project are designed to identify determine how stabilization of the rDNA array by SIR2 and cohesin leads to improved fidelity of chromosome segregation. We hypothesize that rDNA interactions with the genome, including centromeres, play a significant role in the age-induced CIN phenotype. Therefore, we will develop a method to define genome-wide rDNA contacts, and test how these contacts change with age or when rRNA synthesis is compromised. Mechanistic experiments will also address why certain nuclear proteins are depleted or destabilized with aging, and identify additional dose dependent longevity factors. We anticipate these studies in yeast will provide a paradigm for future structural-function studies of rDNA heterochromatin during aging in mammalian cells.