Single-molecule dynamics of human transcription regulation

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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 interact with 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.