Award details

Single-molecule dynamics of human transcription regulation

Reference	BB/L021730/1
Principal Investigator / Supervisor	Dr Andrey Revyakin
Co-Investigators / Co-Supervisors
Institution	University of Leicester
Department	Biochemistry
Funding type	Research
Value (£)	768,736
Status	Completed
Type	Research Grant
Start date	04/11/2014
End date	03/11/2018
Duration	48 months

Abstract

Transcription of messenger RNA in the human cell begins with the assembly of RNA Polymerase II (Pol II) and General Transcription Factors (GTFs) on a promoter into a so-called preinitiation complex (PIC). Formation of the PIC is the step at which the cell commits to express a gene, and therefore is the main control panel for gene regulation. Although the key components of the PIC have been identified, the dynamics of PIC assembly, and the mechanism of PIC regulation are poorly understood. We propose to apply a cutting-edge imaging technology to obtain a real-time, single-molecule 'movie' of PIC assembly and regulation. This new approach will reveal the full spectrum of dynamic interactions between Pol II and GTFs without introducing population-averaging effects, and will preserve the inherent stochastic, 'single-molecule' aspect of transcription in living cells. The project has two specific objectives. In Objective 1, we will use a model cell-free system for 'basal transcription,' comprised of purified human Pol II and five GTFs (IIB, IID, IIF, IIE, and IIH) to determine in what order and at what rates GTFs and Pol II assemble on the promoter, which will pinpoint the rate-limiting steps of PIC formation. Furthermore, we will directly test whether GTFs remain at the promoter after Pol II starts elongation (a prototypical model for 'gene bookmarking'). In Objective 2, we will use a model cell-free system for regulated transcription, comprised of Pol II, GTFs, a prototypical human activator Sp1 and its co-coactivator IIA, to determine which step of PIC formation is targeted by activators to stimulate transcription, and how dynamic interactions between activator and co-activator molecules are integrated into the stimulatory signal. This study will produce the first quantitative model of human transcription regulation, and will establish the benchmarks, technology and reagents for future studies of regulation of native genes in single living cells.

Summary

My lab is interested in understanding how cells 'decide' what genes, and when, to switch on. Knowing how genes are regulated could, for example, provide clinicians with new drugs to cure genetic diseases, and scientists with new tools to turn regular cells into stem cells. Previously, scientists had to infer how genes work from genetics (by following inherited traits), or from biochemical experiments (by grinding up cells and analyzing their chemical composition). However, no one has ever 'seen' a human gene molecule switch on, which limited our understanding of gene regulation. Recently, our lab has developed an imaging technology to directly 'see' gene molecules being switched on by single enzyme molecules. In this proposal, we will use our technology to reconstruct a minimal circuit of gene activation at single-molecule resolution, which will give us fundamentally new ideas on how genes are regulated. Physically, genes are molecules of DNA located in the cell nucleus; most cells contain only two molecules of each gene. To switch a gene on means to make a protein based on the information encoded in the DNA. The cell decides to switch on a gene by recruiting an enzyme, called Pol II, to the gene molecule. Pol II then runs along the gene while making (transcribing) an 'active copy' of the gene, called RNA, which is then used as a template for making protein. The focus of this proposal is to find out how Pol II 'decides' to bind to a gene in the first place. This is an important question, because many genetic diseases (e.g. cancer) can be traced back to Pol II binding and copying a wrong set of genes. Binding of Pol II to genes requires several other molecules, called transcription factors. Although most of transcription factors are known, the order in which they interact with each other to bring Pol II to a gene is not clear. By analogy, to understand football, it is not enough to identify each player in the field: one has to understand how the players interactwith each other to get the ball (Pol II) between the goalposts (to a gene). We propose to elucidate how transcription factors bring Pol II to a gene, literally, by watching the entire molecular game live. To do that, we will isolate a minimal team of players -- Pol II and five transcription factors -- from cells and mix them together in a drop of physiological solution. We will then put a single gene molecule on a microscope slide, cover it with the drop, and watch what is going on at the gene under a microscope in a dark room. To see the molecules of Pol II and transcription factors, we will label them with dyes of different colors (e.g. blue, green, and red), which will make the molecules glow against dark background like stars. By watching in what order the blue, green, and red stars bind to the gene, we will determine how the transcription factors bring Pol II to the gene. After we elucidate how the minimal team of five factors plays the game, we will add one more player -- transcription 'activator' Sp1, which is present at high levels in rapidly dividing and cancerous cells -- and determine how Sp1 changes the game (e.g. with which transcription factors it interacts) to make Pol II produce more RNA copies of a gene. Our analysis of how single molecules switch genes on may fundamentally change the way scientists think about gene regulation. In textbooks, switching a gene on is often shown as flipping on a switch. However, because each gene in a cell is represented by only two molecules, switching a gene on could be a stochastic ('sloppy') event, due to the Brownian motion of molecules in the microscopic world. Therefore, all decisions made by the cell (for instance, a decision to turn into a cancer cell) could be affected by stochastic collisions between Pol II, transcription factors and genes -- which may explain why some cell behaviors are difficult to control.

