A mathematical and experimental study of feedback in the single-cell dynamics of a transcriptional network in budding yeast

Cells respond to environmental changes through corresponding changes in the biochemical networks they use to sense the environment. These networks are complex with genes that are activated often feeding back to affect levels of the proteins that first activated the genes. By studying how the single-celled organism, budding yeast, responds to changes in extracellular sugars we will determine the role of three of these feedbacks that occur in the genetic network that controls sensing of sugar. Using microfluidic technology and fluorescent proteins, we will quantify the responses of individual yeast cells to environments whose sugar levels fluctuate in ways we can define. By genetically engineering the cells so that we can set the level of each of the feedbacks by a chemical we supply, we will determine how the cell's response to fluctuating signals changes when the feedback changes. We will use mathematical modelling to predict the most informative signals to apply to discover how feedback determines the cell's response. In addition to its insights into nutrient sensing in an industrially valuable organism, our study is important because it will develop experimental and mathematical techniques to study the dynamic response of a biochemical network in living cells. All cells, whether in our own bodies or a single bacterium, have evolved in fluctuating environments where dynamic responses are often just as relevant as the long-term behaviours commonly studied.

All cells, but particularly micro-organisms, live in dynamic, fluctuating environments. To reliably sense and respond to such extracellular changes, signalling networks are complex with downstream proteins almost always feeding back to affect the activation of those upstream. We propose to determine the role of three transcriptional feedbacks in the dynamic response of a genetic network in budding yeast. We will study the dynamics of the GAL network in single cells using fluorescent protein reporters and quantitative microfluidic assays to control and stochastically vary the extracellular environment. We will develop a mathematical model of the network, fit this model to data generated by perturbing the network through varying both the sugar signals it senses and the levels of its protein components, and use this model to predict the statistics of signals most informative for determining the function of feedback in the network. Using microfluidics, we will apply these signals to living cells and quantitatively compare their dynamic response to our in silico predictions. Further, by placing the feedback genes under inducible promoters, we will `break' the feedbacks, both individually and in pairs, and determine if the cellular response changes in accord with the hypothesized function of the feedbacks being tested. The GAL network is the paradigmatic example of genetic regulation in eukaryotes and understanding how feedback controls its dynamics will have far-reaching implications, particularly because the structure of its feedback occurs in other organisms, including mammals.

The most economically important non-academic beneficiaries of our proposal are the biotechnology and pharmaceutical industries, particularly those adopting approaches from systems biology and synthetic biology. Our research is, however, perhaps best positioned to benefit those companies that provide tools and expertise to these industries. For example, we are working with engineers at the Scottish Microelectronics Centre, which is co-funded by Scottish Enterprise and the University of Edinburgh, to promote the uptake of microfluidics technology by other bioscientists through providing examples of its use to quantitatively study responses in single cells. Given the enormous costs of successfully developing a drug, systems biology, with its potential to predict the most effective molecules or cellular processes to target, is now a realistic alternative to traditional drug discovery. The mathematical model of the GAL network we will develop should contribute to models of signal transduction being developed by the pharmaceutical industry, particularly given the GAL network's status as a paradigm of genetic regulation. Yeast is also often the organism of choice to demonstrate proof-of-principle. For example, Novartis is interested in modelling nitrogen-sensing in budding yeast, and one of Swain's final-year Ph.D. students is interviewing with Novartis to work on this research. We have already had a successful collaboration with a biotechnology company, Gene Network Sciences, in North America: our modelling contributed to their efforts to predict potential drugs that do not also prolong QT intervals, a cardiotoxic side-effect. Several convenient and effective ways to make new connections to industry are in place. Both Swain and Tyers are members of the Centre for Systems Biology at Edinburgh (CSBE), which has a Commercialisation Team led by Dr. E. Elliot whose office is next-but-one to Swain's. Dr. Elliot has previous board-level experience in the private sector biotechnology industry with a strong track record in the commercialisation of research. This team also includes members of Edinburgh Research and Innovation, which promotes the commercialisation of the university's research. Both Swain and Tyers are hired through the Scottish Universities Life Sciences Alliance (SULSA) and have access to their Knowledge Exchange Committee made up of representatives from six Scottish universities. We also intend to publicize our work on cellular decision-making outside of the industrial and scientific communities. The subject is particularly topical because of the current public interest in stem cells. Swain plans to talk about his research at Edinburgh's Cafe scientifique, at which several of our colleagues have already presented. Further, we encourage our lab members to take part in outreach. For example, within the last year, our postgraduate students have participated in Edinburgh's International Science Festival, talked about their research at local secondary schools, and one will present their work at the National Museum of Scotland in July.