In meiosis, homologous recombination promotes genetic diversity and ensures the proper segregation of chromosomes in the first meiotic division. Defects in recombination trigger aberrant chromosome segregation and are the primary cause of spontaneous pregnancy loss (~35% of clinically recognized pregnancies), congenital birth defects like Downs syndrome (~1/300 live births) and intellectual disability. The overall goa of this project is to define mechanisms of meiotic recombination, which has implications for the etiology of meiotic aneuploidies, for linkage mapping, and for the evolutionary dynamics of genomes. The meiotically induced, topoisomerase II-like protein Rec12 (Spo11) catalyzes the formation of DNA double- strand breaks (DSBs) that initiate recombination. Intriguingly, recombination is clustered preferentially at hotspots that regulate its frequency and distribution in the genome. The fission yeast Schizosaccharomyces pombe, with its highly synchronous meiosis and well-defined hotspots (coupled with excellent genetics, molecular biology and protein biochemistry) provides a powerful system for dissecting mechanisms of recombination. In the previous (first) funding period, we defined the structure and function of Rec12; we characterized a large, multisubunit meiotic recombination complex (MRC) that contains Rec12; and we further defined pathway mechanisms that regulate recombination. In the second funding period, we will focus on mechanisms that direct Rec12-initiated recombination to hotspots. An emerging view is that post-translational modifications (PTMs) of histones have a key role in regulating recombination hotspots in diverse species. However, there are more than a hundred different histone PTMs and few have even been interrogated for a possible role in recombination. We developed and validated an approach called Mini-Chromosome Affinity Purification with Mass Spectrometry (MiniCAP-MS) that allows us to enrich and characterize the constituents of a discrete segment of chromatin. We will use this revolutionary technology to identify systematically, in an unbiased way, histone PTMs and proteins that regulate hotspot activation. We shall apply this technology to matching hotspot and basal control alleles to identify hotspot- specific binding proteins and histone PTMs. A combination of genetic, molecular and ChIP-seq methods are in place to determine functional significance. We also developed and validated a way to tether hotspot-activating proteins to the chromosome. In addition to confirming cis-acting specificity of individual components (e.g., histone modifying enzymes), this system will be used for epistasis analyses to elucidate order of function within pathways of chromatin remodeling that regulate meiotic recombination.
The most common forms of congenital birth defects (e.g., Downs syndrome) and about 35% of miscarriages are attributable to defects in the positioning of homologous recombination during meiosis in one of the parents. Dr. Wahls and his colleagues are using fission yeast as a model organism to determine what regulates the positioning of meiotic recombination. This work will help us to understand the causes of Downs syndrome and miscarriage and will identify targets of potential diagnostic value.
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