The role of non-histone proteins in chromosome structure and function during mitosis
Our research focuses on three main aims.
1. Making mitotic chromosomes: How do mitotic chromosomes condense, and what is the role of histones and non-histone proteins in shaping them?
2. Segregating the chromosomes: How are centromere specification and kinetochore assembly controlled epigenetically?
3. Controlling the process: How does the chromosomal passenger complex (CPC) regulate chromosome segregation?
This year, a collaboration exploiting Hi-C technology (with Job Dekker at U. Mass. Medical School in Worcester) with mathematical modelling (with Leonid Mirny at M.I.T.) allowed us to propose a new model for the pathway of mitotic chromosome formation. Our lab did all the cell biology and imaging. The resulting model integrates all previous major models of chromosome structure, showing that chromosomes are built of nested dynamic loops emanating from a spiral scaffold structure composed of condensin II. The key advance enabling this study was development of a chemical biology protocol yielding an almost perfectly synchronous entry of DT40 cells into mitosis. We also completed a study showing that rapid depletion of condensin leads to novel and much more dramatic phenotypes than seen previously. Earlier this year, we published a study strongly suggesting that histone posttranslational modifications may be a key factor driving mitotic chromatin compaction.
Studies of chromosome segregation focused on using human synthetic artificial chromosomes to probe the role of epigenetics and mitotic transcription in centromere stability and function. We expanded our approach to removing histone marks from centromeres, showing that we can target multiple competing activities simultaneously to perform what are essentially in situ epistasis studies. This led to a hypothesis that centromeric H3K9ac defends centromeres against invasion by surrounding heterochromatin. We also began work on a new generation of synthetic human chromosomes containing separate heterochromatin and centromeric arrays that will allow us to systematically probe interactions between these two centromeric domains.
Our work on the CPC revealed that interactions between heterochromatin protein HP1 and the CPC play a key role in targeting and activation of this important mitotic kinase complex during mitotic entry. Other studies probed the role of nucleoporin Seh1 in targeting regulators of Tor kinase signaling to mitotic chromosomes and examined the mitotic phenotypes resulting from treatment of cells with p53-activating inhibitors of the enzyme DHODH in one study and inhibitors of telomerase in a second.
Our work is supported by a Wellcome Principal Research Fellowship and by the Centre for Mammalian Synthetic Biology.
Vargiu, G., Makarov, A.A., Allan, J., Fukagawa, T., Booth, D.G., and Earnshaw, W.C. (2017). Stepwise unfolding supports a subunit model for vertebrate kinetochores. PROC. NATL. ACAD. SCI. U.S.A. 114:3133-3138. PMID: 28265097; DOI: 10.1073/pnas.1614145114.
Zhiteneva, A., Bonfiglio, J.J., Makarov, A., Colby, T., Vagnarelli, P., Schirmer, E.C., Matic, I., and Earnshaw, W.C. (2017). Mitotic post-translational modifications of histones promote chromatin compaction in vitro. OPEN BIOL. 7, pii: 170076. PMID: 28903997; PMC5627050; DOI: 10.1098/rsob.170076.
Gibcus, J.H., Samejima, K., Goloborodko, A., Samejima, I., Naumova, N., Nuebler, J., Kanemaki, M., Xie, L., Paulson, J.R., Earnshaw, W.C., Mirny L.A., Dekker, J. (2018). A pathway for mitotic chromosome formation. SCIENCE 9:359. PMID: 29348367; DOI: 10.1126/science.aao6135.