Investigating the roles of a Topoisomerase complex in autophagy and lifespan regulation

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This new proposal is to investigate how a RNA/DNA dual-activity topoisomerase regulates autophagy and affects lifespan in animal models. Autophagy is an evolutionarily conserved self eating process that is activated when cells and organisms are under starvation3. The optimal autophagic activity is critical to stress survival by removing damaged organelles and recycling necessary proteins and nutrients. Notably, compromised autophagy is a hallmark of aging (Aman et al., Nature Aging, 2021), and has been observed in multiple age related neurodegenerative and autoimmune diseases4,5. In model organisms, reduced autophagy is associated with shortened lifespan, whereas increased autophagy is associated with delayed aging (Aman et al., Nature Aging, 2021). Autophagy is a therapeutic target, as compounds that increase autophagy can improve lifespan and reduce age-associated disease conditions in model organisms. Some of these compounds are being examined in clinical trials (Aman et al., Nature Aging, 2021). Our group has discovered the first DNA/RNA dual activity topoisomerase complex in eucaryotes, Topoisomerase 3 beta (Top3b)-TDRD3. This complex can resolve both DNA and RNA topological complexity. Top3b-Tdrd3 complex can regulate neuronal activity-activated transcription6, mRNA translation and turnover7,8. Notably, the Top3b mutation has been linked to multiple mental disorders including schizophrenia, autism, and intellectual disability6,9,10. Moreover, mouse carrying Top3b mutations exhibits shorter lifespans11. However, the mechanisms underlying how Top3b inactivation can lead to reduced lifespan and neurological disorders remain unclear. One possible mechanism is that Top3b-Tdrd3 may prevent early aging by silencing transposons12,13,the jumping genetic elements that can be a driver of aging and aging associated diseases. Here we propose another possible mechanism-- TOP3B-TDRD3 may prevent premature aging and age associated conditions by promoting autophagy. We will investigate this proposal by using combined expertise of three groups, Wang (Top3b, Drosophila, genomics), DeCabo (starvation, mouse), and Cai (neurological diseases, mouse). It is known that in response to starvation, cellular transcription and translation are both reprogramed (Feng et al., Trends Cell Biol., 2015)(Fullgrabe et al., NRMCB 2014); and expression of many critical autophagy-related genes (ATGs) are activated (Peeters, et al., Autophagy 2019)14. Disruption of activation of autophagy genes can lead to shortened lifespan. Because Top3b-TDRD3 can regulate both transcription and translation, we hypothesize that this complex may also facilitate starvation-induced expression of autophagy genes at either transcription or translation steps. We plan to investigate whether cells or animal models inactivated of TOP3B-TDRD3 exhibit defective expression of autophagy genes, as well as reduced autophagy in three specific aims. Top3b promotes starvation-activated and repressed transcription (Aim 1) We have established the TOP3B-KO and TDRD3-KO HCT116 cell culture system for studies of starvation-induced gene expression and autophagy (Su et al., manuscript in revision). Our RNA-seq shows that many genes are up- or down-regulated in response to starvation, and we named them SAGs (starvation activated genes) and SRGs (starvation repressed genes), respectively. Notably, our ChIP-seq using RNAPII-ser2p antibodies, a transcription elongation marker, revealed that many of those SAGs and SRGs display altered transcription, suggesting that Top3b is critical for starvation induced transcriptional activation and repression. TOP3B-KO alters the transcription of autophagy-associated genes (Aim 1) Our RNA-seq and RNAP2-ser2p ChIP-seq revealed that several autophagy-associated genes show reduced transcription in Top3b-KO HCT116 cells upon starvation. For instance, with a key active autophagy marker, MAP1LC3B transcription is increased by starvation in RNA-seq and RNAPII-ser2p ChIP-seq in WT, but this increase is diminished in Top3b-KO. Western blotting confirmed the reduction of one isoform of this protein. We have also performed lysotracker staining, a common method to detect autophagy; and observed reduced staining in TOP3B-KO cells at 6 hours post starvation. Drosophila exhibits reduced autophagy and shorter lifespan responding to starvation (Aim 2,3) We tested whether autophagy activation in response to starvation is defective in TOP3b-KO Drosophila. Six hours of starvation induced a significant increase of LysoTracker signals in the fat bodies of WT Drosophila 3rd instar larva, however, this activation is nearly completely absent in Top3b-/-, implying that Top3b is necessary for starvation-induced autophagy formation. Notably, we found that the Top3b-/- flies show a significantly shorter lifespan upon starvation vs. WT flies. The data support our hypothesis that Top3b facilitates normal lifespan by promoting autophagy. We have published one paper showing that Top3b-TDRD3 complex promotes transcription starvation-induced genes and autophagy per se in human cancer cells. We also demonstrate that Top3b-KO flies show reduced autophagy (Su et al., NAR 2023).