Exploring the sequence identity of cytoplasmic chromatin in senescence

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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.