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Imaging Transcription in Living Cells ANRV376-BB38-09 ARI 27 March 2009 10:13 Imaging Transcription in Living Cells Xavier Darzacq,1,2 Jie Yao,1,3 Daniel R. Larson,1,4 Sebastien´ Z. Causse,1,2 Lana Bosanac,1,2 Valeria de Turris,1,4 Vera M. Ruda,1,2 Timothee Lionnet,1,4 Daniel Zenklusen,1,4 Benjamin Guglielmi,1,3 Robert Tjian,1,3 and Robert H. Singer1,4 1Janelia Farm Research Consortium on Imaging Transcription, Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147 2Imagerie Fonctionelle de la Transcription, Ecole Normale Superieure´ CNRS UMR 8541, 75230 Paris cedex 05, France; email: [email protected] 3Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720; email: [email protected] 4Department of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461; email: [email protected] Annu. Rev. Biophys. 2009. 38:173–96 Key Words Annu. Rev. Biophys. 2009.38:173-196. Downloaded from arjournals.annualreviews.org by Yeshiva Univ-Albert Einstein College of Medicine on 08/28/09. For personal use only. First published online as a Review in Advance on modeling, RNA synthesis in live cells, polymerase kinetics February 3, 2009 The Annual Review of Biophysics is online at Abstract biophys.annualreviews.org The advent of new technologies for the imaging of living cells has made This article’s doi: it possible to determine the properties of transcription, the kinetics of 10.1146/annurev.biophys.050708.133728 polymerase movement, the association of transcription factors, and the Copyright c 2009 by Annual Reviews. progression of the polymerase on the gene. We report here the current All rights reserved state of the field and the progress necessary to achieve a more complete 1936-122X/09/0609-0173$20.00 understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding. 173 ANRV376-BB38-09 ARI 27 March 2009 10:13 and measure photons. The components of the Contents transcription reaction can then be assigned rate constants describing their forward and reverse INTRODUCTION .................. 174 rates. As a result of these analyses, new mod- IMAGING GENE ARRAYS .......... 174 els have arisen to fit these data. Among those Drosophila melanogaster Polytene are the observations that for some models the Chromosomes .................. 175 transcriptional complex can be transient, exist- Natural Tandem Repeated Genes . 178 ing only for a few seconds, and that the entire Artificial Gene Arrays .............. 179 process is inefficient, yet other factors can be Viruses ............................ 180 stably associated for hours. Understanding the NUCLEAR ORGANIZATION OF kinetic components that give rise to these dis- TRANSCRIPTION ............... 182 parate time constants will be an important func- Transcription Factories............. 182 tion of the new technologies. Gene Positioning .................. 183 This review is dedicated to exploring the IMAGING ENDOGENOUS work that is contributing to the real-time anal- GENES ........................... 185 ysis of transcription, an aspect that contributes Steroid Receptors .................. 185 data not addressed by chromatin immunopre- Imaging a Single Gene ............. 186 cipitation, microarray studies, or other bulk as- ANALYSIS OF KINETICS ........... 190 says that cannot resolve the events occurring Mobility of Transcription Factors . 190 in single cells. Many cell and tissue models are Dynamics ......................... 190 described including bacteria, polytene chromo- Modeling .......................... 190 somes, various reporter genes, and cell lines. CONCLUSION ..................... 191 Different approaches include fluorescence re- THE FUTURE ...................... 191 covery after photobleaching (FRAP), fluores- cence correlation spectroscopy (FCS), fluores- cence resonance energy transfer (FRET), and INTRODUCTION multiphoton microscopy (MPM) (see sidebar, With the uncovering of the ever-growing frac- Methods Used to Analyze Transcriptionin Liv- tion of the animal genome that is transcribed, ing Cells). This is a new field that is rapidly transcription is more than ever the centerpiece emerging, and these initial forays represent the of cell metabolism. Through biochemical anal- beginning of a new territory in the area of gene ysis and genetics, many if not most of the expression research. proteins implicated in transcription have been The review is organized by the model sys- identified. Decades of in vitro studies deter- tems that have contributed to studies in live mined that the transcription process could be cell imaging: naturally occurring and artificial separated into three steps: preinitiation com- gene arrays, viral genes, steroid receptor re- Annu. Rev. Biophys. 