Transcription is the first step of gene expression and therefore determines how a cell functions and eventually its fate. However, monitoring and understanding transcription in live cells is a daunting task: a single transcription event requires the coordinated activity of a vast number of factors that sometimes interact for less than a second; because genes are present in at most few copies per cell, transcription is inherently stochastic and individual cells can display highly variable expression profiles; finally, the nuclear environment plays an important regulatory role through chromatin architecture and crowding. All these factors not only set constraints on the way cells can regulate the expression of their genes; they also constitute tremendous experimental challenges.
In order to tackle this problem, we decided to bring together a vast range of expertise ranging from biochemistry to cell biology, optics, biophysics and theoretical physics. Through a combination of cutting edge microscopes, labeling reagents, innovative experimental approaches and analysis techniques, we aim to decipher the regulation of eukaryotic transcription through single-cell, image-based techniques. The Consortium consists of a Janelia-based core team working in close collaboration with the labs of Robert Tjian (Janelia Farm and UC Berkeley), Robert Singer (Janelia Farm and Albert Einstein College of Medicine), Xavier Darzacq (IBENS, Paris), Carl Wu (Janelia Farm and NCI,NIH) and Maxime Dahan (Institut Curie, Paris). By extensively sharing ideas, technology and reagents, we aim to accomplish progress that would be impossible in isolated labs. In addition to establishing and publishing novel approaches, the Transcription Imaging Consortium will make technology and reagents available to the scientific community worldwide.
We are welcoming applications from talented and motivated scientists. Interested candidates should contact Timothee Lionnet about possible positions.
The goals of the Transcription Imaging Consortium are:
- Understanding the in vitro assembly and regulation of the preinitiation complex
- Analyzing the nuclear dynamics of elements of the transcription machinery in live cells
- Measuring the transcriptional activity in live cells and tissues to understand its role in gene expression regulation and variability
- Developing new labeling and imaging tools for the study of gene expression
We are probing basic mechanisms of human RNA polymerase II transcription and regulation using an in vitro, single-molecule approach. We have built an ultrastable total internal reflection fluorescence (TIRF) microscope allowing multicolor imaging at up to 30 Hz of thousands of immobilized DNA templates in a single field simultaneously. We have adopted modified imaging surfaces and fluidics systems to support a fully reconstituted Pol II transcription system that efficiently utilizes surface-immobilized DNA templates. With this experimental approach, we have observed multiple rounds of promoter-dependent Pol II transcription per DNA template. The efficiency of transcription reinitiation was found to be much higher than the efficiency of the first transcription round. We have further observed that TFIID and a subset of other factors may provide an assembled “scaffold” at the core promoter to direct efficient reinitiation.
We are addressing the formation of the transcription preinitiation complex at the naturally amplified histone gene locus (His) of Drosophila. Using live-cell microscopy and fluorescence recovery after photo-bleaching, we have been able to follow the movement of GFP-tagged general transcription factors and RNA polymerase II during the transcription of His genes during S-phase. We have also designed a single-cell quantitative measurement of the mRNA production for the His genes. With this quantitative FISH assay, we discovered that the different His genes are not all expressed at the same time. We devised a double-labeling assay to time the cells in S-phase using two nucleotide analogs. Combining both methods we showed for the first time that the core histone genes (e.g., H2A) are transcribed as a short burst early in S-phase, whereas the linker histone H1 is expressed continuously during S-phase.
We are studying the mechanisms governing the nuclear dynamics of nuclear factors. By using a combination of techniques such as FRAP, FLIP, individual molecules tracking (SMT) or FCS as well as engineered cell lines, we aim at determining how transcription factors can find their activity site and how their large-scale mobility is regulated. While noncompartmentalized by membranes, the nucleus is highly organized. The highly compacted DNA polymer chain and the free available space in the nucleus have been described as structures showing a multiscale high degree organization reminiscent of a fractal organization. Within this complex environment, biochemical reactions cannot be seen as occurring in a well-mixed reactor and molecule availability needs to be taken into account. This study focuses on the transcriptional machinery itself as well as specific transcription factors.
