Investigating mechanisms of cohesin function in the Caenorhabditis elegans germ line

Abstract

The cohesin complex is essential for both mitosis and meiosis because of its vital roles in sister chromatid cohesion, DNA damage repair, and chromosome architecture. During meiosis, chromosomes must undergo a series of processes that do not occur in mitotic cells, most notably pairing and recombination between homologous chromosomes during the long prophase that precedes the first meiotic division. This results in the formation of chiasmata, which are required for correct chromosome orientation on the first meiotic spindle. Pairing and recombination depend on the formation of proteinaceous axial elements containing meiosis-specific cohesin at the onset of meiosis. In C. elegans meiotic cohesin complexes are defined by the substitution of the mitotic kleisin subunit SCC-1 by REC-8, or COH-3/4. These complexes have been shown to carry out overlapping, but also distinct functions during meiotic progression. Why meiosis in higher eukaryotes requires multiple cohesin complexes is unclear. This thesis aims to further characterize the functions of meiotic cohesin complexes and to understand the mechanisms driving their functional diversity. Initial experiments revealed differential binding dynamics between REC-8 and COH-3/4 complexes, a larger contribution of REC-8 to DSB repair, and a surprising role for REC-8 in the proliferation of mitotic germ cells. A CRISPR-Cas9 deletion screen revealed that REC-8 tolerates removal of several regions up to 59 amino acids and identified two, short, conserved regions required for REC-8 and COH-3 function. Removing region 1 (REC-8 I497-L501; COH-3 I385-L389) affected formation of the meiotic axis, and in the case of REC-8, impaired SCC maintenance during late prophase. Deletion of region 2 (REC-8 E176- P185; COH-3 Q179-S189) hindered chromosome segregation during the meiotic divisions (REC-8) and had a detrimental effect in homologue pairing (COH-3). My results highlight the modularity of meiotic kleisins and have started identifying key functional regions in the central domain of these proteins.