2009.38:173-196. Downloaded from arjournals.annualreviews.org by Yeshiva Univ-Albert Einstein College of Medicine on 08/28/09. For personal use only. plex formation, initiation, and elongation. Each sponsive genes, and single copy endogenous FRAP: fluorescence one of these steps may be subjected to regula- genes. There are four sections: imaging gene ar- recovery after tion, accounting for the fine-tuning of gene ex- rays, imaging the nuclear organization of tran- photobleaching pression. But biochemistry tells us only what is scription, imaging single copy genes, and the FCS: fluorescence possible, not what actually happens, in the very analysis of imaging data. correlation specific milieu of a living cell. spectroscopy It has become feasible, using the new ad- FRET: fluorescence vances in microscopy, to interrogate the pro- IMAGING GENE ARRAYS resonance energy transfer cesses that make up transcription and break Some gene families or duplicated genes in them down into their component parts. Accu- vertebrate genomes have naturally regrouped MPM: multiphoton microscopy rate quantification is possible due to technol- into tandem arrayed genes. These genes ogy that has evolved over the years to detect therefore lie as neighbors on the same locus 174 Darzacq et al. ANRV376-BB38-09 ARI 27 March 2009 10:13 in the genome and are often under the control of the same transcriptional regulators. In METHODS USED TO ANALYZE addition, the fruit fly (Drosophila melanogaster) TRANSCRIPTION IN LIVING CELLS contains giant polytene chromosomes in cer- tain larval cell types where the entire genome Image Sampling is multiplied. This spatial clustering of genes Cells imaged under wide-field fluorescence microscopy using offers researchers many opportunities to study long exposure times can yield images of transcription factors transcription, especially when using fluores- specifically bound to the relatively stationary DNA in contrast to cence microscopy, in which accumulation of the unresolved undersampled free transcription factors. Tracking fluorescence means a better signal-to-noise of individual transcription factors was possible using stroboscopic ratio. Another advantage of such arrays is that laser excitation. when studying a fluorescent molecule that interacts directly or indirectly with the arrays, FRAP the majority of the fluorescence will be due to interacting molecules (in contrast to freely dif- FRAP involves the irreversible photobleaching of a specified area fusing molecules, which might be preponderant in the nucleus by a focused laser of biologically nonabsorptive at a nonamplified locus). This increases the wavelength. The diffusion of surrounding fluorescent molecules resolution on binding events that reflect cat- into the bleached spot portrays a recovery characteristic of the alytic activities. So far three cases of amplified movement of tagged fluorophores. genes have been used to study transcription as illustrated in Figure 1: polytene salivary gland FLIP chromosomes in Drosophila (95), the ribosomal By the method of FLIP,a specified cell area is repeatedly bleached DNA nucleolar clusters (36), and artificially and the loss of fluorescence of surrounding areas are monitored. developed mammalian gene arrays (68). These This gives a more exact way to analyze the mobility of a pro- examples are covered more fully below. tein which is likewise involved in binding. Dissociation kinetics of proteins from compartments can thus be determined more precisely. Drosophila melanogaster Polytene Chromosomes FCS polytene chromosomes are found in Drosophila FCS is a tool for binding measurements. A laser beam is focused many larval cell types formed by endoredupli- in the cell within a femtoliter range volume. Fluctuations in flu- cation during development, i.e., these cells un- orescence signals are measured over short periods of time, the dergo DNA replication without cell division. output reflecting movement of labeled proteins through the vol- Polytene chromosomes from salivary gland ume. This gives a direct measurement of concentration, diffusion cells contain approximately 1000 copies of constants and binding constants. Generally, FCS is well suited DNA. Condensed and decondensed chromatin Annu. Rev. Biophys. 2009.38:173-196. Downloaded from arjournals.annualreviews.org to fast processes on a scale of milliseconds, whereas FRAP is by Yeshiva Univ-Albert Einstein College of Medicine on 08/28/09. For personal use only. form unique band and interband structures better suited to slower processes on the scale of seconds. Cross- that can be distinguished with a light micro- correlation FCS can likewise be used to determine
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