Summary Cellular senescence, the irreversible cessation of the cell cycle, has emerged as an important regulator of age- associated pathologies. Senescence is linked to compromised genome organization, loss of coordinated gene expression, and activation of a senescence-associated secretory phenotype (SASP), an inflammatory cascade that can affect neighboring cells. Aspects of senescence are likely beneficial to tissue homeostasis through immune-mediated clearance of damaged cells, while other aspects and the SASP are likely deleterious and have been linked to chronic inflammation and age-related pathologies. Thus, dissecting the molecular mechanisms that direct the emergence of senescence remains of interest to a wide variety of scientists, including those studying aging, age-linked disease, and cellular identity. Nuclear architecture is a powerful mechanism underlying coordinated gene expression. While senescence is associated with a loss of peripheral chromatin organization, it is unclear if these changes to genome organization result in loss of spatial LAD positioning; if so, do these positioning changes precede senescence or are they a byproduct of senescence? Moreover, because of the population-based approach of genomics assays used to probe senescence, it remains unknown if there is heterogeneity of genome organization as cells senesce or if synchronous changes occur in all cells. The importance of this question is highlighted by studies using state-of-the-art imaging technologies, similar to the ones we propose, demonstrating remarkable heterogeneity in genome organization across single cells that have been masked by bulk assays. In the proposed studies, we seek to test the hypothesis that compromised transcriptional mechanisms that maintain the spatial positioning of loci relative to the nuclear lamina underlie senescence phenotypes. We will use a combination of Oligopaints and super- resolution microscopy to identify and dissect the molecular players that guide spatial positioning and their relationship to chromatin structure and senescence at single-cell resolution. Our interdisciplinary team will reveal how locus positioning changes are linked to senescence and will manipulate regulators of genome spatial positioning that we have started to identify to determine if senescence phenotypes are accelerated by their alteration. Given the emerging importance of nuclear architecture in several human diseases, our studies will provide critical insights into the molecular rationale for targeting genome organization changes in senescence, knowledge that can be applied to other diseases including aging-associated conditions and cancer.