Cell Cycle Regulation In Oogenesis

卵子发生中的细胞周期调控

• 批准号: 10007491
• 负责人: MARY A LILLY
• 金额: $ 140.13万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10007491
• 关键词: Amino Acids; Animal Model; Animals; Attenuated; CRISPR/Cas technology; Cell Cycle; Cell Cycle Regulation; Cells; Communities; Complex; Congenital Abnormality; DNA Damage; DNA Double Strand Break; Dangerousness; Data; Defect; Development; Down-Regulation; Drosophila genus; Drosophila melanogaster; Epilepsy; Epitopes; Event; Exhibits; Female; Gametogenesis; Generations; Genes; Genetic Epistasis; Genome; Genome Stability; Genotoxic Stress; Germ Cells; Goals; Growth; Growth and Development function; Homologous Gene; Human; Inherited; Laboratories; Light; Long Interspersed Elements; Malignant Neoplasms; Mediating; Meiosis; Meiotic Recombination; Metabolic; Metabolism; Modeling; Oocytes; Oogenesis; Organism; Ovarian; Ovary; Pathology; Pathway interactions; Phenocopy; Phenotype; Phosphorylation; Physiological; Proteins; RNA Interference; Recovery; Regulation; Regulatory Pathway; Reproductive Biology; Reproductive Medicine; Retrotransposon; Ribosomal Protein S6 Kinase; Role; Saccharomyces cerevisiae; Signal Pathway; Spontaneous abortion; Stress; Study models; System; TP53 gene; TSC1 gene; Testing; Time; Tissues; Work; chromosome missegregation; egg; event cycle; genetic analysis; high throughput screening; homologous recombination; hormonal signals; in vivo; in vivo Model; in vivo evaluation; insight; live cell imaging; member; mutant; novel; programs; repaired; response; tissue/cell culture; tool; transcription factor

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.

TORC1根据大量上游输入来调节新陈代谢和生长。 Gator Complex是TORC1活性的上游调节剂，包含两个子复合物，Gator1和Gator2。 Trimeric Gator1复合物可抑制TORC1对氨基酸限制的活性。在人类中，Gator1复合体已与包括癌症和遗传形式的癫痫病在内的各种各样的病变有关。 Gator2复合物抑制Gator1的活性促进TORC1活性。相对于Gator1，对Gator2的调节或作用机理知之甚少。在过去的一年中，我的实验室研究了使用模型有机体果蝇Melanogaster研究Gator复合物在代谢和卵母细胞发育中的作用。我们的工作为Gator/TSC途径对TORC1活性的组织特异性调节提供了新的见解。 我们先前证明，在Gator2成分MIO的突变体中，Gator1复合物的组成型激活导致雌性种系中Torc1活性的永久下调，并导致卵母细胞生长和发育的障碍。正如MIO，NPRL3双突变体，救援，MIO突变体表型中观察到的那样，禁用Gator1功能。令人惊讶的是，通过阻止减数分裂双链断裂（DSB）的形成，也可以抑制MIO突变体。一个解释这一观察结果的模型是，减数分裂DSB会在早期减数分裂循环中触发Gator1依赖性TORC1活性的下调，并且需要MIO在后来的卵子发生中减弱这种反应。为了测试这个想法，我们检查了在mio突变体背景下阻止减数分裂DSB的形成增加的Torc1活性。为此，我们比较了MIO单突变体的卵巢中S6激酶（一个下游Torc1）的S6激酶的磷酸化状态，而MEI-W68双突变体的卵巢中的卵巢酶状态。 MEI-W68（SPO11同源物）是减数分裂DSB所必需的。值得注意的是，我们发现MIO，MEI-W68双突变体相对于MIO单突变体具有增加的TORC1活性。这项工作得出了以下结论：减数分裂DSB触发了gator1依赖TORC1活性的下调。 通过编程的DNA DSB来启动同源重组是减数分裂的普遍特征。 DSB代表了DNA损伤的危险形式，可能导致生殖线基因组发生巨大变化。为了最大程度地减少这种破坏性的潜力，减数分裂DSB的产生和修复在时空中受到严格控制。减数分裂DSB促进Gator1依赖TORC1活性下调的观察结果表明，较低的Torc1活性可能对减数分裂DSB的有效修复可能很重要。 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.重要的是，TSC1的RNAi耗竭将Gator1卵巢缺陷表达。这些数据证实，在Gator1突变卵母细胞中观察到的减数分裂DSB的正直是由于TORC1活性高，而不是Gator1复合物的TORC1独立函数。进一步的遗传分析表明，Gator1会影响减数分裂DSB的修复而不是产生。鉴于在酿酒酵母中NPR3突变体中观察到的类似的减数分裂缺陷，这些数据尤其引人入胜。这些结果提出了一种有趣的可能性，即Gator1介导的TORC1活性的减少调节可能是许多生物中早期减数分裂周期的常见特征。这项工作得出了以下结论：在早期减数分裂周期中的TORC1活性对于修复果蝇期间的减数分裂DSB和种系基因组稳定性至关重要。 遗传毒性应激与包括果蝇在内的多种生物体中的逆转座子表达有关。与这些研究一致，我们发现在Gator1突变体中，启动减数分裂重组的DSB会触发逆转录子表达的放松管制。通过上位分析，我们确定p53和Gator1通过独立的途径作用，以抑制雌性种系中的逆转录子表达。令人惊讶的是，雌性种系中的TSC耗竭几乎没有增加逆转录盆地表达。这些数据提出了一种有趣的可能性，即Gator1调节返回跨座子的表达与TORC1活性无关。值得注意的是，最近在抑制哺乳动物组织培养细胞中抑制（长散布的元素1）line1表达的基因的高吞吐量筛选中发现了Gator1成分，而不是TSC成分。这项工作得出了以下结论：Gator1复合物在减数分裂过程中相反，在果蝇的女性种系中与P53并行起作用的途径。 当前，我们在果蝇中的工作代表了多细胞动物中Gator2功能的唯一体内检查。值得注意的是，我们的研究挑战了Gator2复合物对TORC1调控的主要模型的几个预测。最重要的是，我们的数据表明，Gator2复合函数的多个成员在从应力中恢复中恢复，并且在大多数代谢条件下通常不需要TORC1激活。但是，目前由于缺乏在体内研究Torc1的适当工具，因此无法测试我们的模型。为了解决这种情况，我们正在努力开发果蝇作为研究细胞和亚细胞水平的TORC1调节的体内模型。 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将允许我们直接测试Gator2功能的基本模型。此外，我们认为我们的系统将为果蝇社区提供强大的工具，以研究Torc1调节和功能在无数生理和发育环境中的重要性。