What are we doing ?

The zenklusen lab studies how individual cells execute gene expression programs. We are interested in the mechanism that regulate the different steps along the gene expression pathway, in particular transcription and mRNA export. We therefore combine single molecule techniques and quantitative analysis methods with genetics, biochemistry and mathematical modeling.

Why study gene expression in single cells ?

Dissecting the regulation of gene expression processes is fundamental to understand how cells function and has been extensively studied for many years. However, most methods used to obtain our current view of gene expression rely on isolating mRNAs from large numbers cells, which is often associated with a loss or damage of the material, as well as the loss of spatial information. Moreover, individual cells within a population are unlikely to behave all in the same way and current standard techniques are unable to detect cell-to-cell differences that can result from genetic variation, biological noise and/or different characteristics of genes within a population. Due to these limitations, more direct tools are required that allow analyzing these processes in their natural environment, the individual cell. Therefore, we are doing our experiments not in a test tube, but in intact, individual cells, basically performing biochemistry of life.

Why single molecules ?

Many biological processes, and in particular gene expression regulation, can be reduced to a single molecule problem. In a haploid cell, only a single copy of each gene exists, and the cellular 'machinery' has to ensure that proper regulation is achieved for each of these individual entities (genes). For a gene A to be transcribed, a transcription factor has to find its promoter, bind to it and initiate downstream processes that lead to the production of an mRNA. To truly understand the regulation of a gene, or any biological process, we have to be able to monitor these individual events, see how and when they happen. This, in turn, requires that we visualize individual molecules within a single cells.

And how do we do this ?

Our main tool is, obviously, a microscope. We have developed microscopy methods that allow the detection of single mRNA molecules in cells, allowing direct monitoring of gene expression and therefore a quantitative description of the different steps of the gene expression pathway.
Two methods are frequently used in our lab, fluorescent in situ hybridization (FISH) and GFP-tagging of mRNAs. We use GFP-tagging to follow individual mRNAs from their site of synthesis and during different steps in the gene expression pathway, trying to understand how these processes are executed in real time. The FISH requires fixation and is therefore not used to study highly dynamic processes, however, it is very powerful to determine expression levels, transcriptional activity and localization of mRNAs in cells.

mRNA detection
mRNA detection in single cells. Two common methods for single cell gene expression analysis using imaging. (Top) Single-molecule-resolution fluorescence in situ hybridization (FISH) uses synthetic oligonucleotides labeled at multiple positions with fluorescent dyes to detect single mRNAs. Multiple fluorescent probes are hybridized to paraformaldehyde-fixed cells. FISH allows the detection of single mRNAs in the cytoplasm as well as nascent mRNAs at the site of transcription. On the right, yeast cells expressing MDN1 mRNA (Zenklusen et al. NSMB 2008). (Bottom) The GFP-tagging system uses the specific interaction between a phage MS2/PP7 RNA hairpin and a fusion of a fluorescent protein and the MS2/PP7 phage coat protein to create a fluorescent labeled mRNA. Inserting multiple binding sites into an mRNA allows the detection of single mRNAs in living cells. The MS2 system has been used to detect single mRNAs in different organisms, for example in yeast to study transcription kinetics (Larson, Zenklusen et al., Science 2011). Sites of transcription are marked by arrows.

Our projects

We study two key processes of gene expression pathway, transcription and mRNA export. As these two steps are strongly interconnected, we are also interested in the mechanisms that mediate this coupling. Our main goals are 1) To understand how coordination of transcription regulation is achieved in single cells. We therefore focus on the question of how cells can achieve coordinated expression despite the stochastic nature of many cellular reactions. 2) Determine how ncRNAs participate in transcription regulation in yeast. 3) To know how an mRNA finds its way out of the nucleus. Many factors involved in mRNA export are recruited co-transcriptionally to the nascent mRNA and genes are tethered to the nuclear periphery upon activation. We want to understand how this coupling affects the ability of an mRNA to be efficiently exported to the cytoplasm.