Abstract Evolutionarily conserved stress-responsive transcription factors (TFs) are required for longevity in diverse genetic pathways. Their activities are repressed in early adulthood, preceding physiological decline in animal models, implicating their central roles in aging. It is poorly understood, however, how they control transcription to promote longevity, and how their activities are regulated in different tissues at critical life stages to dictate organismal aging. Our long-term goal is to identify the causes of aging and extend the human healthspan. The objective of this grant is to understand the function and regulation of stress-responsive TFs in longevity. We use the nematode C. elegans as a model and focus on two highly conserved stress-responsive TFs, HSF-1 (heat shock factor 1) and DAF-16 (the C. elegans ortholog of FOXO). Our central hypothesis is that the spatiotemporal regulation of HSF-1 and DAF-16 dictates a neuron-to-intestine signaling pathway to regulate longevity. Our hypothesis is based on the following findings: 1) JMJD-3.1, a conserved H3K27 demethylase that facilitates HSF-1 target binding in stress responses, decreases expression upon reproductive maturity. 2) Inhibition of germline stem cells preserves JMJD-3.1 expression and extends lifespan in a manner that requires HSF-1. 3) Expression of JMJD-3.1 is highly enriched in neurons. 4) Over-expression of HSF-1 solely in neurons is sufficient to promote longevity in a manner that depends on intestinal DAF-16. Our specific aims will test the following hypotheses: (Aim 1) JMJD-3.1 enhances HSF-1 activity in neurons to promote longevity; (Aim 2) Neural HSF-1 contributes to a neuron-to-intestine signaling pathway involving specific neuropeptides to activate DAF-16 in the intestine; (Aim 3) Intestinal DAF-16 creates chromatin accessibility for other co-factors to activate diverse longevity-promoting pathways. Upon completion, our results will reveal the functions and regulation of HSF-1 and DAF-16 in promotion of longevity at the molecular, cellular and organismal levels. This contribution is significant because it advances our mechanistic understanding of stress-responsive TFs in longevity and their functional interactions with two hallmarks of aging, namely `epigenetic alterations' and `altered intercellular communication'. Furthermore, this study will provide a basis for new ideas of modulating stress-responsive TFs to ameliorate age-related declines. This study is innovative because we investigate HSF-1 and DAF-16 in longevity beyond their canonical roles in stress responses, and we apply unbiased genomic analyses of transcription and chromatin in a tissue-specific manner to understand their mechanistic roles in organismal aging.