Exploring the sequence identity of cytoplasmic chromatin in senescence

项目摘要 细胞衰老是与炎症反应相关的细胞周期停滞的稳定形式。衰老 细胞积聚在老化和患病的组织中，并被认为是导致 慢性炎症，涉及大多数（如果不是全部）与年龄相关的疾病。与这一概念相一致， 衰老细胞的遗传或药物清除延长了小鼠的寿命和健康期。 衰老细胞分泌大量的促炎细胞因子、趋化因子、生长因子， 蛋白酶，统称为衰老相关分泌表型（SASP）。SASP计划 改变组织微环境并募集免疫细胞，最终导致炎症。我们组最近 结果显示，衰老细胞在细胞质中显示出基因组DNA，这被细胞解释为“危险”， 通过触发促进SASP的先天免疫细胞溶质DNA传感cGAS-STING途径， 衰老的程序这些发现已经被几个实验室独立复制， cGAS-STING途径被认为是SASP程序的中心机制。 细胞质基因组DNA的遗传起源是衰老中一个尚未解决的问题。我们的成像 结果表明，胞质DNA来源于染色质片段，由核质介导 运输，通过核膜泡分配到细胞质中。但是基因组的哪些部分 穿梭于细胞质中这些区域的染色质状态如何？基因组会丢失基因吗？这些 问题需要无偏见的排序方法来解决。本申请提出了两种新颖的策略 对衰老细胞的细胞质DNA进行测序。首先，我们的目标是鉴定cGAS相关的胞质DNA， 衰老，通过进行cGAS DNA免疫沉淀。第二，我们的目标是生物化学压裂 来自衰老细胞的细胞质的DNA。DNA样本将进行下一代测序， 计算分析以探索染色质标记和基因表达状态。这些结果将允许 我们可以直接操纵基因组DNA，以询问细胞经历基因组改造后的功能结果。 DNA运输到细胞质。 这项研究将有助于衰老领域了解衰老的关键机制- 相关的炎症，并可能揭示以前未知的知识的遗传改变， 衰老和老化。这项研究有可能促进新的方法来靶向和抑制慢性 炎症，以促进健康衰老和抑制与年龄相关的疾病。

PROJECT SUMMARY Cellular senescence is a stable form of cell cycle arrest associated with inflammatory responses. Senescent cells accumulate in aged and diseased tissues and are considered as one of the major sources contributing to chronic inflammation that is implicated in most, if not all, age-associated disorders. Consistent with this notion, genetic or pharmaceutical clearance of senescent cells extend lifespan and healthspan of mice. Senescent cells secret a large array of pro-inflammatory cytokines, chemokines, growth factors, and proteases, collectively referred to as senescence-associated secretory phenotype (SASP). The SASP program alters tissue microenvironment and recruits immune cells, ultimately leading to inflammation. Our group recently showed that senescent cells exhibit genomic DNA in the cytosol, which is interpreted by the cells as a “danger signal” by triggering the innate immunity cytosolic DNA sensing cGAS-STING pathway that promotes the SASP program of senescence. These findings have been independently reproduced by several laboratories, and collectively the cGAS-STING pathway is considered as a central mechanism for the SASP program. A major unaddressed question in senescence is the genetic origin of cytosolic genomic DNA. Our imaging results suggest that the cytosolic DNA is derived from fragments of chromatin, mediated by nucleus-to-cytoplasm trafficking, via nuclear membrane blebs that partition into the cytoplasm. But which parts of the genome are shuttled to the cytoplasm? What is the chromatin status of those regions? Does the genome lose genes? These questions require unbiased sequencing approaches to address. This application proposes two novel strategies to sequence cytoplasmic DNA in senescent cells. First, we aim to identify cGAS-associated cytosolic DNA in senescence, by performing cGAS DNA-immunoprecipitation. Second, we aim to biochemically fractionate the DNA from the cytoplasm of senescent cells. The DNA samples will be subjected to next-gen sequencing and computational analyses to explore the chromatin marks and gene expression status. These results will permit us to directly manipulate the genomic DNA to inquire the functional consequences of cells undergoing genomic DNA trafficking to the cytoplasm. This study will help the senescence field understand a critical mechanism underlying senescence- associated inflammation, and may reveal previously unknown knowledge of the genetic alterations of senescence and aging. This study has the potential to facilitate new approaches to target and inhibit chronic inflammation to promote healthy aging and to suppress age-associated diseases.