Digital nature of the immediate-early transcriptional response

Stevense, Michelle; Muramoto, Tetsuya; Müller, Iris; Chubb, Jonathan R.

doi:10.1242/dev.043836

Cited by 38 publications

(46 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This finding implies the transcriptional response for developmental genes is binary or all-or-nothing, with parameters of pulses remaining relatively constant but the number of pulsing cells changing. This finding has been observed at the protein level in viral (23), prokaryotic (24), and synthetic (25,26) gene-induction contexts, and is apparent for pulsing of two additional developmental genes in Dictyostelium (1,27). In contrast, pulse durations for two strongly expressed housekeeping genes, act5 and scd, diminished strongly during development, with pulses two-to threefold shorter for these genes at 5 h than at 0 h. This finding indicates an alternative, nonbinary response, where individual cells tune the level of transcript produced per transcriptional event during differentiation.…”

Section: Resultssupporting

confidence: 57%

Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation

Muramoto¹,

Cannon²,

Gierliński

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

119

116

View full text Add to dashboard Cite

Transcription of genes can be discontinuous, occurring in pulses or bursts. It is not clear how properties of transcriptional pulses vary between different genes. We compared the pulsing of five housekeeping and five developmentally induced genes by direct imaging of single gene transcriptional events in individual living Dictyostelium cells. Each gene displayed its own transcriptional signature, differing in probability of firing and pulse duration, frequency, and intensity. In contrast to the prevailing view from both prokaryotes and eukaryotes that transcription displays binary behavior, strongly expressed housekeeping genes altered the magnitude of their transcriptional pulses during development. These nonbinary "tunable" responses may be better suited than stochastic switch behavior for housekeeping functions. Analysis of RNA synthesis kinetics using fluorescence recovery after photobleaching implied modulation of housekeeping-gene pulse strength occurs at the level of transcription initiation rather than elongation. In addition, disparities between single cell and population measures of transcript production suggested differences in RNA stability between gene classes. Analysis of stability using RNAseq revealed no major global differences in stability between developmental and housekeeping transcripts, although strongly induced RNAs showed unusually rapid decay, indicating tight regulation of expression.transcriptional bursting | RNA turnover | stochastic gene expression T ranscription is not adequately described by the smooth, seamless process implied by standard measures of RNA level. Within individual cells, transcription occurs as a series of irregular pulses, interspersed by long, irregular periods of inactivity. Pulsing (or bursting) is a fundamental feature of transcription, conserved from prokaryotes to mammalian cells (1-5). These phenomena have strong implications for our understanding of transcriptional mechanism and may provide a major source of stochasticity in gene expression (6), a driver of cell diversity in differentiation and disease (7,8). However, it is unclear how pulsing behaves for different genes with different functional properties.Pulsing dynamics, and therefore deeper understanding of underlying transcriptional mechanics and regulation of different genes, are masked when averaged over millions of dead cells, as occurs with standard bulk RNA measurement techniques, from Northern blotting to RNA sequencing (RNAseq). The readout from these methods also has a variable contribution from RNA stability. Although strong inferences can be made from heterogeneities in transcript number using hybridization against RNA in single cells (RNA-FISH) (9-11), an erroneous inference of strong transcription from both bulk and fixed-cell RNA techniques emerges if RNA is stable. To appreciate how transcription is regulated and how the process differs between different genes, it is crucial to look at the process itself in living cells at the singlegene level. Live-cell methods using fluorescent protein...

show abstract

Section: Resultssupporting

confidence: 57%

Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation

Muramoto¹,

Cannon²,

Gierliński

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

119

116

View full text Add to dashboard Cite

show abstract

“…For long-term 2i treatment, H2B-RFP TNGA cells were co-cultured at a 50:50 ratio with parental TNGA cells for several passages prior to imaging, to facilitate cell tracking. We used a widefield fluorescence system designed for fast imaging of photosensitive samples (Stevense et al, 2010;Corrigan and Chubb, 2014). Images were captured using a GFP/mCherry filter set (Chroma 59022), 40×1.30 NA objective, UV (GG420, Schott) and neutral density (Chroma) filters to attenuate illumination.…”

