TORC1 regulates metabolism and growth in response to a large array of upstream inputs. The GATOR complex, an upstream regulator of TORC1 activity, contains two sub-complexes, GATOR1 and GATOR2. The trimeric GATOR1 complex inhibits TORC1 activity in response to amino acid limitation. In humans, the GATOR1 complex has been implicated in a wide array of pathologies including cancer and hereditary forms of epilepsy. The GATOR2 complex inhibits the activity of GATOR1 to promote TORC1 activity. Relative to GATOR1, little is known about the regulation or mechanism of action of GATOR2. Over the last year, my laboratory has examined the role of the GATOR complex in the regulation of metabolism and oocyte development using the model organism Drosophila melanogaster. Our work has provided novel insights into the tissue specific regulation of TORC1 activity by the GATOR/TSC pathway. We previously demonstrated that in mutants of the GATOR2 component mio, the constitutive activation of the GATOR1 complex results in the permanent downregulation of TORC1 activity in the female germline and a block to oocyte growth and development. Disabling GATOR1 function, as is observed in mio, nprl3 double mutants, rescues, the mio mutant phenotype. Surprisingly, mio mutants are also suppressed by blocking the formation of meiotic double-stranded breaks (DSBs). One model to explain this observation is that meiotic DSBs trigger the GATOR1 dependent downregulation of TORC1 activity in the early meiotic cycle and that mio is required to attenuate this response later in oogenesis. To test this idea, we examined if blocking the formation of meiotic DSBs in the mio mutant background increased TORC1 activity. Towards this end, we compared the phosphorylation status of S6 kinase, a downstream TORC1 target, in ovaries from mio single mutants versus ovaries from mio, mei-W68 double mutant. mei-W68 (SPO11 homolog) is required for the generation of meiotic DSBs. Notably, we found that mio, mei-W68 double mutants have increased TORC1 activity relative to mio single mutants. This work resulted in the following conclusion: Meiotic DSBs trigger the GATOR1 dependent downregulation of TORC1 activity. The initiation of homologous recombination through the programmed generation of DNA DSBs is a universal feature of meiosis. DSBs represent a dangerous form of DNA damage that can result in dramatic and permanent changes to the germline genome. To minimize this destructive potential, the generation and repair of meiotic DSBs is tightly controlled in space and time. The observation that meiotic DSBs promote the GATOR1 dependent downregulation of TORC1 activity, suggested that low TORC1 activity may be important to the efficient repair of meiotic DSB. In line with this hypothesis, we found that GATOR1 mutant ovaries exhibit multiple phenotypes consistent with the misregulation of meiotic DSBs including an increase in the steady state number of meiotic DSBs, the retention of meiotic DSBs into later stages of oogenesis and the hyper-activation of p53, a transcription factor that mediates a highly-conserved response to genotoxic stress. Importantly, RNAi depletions of Tsc1 phenocopied the GATOR1 ovarian defects. These data confirm that the misregulation of meiotic DSBs observed in GATOR1 mutant oocytes are due to high TORC1 activity and not to a TORC1 independent function of the GATOR1 complex. Further genetic analysis demonstrated that GATOR1 impacts the repair, rather than the generation, of meiotic DSBs. These data are particularly intriguing in light of similar meiotic defects observed in npr3 mutants in Saccharomyces cerevisiae. These results raise the intriguing possibility that GATOR1 mediated down regulation of TORC1 activity may be a common feature of the early meiotic cycle in many organisms. This work resulted in the following conclusion: Constraining TORC1 activity in the early meiotic cycle is essential for the repair of meiotic DSBs and germline genome stability during Drosophila oogenesis. Genotoxic stress has been implicated in the deregulation of retrotransposon expression in multiple organisms including Drosophila. In line with these studies, we find that in GATOR1 mutants, the DSBs that initiate meiotic recombination trigger the deregulation of retrotransposon expression. Through epistasis analysis we determined that p53 and GATOR1 act through independent pathways to repress retrotransposon expression in the female germline. Surprisingly, TSC depletions in the female germline resulted in little to no increase in retrotransposon expression. These data raise the interesting possibility that GATOR1 regulates retrotransposon expression independent of TORC1 activity. Notably, GATOR1 components, but not TSC components, were recently identified in a high throughput screen for genes that suppress (Long Interspersed Element-1) LINE1 expression in mammalian tissue culture cells. This work resulted in the following conclusion: The GATOR1 complex opposes retrotransposon expression during meiosis in a pathway that functions in parallel to p53 in the female germline of Drosophila. Currently, our work in Drosophila represents the only in vivo examination of GATOR2 function in a multicellular animal. Notably, our studies challenge several predictions of the prevailing model of TORC1 regulation by the GATOR2 complex. Most importantly, our data suggest that multiple members of the GATOR2 complex function in the recovery from stress and may not be generally required for TORC1 activation in most metabolic conditions. However, currently it is impossible to test our models because of a lack of appropriate tools to study TORC1 in vivo. To remedy this situation, we are working to develop Drosophila as an in vivo model for the study of TORC1 regulation at the cellular and subcellular level. Specifically, we are tagging multiple proteins in the TORC1 regulatory pathway at their endogenous loci using CRISPR/Cas9 gene editing with both a small epitope tag that can be used for localization studies on fixed tissue and a fluorescent tag that can be used for live cell imaging A system for the in vivo evaluation of TORC1 regulation will greatly enhance our abilities to examine the role of TORC1 during meiotic progression and oocyte development and will allow us to directly test basic models of GATOR2 function. Additionally, we believe our system will provide a powerful tool for the Drosophila community to examine the importance of TORC1 regulation and function in myriad physiological and developmental contexts.
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