We have developed a novel quantitative analysis of live transcription based on MS2 imaging. We have derived an MEF cell line from a mouse model where the endogenous β-actin mRNA is fluorescently labeled. Using high-resolution 4D fluorescence imaging of the cells, we have been able to quantify the absolute number of nascent chains being produced at each allele of a single cell over time. This gives us unprecedented access to transcription regulation (e.g., coordinated expression within a cell vs. stochasticity of initiation, transcription memory and factors regulating the transcription). We are investigating how transcription correlates with the nuclear concentration of upstream regulators, both in unperturbed state and when signaling pathways are activated. We are also using two-photon FCS to determine the interactions of the various components involved in transcriptional signaling.
We have developed a mouse model in which all β-actin mRNA in every cell within tissue is fluorescently labeled. This allows an unprecedented ability to view the transcriptional regulation of any tissue of choice. We have chosen neurons as our first tissue to investigate. We started measuring the transcriptional activity in response to pharmacological agents known to induce actin dynamics and neuronal activity. This constitutes an example of the dynamic regulation of gene expression that may be revealed in the native brain tissue environment.
Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.
Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.Nature methods 2013
S. Abrahamsson, J. Chen, B. Hajj, S. Stallinga, A. Y. Katsov, J. Wisniewski, G. Mizuguchi, P. Soule, F. Mueller, C. Darzacq, X. Darzacq, C. Wu, C. I. Bargmann, D. A. Agard, M. Dahan, and M. G L. Gustafsson Nature methods, 10:60-63 (2013)
Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.
Real-time dynamics of RNA polymerase II clustering in live human cells.Science (New York, N.Y.) 2013
I. I. Cisse, I. Izeddin, S. Z. Causse, L. Boudarene, A. Senecal, L. Muresan, C. Dugast-Darzacq, B. Hajj, M. Dahan, and X. Darzacq Science (New York, N.Y.), 341:664-7 (2013)
Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell's ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.
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--including initiation, elongation, and termination--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.
Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3' untranslated region of the essential β-actin gene. As β-actin-MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.
Recent findings implicate alternate core promoter recognition complexes in regulating cellular differentiation. Here we report a spatial segregation of the alternative core factor TAF3, but not canonical TFIID subunits, away from the nuclear periphery, where the key myogenic gene MyoD is preferentially localized in myoblasts. This segregation is correlated with the differential occupancy of TAF3 versus TFIID at the MyoD promoter. Loss of this segregation by modulating either the intranuclear location of the MyoD gene or TAF3 protein leads to altered TAF3 occupancy at the MyoD promoter. Intriguingly, in differentiated myotubes, the MyoD gene is repositioned to the nuclear interior, where TAF3 resides. The specific high-affinity recognition of H3K4Me3 by the TAF3 PHD (plant homeodomain) finger appears to be required for the sequestration of TAF3 to the nuclear interior. We suggest that intranuclear sequestration of core transcription components and their target genes provides an additional mechanism for promoter selectivity during differentiation.
Commentary: Jie Yao in Bob Tijan's lab used a combination of confocal microscopy and dual label PALM in thin sections cut from resin-embedded cells to show that certain core transcription components and their target genes are spatially segregated in myoblasts, but not in differentiated myotubes, suggesting that such spatial segregation may play a role in guiding cellular differentiation.
Nuclear physics: quantitative single-cell approaches to nuclear organization and gene expression.Cold Spring Harbor Symposia on Quantitative Biology 2010
T. Lionnet, B. Wu, D. Grünwald, R H. Singer, and D R. Larson Cold Spring Harbor Symposia on Quantitative Biology, 75:113-26 (2010)
The internal workings of the nucleus remain a mystery. A list of component parts exists, and in many cases their functional roles are known for events such as transcription, RNA processing, or nuclear export. Some of these components exhibit structural features in the nucleus, regions of concentration or bodies that have given rise to the concept of functional compartmentalization--that there are underlying organizational principles to be described. In contrast, a picture is emerging in which transcription appears to drive the assembly of the functional components required for gene expression, drawing from pools of excess factors. Unifying this seemingly dual nature requires a more rigorous approach, one in which components are tracked in time and space and correlated with onset of specific nuclear functions. In this chapter, we anticipate tools that will address these questions and provide the missing kinetics of nuclear function. These tools are based on analyzing the fluctuations inherent in the weak signals of endogenous nuclear processes and determining values for them. In this way, it will be possible eventually to provide a computational model describing the functional relationships of essential components.
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.
Team Members Groups