Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation Tetsuya Muramotoa,1, Danielle Cannona,2, Marek Gierlinski b,c, Adam Corrigana,2, Geoffrey J. Bartonb,c, and Jonathan R. Chubba,2,3 aDivision of Cell and Developmental Biology, bDivision of Biological Chemistry and Drug Discovery, and cWellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom Edited by Sanjay Tyagi, University of Medicine and Dentistry of New Jersey, Newark, NJ, and accepted by the Editorial Board March 21, 2012 (received for review October 25, 2011) Transcription of genes can be discontinuous, occurring in pulses or have different fluctuation kinetics. However, available data on bursts. It is not clear how properties of transcriptional pulses vary transcription bursts and pulses imply active transcriptional states between different genes. We compared the pulsing of five house- last more in the range of minutes, even for strongly transcribed keeping and five developmentally induced genes by direct imaging genes (1). To describe the dynamics of transcription pulses it is of single gene transcriptional events in individual living Dictyoste- therefore necessary to directly observe pulses of RNA production, lium cells. Each gene displayed its own transcriptional signature, which requires using high-affinity RNA–protein interactions to differing in probability of firing and pulse duration, frequency, and deliver fluorescent signals to nascent RNA (15, 16). The resultant intensity. In contrast to the prevailing view from both prokary- accumulation of fluorescence at the site of transcription is viewed otes and eukaryotes that transcription displays binary behavior, under a microscope as a fluorescent spot, which appears and dis- strongly expressed housekeeping genes altered the magnitude of appears (pulses) at irregular intervals when imaged in living cells. their transcriptional pulses during development. These nonbinary How is pulsing different for genes with different functions and “tunable” responses may be better suited than stochastic switch expression requirements? How is pulsing reactive to cell context behavior for housekeeping functions. Analysis of RNA synthesis for different genes? To address these questions, we compared kinetics using fluorescence recovery after photobleaching implied pulsing of a set of housekeeping and developmentally induced BIOLOGY modulation of housekeeping-gene pulse strength occurs at the genes in Dictyostelium cells. Dictyostelium are social amoebae, DEVELOPMENTAL level of transcription initiation rather than elongation. In addition, existing as single feeding cells that, upon starvation, initiate a disparities between single cell and population measures of tran- developmental program resulting in chemotactic cell aggrega- script production suggested differences in RNA stability between tion, followed by differentiation and remodeling of the aggregate gene classes. Analysis of stability using RNAseq revealed no major into a spore-containing mass suspended above the substrate by a global differences in stability between developmental and house- stalk. As with many differentiation steps in disease and devel- keeping transcripts, although strongly induced RNAs showed un- opment, from prokaryotes to stem cells, initial differentiation is usually rapid decay, indicating tight regulation of expression. scattered or stochastic, not determined by cell position (7, 17). We measured pulsing of five developmental and five house- transcriptional bursting | RNA turnover | stochastic gene expression keeping genes at different stages during preaggregative devel- opment. Each gene showed its own pulsing properties, measured ranscription is not adequately described by the smooth, seam- directly using a variety of parameters. Housekeeping genes Tless process implied by standard measures of RNA level. strongly modulate pulsing strength during development, allowing Within individual cells, transcription occurs as a series of irregular greater tunability of transcription at the single-cell level. In con- pulses, interspersed by long, irregular periods of inactivity. Pulsing trast, most developmental genes showed binary behavior. Dif- (or bursting) is a fundamental feature of transcription, conserved ferences in pulsing at different developmental time points are from prokaryotes to mammalian cells (1–5). These phenomena controlled at the level of transcription initiation. Finally, we have strong implications for our understanding of transcriptional demonstrate strongly induced developmental transcripts tend to mechanism and may provide a major source of stochasticity in show higher turnover than other transcripts, indicative of tight gene expression (6), a driver