Coordination of ATR Signaling for Genetic Quality Control, Silencing, and DNA Repair During Meiosis

在减数分裂过程中，由于遗传质量控制不当，经常出现不育和出生缺陷。为了确保配子的产生没有遗传缺陷，减数细胞的基因组由一组进化上保守的激酶监测，称为检查点激酶。这些激酶感知DNA损伤或染色体配对问题，一旦激活，可阻断减数分裂进程并诱导细胞死亡。但是检查点激酶的作用不仅仅是作为一种质量控制机制。程序性双链断裂（DSB）的形成是正常减数分裂过程中丰富而必要的事件，而检查点激酶需要协调重组修复、交叉调控和转录沉默中的关键事件。减数分裂检查点信号是如何调控的尚不清楚，特别是在哺乳动物中。特别是，对于检查点激酶如何作为正常减数分裂过程的基本调节因子以及导致细胞死亡的质量控制机制的效应器，人们知之甚少。这种知识差距对理解遗传质量控制的决定因素以及检查点信号的错误调节如何异常地阻断减数分裂进程和促进不孕症构成了主要障碍。同样的途径也介导有丝分裂细胞的基本DNA修复和检查点功能，有时在癌症中不受控制，并且作为一种新兴的癌症治疗策略被临床靶向。该建议应用创新的方法来克服哺乳动物减数分裂检查点信号研究的长期障碍。拟议的研究重点是必需的检查点激酶ATR，它在减数分裂期间对DSB修复和未突触染色质的转录沉默很重要，并且部分受支架蛋白TOPBP1和其他上游调节因子如RAD9A-RAD1-HUS1复合物的调节。小鼠基因组编辑的前沿方法将用于产生合理设计的功能分离小鼠突变体，目的是揭示在减数分裂中起作用的新的检查点调节机制。为了指导和补充遗传和功能实验，睾丸提取物的质谱分析将用于精母细胞中检查点信号的定量和无偏定性。总的来说，这些研究有望揭示减数分裂ATR信号如何协调以实现结构特异性信号输出，以响应未修复的dsb或染色体失联，而不诱导细胞死亡。除了对几乎在所有细胞中起作用的基因组维持途径的作用提供基本的见解之外，这项工作的结果将对不孕症和出生缺陷的分子起源以及ATR抑制在临床环境中的影响具有重要意义。

Infertility and birth defects often arise due to improper genetic quality control during meiosis. To ensure the production of gametes without genetic defects, the genome of meiocytes is monitored by a set of evolutionarily conserved kinases, known as checkpoint kinases. These kinases sense damage to DNA or problems in chromosome pairing and, upon activation, can block meiotic progression and induce cell death. But the action of checkpoint kinases is not solely utilized as a quality control mechanism. Programmed double strand break (DSB) formation is an abundant and essential event for normal meiotic progression, and checkpoint kinases are required to coordinate key events in recombination repair, crossover regulation and transcriptional silencing. How meiotic checkpoint signaling is regulated is not understood, especially in mammals. In particular, little is known about how checkpoint kinases can act both as essential regulators of normal meiotic progression as well as effectors of quality control mechanisms that lead to cell death. This knowledge gap poses a major barrier for understanding the determinants of genetic quality control and how mis-regulation of checkpoint signaling may aberrantly block meiotic progression and promote infertility. The same pathways also mediate fundamental DNA repair and checkpoint functions in mitotic cells, are sometimes deregulated in cancers, and are being targeted clinically as an emerging strategy for cancer treatment. This proposal applies innovative approaches to overcome long-standing barriers for the study of meiotic checkpoint signaling in mammals. The proposed studies focus on the essential checkpoint kinase ATR, which is important for DSB repair and transcriptional silencing of unsynapsed chromatin during meiosis and is regulated in part by the scaffolding protein TOPBP1 and other upstream regulators such as the RAD9A-RAD1-HUS1 complex. Cutting edge approaches for genome editing in the mouse will be used to generate rationally designed separation-of- function mouse mutants with the goal of revealing novel checkpoint regulatory mechanisms operative in meiosis. To guide and complement genetic and functional experiments, mass spectrometry analysis of testis extracts will be used for quantitative and unbiased characterization of checkpoint signaling in spermatocytes. Collectively, these studies are expected to reveal how meiotic ATR signaling is coordinated to achieve structure-specific signaling outputs in response to unrepaired DSBs or chromosome asynapsis, without inducing cell death. Beyond providing fundamental insights into the actions of genome maintenance pathways that function in virtually all cells, the results from this work will carry important implications related to the molecular origins of infertility and birth defects as well as the impact of ATR inhibition in clinical settings.