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Tanenbaum: Gene expression dynamics

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The Tanenbaum group uses cutting-edge live-cell single molecule microscopy and new types of genetic engineering to uncover the molecular mechanisms of gene expression control in individual cells.

Our work focuses both on regulation of human genes in health and disease and on gene regulation of RNA viruses. Using our single-molecule approaches, we aim to uncover how the dynamics and heterogeneity in gene expression affects cell fate. In addition, we are developing new imaging technologies to visualize gene expression dynamics with ever increasing resolution to achieve a deep molecular understanding of these processes.

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Figure 1. Single molecule imaging of mRNA translation using the SunTag system. (A) Schematic of the SunTag fluorescence labeling strategy. (B) Imaging translation on single mRNA molecules in live cells.

Single-molecule analysis of gene expression in living cells

Previously, we have developed an imaging technique called “SunTag” (Tanenbaum et al., 2014, Cell), which allows us to link many GFPs to a protein molecule of interest (Fig. 1A). This GFP multimerization approach makes the fluorescence tags far 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, we have developed a method to visualize translation and degradation of single mRNA molecules in space and time (Fig. 1B) (Yan et al., 2016, Cell; Hoek et al., 2019, Mol Cell). We are employing these methods to visualize gene expression control in living cells with incredible precision and to uncover how regulatory mechanisms function at the single-molecule level. 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 and how differences in gene expression affect cell fate. For example, we have applied the SunTag translation imaging technique to study RNA quality control (Hoek et al., 2019, Mol Cell), heterogeneity in translation start site selection (Boersma et al., 2019, Cell)  and post-transcriptional regulation of gene expression by small RNAs (Ruijtenberg et al., 2020, Nat Struct Mol Biol). We are currently exploring additional mechanisms of post-transcriptional gene regulation and are developing new technologies to examine gene expression control with even greater precision.

Visualizing translation, transcription and replication of single RNA viruses

RNA viruses are among the most prevalent pathogens worldwide and are a major burden on society. RNA viruses have been studied extensively, but surprisingly little is known about the processes that occur during early viral infection. The first hours of infection are likely very important, as the outcome of the infection (e.g. is the virus able to replicate? Can the host cell detect the virus and prevent its spreading before viral replication can occur?) may be determined at the early stages of infection. Lack of understanding of early infection is largely due to a lack of highly sensitive assays for viral detection, as during the early stages only one or a few viral RNAs are present per host cell. We have developed a single-molecule imaging assay based on our SunTag technology, called virus infection real-time imaging (VIRIM) that allows detection of single viral RNAs, which is therefore uniquely suitable to study early viral infection (Boersma et al, 2020, Cell). Using VIRIM we can detect the moment of host entry of individual viruses and follow viral translation and replication in single cells. Surprisingly, we observe a large degree of heterogeneity in viral infection dynamics in different cells, with viruses rapidly replicating to high numbers in some cells, while showing no replication at all in other cells. A similar heterogeneity is observed in the host response to the infection, which is critical to fight off the virus. We are employing VIRIM to uncover the mechanisms underlying heterogeneity in infection and are trying to understand how viral and host gene expression is coordinated to optimize viral replication and anti-viral signaling, respectively. Together, these studies can serve as a starting point for the development of new targeted therapies against virus infections.

figure 3. Gene activation using synthetic transcription factors. (A) A nuclease-dead CRISPR/Cas9 protein is fused to an array of transcriptional activation domains through SunTag and targeted to an endogenous gene promoter to turn on transcription. (B) K562 cells in which the membrane receptor CXCR4 (red) is turned on in the bottom two cells using CRISPRa. (C) K562 cells in which CXCR4 gene activity is artifically modulated show increased cell migration.

Studying gene expression using novel genome engineering approaches

Control of gene expression is critical for cell fate and homeostasis, and is often de-regulated 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. In collaboration with the lab of Jonathan Weissman, we have developed a new system to modulate transcription rates of endogenous genes called CRISPR activation (CRISPRa), which can be applied both to study regulation of individual genes and to perform genome-wide functional screens. We are also developing innovative methods based on CRISPR gene targeting to study transcriptional and post-transcriptional gene regulation.