Abstract Based on the importance of defining new insights into cellular senescence, we initiated studies to investigate whether there might be a specific enhancer activation “code” that underlies cellular senescence for identifying the responsible DNA binding transcription factors. While there is rapidly-emerging, and now unassailable evidence, on the role of the 40-70,000 enhancers in each cell type in development, homeostasis and, often, pathological events, their role in cellular senescence remains undefined. Furthermore, while cellular senescence represents a fundamental process of aging and a known driver of pathologies, the causative role of newly activated enhancer cohorts underlying progression of senescence remain poorly understood. Therefore, the goal of this proposal, supported by extensive preliminary data, is to test a novel hypothesis that the de novo appearance of two specific cohorts of enhancers sets into motion a progressive, functionally- important, alteration in gene transcription programs. Based on our study of the altered enhancer and chromosomal landscape during replicative senescence, we have begun to establish that the geroprotective mTOR inhibitor, Rapamycin, markedly delays all aspects of cellular senescence, including the appearance of new, functional, enhancers. Our focus is to elucidate the functional importance of a gained enhancer program underlying cellular senescence, and identify the critical DNA binding transcription factors underlying the transcriptional programs that are determinants of replicative senescence, based on the complementary expertise of the Suh and Rosenfeld laboratories. Specifically: i) We will use unbiased screens to document that at least two distinct activated enhancer networks independently regulate the proliferation arrest and SASP aspects of replicative senescence, respectively. ii) We will identify combinatorial factors synergizing with the previously-unrecognized transcription factors, NFI-A, NFI-C, to regulate the gained enhancers underling proliferation arrest, and those that, with SMAD2/3 and NFkB, to regulate the SASP program. In parallel, we can implicate the underlying signaling pathways. iii) We will identify previously unrecognized histone modification signatures of, and their functional importance in replicative senescence . iv) We will Identify Activin and Tgf2 as inhibitors of the proliferation and SASP enhancer programs, respectively. Our proposal promises to provide transformative insights into molecular events that initiate and perpetuate the senescent cell phenotypes, and help elucidate potential novel therapeutic modalities against the deleterious SASP program.