Efficient chromosomal movements are important for the fidelity of chromosome segregation during mitosis; however, movements are constrained during interphase by tethering of multiple domains to the nuclear envelope (NE). Higher eukaryotes undergo open mitosis accompanied by NE breakdown, enabling chromosomes to be released from the NE, whereas lower eukaryotes undergo closed mitosis, in which NE breakdown does not occur. Although the chromosomal movements in closed mitosis are thought to be restricted compared to open mitosis, the cells overcome this problem by an unknown mechanism that enables accurate chromosome segregation. Here, we report the spatiotemporal regulation of telomeres in Schizosaccharomyces pombe closed mitosis. We found that the telomeres, tethered to the NE during interphase, are transiently dissociated from the NE during mitosis. This dissociation from the NE is essential for accurate chromosome segregation because forced telomere tethering to the NE causes frequent chromosome loss. The phosphorylation of the telomere protein Rap1 during mitosis, primarily by Cdc2, impedes the interaction between Rap1 and Bqt4, a nuclear membrane protein, thereby inducing telomere dissociation from the NE. We propose that the telomere dissociation from the NE promoted by Rap1 phosphorylation is critical for the fidelity of chromosome segregation in closed mitosis.

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Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues.

The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1 + have revealed that the long N-terminal region (1–456 a.a. [amino acids]) of Rap1 (full length: 693 a.a.) is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457–693 a.a.) containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus) and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.