Scraping the tip of Zip1's role in meiotic chromosome dynamics: Using lacO/LacI Corecruitment to identify crossover promoting factors that interface with the N‐terminus of a synaptonemal complex protein
Authors: Feil Arden , DeMuyt Arnaud , Borde Valérie , Voelkel‐Meiman Karen , MacQueen Amy 
Published: June 2021

During the first division of meiosis, diploid nuclei divide to produce nuclei with only one set of chromosomes. Accurate chromosome segregation during this so‐called “reductional” division relies on the prior establishment of recombination‐based associations between homologous chromosomes (homologs). Typically, homolog pairing and interhomolog recombination take place in conjunction with the assembly of a conserved tripartite chromosomal structure, the Synaptonemal Complex (SC), and interhomolog crossover recombination intermediates are processed within the physical context of SC. However, the molecular underpinnings of the relationship between SC and meiotic recombination remain obscure. The SC is assembled by rod‐like transverse filament proteins that bridge aligned homolog axes; transverse filaments interface with a central element substructure at the midline of the SC. In the yeast Saccharomyces cerevisiae, a major component of the SC transverse filament is the coiled‐coil protein, Zip1. Our prior work found that small, adjacent regions within Zip1's N terminus act to independently promote crossover recombination and SC assembly, and that versions of Zip1 missing residues 2–9 or 10–14 support SC assembly but not SC‐associated crossover formation. Mutants expressing zip1‐[Δ2–9] and zip1‐[Δ10–14] alleles assemble SC, but form remarkably large Zip1 polycomplex structures and display hyper‐SUMOylation of another SC protein, Ecm11, two relatively unusual meiotic phenotypes that are also observed in meiotic cells missing the crossover‐promoting factor, Zip3. These observations suggest that residues 2–14 of Zip1 somehow affect Zip3 behavior. Zip1 has previously been found to be essential for the recruitment of Zip3 protein to sites of recombination initiation on meiotic chromosomes, and chromatin immunoprecipitation experiments revealed that Zip3 fails to localize to all surveyed meiotic recombination initiation sites in zip1‐D1[Δ2–9] and zip1‐D2[Δ10–14] mutants, suggesting that the crossover defect exhibited by these mutants is based at least in part on the inability of the Zip1[Δ2–9] or Zip1[Δ10–14] proteins to recruit Zip3 to recombination initiation sites. Furthermore, cytological analysis indicated that Zip3 is excluded from the polycomplex structures assembled by Zip1‐[Δ2–9] or Zip1‐[Δ10–14] protein, suggesting that Zip1's residues 2–14 somehow interface with Zip3. I am currently creating a lacO/LacI co‐recruitment assay to investigate whether Zip1's N‐terminus is sufficient for recruiting Zip3 to an ectopic site in meiotic nucleus. I have created diploid strains in which the Zip1's N terminal residues 1–17, 1–33, 1–171, or 1–348 are fused to the N‐terminus of GFP‐LacI and that carry lacO on chromosome IV. Using immunofluorescence on surface spread meiotic chromosomes, I will ask whether Zip3‐MYC is recruited to a chimeric Zip1‐GFP‐LacI protein bound to a lacO array positioned on chromosome IV within the meiotic nucleus. The data from this experiment will not only provide strong evidence for a direct interaction between Zip1 and Zip3, but also establish a starting point for future structure‐function studies aimed at identifying residues that are critical for, and the meiotic genes that might regulate, a putative Zip1‐Zip3 interaction.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.