Initial investigation of ubiquitination pathway in mammalian meiosis

Abstract

The Cohen's lab research focuses mainly on the regulation of mammalian meiosis in mouse models, which include several induced mutants that have helped in broadening our knowledge of meiotic recombination and the gametogenesis. The lab also studies several DNA mismatch repair (MMR) proteins and their subsequent effects on meiotic recombination. Thus, our research serves two major purposes; first, to understand the genetics of re-combination and thereby to understand how such events can fail in human gametogenesis and secondly, to further elucidate the mechanisms of DNA repair and genome stability in an important in vivo system. Extending from these aims, one of these genes is the MLH3 gene, evolutionarily conserved in many species such as mice, humans, and worms and which can solve the infertility problems in MLH3 mutant mice. The focus of our project is to investigate the functions of several genes involved in meiotic prophase, using the technique of chromosome spreading. This method allows us to visualize the different stages of prophase I, and see the staining pattern of several antibodies that are specific to meiotic proteins. Using a battery of specific antibodies, we set out to understand the meiotic roles of a number of poorly characterized proteins involved in DNA repair and ubiquitination pathways. These antibodies of interest are those that are involved in the ubiquitination pathway of the sex chromosomes such as antibodies raised against Rnf 8, Rad 18, and Rnf 168, all of which are involved in ubiquitination pathways, as well as other antibodies involved in ubiquitination/deubiquitinatio. A group of other antibodies that include mdc λ, HR9B, MRαB, USP2B, and UBQ 2 is also being studied. Our goal was to first characterize the accumulation of these proteins on meiotic chromosomes in order to identify which, if any of these proteins might be functionally relevant for meiotic recombination and prophase | progression. The project started with studying the staining pattern of the antibodies listed above in adult male wild type mice, then younger male wild type mice to see both the early and late stages of meiosis. The project is progressing to studying the staining pattern in the MLH3 and Ago 4 mutant mice to see any possible co-localizations between MMR genes and the antibodies considered by this study