Real-time dynamics of gene expression at the single-molecule level
We have recently developed a new imaging technique called “SunTag” (Tanenbaum et al., 2014, Cell), which allows us to link many GFPs to a protein molecule or organelle of interest (Fig. 1A). This GFP multimerization approach makes the fluorescence tags much brighter than was previously possible and enables us to visualize complex biological processes with single-molecule sensitivity in real-time in living cells. Using the SunTag technology, we can continuously monitor the translation of single mRNA molecules in space and time (Yan et al., 2016, Cell).
We are employing SunTag technology to visualize gene expression control in living cells with incredible precision to uncover how regulatory mechanisms function at the molecular level, and how regulation of protein expression affects cell fate decisions. We are using a combination of quantitative single-cell and single-molecule fluorescence microscopy and computer simulations to look beyond cell population averages, and study how single cells tune gene expression over time.
Manipulation of gene expression using synthetic transcription factors
Control of gene expression is critical for cell fate and homeostasis and is often deregulated in diseases like cancer. To study the function of gene expression regulation, it is critical to be able to perturb it. However, modulating the expression of endogenous genes has been very challenging. Recently, in collaboration with the lab of Jonathan Weissman at UCSF, we have developed a new system to modulate transcription rates using an artificial, precisely controlled transcription factor-based CRISRP/Cas9 protein fused to transcription activation domains through the SunTag (Fig. 2) (Tanenbaum et al., 2014, Cell).
We are using this new technology to study how transcriptional regulation drives key cell cycle transitions, and how transcriptional control interplays with other gene expression regulatory mechanisms, like mRNA stability and translation.
Post-transcriptional regulation of the cell cycle in single cells
Hundreds of proteins show altered expression as cells progress through the cell cycle, but the mechanisms underlying these changes remain poorly understood. While a significant body of work has focused on the regulation of protein degradation, very little is known about the control of protein synthesis, even though protein levels are equally dependent on protein degradation and synthesis. Protein synthesis rates can be regulated through many different regulatory mechanisms, including transcriptional and translational control, mRNA localization, and mRNA stability.
We have developed new techniques to visualize different steps in gene expression in single cells to understand how these different mechanisms that alter gene expression activity are controlled as cells progress through the cell cycle (Tanenbaum et al., 2015, eLife). Using these techniques, we aim to understand how protein levels are modulated over time and how control of gene expression ensures reliable cell cycle decisions in single cells.