Dynamic Analysis of Stochastic Transcription Cycles

Abstract

Cycling of gene expression in individual cells is controlled by dynamic chromatin remodeling.

Abstract

In individual mammalian cells the expression of some genes such as prolactin is highly variable over time and has been suggested to occur in stochastic pulses. To investigate the origins of this behavior and to understand its functional relevance, we quantitatively analyzed this variability using new mathematical tools that allowed us to reconstruct dynamic transcription rates of different reporter genes controlled by identical promoters in the same living cell. Quantitative microscopic analysis of two reporter genes, firefly luciferase and destabilized EGFP, was used to analyze the dynamics of prolactin promoter-directed gene expression in living individual clonal and primary pituitary cells over periods of up to 25 h. We quantified the time-dependence and cyclicity of the transcription pulses and estimated the length and variation of active and inactive transcription phases. We showed an average cycle period of approximately 11 h and demonstrated that while the measured time distribution of active phases agreed with commonly accepted models of transcription, the inactive phases were differently distributed and showed strong memory, with a refractory period of transcriptional inactivation close to 3 h. Cycles in transcription occurred at two distinct prolactin-promoter controlled reporter genes in the same individual clonal or primary cells. However, the timing of the cycles was independent and out-of-phase. For the first time, we have analyzed transcription dynamics from two equivalent loci in real-time in single cells. In unstimulated conditions, cells showed independent transcription dynamics at each locus. A key result from these analyses was the evidence for a minimum refractory period in the inactive-phase of transcription. The response to acute signals and the result of manipulation of histone acetylation was consistent with the hypothesis that this refractory period corresponded to a phase of chromatin remodeling which significantly increased the cyclicity. Stochastically timed bursts of transcription in an apparently random subset of cells in a tissue may thus produce an overall coordinated but heterogeneous phenotype capable of acute responses to stimuli.

Author Summary

Timing of biological processes such as gene transcription is crucial to ensure that cells and tissues respond appropriately to their environment. Until recently it was assumed that most cells in a tissue responded in a similar way, and that changes in cellular activity were relatively stable. However, studies of messenger RNA and protein levels in single cells have shown the presence of considerable heterogeneity. This suggested that transcription in single cells may be highly dynamic over time. Using a combined experimental and theoretical approach, with time-lapse imaging of reporter gene expression over 25 h periods, we measured the rate of prolactin gene transcription in single pituitary cells and detected clear cycles of transcriptional activity. Mathematical analysis, using a binary model that assumed transcription was on or off, showed that these cycles were characterized by a minimum refractory period that involved chromatin remodeling. The timing of transcription from two different reporter constructs driven by identical promoters in the same cell was out-of-phase, suggesting that the pulses of gene expression are due to processes intrinsic to expression of a particular gene and not to environmental effects. We further show that the pulses of transcription are independent chromatin cycles at each gene locus. Therefore, heterogeneous patterns of gene expression may facilitate flexible transcriptional responses in cells within intact tissue, while maintaining a well-regulated average level of gene expression in the resting state.