Directional chromosome movement during mitosis depends on assembly of dynamic microtubule polymers around chromosomes, and on attachment of microtubule ends in the correct orientation to the kinetochore apparatus, which forms the centromere locus of each chromosome. We aim to understand the molecular mechanisms that ensure accurate chromosome segregation, focusing on mechanistic dissection of the chromosomal passenger complex (CPC), which contains the protein kinase Aurora B and regulates a variety of mitotic processes including microtubule dynamics, kinetochore assembly, and kinetochore-microtubule attachment.
We have discovered that binding of chromatin (particularly chromatin with mitotic specific phosphorylation at histone H3 Thr3) or microtubules stimulates the kinase activity of Aurora B, and that these processes are important for spindle microtubule assembly and proper kinetochore functions including activation of the spindle assembly checkpoint. It has been hypothesized that Aurora B and the CPC play a central role in sensing and correcting aberrant microtubule attachment kinetochores. The mechanism that senses the aberrant attachment remains a big mystery.
- What are the structural bases for chromatin- and microtubule-dependent activation of Aurora B?
- How the coupling of Aurora B activation to chromatin and microtubule contributes to proper kinetochore-microtubule attachment regulation?
Sampath, S. C., Ohi, R., Leismann, O., Salic, A., Pozniakovski, A., and Funabiki, H. (2004). The chromosomal passenger complex is required for chromatin-induced microtubule stabilization and spindle assembly. Cell 118, 187-202.
Kelly, A. E., Sampath, S. C., Maniar, T. A., Woo, E. M., Chait, B. T. and Funabiki, H. (2007). Chromosomal enrichment and activation of the Aurora B pathway are coupled to spatially regulate spindle assembly. Dev. Cell, 12, 31-43.
Tseng, B. S., Tan, L., Kapoor T. M. and Funabiki, H. (2010) Dual detection of chromosomes and microtubules by the chromosomal passenger complex drives spindle assembly. Dev. Cell., 18, 903-912
Kelly, A. E., Ghenoiu, C., Xue, J. Z., Zierhut, C., Kimura, H., and Funabiki, H. (2010) Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B. Science, 330, 235-239
Ghenoiu, C., Wheelock, M. S., and Funabiki, H. (2013) Autoinhibition and Polo-dependent multisite phosphorylation restrict activity of the histone H3 kinase Haspin to mitosis. Mol Cell, 52, 734-745
Wynne, D. J., and Funabiki, H. (2015) Kinetochore function is controlled by a phospho-dependent co-expansion of inner and outer components. J. Cell Biol., 210, 899-916.
Wynne, D. J., and Funabiki, H. (2016) Heterogeneous architecture of vertebrate kinetochores revealed by 3D super-resolution fluorescence microscopy. Mol. Biol. Cell. 27, 3395-3404
Wheelock, M. S., Wynne, D. J., Tseng, B. S. and Funabiki, H. (2017) Dual recognition of chromatin and microtubules by INCENP is important for mitotic progression. J. Cell Biol, 216, 925-941
Chromosomes in interphase are relatively decondensed and support DNA replication and transcription during interphase. In contrast, mitotic chromosomes are more condensed and individualized to support chromosome segregation. Moreover, chromosomes in interphase stimulate distinct signals to assemble the nuclear envelope, but in mitosis, the nuclear envelope breaks down and chromosomes promote assembly of the spindle microtubules. We aim to understand molecular mechanisms behind these structural and functional changes of chromosomes at the cell cycle transitions between interphase and mitosis.
The nucleosome, where ~146 bp DNA wraps around core histone octamers, is the fundamental unit of chromatin. The linker histone interacts with the entry and exit points of nucleosomal DNAs. While these core and linker histones receive a number of mitosis-specific modifications, little is known about their functional significances. Using Xenopus egg extracts, we have developed a strategy to directly manipulate histone compositions, specific residues and modifications, and monitor functional, compositional and structural consequences.
- How are chromosome shape and size mechanistically defined? Do distinct mechanisms engage to control folding of DNA in a kilo/megabase scale and to change local compaction of DNA/nucleosome? We are addressing this question through developing new imaging-based methods.
- What are constituents of mitotic chromatin and how they are built? Combining quantitative mass spectrometry and Xenopus egg extracts, we are in the unique position to examine the direct role of specific histone modifications and test their roles in chromatin composition and functions.
Zierhut C., Jenness C., Kimura, H., and Funabiki, H. (2014) Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion. Nat Struct Mol Biol, 21, 617-625
Zierhut, C. and Funabiki, H. (2015) Nucleosome functions in spindle assembly and nuclear envelope formation. Bioessays, 37, 1074-1085.
Visualizing centromere-associated repeats using CO-FISH in human cells. Credit: Simona Giunta.
Human centromeres are composed of large tandem arrays of alpha-satellite repetitive DNA elements, spanning up to ~ 5 Mb in size. Our recent results indicate that this repetitive organization becomes unstable in cancer cells and during replicative senescence. In addition, arrays of satellite 2 and 3 repeats at juxtacentromere of chromosome 1 and 16 are fragile in Immunodeficiency–Centromeric instability–Facial anomalies (ICF) syndrome. Underlining mechanisms behind the ICF syndrome is poorly understood.
- What are the mechanisms that maintain integrity and copy number of centromere-associated satellite DNAs? Using CO-FISH and quantitative FISH techniques, we have established a method to quantitatively measure those instabilities to address this question.
- What are the functional consequences upon induced instabilities of centromere-associated repetitive sequences?
Giunta, S. and Funabiki, H. (2017) Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T. Proc. Natl. Acad. Sci. U.S.A. 114, 1928-1933
Many cancer cells exhibit a “chromosome instability (CIN)” phenotype, where chromosomes frequently fail to equally distribute to daughter cells. How chromosome missegregation contributes to tumorigenesis remains unclear. In addition, anti-microtubule drugs, such as taxol, which cause mitotic arrest and chromosome missegregation, are commonly used to treat breast and other cancers but their underlining mechanism is not established. We hypothesize that the innate immune system, which normally responds to pathogenic DNAs, senses aberrant mitosis and affect fates.
- How can the innate immune system distinguish between pathogenic DNA and normal genomic DNA?
- What is the consequence of activation of the innate immune system in cells undergoing aberrant mitoses in cancers.
Zierhut C., Yamaguchi N., Paredes M., Luo JD., Carroll T., Funabiki H. (2019) The cytoplasmic DNA sensor cGAS promotes mitotic cell death. bioRxiv 168070; doi: https://doi.org/10.1016/j.cell.2019.05.035