PROJECT SUMMARY Hematopoietic stem cells (HSCs) regenerate all blood and immune cells throughout life. Aging HSCs exhibit diminished regenerative function, reduced lymphoid potential, and clonal outgrowth that is associated with compromised immunity as well as an increased incidence of anemia, bone marrow failure, and hematological malignancies in older adults. The regulation of protein homeostasis (proteostasis) has recently emerged as a fundamental process required to promote HSC self-renewal. Loss of proteostasis is considered one of the hallmarks of aging, but to what extent it contributes to stem cell aging is largely unknown. An essential pathway in maintaining proteostasis is the Heat Shock Response, which is regulated by the master transcription factor Heat shock factor 1 (Hsf1). Hsf1 induces expression of heat shock proteins that aid in proper protein folding, trafficking, and degradation. At steady state, Hsf1 is typically sequestered in the cytoplasm, but translocates into the nucleus in response to cellular and proteotoxic stress. Previously, we demonstrated that HSCs undergo cellular stress when cultured ex vivo and Hsf1 activation can alleviate this stress to maintain HSC regenerative activity. Hsf1 is highly expressed in young and old adult HSCs but is specifically activated during aging in middle- aged and old adult HSCs. Aging is a notably stressful process associated with the accumulation of genetic mutations, inflammation, and oxidative stress. Based on these preliminary results, the central hypothesis of this proposal is that Hsf1 activation promotes HSC function and proteostasis during aging. To test this hypothesis, Aim 1 will examine the role of Hsf1 in aging HSC function and proteostasis using conditional Hsf1 knockout mice. HSC function will be assessed in competitive transplantation assays and proteostasis will be assessed by quantifying protein synthesis, proteasome activity, misfolded protein, unfolded protein, and protein aggregate abundance. I expect that there will be less reconstitution in aged Hsf1-deficient HSCs and more protein synthesis, misfolded and unfolded proteins, and aggregates. While Hsf1 activation is hypothesized to be important for HSC function during aging, the mechanism underlying heterochronic Hsf1 activation is unknown. Preliminary RNA-sequencing results revealed that Transglutaminase 2 (Tgm2), involved in Hsf1 activation, is significantly upregulated in old adult HSCs. Thus, Aim 2 will examine if age-related Hsf1 activation depends on Tgm2 upregulation. Hsf1 activation, HSC function, and proteostasis will be assessed in conditional Tgm2 knockout mice. I expect that loss of Tgm2 in aging HSCs will disrupt proteostasis and exhibit an associated decline in fitness and function due to a decrease in Hsf1 activation. Collectively, these studies will provide deeper insights into mechanisms that regulate proteostasis during stem cell aging. These findings will uncover new therapeutic targets to promote ...