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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM075249-09
Application #
8708102
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Carter, Anthony D
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Anatomy/Cell Biology
Type
Graduate Schools
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10065
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; Funabiki, Hironori (2014) Nuclear assembly shaped by microtubule dynamics. Nucleus 5:40-6
Postow, Lisa; Funabiki, Hironori (2013) An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation. Cell Cycle 12:587-95
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
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
Funabiki, Hironori; Wynne, David J (2013) Making an effective switch at the kinetochore by phosphorylation and dephosphorylation. Chromosoma 122:135-58
Du, Jiamu; Kelly, Alexander E; Funabiki, Hironori et al. (2012) Structural basis for recognition of H3T3ph and Smac/DIABLO N-terminal peptides by human Survivin. Structure 20:185-95
Rivera, Teresa; Ghenoiu, Cristina; Rodriguez-Corsino, Miriam et al. (2012) Xenopus Shugoshin 2 regulates the spindle assembly pathway mediated by the chromosomal passenger complex. EMBO J 31:1467-79
Rosenberg, Jessica S; Cross, Frederick R; Funabiki, Hironori (2011) KNL1/Spc105 recruits PP1 to silence the spindle assembly checkpoint. Curr Biol 21:942-7
Kelly, Alexander E; Ghenoiu, Cristina; Xue, John Z et al. (2010) Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B. Science 330:235-9

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