Domains of unknown function are essential in yeast
- 1. Domains of unknown function are essential in yeast Norman Goodacre FDA CBER
- 2. The world is a man-made oyster • No clear limit to the potential of Synthetic Biology • Nascent technology, much like IT in the 50s • The short-term benefits already seem promising • Engineering metabolic networks for food and drug production • Environmental clean-up • Balancing human microbiomes • “DNA” computers Image credit: Nielsen, A.A., Der, B.S., Shin, J., Vaidyanathan, P., Paralanov, V.,Strychalski, E.A., Ross, D., Densmore, D. and Voigt, C.A., 2016. Genetic circuit design automation. Science, 352(6281), p.aac7341.
- 3. Overview of talk • Protein-protein interactions (PPIs) drive the majority of cellular processes • Many PPIs may be redundant • Engineering new cellular processes can be streamlined by eliminating non-essential PPIs
- 4. What is an essential PPI? • If an essential gene or protein is deleted from the genome, the organism cannot form • Many genes or proteins may be conditionally-essential • These ‘accessory’ or conditional genes may not be relevant to the engineered purpose of the organism • More interactions = more potential for cross-talk • Modular components need minimal cross-talk
- 5. Design of minimal genomes Hutchison, C.A., Chuang, R.Y.,Noskov, V.N., Assad-Garcia, N., Deerinck, T.J., Ellisman, M.H., Gill, J., Kannan, K., Karas, B.J., Ma, L. and Pelletier, J.F., 2016. Design and synthesis of a minimal bacterial genome. Science, 351(6280), p.aad6253.
- 6. Native protein-protein interaction (PPI) network RpoD RpoS GadX ArcA CpxR Crp RpoE CytR RpoH LexA CpxR Crp Fur Fur DnaA DnaA LexA redox, carbon metab global, carbonmetab replinitiator transport, carbonmetab membrane stress RpoD/S/E/H are transcription initiation factors, recruiting RNA pol to promoters ArcA aerobic metab, membraneproteins acid resistance Non-essential interactions Essential interactions
- 7. PPI network with removal of non-essential proteins RpoD RpoS GadX ArcA CpxR Crp CytR RpoH LexA CpxR Fur Fur DnaA LexA redox, carbon metab global, carbonmetab replinitiator transport, carbonmetab membrane stress RpoD/S/E/H are transcription initiation factors, recruiting RNA pol to promoters ArcA aerobic metab, membraneproteins acid resistance Non-essential interactions Essential interactions
- 8. PPI network with removal of non-essential interactions RpoD RpoS GadX ArcA CytR RpoH Fur DnaA replinitiator LexA redox, carbon metab RpoD/S/E/H are transcription initiation factors, recruiting RNA pol to promoters acid resistance Essential interactions CpxR membrane stress similardomain architectures similardomain architectures aerobic metab, membraneproteins transport, carbonmetab
- 9. PPI network with removal of non-essential domains RpoD GadX Fur DnaA LexA redox, carbon metab acid resistance Essential domain interactions ArcA aerobic metab, membrane proteins CytR transport, carbonmetab replinitiator Is this possible?
- 10. Overview of protein domains • Protein domains are the independently-folding units of tertiary structure • Each has a specific function or (sometimes) set of functions • Of the > 17,000 known domains, ~3,000 are domains of unknown function (DUFs) • ~6,000 enzymes 1° 2° 3° (domains) 4°
- 11. DUFs – Protein domains of unknown function Goodacre, N.F., Gerloff, D.L. and Uetz, P., 2014. Protein domains of unknown function are essential in bacteria. MBio, 5(1), pp.e00744-13.
- 12. eDUFs – essential protein domains of unknown function These are all in JCVI’s Syn3.0, based on M. mycoides Hutchison, C.A., Chuang, R.Y., Noskov, V.N., Assad- Garcia, N., Deerinck, T.J., Ellisman, M.H., Gill, J., Kannan, K., Karas, B.J., Ma, L. and Pelletier, J.F., 2016. Design and synthesis of a minimal bacterial genome. Science, 351(628 0), p.aad6253. Goodacre, N.F., Gerloff, D.L. and Uetz, P.,2014. Protein domains of unknown function are essential in bacteria. MBio, 5(1), pp.e00744-13.
