Understanding and targeting chromatin mechanisms of cytoplasmic chromatin fragments during senescence and aging

Senescence and aging are associated with cytosolic DNA responses that drive chronic inflammation in the absence of infection. Chronic inflammation is associated with the infiltration of immune cells, disrupted cellular activities, an altered tissue microenvironment, and ultimately promotes many age-associated pathologies. Understanding and targeting senescence and aging-associated inflammation is a major biomedical objective. Our group has contributed to the understanding of chronic inflammation by showing that the cytosolic DNA sensing cGAS-STING pathway is involved. In senescent cells and aged mouse tissues, chromatin fragments undergo nucleus-to-cytoplasm trafficking, forming cytoplasmic chromatin fragments (CCF). CCF activate cGAS and eventually the senescence-associated secretory phenotype (SASP). These results have been independently reproduced by other groups and collectively, the CCF-cGAS-STING pathway is considered one of the central mechanisms contributing to chronic inflammation in senescence and aging. The genetic identity of CCF remains a major unaddressed question. We do not know from which chromatin regions CCF are derived and whether the formation of CCF is associated with the loss of genes, and if so, any actively transcribed genes. Unraveling the genetic identity of CCF is key to our understanding of the genetic alterations in senescence and aging as well as the origins of chronic inflammation. In this study, we propose to use cytosolic cGAS to pull down CCF followed by unbiased sequencing. Our preliminary results show that cGAS-bound CCF in senescent cells are comprised of specific chromatin regions enriched in tri-methylation of lysine 27 on histone H3 protein (H3K27me3), a heterochromatic mark associated with gene repression. Furthermore, we found that the H3K27me3 chromatin reader protein, CBX8, and not other H3K27me3 readers, is required for CCF formation. This application will examine a central hypothesis that CCF are derived from specific, rather than random, chromatin regions. We will test the specific hypothesis that the H3K27me3-CBX8 pathway mediates CCF formation, and reason that targeting CBX8 holds promise in blunting senescence and aging-associated inflammation. In Aim 1, we will test the role of the H3K27me3-CBX8 pathway in promoting CCF formation and the SASP in senescence of primary cells in vitro. In Aim 2, we will examine the role of CBX8 in promoting CCF and the SASP in vivo, employing oncogene-induced senescence in mouse liver as a model. In Aim 3, we will test this pathway in naturally aged mice, studying CBX8-mediated CCF formation and inflammaging. If the major goals are met, this study will offer novel mechanistic insights into CCF, a key event that promotes the SASP and inflammaging. The revealed chromatin mechanisms of CCF will also pave the way for future studies on the deeper biology of CCF. In addition, the study will reveal new therapeutic targets to block CCF, guiding the targeted design of therapies to address inflammaging and promote healthy aging. Project Number: 1R01AG094861-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: Zhixun Dou | Institution: MASSACHUSETTS GENERAL HOSPITAL, BOSTON, MA | Award Amount: $463,000 | Activity Code: R01 | Study Section: Cellular Mechanisms in Aging and Development Study Section[CMAD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11364288