Regulation and cell-to-cell variability of estrogen-dependent transcription
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Abstract
Transcription is the major control point for the production of cellular components, and as such, it governs fate and function of cells. Surprisingly, this critical step in information transmission is rather unreliable: The low copy-number of molecules controlling transcription, leads to stochastic effects in the production of RNAs. Discontinuities in transcriptional permissiveness of gene promoters, witnessed as “transcriptional bursts”, amplify this effect and lead to variability in gene expression between cells. Furthermore, the cellular state influences the capacity of transcription in a cell-specific manner, hereby affecting transcriptional output on long timescales. Variegated expression can be beneficial in development and cellular decision-making. However, it is also exploited by cancerous tissue, as intra-tumor diversity lowers therapeutic effectiveness. It is essential to study stochastic transcriptional regulation at the single-cell level, to comprehend sources and consequences of cellular heterogeneity in health and disease.
This thesis examines how cells control transcriptional bursts to adapt gene expression output to environmental signals and how the cellular state influences this process. An estrogen-sensitive locus served as a model system as it allows for tight experimental control of expression through varying estrogen levels. Fluorescent labeling of nascent transcripts in combination with time-resolved microscopy in living cells enabled quantitative measurements of single-cell transcriptional dynamics. Transcription occurred in stochastically timed bursts with a strong cell-to-cell variability in long-term transcriptional output. Stochastic mathematical models of promoter progression and transcription were fitted to the acquired datasets to discriminate alternative hypotheses of promoter regulation. This revealed that estrogen adjusts the frequency of transcriptional bursts by controlling the transition to a transcriptionally permissive promoter. The cellular state, however, alters longterm transcriptional output through initiation and elongation kinetics. This effect is mediated through a diffusible factor, as two alleles within the same cell were similarly affected and recently divided daughter cells correlated in time-averaged transcription.
To infer whether the chromatin environment influences burst characteristics and transcriptional noise, perturbations using inhibitors of chromatin modifying enzymes were performed. Interestingly, the inhibition of histone deacetylation reduced noise in gene expression, highlighting that chromatin permits noise regulation at the level of nascent transcription. In conclusion, this thesis provides a quantitative description of estrogen-dependent transcription, which incorporates transcriptional bursting, the influence of cellular state, and gene-specific tuning through chromatin.