- Regulation of replication-independent chromatin assembly in yeast
All living cells have evolved mechanisms to ensure that the information stored within their chromosomes is correctly translated, replicated and passed onto daughter cells during each round of growth. The vast majority of the proteins required to faithfully carry out these mechanisms are conserved from yeast to human, allowing us to draw conclusions about how higher eukaryotic systems work by manipulating simple systems, such as the brewing yeast,Saccharomyces cerevisiae. Our work utilizes yeast to address how the basic unit of the chromosome (chromatin) is assembled and how this process is regulated. Chromatin is a highly dynamic structure composed of histones and DNA. The histones that make up chromatin can be post-translationally modified and these modifications control transcription, recombination, entrance into mitosis and chromosome segregation. An array of factors encoded within the yeast genome, called chromatin assembly factors, or CAFs, function to deposit histones onto DNA. Why the cell would encode multiple CAFs and how they, and the many histone modifying activities, are controlled throughout the cell cycle are just the beginning of the questions that are without answers.
We have developed an in vitro system that allows us to assay chromatin assembly in a yeast whole cell extract. This assay is histone and cell cycle dependent with peak activity occurring during mitosis. We have identified two mutations that compromise this activity. The mutations were found in the genes encoding APC5 and RSP5, which are both components of the ubiquitin-dependent targeting pathway. Additional studies have shown that the APC5 defect reflects compromised Anaphase Promoting Complex (APC) activity. These intriguing results have directed our focus to how the cell cycle and signal transduction pathways modulate chromatin assembly and histone post-translational modifications.
How lifespan in eukaryotic organisms is determined
A second project is aimed at understanding how lifespan in eukaryotic organisms is determined. We have recently shown that the APC is required for extended lifespan in yeast. Our studies have shown that two aging pathways, Snf1 (lifespan extension) and Ras (lifespan reduction), converge on the APC to influence aging. These results suggest possible mechanisms controlling lifespan in higher eukaryotes, as all factors under investigated are evolutionarily conserved.
Our present and future work is directed at i) the role played by the RAS/PKA and Snf1 signaling pathways in determining APC-dependent lifespan, ii) identifying other components of the ubiquitin-dependent protein targeting cascade involved in chromatin metabolism, iii) determining the molecular basis of APC-dependent chromatin assembly, and iv) determining the effects on higher eukaryotic systems, such as humans and mouse, when the mitotic-specific chromatin assembly activity is compromised.
- Postnikoff SD, Harkness TA. Replicative and chronological life-span assays. Methods Mol Biol. 2014;1163:223-7. doi: 10.1007/978-1-4939-0799-1_17.
- Davies GF, Berg A, Postnikoff SD, Wilson HL, Arnason TG, Kusalik A, Harkness TA. TFPI1 mediates resistance to doxorubicin in breast cancer cells by inducing a hypoxic-like response. PLoS One. 2014 Jan 28;9(1):e84611. doi: 10.1371/journal.pone.0084611. eCollection 2014.
- Menzel J, Malo ME, Chan C, Prusinkiewicz M, Arnason TG, Harkness TA. The anaphase promoting complex regulates yeast lifespan and rDNA stability by targeting Fob1 for degradation. Genetics. 2014 Mar;196(3):693-709. doi: 10.1534/genetics.113.158949. Epub 2013 Dec 20.
- Titorenko VI, Harkness TA. The spatiotemporal dynamics of longevity-defining cellular processes and its modulation by genetic, dietary, and pharmacological anti-aging interventions. Front Physiol. 2012 Oct 31;3:419. doi: 10.3389/fphys.2012.00419. eCollection 2012.
- Postnikoff SD, Harkness TA. Mechanistic insights into aging, cell-cycle progression, and stress response. Front Physiol. 2012 Jun 4;3:183. doi: 10.3389/fphys.2012.00183. eCollection 2012.
- Postnikoff SD, Malo ME, Wong B, Harkness TA. The yeast forkhead transcription factors fkh1 and fkh2 regulate lifespan and stress response together with the anaphase-promoting complex. PLoS Genet. 2012;8(3):e1002583. doi: 10.1371/journal.pgen.1002583. Epub 2012 Mar 15.
- >Goldberg AA, Beach A, Davies GF, Harkness TA, Leblanc A, Titorenko VI. Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells. Oncotarget. 2011 Oct;2(10):761-82.
