Transcription in single cells is a stochastic process that arises from the random collision of molecules, resulting in heterogeneity in gene expression in cell populations. This heterogeneity in gene expression can influence cell fate decisions and disease progression. The projects in our lab are focused on understanding the dynamics and mechanisms of stochastic transcription at a molecular level as well as their effect on the organism. We are interested in every cellular component or process that may regulate transcription, including promoter and enhancer sequences, gene-specific transcription factors, chromatin regulators, 3D genome architecture, ncRNA transcription, and the binding kinetics of the transcriptional machinery to the DNA. We develop and apply single-molecule microscopy techniques in both yeast and human model systems, to dissect the mechanism of gene regulation and connect them to human disease relevance. Examples of our recent studies are described below.
Single-molecule imaging inside living cells has revealed that transcription factors (TFs) bind to DNA transiently, but a long-standing question is how this transient binding is related to transcription activation. We devised a microscopy method to simultaneously measure transient TF binding at a single locus and the effect of these binding events on transcription. We show that DNA binding of the yeast TF Gal4 activates transcription of a target gene within a few seconds, with at least ∼20% efficiency and with a high initiation rate of ∼1 RNA/s. Gal4 DNA dissociation decreases transcription rapidly. Moreover, at a gene with multiple binding sites, individual Gal4 molecules only rarely stay bound throughout the entire burst, but instead frequently exchange during a burst to increase the transcriptional burst duration. Our results suggest a mechanism for enhancer regulation in more complex eukaryotes, where TF cooperativity and exchange enable robust transcription activation.
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DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how DNA supercoiling impacts transcription dynamics in eukaryotes is unexplored. Using single-molecule dual-color nascent transcription imaging in budding yeast, we show that transcriptional bursting of divergent and tandem GAL genes is coupled. Temporal coupling of neighboring genes requires rapid release of DNA supercoils by topoisomerases. When DNA supercoils accumulate, transcription of one gene inhibits transcription at its adjacent genes. Transcription inhibition of the GAL genes results from destabilized binding of the transcription factor Gal4. Moreover, wild-type yeast minimizes supercoiling-mediated inhibition by maintaining sufficient levels of topoisomerases. Overall, we discover fundamental differences in transcriptional control by DNA supercoiling between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.
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To undersatnd how transcriptional bursting is regulated by the remodeling of promoter nucleosomes, we use single-molecule live-cell imaging of GAL10 transcription in Saccharomyces cerevisiae to measure how bursting changes upon combined perturbations of chromatin remodelers, the transcription factor Gal4 and preinitiation complex components. Using dynamic epistasis analysis, we reveal how the remodeling of different nucleosomes regulates transcriptional bursting parameters. At the nucleosome covering the Gal4 binding sites, RSC and Gal4 binding synergistically facilitate each burst. Conversely, nucleosome remodeling at the TATA box controls only the first burst upon galactose induction. At canonical TATA boxes, the nucleosomes are displaced by TBP binding to allow for transcription activation even in the absence of remodelers. Overall, our results reveal how promoter nucleosome remodeling together with Gal4 and preinitiation complex binding regulates transcriptional bursting.
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In order to study the role of antisense non-coding RNA transcription in transcription regulation, we
devised a method to visualize the dynamic interplay between sense and antisense transcription at
single-molecule resolution in living yeast cells. We found that during galactose induction,
transcription of the sense gene GAL10 occurs in transcriptional bursts, which are unaffected by
stochastic transcription in the antisense direction. However, strand-specific inhibition of antisense
transcription using CRISPR/dCas9 showed that in non-induced conditions antisense ncRNA transcription is
critical to prevent transcriptional leakage of GAL1 and GAL10. Transcription of the same
ncRNA is thus functional under repressive conditions but spurious under activating conditions,
highlighting the nuanced roles that ncRNA can play in gene regulation.
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