- 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.
- Belak, Z, Harkness, T, Eskiw, C. A rapid, high- throughput method for determining chronological lifespan in budding yeast. J Biol Methods. 2018 Dec;5:e106.
- Hu S, Tomoiaga AL, Postnikoff SDL, Harkness TAA, Seol JH, Xia B, Li W, Chen K, Tyler JK. Ssd1 and Gcn2 suppress global translational efficiency in replicatively aged yeast, while their activation in young cells extends lifespan. eLife. 2018 Aug;7:pii: e35551.
- Ghavidel. A, Baxi K, Prusinkiewicz M, Swan C, Belak ZR, Eskiw CH, Carvalho CE, Harkness TAA. Rapid Nuclear Exclusion of Hcm1 in Aging Saccharomyces cerevisiae Leads to Vacuolar Alkalization and Replicative Senescence. G3. 2018 May;8(5):1579- 1592.
- Davies GF, Lobanova L, Dawicki W, Groot G, Gordon JR, Bowen M, Harkness TAA, Arnason TG. Metformin inhibits the development, and promotes the resensitization, of treatment-resistant breast cancer. PLoS ONE. 2017 Dec;12(12):e0187191.
- Baxi K, Ghavidel A, Waddell B, Harkness TA, Carvalho C. Regulation of Lysosomal Function by the DAF-16 Forkhead Transcription Factor Couples Reproduction to Aging in Caenorhabditis elegans. Genetics. 2017 Sep.
- Kakish J, Allen KJ, Harkness TA, Krol ES, Lee JS. Novel Dimer Compounds That Bind -Synuclein Can Rescue Cell Growth in a Yeast Model Overexpressing - Synuclein. A Possible Prevention Strategy for Parkinson's Disease. ACS Chem Neurosci. 2016 Dec;7(12):1671-1680.
- Jiao R, Lobanova L, Waldner A, Fu A, Xiao L, Harkness TA, Arnason TG. The ubiquitin-conjugating enzyme, Ubc1, indirectly regulates SNF1 kinase activity via Forkhead-dependent transcription. Microb Cell. 2016 Nov;3(11):540-553.
- Li Q, Shi X, Ye S, Wang S, Chan R, Harkness T, Wang H. A short motif in Arabidopsis CDK inhibitor ICK1 decreases the protein level, probably through a ubiquitin-independent mechanism. Plant J. 2016 Sep.
- Malo M, Postnikoff S, Arnason T, Harkness T. Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance. Aging. 2016 Apr;8(4):810-30.
- Ghavidel A, Baxi K, Ignatchenko V, Prusinkiewicz M, Arnason T, Kislinger T, Carvalho C, Harkness T. A Genome Scale Screen for Mutants with Delayed Exit from Mitosis: Ire1-Independent Induction of Autophagy Integrates ER Homeostasis into Mitotic Lifespan. PLoS Genetics. 2015 Jul;11:e1005429.
- Jiao R, Postnikoff S, Harkness T, Arnason T. The SNF1 Kinase UBA Domain Restrains its Activation, Activity, and the Yeast Lifespan. Journal of biological Chemistry. 2015 Jun 19.
- Harkness T. Living longer through mitochondrial housekeeping. Cell Cycle. 2015 Apr 13.