Régulation de cohésine par le complexe TORC1 chez la levure Schizosaccharomyces pombe

Overview & Synopsis

Cohesin is a protein complex capable of capturing DNA molecules. Cohesin ensures the cohesion of sister chromatids, which is essential for chromosome segregation during nuclear divisions. It is also involved in interphase via the formation of intra-chromosomal DNA loops that shape the functional architecture of the genome. Gene expression is thus regulated by the spatial organisation of chromosomes, particularly during development and differentiation. The many functions of cohesin suggest fine regulation in time and space. The laboratory is addressing this question using a genetic approach in the model organism Schizosaccharomyces pombe. DNA capture by cohesin requires the intervention of a loading complex, Mis4/Ssl3 (hNIPBL/MAU2). The mis4-G1487D mutant is thermosensitive to growth at 36°C, has a defect in cohesin loading on chromosomes and a high frequency of chromosome segregation defects during mitosis. A genetic screen identified extragenic mutations capable of restoring the growth of mis4-G1487D at 36°C. Five of these mutations affect the mip1 gene and one the tor2 gene. Mip1 and Tor2 are components of the TORC1 complex, the equivalent of mTOR (Mammalian Target Of Rapamycin), which is a major regulatory kinase for cell metabolism and growth. Its activity is stimulated by signals such as the availability of nutrients, energy levels, hormones and growth factors. In S. pombe, Tor2 is the catalytic subunit and Mip1 (hRaptor) is involved in substrate selection. The tor2 and mip1 genes are essential for cell viability, indicating that the alleles produced by the screen are hypomorphic. We focused our work on mip1-R401G, which causes virtually no growth defects while being an excellent suppressor. Remarkably, mip1-R401G restored cohesin association with chromosomes and reduced the frequency of abnormal chromosome segregation in the mis4-G1487D mutant at 36°C. In the mis4+ background, mip1-R401G increased the amount of cohesin associated with chromosomes. Similar results were obtained by treating the cells with Rapamycin, a TORC1 inhibitor. These data suggest that TORC1 activity negatively regulates the cohesin loading complex in S. pombe.All subunits of the TORC1 complex co-purify with cohesin and Mis4. The Psm1 subunit of cohesin and Mis4 are hypophosphorylated in the mip1-R401G background. The combination of mutations mimicking the non-phosphorylated state reduces the frequency of mis4-G1487D segregation defects. Conversely, segregation defects are exacerbated by mutations mimicking the phosphorylated state. These data indicate that TORC1 controls the phosphorylation state of Psm1 and Mis4. Given that TORC1's known function is to adapt the cell to environmental changes, we carried out a transcriptome analysis in various experimental situations (culture medium composition, temperature, cell cycle phase). Taking all experiments together, 337 genes were differentially expressed in the mis4-G1487D background compared with the wild-type control. Remarkably, the genes affected differed widely from one condition to another, suggesting that mis4-G1487D cells have a defective adaptive response. Almost all the genes deregulated by mis4-G1487D were also deregulated by mip1-R401G. These genes are preferentially located at the ends of chromosomes and are involved in the stress response and sexual differentiation.Taken together, the data suggest that cohesin is an effector of the TORC1 pathway for adapting the cell to environmental changes. Mechanistically, this might involve a change in gene expression induced by a modification in the spatial organization of the genome.