Exploring the sequence identity of cytoplasmic chromatin in senescence

项目总结 细胞衰老是与炎症反应相关的细胞周期停滞的一种稳定形式。衰老 细胞聚集在老化和患病的组织中，被认为是导致 与大多数(如果不是全部)年龄相关疾病有关的慢性炎症。与这一概念相一致， 基因或药物清除衰老细胞可延长小鼠的寿命和健康时间。 衰老细胞分泌大量促炎细胞因子、趋化因子、生长因子和 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，招募免疫细胞，最终导致炎症。我们的小组最近 显示衰老的细胞在胞浆中显示基因组DNA，这被细胞解释为一种“危险” 通过触发先天免疫胞浆DNA感应cGAS-STING通路促进SASP 衰老程序。这些发现已经由几个实验室独立复制，并且 总体而言，cGAS-STING途径被认为是SASP计划的中心机制。 衰老过程中一个尚未解决的主要问题是细胞质基因组DNA的遗传起源。我们的影像 结果表明，胞质dna是由染色质片段经核质传递而来。 通过核膜气泡进入细胞质进行运输。但是基因组的哪些部分是 穿梭到细胞质？这些区域的染色质状况如何？基因组会丢失基因吗？这些 问题需要不偏不倚的排序方法来解决。该应用程序提出了两种新的策略 对衰老细胞的胞质DNA进行测序。首先，我们的目标是鉴定cGAS相关的胞浆DNA。 衰老，通过执行cGAS DNA免疫沉淀。第二，我们的目标是从生物化学上分离出 从衰老细胞的细胞质中提取的DNA。DNA样本将接受下一代测序和 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 美国将直接操纵基因组DNA以查询细胞接受基因组后的功能后果 DNA转运到细胞质。 这项研究将帮助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变化 衰老和衰老。这项研究有可能促进新的方法来靶向和抑制慢性 促进健康衰老和抑制与年龄相关的疾病的炎症。

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.