- Islam A, Turner EL, Menzel J, Malo ME, Harkness TA. Antagonistic Gcn5-Hda1 interactions revealed by mutations to the Anaphase Promoting Complex in yeast. Cell Div. 2011 Jun 8;6(1):13.
- Lindsay DL, Bonham-Smith PC, Postnikoff S, Gray GR, Harkness TA. A role for the anaphase promoting complex in hormone regulation. Planta. 2011 Jun;233(6):1223-35. Epub 2011 Feb 17.
- Wang, J., A.P. Hitchcock, C. Karunakaran, A. Prange, B. Franz, T. Harkness, Y. Lu, M. Obst and J. Hormes. 2010. 3D Chemical and Elemental Imaging by STXM Spectrotomography. American Institute of Physics Conference Proceedings, accepted Oct. 18, 2010.
- Feser, J., D. Truong, C. Das, J.J. Carson, J. Kieft, T. Harkness, and J.K. Tyler. 2010. Elevated histone expression promotes life span extension. Mol Cell 39: 724-35. Featured article.
- Turner, E.L., M.E. Malo, M.G. Pisclevich, M.D. Dash, G.F. Davies, T.G. Arnason, and T.A. Harkness. 2010. TheSaccharomyces cerevisiae Anaphase-Promoting Complex interacts with multiple histone-modifying enzymes to regulate cell cycle progression. Eukaryot Cell 9: 1418-1431.
- Harkness, T.A.A. 2010. Longevity as a matter of housekeeping. Aging 2: 392.
- Davies, G.F., A.R. Ross, T.G. Arnason, B.H.J. Juurlink, and T.A.A. Harkness. 2010. Troglitazone inhibits histone deacetylase activity in breast cancer cells. Cancer Lett., 288: 236-250.
- Davies, G.F., B.H.J. Juurlink and T.A.A. Harkness. 2009. Troglitazone reverses the multiple drug resistant phenotype in cancer cells. Drug Des. Dev. and Ther., 3: 79-88.
- Noyan-Ashraf, M.H., Z. Sadeghinejad, G.F. Davies, A.R. Ross, D. Saucier, T.A.A. Harkness and B.H.J. Juurlink. 2008. Phase 2 protein inducers in the diet promote healthier aging. Journal of Gerontology, Series A: Biological Sciences Medical Science, 63: 1168-76.
- Rostek, C., E.L., Turner, M., Robbins, S., Richnar, W., Xiao, A. Obenaus, and T.A.A., Harkness. 2008. Involvement of homologous recombination repair in proton-induced DNA damage. Mutagenesis, 23: 119-29.
- Harkness, T.A.A. 2006. Decondensation of Xenopus sperm chromatin using Saccharomyces cerevisiae whole-cell extracts. Canadian Journal of Pharmacology and Physiology, 84: 451-458.
- Harkness, T.A.A. 2006. The anaphase promoting complex and aging: The APCs of longevity. Curr. Genomics, 7: 263-272.
- Harkness, T.A.A. 2005. Chromatin assembly from yeast to man: Conserved factors and conserved molecular mechanisms. Curr. Genomics, 6: 227-240.
- Davies, G.F., Roesler, W.J., Juurlink, B.H.J. and Harkness, T.A.A. 2005. Troglitazone overcomes doxorubicin-resistance in resistant K562 leukemia cells. Leuk. Lymphoma, 46: 1199-1206.
- Harkness, T.A.A., Arnason, T.G., Legrand, C., Pisclevich, M.G., Davies, G.F. and Turner, E.L. 2005. Contribution of CAF-I to anaphase-promoting-complex-mediated mitotic chromatin assembly in Saccharomyces cerevisiae.Eukaryot. Cell, 4: 673-684.
- Arnason, T.G., Pisclevich, M.G., Dash, M.D., Davies, G.F. and Harkness, T.A.A. 2005. Novel interaction between Apc5p and Rsp5p in an intracellular signaling pathway in Saccharomyces cerevisiae. Eukaryot. Cell, 4: 134-146.
- Harkness, T.A.A., Shea, K.A., Legrand, C., Brahmania, M. and Davies, G.F. 2004. A functional analysis reveals dependence on the anaphase-promoting complex for prolonged life span in yeast. Genetics, 168: 759-774.