Generation and characterization of a novel mouse line to elucidate CHTF18 function in male and female meiosis

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SUMMARY Chromosome segregation errors are a leading cause of aneuploidy in humans that can have catastrophic clinical consequences. The fidelity of meiotic chromosome segregation requires well-orchestrated DNA double-strand break repair. During this critical process, DNA crossovers and the synaptonemal complex tether homologous chromosomes, and sister-chromatid cohesion keeps sister chromatids together. However, there is a gap in our knowledge of the mechanisms that establish and maintain the tethering of homologous chromosomes during meiosis. Our long-term goal is to define the mechanisms that regulate normal chromosome linkage and disjunction and identify molecular defects that cause chromosome mis-segregation. Previously, we identified CHTF18 as a crucial regulator of mammalian meiosis. CHTF18 is a conserved DNA replication protein essential for sister chromatid cohesion and proper chromosome segregation in yeast, Male and female mice lacking Chtf18 (Chtf18-/-) are subfertile with impaired gametogenesis. In Chtf18-/- meiotic cells, DNA double-strand breaks (DSBs) persist, DNA crossovers are decreased, and homologous chromosomes separate prematurely. We have recently discovered that CHTF18 is required for meiotic chromosome cohesion, a process mediated by cohesins. We showed that CHTF18 physically interacts with cohesins and regulates their association with chromatin and their composition during prophase I. Our data support a model in which CHTF18 mediates cohesins during meiotic DNA replication and recombination to promote crossover formation. However, the distribution and timing of CHTF18 throughout meiosis is not known because current antibodies pose a significant limitation. Functional partners with which CHTF18 interact are also not known. This foundational information is essential to discover how CHTF18 functions at the molecular level. Thus, the objective of this proposal is to generate and characterize a unique mouse line as a tool to delineate the role of CHTF18 in meiosis. This novel mouse line will permit us to overcome the limitation of elucidating the CHTF18 mechanism of action caused by a lack of specific antibodies. We propose to: 1) generate and validate a mouse line that contains an epitope- tagged allele of Chtf18 (Chtf18FH) and determine the spatiotemporal distribution of CHTF18 in meiosis; and 2) identify and validate functional partners that interact with CHTF18 in meiosis. Completing this project will generate an invaluable experimental tool and essential information about CHTF18 function, providing the basis for studies that investigate how chromosome cohesion is necessary for meiotic recombination and chromosome segregation. The use of male and female mice will allow us to precisely define the sexually dimorphic roles of CHTF18 in the regulation and timing of meiosis. By providing novel information about the distribution, timing, and interacting partners of CHTF18 during key steps of meiosis in spermatocytes and oocytes, these studies will provide a firm basis to define the molecular mechanisms of CHTF18 roles in this critical developmental process. This will allow us to establish underlying causes of chromosome mis-segregation and aneuploidy.