Transcription Imaging Consortium

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 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.

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.