Crossing over during meiotic prophase I is essential for tethering homologous chromosomes together until the first meiotic division (MI). The localization of crossovers (CO) at the correct temporal and spatial frequency is critical for ensuring equal segregation of homologous chromosomes at MI, the importance of which is underscored by the observation that 50% of all spontaneous miscarriages in humans are due to chromosome mis-segregation errors at this stage. Not surprisingly, therefore, CO formation is tightly regulated, such that, of the 10-fold excess of initiating double strand break (DSB) events that arise during early prophase I, very few final COs are achieved. CO designation of DSB intermediates is largely controlled by the "ZMM" genes that include the meiosis-specific DNA mismatch repair heterodimeric complexes, MutSc (MSH4/MSH5) and MutLc (MLH1/MLH3). MutSc first binds to 150 of the 250+ DSBs and, of these, approximately 24-28 subsequently accumulate MutLc to become class I COs. How this specific subset of MutSc sites are designated is unclear, but recent studies in our lab have revealed that Cyclin N-terminal domain-containing protein-1 (CNTD1) plays a crucial role in this process, since MutSc focus frequency remains persistently elevated throughout prophase I in mouse mutants bearing a mutation in the Cntd1 gene. Moreover, MutLc fails to load on chromosomes during late prophase I in Cntd1 mutants, suggesting that CO designation and maturation are intimately coupled events regulated by CNTD1. Together with other regulators of crossover designation, including the Zip3/ZHP- 3 ortholog, RNF212, and human enhancer of invasion-10 (HEI10), we hypothesize that CNTD1 acts to ensure crossover designation in the class I CO pathway, either by promoting the processing/maturation of a finite set of MutSc events, or by removing excess DSB repair intermediates. Studies in this proposal are aimed at elucidating this novel crossover designation regulatory circuit.
In aim 1, we will ask how and when CNTD1 is recruited to meiotic chromosome cores using a novel Cntd1-V5-tagged mouse, and by assessing CNTD1 localization in the presence of reduced levels of MutSc.
In aim 2, we will identify key functional interactions that mediate CNTD1 function and we will determine whether CNTD1 acts in concert with one or several cyclin- dependent kinases, as its status as a cyclin family member would suggest.
In aim 3, we will elucidate the mechanism by which loss of CNTD1 results in the absence of MutLg foci despite persistent MutSg complexes, asking whether the designation of COs in the mouse genome requires a dose-dependent threshold level of MutSc at specific DSB intermediate sites, whose accumulation and/or stability is ensured through the loading and activity of CNTD1. These studies take advantage of the impressive repertoire of mouse mutants available in the PI's lab, together with innovative experimental approaches that take advantage of advances in mouse transgenesis and high-resolution 3-dimensional imaging of prophase I events in mammalian germ cells.
Meiosis is the process by which normal cells undergo a double cell division process to halve their DNA content in preparation for fertilization and sexual reproduction. Errors in meiosis are common in humans, and are the leading cause of prenatal loss and birth defects. The majority of errors arise during the period of prophase I, when pairs of chromosomes must exchange DNA in a process called crossing over. This grant seeks to understand how crossing over is regulated at the molecular level, and what defective processes might give rise to the high error rates seen in higher mammals.
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