Life science
Medical & healthcare, Drug design

Structural analysis on the protein complexes required forchromosome segregation
Graduate School of Frontier Biosciences
Professor Tatsuo Fukagawa
Researchmap https://researchmap.jp/fukagawa_tatsuo?lang=en

Life science

Abstract
To maintain life all genome information must be transmitted
accurately into next generation-cells. Inaccurate transmission ofchromosomes including genome information causes genome instabilities, which lead to cancer formation. Therefore, it is essential
to study mechanisms how chromosomes are accurately segregated. Chromosome segregation is performed through attachment of
spindle microtubules to a special structure on chromosome called“kinetochore”. We are extensively studying kinetochore architecture and function to understand molecular mechanisms for chromosome segregation. Recently, we have shown a key structuralchange for kinetochore proteins.

▍ Background & Results
The centromere is an essential genomic region for accurate chromosome segregation. In most organisms, the centromere is specified at a particular locus on each chromosome by sequence independent epigenetic mechanisms, and histone H3 variant CENP-A
is a key epigenetic marker for centromere formation. Once theCENP-A nucleosome is established in the centromere, additional
proteins recognize the CENP-A nucleosome to form a kinetochore.
CENP-C and CENP-N are known as CENP-A binding proteins. We
have previously demonstrated that CENP-C binding to the CENP-A
nucleosome is regulated by CENP-C phosphorylation. However, it
is still unknown how CENP-C phosphorylation regulates its bindingto CENP-A. Further, it is not completely understood how and whether CNPE-C and CENP-N act on the CENP-A nucleosome together.
In our recent study, we reveal a stable CENP-A nucleosome binding mode of CENP-C through the unique structured region, usingcryo-EM in combination with a biochemical approach. We foundthat the CENP-C structure bound to the CENP-A nucleosome wasstabilized by an intramolecular link through the phosphorylated residue. Furthermore, the stable CENP-A-CENP-C complex excluded
CENP-N from the CENP-A nucleosome. These structural outcomes
provide a mechanistic insight into the dynamic kinetochore assembly regulated by CDK1 mediated CENP-C phosphorylation.

Fig. 1 Chromosome segregation during mitosis. Spindles bind to a centromere
region, and chromosomes are divided into daughter cells.

Fig. 2 Cryo-EM analysis of the CENP-A nucleosome complexed with CENP-C or
CENP-N. Phosporylated CENP-C dominatly binds to the CENP-A nucleosome.

▍ Significance of the research and Future perspective
Because defects in chromosome segregation cause various diseases including cancer, it is significant to examine structures of kinetochore complexes with high resolution. In addition, mutations oroverexpression of kinetochore proteins are known to induce cancer
formation, and the kinetochore proteins are good target for designof anti-cancer drug. Our structural study would contribute such adrug design.

P a t e n t Japanese Patent No. 4820995, No. 4787960
Ariyoshi, Mariko; Fukagawa, Tatsuo et al. Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C, EMBO Journal. 2021;
40(5): e105671. doi: 10.15252/embj.2020105671
Tr e a t i s eHori, Tetsuya; Fukagawa, Tatsuo et al. Essentiality of CENP-A depends on its binding mode to HJURP, Cell Reports, 2020; 33(7): 108388. doi: 10.1016/j.celrep.2020.108388
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L https://www.fbs.osaka-u.ac.jp/labs/fukagawa/research_j.html
K e y w o r d chromosome, mitosis, centromere, kinetochore, cancer