Co-workers:

Our studies presently aim to answer the following three questions: What are the structural proteins of the mitotic chromosome and kinetochore and how do they direct chromosome segregation in mitosis? What is the chromatin environment of the centromere that provides an epigenetic landscape permissive for kinetochore assembly? How does the chromosomal passenger complex (CPC) regulate mitotic events? In an ongoing collaboration with Juri Rappsilber, we have defined the complete proteome of vertebrate mitotic chromosomes (>4000 proteins) using a novel approach that we term Multi-Classifier Combinatorial Proteomics (MCCP). We are presently defining a new series of classifiers, based on isolating chromosomes from cells genetically deficient in key chromosomal proteins. Our present studies focus on mapping the roles of all key SMC protein-containing complexes (condensin, cohesin and the SMC5/6 complex) in chromosome assembly – i.e. determining how depleting key members of each complex affects the global chromosome proteome. We are also beginning a series of studies aimed at using MCCP to determine the comprehensive assembly pathway for the vertebrate kinetochore. Our studies of the chromatin environment within and surrounding the kinetochore of a synthetic human artificial chromosome (HAC) revealed that the histone modification H3K4me2 (histone H3 dimethylated on lysine 4) is essential for the long-term stability of the kinetochore. H3K4me2 may act by providing a chromatin ‘memory” that promotes continued low-level transcription of the kinetochore. Current studies of our synthetic HAC have focused on analysis of the role of transcription and “open chromatin” in kinetochore function. We have done this by targeting the activation domains of Herpes virus VP16 and NF-kB component p65 into the kinetochore. Interestingly, VP16 promotes rapid disassembly of the kinetochore, whereas p65 has no discernable effect. Both factors promote “open” acetylated chromatin, but VP16 is a far more potent transcriptional activator. Current studies therefore focus on the role of low level transcription in kinetochore function. Lastly, our genetic studies of the CPC led to the demonstration that INCENP appears to act as a scaffold integrating the activities of the two key mitotic kinases Polo and Aurora B. Combining genetic and biochemical studies in Drosophila, we have shown that Aurora B is responsible for Polo activation at kinetochores. This is extremely interesting because the two kinases have opposite effects: Polo promoting microtubule binding and Aurora B promoting microtubule release. We believe that INCENP integrates and coordinates the activity of these two kinases, thereby enabling accurate chromosome segregation.

Selected publications:

Ohta S., J.-C. Bukowski-Wills., L. Sanchez-Pulido, F. de L. Alves, L. Wood, Z.A. Chen, M. Platani, L. Fischer, D.F. Hudson, C.P. Ponting, T. Fukagawa, W.C. Earnshaw* and J. Rappsilber*. (2010). The protein composition of mitotic chromosomes determined using multi-classifier combinatorial proteomics. CELL 142: 810-821.
Xu Z, Vagnarelli P, Ogawa H, Samejima K, Earnshaw WC. (2010) A gradient of increasing Aurora B activity is required for cells to execute mitosis. J BIOL CHEM. 285: 40163-40170. Bergmann J.H., M. Gomez Rodriguez, N.M.C. Martins, H. Kimura, D.A. Kelly, H. Masumoto, V. Larionov, L.E.T. Jansen and W.C. Earnshaw. (2011). Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore. EMBO J. 30: 328-340.