of cell diversity in differentiation and control of expression. disease (7, 8). However, it is unclear how pulsing behaves for Results and Discussion different genes with different functional properties. Pulsing dynamics, and therefore deeper understanding of un- To visualize transcriptional dynamics of single housekeeping and derlying transcriptional mechanics and regulation of different developmentally induced genes, we used MS2 stem loops for na- genes, are masked when averaged over millions of dead cells, as scent RNA detection (15). A 1.3-kb array of 24 MS2 loops was ′ occurs with standard bulk RNA measurement techniques, from integrated into 5 coding sequences of genes in Dictyostelium cells Northern blotting to RNA sequencing (RNAseq). The readout (Fig. 1A). Selection of recombinants used blasticidin resistance from these methods also has a variable contribution from RNA stability. Although strong inferences can be made from hetero- geneities in transcript number using hybridization against RNA in Author contributions: T.M. and J.R.C. designed research; T.M. and D.C. performed re- – search; T.M., M.G., G.J.B., and J.R.C. contributed new reagents/analytic tools; T.M., D.C., single cells (RNA-FISH) (9 11), an erroneous inference of strong M.G., A.C., and J.R.C. analyzed data; and T.M., D.C., M.G., and J.R.C. wrote the paper. fi transcription from both bulk and xed-cell RNA techniques The authors declare no conflict of interest. emerges if RNA is stable. To appreciate how transcription is This article is a PNAS Direct Submission. S.T. is a guest editor invited by the Editorial Board. regulated and how the process differs between different genes, it 1Present address: Quantitative Biology Center, RIKEN, Osaka 565-0874, Japan. is crucial to look at the process itself in living cells at the single- fl 2Present address: Department of Cell and Developmental Biology and Medical Research gene level. Live-cell methods using uorescent proteins or lucif- Council Laboratory for Molecular Cell Biology, University College London, London WC1E erase have been very useful in giving us a sense of the instability 6BT, United Kingdom. of transcriptional states, with expression heterogeneity between 3To whom correspondence should be addressed. E-mail: [email protected]. fl cells and slow uctuations (hours to days) between different This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. states (12–14). These methods also reveal that different genes 1073/pnas.1117603109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1117603109 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 A MS2-GFP gene bsr MS2 RNA 24xMS2 (1.3kb) (1.3kb) B act5-MS2 carA-MS2 0:00 2:30 5:00 7:30 10:00 12:30 Fig. 1. Visualization of nascent RNA pulses during development. (A) Schematic of RNA detection system. MS2-GFP protein binds with high affinity to MS2 RNA. Twenty-four MS2 repeats and selection cassette (bsr) were knocked into 5′ regions of target genes by homologous recombination in Dictyostelium cells. (B) Examples of cells displaying pulses for housekeeping (act5) and development (carA) genes are shown. Arrows indicate transcription spots. Timing (minute: second). Maximum-intensity projections of 3D stacks. Cells can move several microns between frames. Field of view in each image is 36 × 36 μm. cassettes (bsr) downstream of MS2 repeats. To ensure similar (Fig. 2A), from 90% of cells for act5 and scd, to 15% for abpE transcript length between genes, termination of transcription and rpl15. The reverse was true for development genes, with 3% after MS2 repeats used the bsr terminator, which directly fol- of cells with carA spots at 0 h increasing to 80% at 5 h of de- lows the repeats. The RNA contained only the 1.3-kb MS2 array, velopment (Fig. 2A). Weaker expressed development genes, plus standard short 3′ and short variable 5′ extensions. We studied cofC and zfaA, barely reached 1% of cells showing spots, al- transcription of five housekeeping genes [abpE (actin-binding), though to preserve viability we did not image with single-mole- act5 (β-actin), cinD (putative transcription factor), rpl15 (ribosome cule sensitivity, so would not detect weak transcription. Under no protein), and scd (fatty acid desaturase)] and five development conditions did any gene show spots in 100% of cells. Four of five genes [carA (chemoattractant receptor), csaA (cell adhesion), development genes showed spots in a small percentage (<5%) of cofC (actin-severing), hspF (heat-shock), and zfaA (early devel- undifferentiated cells. Do these rare events imply all cells ex- opment marker)]. Developmental genes show strong increases in pressed, masked by use of a limited capture period? Or do they fi mRNA