Cohesin is a ring-shaped protein complex highly conserved in evolution that is composed in all eukaryotes of at least two SMC proteins (Structural Maintenance of Chromosomes) SMC1 and SMC3 in humans (Psm1 and Psm3 in fission yeast), and the kleisin RAD21 (Rad21 in fission yeast). Mutations in its components or its regulators cause genetic syndromes (known as cohesinopathies) and several types of cancer. It has been shown in a number of organisms that only a small fraction of each subunit is assembled into complexes. Therefore, the presence of an excess of soluble components hinders dynamic chromatin loading/unloading studies using fluorescent fusions in vivo. Here, we present a system based on bimolecular fluorescent complementation in the fission yeast Schizosaccharomyces pombe, named Bi-molecular Fluorescent Cohesin (BiFCo) that selectively excludes signal from individual proteins to allow monitoring the complex assembly/disassembly within a physiological context during a whole cell cycle in living cells. This system may be expanded and diversified in different genetic backgrounds and other eukaryotic models, including human cells.
Cohesin is a highly conserved, ring-shaped protein complex found in all eukaryotes. It consists of at least two structural maintenance of chromosomes (SMC) proteins, SMC1 and SMC3 in humans (Psm1 and Psm3 in fission yeast), and the kleisin RAD21 (Rad21 in fission yeast). Mutations in its components or regulators can lead to genetic syndromes, known as cohesinopathies, and various types of cancer. Studies in several organisms have shown that only a small fraction of each subunit assembles into complexes, making it difficult to investigate dynamic chromatin loading and unloading using fluorescent fusions in vivo because of excess soluble components. In this study, we introduce bimolecular fluorescent cohesin (BiFCo), based on bimolecular fluorescent complementation in the fission yeastSchizosaccharomyces pombe. BiFCo selectively excludes signals from individual proteins, enabling the monitoring of complex assembly and disassembly within a physiological context throughout the entire cell cycle in living cells. This versatile system can be expanded and adapted for various genetic backgrounds and other eukaryotic models, including human cells.
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