@article {46707, title = {Direct observation of frequency modulated transcription in single cells using light activation.}, journal = {eLife}, volume = {2}, year = {2013}, month = {2013 Sep 24}, pages = {e00750}, abstract = {

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus. DOI:http://dx.doi.org/10.7554/eLife.00750.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.00750}, author = {Larson, Daniel R and Fritzsch, Christoph and Sun, Liang and Meng, Xiuhau and Lawrence, David S and Singer, Robert H} } @article {46709, title = {Eukaryotic transcriptional dynamics: from single molecules to cell populations.}, journal = {Nature Reviews Genetics}, volume = {14}, year = {2013}, month = {2013 Aug}, pages = {572-84}, abstract = {

Transcriptional regulation is achieved through combinatorial interactions between regulatory elements in the human genome and a vast range of factors that modulate the recruitment and activity of RNA polymerase. Experimental approaches for studying transcription in vivo now extend from single-molecule techniques to genome-wide measurements. Parallel to these developments is the need for testable quantitative and predictive models for understanding gene regulation. These conceptual models must also provide insight into the dynamics of transcription and the variability that is observed at the single-cell level. In this Review, we discuss recent results on transcriptional regulation and also the models those results engender. We show how a non-equilibrium description informs our view of transcription by explicitly considering time- and energy-dependence at the molecular level.

}, keywords = {DNA-Directed RNA Polymerases, Gene Expression Regulation, Genome, Human, Humans, Transcription Factors, Transcription, Genetic}, issn = {1471-0064}, doi = {10.1038/nrg3484}, author = {Coulon, Antoine and Chow, Carson C and Singer, Robert H and Larson, Daniel R} } @article {46725, title = {Single-mRNA counting using fluorescent in situ hybridization in budding yeast.}, journal = {Nature Protocols}, volume = {7}, year = {2012}, month = {2012 Feb 2}, pages = {408-19}, abstract = {

Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for single-molecule analysis. Diffraction-limited labeled mRNAs are observed as bright fluorescent spots and can be quantified using a spot-detection algorithm. FISH preserves the spatial distribution of cellular RNA distribution within the cell and the stochastic fluctuations in individual cells that can lead to phenotypic differences within a clonal population. This information, however, is lost if the RNA content is measured on a population of cells by using reverse transcriptase PCR, microarrays or high-throughput sequencing. The FISH procedure and image acquisition described here can be completed in 3 d.

}, keywords = {Algorithms, Antisense Elements (Genetics), In Situ Hybridization, Fluorescence, RNA, Fungal, RNA, Messenger, Saccharomyces cerevisiae}, issn = {1750-2799}, doi = {10.1038/nprot.2011.451}, author = {Trcek, Tatjana and Chao, Jeffrey A and Larson, Daniel R and Park, Hye Yoon and Zenklusen, Daniel and Shenoy, Shailesh M and Singer, Robert H} } @article {6596, title = {Real-time observation of transcription initiation and elongation on an endogenous yeast gene.}, journal = {Science}, volume = {332}, year = {2011}, month = {2011 Apr 22}, pages = {475-8}, abstract = {

Cellular messenger RNA levels are achieved by the combinatorial complexity of factors controlling transcription, yet the small number of molecules involved in these pathways fluctuates stochastically. It has not yet been experimentally possible to observe the activity of single polymerases on an endogenous gene to elucidate how these events occur in vivo. Here, we describe a method of fluctuation analysis of fluorescently labeled RNA to measure dynamics of nascent RNA{\textendash}including initiation, elongation, and termination{\textendash}at an active yeast locus. We find no transcriptional memory between initiation events, and elongation speed can vary by threefold throughout the cell cycle. By measuring the abundance and intranuclear mobility of an upstream transcription factor, we observe that the gene firing rate is directly determined by trans-activating factor search times.

}, keywords = {Adenosine Triphosphatases, Cell Cycle, Cell Nucleus, DNA Polymerase I, Facilitated Diffusion, Fluorescence, Fungal, Genes, Genetic, Glutamate Synthase, Green Fluorescent Proteins, Kinetics, Messenger, Microscopy, Models, Promoter Regions, RNA, RNA Polymerase II, RNA Precursors, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spectrometry, Transcription, Transcription Factors, Transcriptional Activation}, issn = {1095-9203}, doi = {10.1126/science.1202142}, author = {Larson, Daniel R and Zenklusen, Daniel and Wu, Bin and Chao, Jeffrey A and Singer, Robert H} } @article {46734, title = {Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast.}, journal = {Cell}, volume = {147}, year = {2011}, month = {2011 Dec 23}, pages = {1484-97}, abstract = {

Messenger RNA decay measurements are typically performed on a population of cells. However, this approach cannot reveal sufficient complexity to provide information on mechanisms that may regulate mRNA degradation, possibly on short timescales. To address this deficiency, we measured cell cycle-regulated decay in single yeast cells using single-molecule FISH. We found that two genes responsible for mitotic progression, SWI5 and CLB2, exhibit a mitosis-dependent mRNA stability switch. Their transcripts are stable until mitosis, when a precipitous decay eliminates the mRNA complement, preventing carryover into the next cycle. Remarkably, the specificity and timing of decay is entirely regulated by their promoter, independent of specific cis mRNA sequences. The mitotic exit network protein Dbf2p binds to SWI5 and CLB2 mRNAs cotranscriptionally and regulates their decay. This work reveals the promoter-dependent control of mRNA stability, a regulatory mechanism that could be employed by a variety of mRNAs and organisms.

}, keywords = {Cell Cycle, Cell Cycle Proteins, Cyclin B, Gene Expression Regulation, Fungal, In Situ Hybridization, Fluorescence, Kinetics, Promoter Regions, Genetic, Protein-Serine-Threonine Kinases, RNA Stability, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors, Transcription, Genetic}, issn = {1097-4172}, doi = {10.1016/j.cell.2011.11.051}, author = {Trcek, Tatjana and Larson, Daniel R and Mold{\'o}n, Alberto and Query, Charles C and Singer, Robert H} } @article {2390, title = {Imaging transcription in living cells.}, journal = {Annual Review of Biophysics}, volume = {38}, year = {2009}, month = {2009 Feb 3}, pages = {173-96}, abstract = {

The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.

}, keywords = {Computer-Assisted, Fluorescence, Gene Expression Profiling, Image Interpretation, Microscopy, Transcription Factors, Transcriptional Activation}, issn = {1936-122X}, doi = {10.1073/pnas.1100640108}, author = {Darzacq, Xavier and Yao, Jie and Larson, Daniel R and Causse, Sebastien Z and Bosanac, Lana and de Turris, Valeria and Ruda, Vera M and Lionnet, Timoth{\'e}e and Zenklusen, Daniel and Guglielmi, Benjamin and Tjian, Robert and Singer, Robert H} }