Section: Cell Culture and Imagingmentioning

confidence: 99%

Multiple cell and population-level interactions with mouse embryonic stem cell heterogeneity

et al. 2015

Self Cite

View full text Add to dashboard Cite

Much of development and disease concerns the generation of gene expression differences between related cells sharing similar niches. However, most analyses of gene expression only assess population and time-averaged levels of steady-state transcription. The mechanisms driving differentiation are buried within snapshots of the average cell, lacking dynamic information and the diverse regulatory history experienced by individual cells. Here, we use a quantitative imaging platform with large time series data sets to determine the regulation of developmental gene expression by cell cycle, lineage, motility and environment. We apply this technology to the regulation of the pluripotency gene Nanog in mouse embryonic stem cells. Our data reveal the diversity of cell and population-level interactions with Nanog dynamics and heterogeneity, and how this regulation responds to triggers of pluripotency. Cell cycles are highly heterogeneous and cycle time increases with Nanog reporter expression, with longer, more variable cycle times as cells approach ground-state pluripotency. Nanog reporter expression is highly stable over multiple cell generations, with fluctuations within cycles confined by an attractor state. Modelling reveals an environmental component to expression stability, in addition to any cell-autonomous behaviour, and we identify interactions of cell density with both cycle behaviour and Nanog. Rex1 expression dynamics showed shared and distinct regulatory effects. Overall, our observations of multiple partially overlapping dynamic heterogeneities imply complex cell and environmental regulation of pluripotent cell behaviour, and suggest simple deterministic views of stem cell states are inappropriate.

show abstract

“…These "transcriptional bursts" amount to "memory" between transcripts: The synthesis of one transcript is likely to be accompanied by the synthesis of another over certain time intervals. Transcriptional bursting has now been observed in yeast (Zenklusen et al 2008;Lenstra et al 2015), slime mold (Chubb et al 2006;Muramoto et al 2010;Stevense et al 2010), fly (Garcia et al 2013), mouse , and human (Yunger et al 2010) cell lines and is a significant source of expression variation between cells. In fact, subsequent steps of gene expression such as RNA export, RNA decay, translation, etc.…”

Section: Transcriptional Burstingmentioning

confidence: 99%

What have single-molecule studies taught us about gene expression?

Chen

Larson

2016

Genes Dev.

View full text Add to dashboard Cite

The production of a single mRNA is the result of many sequential steps, from docking of transcription factors to polymerase initiation, elongation, splicing, and, finally, termination. Much of our knowledge about the fundamentals of RNA synthesis and processing come from ensemble in vitro biochemical measurements. Single-molecule approaches are very much in this same reductionist tradition but offer exquisite sensitivity in space and time along with the ability to observe heterogeneous behavior and actually manipulate macromolecules. These techniques can also be applied in vivo, allowing one to address questions in living cells that were previously restricted to reconstituted systems. In this review, we examine the unique insights that single-molecule techniques have yielded on the mechanisms of gene expression.Single-molecule experiments are now pervasive in biology. What started out as an experimental approach for characterizing ion channels in the 1970s (Neher and Sakmann 1976) has now become a fixture in hundreds of laboratories addressing fundamental questions in biochemistry, cell biology, genetics, and development. The methodology is nearly as diverse as the problems that are addressed and encompasses imaging, optical tweezers, atomic force microscopy, electrophysiology, and cryo-electron microscopy (cryo-EM), to list a few. The unifying principle behind these approaches is straightforward: the ability to observe the heterogeneous, rare, or fleeting behavior of macromolecules that is normally masked by ensemble techniques. In this review, we focus on the role that single-molecule approaches have played in advancing our understanding of the early steps in gene expression.In general, transcription by RNA polymerase (RNAP) in bacteria or RNA polymerase II (Pol II) in eukaryotes begins when transcription factors (TFs) are recruited to the promoter. This leads to the assembly of the preinitiation complex (PIC) that contains a semicompetent polymerase. The PIC is necessary for unwinding duplex DNA and setting the stage for processive elongation by the polymerase. After conformational changes in the PIC, the polymerase escapes the promoter region and enters into productive elongation. In eukaryotes, the nascent RNA undergoes further modifications such as addition of a 5 ′ cap, synthesis of a poly-A tail, and splicing before the mature mRNA is formed. These early steps of gene expression are uniquely suited to elucidation through singlemolecule methods. For example, single-molecule experimental approaches allow one to visualize the order of assembly for molecular complexes (i.e., the PIC) and observe the variety of pathways that can result in initiation of RNAP. The ability to observe kinetics in unperturbed systems can provide clues to the mechanisms of transcription. RNAPs can also be manipulated by singlemolecule optical trapping to reveal the inner workings of force generation by this enzyme. Furthermore, singlemolecule imaging has also revealed the heterogeneity of gene expression that exists among cells in ...

show abstract

Digital nature of the immediate-early transcriptional response

Cited by 38 publications

References 37 publications

Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation

Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation

Multiple cell and population-level interactions with mouse embryonic stem cell heterogeneity

What have single-molecule studies taught us about gene expression?

Contact Info

Product

Resources

About