- 13. Focus of present study – inferring essential domains of unknown function (eDUFs) in two species of yeast • Yeast are model organisms for eukaryotes, and have played key roles in the development of several high-throughput technologies (microarray, yeast two-hybrid, single-gene-knockout essentiality screens) • Saccharomyces cerevisiae (“budding yeast”) – Sc • Schizosaccharomyces pombe (“fission yeast”) - Sp • Sp underwent an ancient genome reduction and duplication event, and has a higher eukaryote – like cell cycle • Cell cycle, chromatin structure, and membrane budding / transport – related genes are known to be different between the two strains • Summary – Sc is the best-characterized eukaryote, but Sp has a minimal genome already and could be interesting for synthetic biologists
- 14. Database of Essential Genes - DEG • DEG is an invaluable resource for gene essentiality information • For prokaryotes, DEG contains essential genes in more than 10 bacteria, such as E. coli, B. subtilis, H. pylori, S. pneumoniae, M. genitalium and H. influenzae, whereas for eukaryotes, DEG contains those in yeast, humans, mice, worms, fruit flies, zebra fish and the plant A. thaliana. • Over 30,000 essential genes (as of November, 2017)
- 15. Methodology for inferring essential domains Strategy to infer yeDUFs using a combined rule-based and expectation-maximization approach. The essentiality of protein domains of unknown function was inferred using a hybrid rule-based and expectation-maximization approach, which was performed separately for S. cerevisiae and S. pombe. Three rules were used to infer essential domains: case 1 were single-domain essential proteins case 2 domains present in essential proteins, but absent in non-essential proteins with otherwise identical architecture case 3 were domains always and only present in essential proteins
- 16. 199 yeDUFs - yeast essential domains of unknown function - were inferred Saccharomyces cerevisiae (Sc) and Schizosaccharomyces pombe (Sp) encode 199 yeDUFs. YeDUFs were inferred for each species of yeast separately, using rule-based inference (top), as well as expectation maximization (EM, middle). EM-inferred yeDUFs were more numerous and mostly contained their respective rule-based predictions as a subset. Approximately one- third of all inferred yeDUFs were shared between Sc and Sp (bottom). 199
- 17. Distribution of yeDUFs across the interactive Tree of Life (iTOL) YeDUFs are highly-conserved in eukaryotes, and broadly conserved across all kingdoms of life. Conservation of total yeDUFs is shown in the innermost ring, while the outer rings (moving outward) show conservation for SceSp- (essential in S. cerevisiae, absent in S. pombe), SceSp+ (essential in S. cerevisiae, present in S. pombe), SceSpe (essential in S. cerevisiae, essential in S. pombe), Sc+Spe (present in S. cerevisiae, essential in S. pombe), Sc-Spe (absent in S. cerevisiae, essential in S. pombe) sub-categories of yeDUFs, respectively.
- 18. Functional clues for inferred yeDUFs YeDUFs essential in both yeast species are suspected to be involved in proteintargeting, protein folding, and translation initiation activities. These were often conserved in prokaryotes as well. YeDUFs essential only in Sp are suspected mostly to be involved in alternative metabolic pathways. YeDUFs essential only in Sc were suspected mostly to be involved in membrane organization and vacuole fusion. Pr = Prokaryotes, Eu = Eukaryotes. Sc-specific Sc + Sp Sp-specific
- 19. Future work • Look at conditionally-essential domains • Does essentiality depend on presence or absence of other domains? • Incorporate synthetic lethality datasets (e.x. Hillenmeyer et al., 2012) • Genes can be essential in pairs • Could help reveal conditionally-essential domains • Determine interactions and function of yeDUFs
- 20. Acknowledgements • Dr. Peter Uetz (VCU CSBC) • Global Engage
- 21. Presented in the Synthetic Biology & Gene Editing strand of the 4Bio Summit. To find out more, visit: www.global-engage.com