Summary Understanding the three-dimensional (3D) genome organization underlying senescence-associated transcriptional regulation remains a major obstacle to overcome. Cellular senescence is accompanied by! radical changes in transcription. For instance, senescent cells are associated with the dramatic upregulation of genes encoding secreted factors such as pro-inflammatory cytokines and chemokines, termed the senescence-associated secretory phenotype (SASP). Moreover, collaborative studies by the P01 team reveal that senescence is characterized by aberrant cryptic transcription, which causes transcription infidelity. Thus, it is critical to understand the 3D genomic basis of transcriptional regulation in senescent cells. Accordingly, the overall goal of this application is to determine 3D genomic basis of transcriptional regulation during senescence. CAPH2 is the regulatory subunit of the condensin II complex. Condensin II is implicated in organizing 3D genomic events ranging from enhancer-promoter contacts to forming topologically associating domains (TADs) to coordinate gene transcription in interphase cells. Notably, senescent cells are often associated with dramatic chromatin structure reorganization. Our preliminary data suggest that CAPH2 orchestrates changes in gene transcription by driving the 3D genome rearrangement during senescence. HMGB2 is a chromatin architecture protein that binds to DNA without sequence specificity. HMGB2 facilitates expression of its target genes by allowing for loop formation. Our published work shows that HMGB2’s redistribution to SASP gene loci promotes their expression by preventing the spreading of heterochromatin. Our unpublished data indicate that HMGB2 interacts with CAPH2 in senescent cells. Both HMGB2 and CAPH2 are associated with SASP gene loci. Our central hypothesis is that CAPH2 organizes the 3D genomic structure to regulate transcription during senescence. We also hypothesize that the CAPH2/HMGB2 complex forms the 3D genomic basis of the SASP. Three specific aims are proposed: Aim 1 will investigate the 3D genome structure basis of transcriptional infidelity during cellular senescence; Aim 2 will characterize the interaction between HMGB2 and condensin II complex; Aim 3 will determine the mechanism by which the CAPH2/HMGB2 complex regulates SASP. This application is highly innovative because this is the first study to explore 3D genomic basis that orchestrates gene transcription during senescence. Thus, our studies are paradigm-shifting in their potential to elucidate the 3D genomic basis of transcriptional reprogramming during senescence. The proposed studies are of high impact because these newly gained insights may ultimately allow for the development of rationale strategies to limit the detrimental side effects of senescence and consequently promotes healthy aging.