The goal of the Transcription Imaging Consortium (TIC) is to develop tools for in vitro and in vivo single molecule imaging and live cell microscopy to investigate the assembly, dynamics, and functional specificity of the eukaryotic transcription machinery.
The consortium was established in 2006 as a collaboration among the labs of Robert Tjian (HHMI at Janelia Farm and at UC Berkeley), Robert Singer (Albert Einstein College of Medicine) and Xavier Darzacq (IBENS, Paris). By bringing together complementary expertise in biochemistry, cell biology, and live cell imaging, the consortium aims at developing innovative and multidisciplinary approaches to address the mechanisms underlying the regulation of gene expression from the molecular to the cellular level.
The goals of the Transcription Imaging Consortium are:
- Probing the in vitro assembly 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
- 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 a 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). As actin protein levels are well known to be highly regulated, we are investigating how G-actin protein concentration regulates transcription levels using cytoskeleton disrupting drugs. Furthermore, in order to correlate actin transcription to upstream regulation points, we have designed a cell line in which a coactivator of actin transcription, MAL, is fluorescent. We are investigating how transcription correlates with the nuclear concentration of the MAL coactivator, both in basal state and in the presence of various stimuli such as high/low serum concentrations. 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.
Darzacq, X., Yao, J., Larson, D.R., Causse, C.Z., Bosanac, L., de Turris, V., Ruda, V.M., Lionnet, T., Zenklusen, D., Guglielmi, B., Tjian, R., and Singer, R.H. (2009). Imaging Transcription in Living Cells, Annual Review of Biophysics 38:173-196
Yao, J., Fetter, R. D., Hu, P., Betzig, E., and Tjian, R. (2011). Subnuclear Segregation of Genes and Core Promoter Factors in Myogenesis, Genes & Development, 25: 569-580
Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells AL, Chao JA, Park HY, de Turris V, Lopez-Jones M, Singer RH (2011). “A transgenic mouse for in vivo detection of endogenous labeled mRNA”. Nat Methods. 2011 8(2):165-70.
Larson, D., D. Zenklusen, B. Wu, J.A. Chao, and R. H. Singer (2011). "Real-Time Observation of Transcription Initiation and Elongation on an Endogenous Yeast Gene." Science April 21.
T. Lionnet, B. Wu, D. Grünwald, R.H. Singer and D.R. Larson (2011), Quantitative Single-Cell Approaches to Nuclear Organization and Gene Expression, Cold Spring Harbor Symposia on Quantitative Biology. qb.2010.75.057; Published in Advance April 18, 2011.
Team Members Groups