Regulation of epigenome stability by SIRT6 during Aging

这个项目的长期目标是确定调节表观基因组的分子机制。 衰老过程中的稳定性以及哺乳动物sirtuin，SIRT6在这一过程中的作用。越来越多的证据表明 这种表观基因组结构随着年龄的增长而受损，这可能是年龄相关性下降的根本原因 在细胞和器官功能上。SIRT6作为与衰老相关的多个途径的关键调节因子出现，例如 基因组和表观基因组稳定性、肿瘤发生、炎症和糖酵解。此外，SIRT6 过度表达可以延长小鼠的寿命。 本实验室已证实SIRT6是DNA双链断裂(DSB)的上游调控因子 修理。我们证明了SIRT6被JNK1/2磷酸化，以响应氨基酸上的氧化应激 S10，这种磷酸化是刺激DSB修复所必需的。我们已经证明，除了 控制DSB修复的SIRT6通过抑制转座元件来维持基因组的稳定性，并且 氧化应激导致SIRT6从转座元件的启动子到DNA断裂的重新定位。 SIRT6具有两种生化活性：脱乙酰酶和单腺苷二磷酸核糖基酶。该功能 SIRT6脱乙酰酶活性的最好表征。相比之下，SIRT6的单-ADP核糖基化活性更高 研究较少。我们的工作表明，这种活性与DNA修复和表观基因组的稳定性有关。我们最近未出版的 分析在百岁老人中发现的SIRT6变异的生化功能的数据显示，百岁老人的SIRT6 降低脱乙酰基活性，增强单-ADP核糖化活性。这表明SIRT6 单-ADP核糖基化活性对长寿很重要。因此，我们着手识别新的功能和 SIRT6单-ADP核糖化的靶点。利用质谱学方法，我们确定了SIRT6单核苷酸的新靶点。 ADP核糖化包括组蛋白H1、H_2A、H_2A、J以及染色质重构体BRG1和SMARCC2。 此外，我们还证明了SIRT6介导的SMARCC2核糖化是激活Nrf2靶标所必需的 基因对氧化应激的反应。 因此，我们处于进行SIRT6单-ADP作用的进一步机械性研究的理想位置 核糖化活性在表观基因组稳定性中的作用。我们将追求以下具体目标：(1)研究 SIRT6在细胞衰老和衰老背景下维持表观基因组稳定性的作用；(2)研究 SIRT6对表观基因组的作用机制；具体地说，H1、H_2A、H_2A、J、BRG1和 SMARCC2单-ADP核糖化；以及(3)确定SIRT6单-ADP核糖化活性在 通过分析表达核糖化缺陷的敲入小鼠模型来研究表观基因组的稳定性和寿命 SIRT6突变。拟议的研究将描绘由SIRT6调控的新途径，这些途径与 表观基因组的稳定性和老化。因此，我们期待这些实验将揭示关键的新信息。 关于衰老过程的研究，将有助于制定治疗年龄相关疾病的新战略。

The long-term goal of this project is to define the molecular mechanisms which regulate epigenome stability during aging and the role of the mammalian sirtuin, SIRT6, in this process. Growing evidence indicates that epigenome structure becomes compromised with age which may be the root cause of age-related decline in cell and organ function. SIRT6 emerged as a critical regulator of multiple pathways related to aging such as genome and epigenome stability, tumorigenesis, inflammation and glycolysis. Additionally, SIRT6 overexpression extends the lifespan of mice. Our laboratory has demonstrated that SIRT6 is an upstream regulator of DNA double strand break (DSB) repair. We demonstrated that SIRT6 is phosphorylated by JNK1/2 in response to oxidative stress on amino acid S10 and this phosphorylation is required for the stimulation of DSB repair. We have shown that, in addition to controlling DSB repair, SIRT6 maintains genome stability by repressing transposable elements, and that oxidative stress causes re-localization of SIRT6 from the promoters of transposable elements to the DNA breaks. SIRT6 has two biochemical activities deacetylase (deacetylase) and mono-ADP ribosylase. The function of SIRT6 deacetylase activity is best characterized. In contrast, mono-ADP ribosylation activity of SIRT6 is much less studied. Our work has implicated this activity in DNA repair and epigenome stability. Our recent unpublished data analyzing biochemical functions of a SIRT6 variant found in centenarians, showed that centenarian SIRT6 has reduced deacetylation activity and enhanced mono-ADP ribosylation activity. This suggests that SIRT6 mono-ADP ribosylation activity is important for longevity. Therefore, we set out to identify the function and new targets of SIRT6 mono-ADP ribosylation. Using mass spectrometry we identified novel targets for SIRT6 mono- ADP ribosylation including histone H1, H2A, H2A.J, and chromatin remodelers BRG1 and SMARCC2. Furthermore, we showed that SIRT6-mediated ribosylation of SMARCC2 is required for activation of Nrf2 target genes in response to oxidative stress. Thus, we are ideally positioned to conduct further mechanistic studies of the role of SIRT6 mono-ADP ribosylation activity in epigenome stability. We will pursue the following specific aims: (1) examine the role of SIRT6 in maintaining epigenome stability in the context of cellular senescence and aging; (2) examine the mechanisms of SIRT6 effect on epigenome; specifically, the biological function of H1, H2A, H2A.J, BRG1 and SMARCC2 mono-ADP ribosylation; and (3) determine the role of SIRT6 mono-ADP ribosylation activity in epigenome stability and longevity by analyzing the knock-in mouse model which expresses ribosylation deficient SIRT6 mutation. The proposed research will delineate new pathways regulated by SIRT6, which are relevant to epigenome stability and aging. As such, we expect that these experiments will reveal critical, new information about the aging process, and will help to develop new strategies for treating age-related diseases.