Chromosomes actively generate a variety of essential signals that promote spindle assembly and chromosome movement during mitosis and meiosis. Failure to properly complete these processes leads to aneuploidy, which is frequently associated with cancer and birth defects. In this proposal, we will focus on a critical regulator of these processes, the chromosomal passenger complex (CPC), consisting of the kinase Aurora B, and its regulatory subunits, INCENP, Dasra and Survivin. The CPC localizes to chromatin and is enriched at centromeres. Chromatin binding is directly coupled to activation of the kinase activity of Aurora B. In Xenopus laevis egg extracts, this activation mechanism is critical for spindle assembly. At centromeres, the CPC mediates spindle assembly checkpoint (SAC) signaling to delay anaphase onset in response to erroneous kinetochore attachments. To reveal the spatiotemporal mechanism that controls the Aurora B pathway, we will: 1) Address how the CPC controls the SAC. We have found that the putative coiled-coil (CC) domain of INCENP is critical for the SAC. To test if the CC domain monitors physical kinetochore changes upon microtubule attachment and converts them into Aurora B signaling, the CC domain will be systematically modulated, and changes in Aurora B-dependent phosphorylation within kinetochores will be monitored. 2) Reveal a mechanism by which a network of protein kinases controls M phase-specific activation of Aurora B by chromatin. Based on our preliminary data, we hypothesize that the histone H3 threonine 3 (H3T3) kinase Haspin is activated by the mitotic kinases Cdk1 and Plx1 to mediate the temporal control of Aurora B activation by chromatin. The precise mechanism by which these mitotic kinases activate Haspin will be analyzed by biochemical methods combined with advanced mass spectrometry. 3) Provide the structural basis for the CPC- chromosome interaction. Localization of the CPC to chromosomes is mediated by Survivin, which directly binds to histone H3 that has been phosphorylated at threonine 3 (H3T3ph). To determine how this phosphorylation regulates binding specificity, crystal structures of the Survivin-H3T3ph complex will be determined. 4) Investigate another chromatin-activated protein that was recently identified in my lab, Vespera, which appears to oppose the Aurora B pathway, inhibit microtubule assembly, and promote nuclear re- formation. Since Vespera is exclusively expressed in pluripotent cells (including eggs), it is possible that its presence is linked to the requirement for the CPC in spindle assembly in these cell types. Therefore we will investigate specific roles for Vespera during these developmental stages. The outcomes of this proposed project will provide molecular explanations for how the chromosome-associated activities of the CPC and Vespera coordinate mitosis, and will help us understand how their misregulation affects the growth of normal and cancer cells.

Public Health Relevance

The distribution of a correct set of chromosomes to dividing cells is thought to be critical for the prevention of tumor development and birth defects. We seek to understand how chromosomes drive this process by studying chemical reactions that are spatially regulated by chromatin. This project will aid the development of therapeutic procedures to specifically identify and kill cancer cells, and improve our understanding of the processes that cause birth defects.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Carter, Anthony D
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Rockefeller University
Anatomy/Cell Biology
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New York
United States
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Wheelock, Michael S; Wynne, David J; Tseng, Boo Shan et al. (2017) Dual recognition of chromatin and microtubules by INCENP is important for mitotic progression. J Cell Biol 216:925-941
Wynne, David J; Funabiki, Hironori (2016) Heterogeneous architecture of vertebrate kinetochores revealed by three-dimensional superresolution fluorescence microscopy. Mol Biol Cell 27:3395-3404
van den Boom, Johannes; Wolf, Markus; Weimann, Lena et al. (2016) VCP/p97 Extracts Sterically Trapped Ku70/80 Rings from DNA in Double-Strand Break Repair. Mol Cell 64:189-198
Wynne, David J; Funabiki, Hironori (2015) Kinetochore function is controlled by a phospho-dependent coexpansion of inner and outer components. J Cell Biol 210:899-916
Zierhut, Christian; Funabiki, Hironori (2015) Nucleosome functions in spindle assembly and nuclear envelope formation. Bioessays 37:1074-85
Xue, John Z; Funabiki, Hironori (2014) Nuclear assembly shaped by microtubule dynamics. Nucleus 5:40-6
Zierhut, Christian; Jenness, Christopher; Kimura, Hiroshi et al. (2014) Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion. Nat Struct Mol Biol 21:617-25
Xue, John Z; Woo, Eileen M; Postow, Lisa et al. (2013) Chromatin-bound Xenopus Dppa2 shapes the nucleus by locally inhibiting microtubule assembly. Dev Cell 27:47-59
Postow, Lisa; Funabiki, Hironori (2013) An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation. Cell Cycle 12:587-95
Ghenoiu, Cristina; Wheelock, Michael S; Funabiki, Hironori (2013) Autoinhibition and Polo-dependent multisite phosphorylation restrict activity of the histone H3 kinase Haspin to mitosis. Mol Cell 52:734-45

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