Meiosis is the specialized cell division that gives rise to haploid gametes for sexual reproduction. During prophase I, homologous chromosomes are physically tethered via the formation of the synaptonemal complex while undergoing DNA double strand break (DSB)-induced recombination. In XY mammals, there is an additional challenge presented by the sex chromosomes, which synapse only at the Pseudoautosomal Region (PAR), leaving vast asynapsed regions that trigger a unique chromosome-wide transcriptional silencing mechanism termed Meiotic Sex Chromosome Inactivation (MSCI). MSCI occurs within the context of the Sex Body (SB), a membrane-less sub-domain of the nucleus that houses the XY. MSCI is a specialized version of the broader process of Meiotic Silencing of Unsynapsed Chromatin (MSUC) that occurs in male and female meiosis when homologs fail to synapse, triggering apoptosis of aberrant germ cells. All of these events are critical to ensure the formation of viable euploid gametes, underscored by the fact that humans show exceptionally high rates of meiotic errors leading to miscarriages and birth defects, with non-disjunction of the sex chromosomes, Klinefelter syndrome, being the most frequent trisomic disorder (1:500 live births). Our labs and others have shown that the kinase ATR is central to many prophase I events, including DSB repair, synapsis, and MSCI. However, these numerous overlapping roles have posed a barrier to understanding the precise mechanistic actions of ATR in meiosis. In the prior funding cycle, we generated novel separation-of-function mouse mutants in ATR regulators that allow us to dissect specific roles for ATR in MSCI with minimal effects on its other meiotic functions. These mice bear mutations in TOPBP1, a key ATR activator that also mediates substrate selectivity, and in RAD9A/B, components of the 911 clamp that helps anchor TOPBP1. Our analysis revealed critical functions of TOPBP1 and 911 in driving ATR functions within the SB, and highlighted essential downstream ATR targets in these processes such as the RNA:DNA helicase Senataxin (SETX). We hypothesize that the ATR-TOPBP1-911 axis plays critical roles in establishing the unique chromatin and transcriptional environment required to initiate, maintain, and terminate MSCI in a temporally restricted manner during meiotic prophase I, and that this function is dependent on downstream ATR targets including SETX. Utilizing our unique mouse models in combination with high-resolution genomic tools and cutting-edge proteomic approaches, we will elucidate the mechanisms by which ATR signaling orchestrates MSCI in male meiosis, and MSUC in male and female meiosis. Finally, based on our findings that ATR signaling is constantly antagonized, indicative of prominent roles for phosphatases during MSCI maintenance and termination, we will determine the mechanisms by which ATR signaling is counteracted by phosphatases to achieve temporally-appropriate shutdown of transcriptional silencing, permitting prophase I completion and enabling the production of high quality gametes.