Impact Summary

This research project has a potential to have a great impact in the academy, industry, and in the wider public. We propose to build the first stochastic model of human transcription regulation based on quantitative data obtained using a cutting-edge single-molecule imaging technology. 1. Academic and industrial. This research will help understand how single cells make decisions to grow, differentiate or de-differentiate despite the stochastic noise of molecular collisions at promoters, and will establish fundamentally new ways of thinking about gene regulation. Thus, the immediate beneficiaries of this research will be researchers studying molecular mechanisms of gene regulation, and researchers using transcription factors as tools to control cell-state decisions. The insights into stochasticity of transcription will be of great interest to researchers studying dynamics of macromolecular complexes involved in nucleic acid transactions (e.g. translation, splicing and replication). Our protocols for fast, specific RNA hybridization will be of interest to researchers developing bottom-up bio-nanostructures. Our methods to generate bio-compatible surfaces have previously attracted, and will continue to attract the interest of companies working on high-throughput DNA sequencing and microfluidics. We will ensure that the academic and industrial beneficiaries are informed of our work by publishing in wide-audience peer-reviewed journals, attending scientific meetings in the UK and abroad, and keeping our lab web page up-to-date with the newest developments and publications. The cutting-edge research that we will bring to the University of Leicester will strengthen the position of the school as a hub for new technology, and attract young investigators from the USA and Europe who work at the interface of biology, chemistry, and physics. 2. Societal. The new insights into the mechanism of gene regulation that we will attain may provide researchers with better toolsto control cell behavior, which may lead to better ways to treat diseases and engineer tissues, and, ultimately, improve the nation's health. The powerful visual aspect of our technology (using lasers to directly 'see' single enzyme molecules switching genes on) is very likely to attract the interest of the general public, in particular, middle- and high-school students. The students will be informed of our work through microscope-building and single-molecule-imaging demonstrations that we will organize with the GENIE Outreach program (see Pathways to Impact). The demonstrations will stimulate students' interest in biomedical sciences and nanotechnology, and, perhaps, encourage them to pursue careers in these high-tech fields -- which, indirectly, will have a long-term impact on the UK economy. 3. Career development. The postdoctoral research assistant and the research technician working on the project will learn a wide range of methods (single-molecule imaging and data analysis, molecular cloning, bioconjugation, surface chemistry, protein purification, instrument design, software development, cell culture, genome editing, and so on) and acquire critical thinking, presentation and writing skills which will prepare them for future careers in academia or industry, or for further education. 4. 'New ways of working'. This multidisciplinary study is within the scope of three strategic priorities of the BBSRC aimed at raising the awareness of novel, alternative methods of doing research: (i) technology development for the biosciences (bioimaging and functional analysis); (ii) data-driven biology (extracting quantitative information from large or complex image sets); and (iii) systems approaches to the biosciences (experimental biology closely-integrated with computational modeling).

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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