Positive and negative regulation of REC-8 cohesin during meiosis

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Project Summary/Abstract Precise reduction of chromosome copy number (the ploidy) from two to one is essential to create healthy sperm and eggs during meiosis. This requires two rounds of meiotic chromosome segregation. Homologous chromosomes separate first, sister chromatids second; this pattern requires two-step destruction of the α- kleisin subunit of the cohesin complexes that mediate sister chromatid cohesion (SCC). In most eukaryotes, replacing the mitotic α-kleisin Scc1/Rad21 with the meiosis-specific α-kleisin Rec8 is critical to establish the meiotic pattern of chromosome segregation (which reduces chromosome number) rather than the mitotic pattern (which stably maintains ploidy). Thus, specialized mechanisms must exist to ensure the establishment of REC-8-dependent SCC during meiosis and to prevent it during mitosis. The objectives of the proposed research are to identify the mediators of these antagonistic mechanisms and to understand their functions. Two specific aims will achieve these objectives. 1) We will identify the genes disrupted in five strains we isolated in an unbiased forward genetic screen for mutations that specifically disrupt REC-8- dependent SCC during C. elegans meiosis. We will then determine how the newly identified factors promote REC-8 cohesin function during normal meiosis. 2) We will determine whether PROM-1, a component of an SCF ubiquitin ligase known to target meiotic proteins for degradation, specifically marks nucleoplasmic, chromosomally unbound REC-8 cohesin for destruction prior to the onset of zygotic mitosis that begins following fertilization. We will also utilize CRISPR/Cas9-dependent genome editing to allow inducible REC-8 expression and determine the consequences of REC-8 expression in mitotically proliferating cells. The proposed research is innovative: although the importance of REC-8 cohesin for gamete formation has been established from yeast to man, the essential regulators that implement REC-8-dependent SCC during meiosis remain largely unidentified. Moreover, it is poorly understood in any organism how Rec8 cohesin function is prevented during mitotic proliferation: while known transcriptional and translational regulation prevent de novo REC-8 expression, additional mechanisms must prevent fertilized zygotes from inheriting REC-8 cohesin from the meiotic germline. We will apply novel approaches, many developed in my lab, to examine cohesin function and to edit the C. elegans genome. The research is significant: Defects in cohesin function are a major factor causing human miscarriage, birth defects, and infertility. Mutations in cohesin subunits and cohesin regulators are found in human cohesinopathies like Cornelia de Lange and Roberts-SC phocomelia syndromes. Cohesin misregulation and ectopic expression of meiosis-specific genes like REC-8 often correlates with poor prognosis in cancers. Thus, the relevance of the proposed research extends to human reproductive health and beyond. This multidisciplinary project will expose undergraduates to molecular biology, genetics, CRISPR-mediated genome editing, and applying the scientific method to understand complex biological problems.