Direct observation of frequency modulated transcription in single cells using light activation

Abstract

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus.

DOI: http://dx.doi.org/10.7554/eLife.00750.001

eLife digest

The process by which a gene is expressed as a protein consists of two stages: transcription, which involves the DNA of the gene being copied into messenger RNA (mRNA); and translation, in which the mRNA is used as a template to assemble amino acids into a protein. Transcription and translation are controlled by many interlinked pathways, which ensures that genes are expressed when and where required.

One of these regulatory pathways involves steroid receptors. The binding of a steroid molecule to its receptor causes the receptor to move into the nucleus and interact with a specific gene, triggering transcription of that gene. When measured at the level of the whole organism, this transcriptional response is dose-dependent—the more steroid molecules that are present, the greater the amount of transcription. However, this is not the case in single cells, in which transcription is either activated or not. This ‘on/off’ behaviour is also seen over time: steroid-activated transcription occurs in bursts, separated by periods of inactivity.

To unravel the molecular mechanism behind this phenomenon, Larson et al. created a light-activated form of the ligand that activates a specific steroid receptor. Using this molecule, they were able to switch transcription of the gene controlled by that receptor on and off. They then used fluorescent proteins to label the mRNA and protein molecules that were produced as a result.

They found that activating the steroid receptor increases the likelihood of transcription occurring inside a cell, but not the duration of individual bursts of transcriptional activity, nor the amount of mRNA produced during each burst. Activation of a steroid receptor seems to control transcription by reducing the length of time each cell spends in the ‘off’ state between bursts.

Larson et al. incorporated their findings into a model that also takes into account the natural variability in levels of transcription between cells, and found that this could explain how the digital (on/off) control of transcription at the cellular level leads to analogue, dose-dependent control at the level of a whole organism. These findings should lead to further insights into how transcription is controlled at the molecular level.

DOI: http://dx.doi.org/10.7554/eLife.00750.002