Lenstra Lab

Netherlands Cancer Institute, Oncode Institute


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. By utilizing a combination of single-molecule microscopy, biophysical, genetic and molecular biology approaches to in both yeast and human model systems, we aim to connect mechanistic insights to human disease relevance

The interplay between transcription factors, nucleosomes and transcriptional bursting

We have developed a novel single-molecule imaging platform to directly visualize both transcription binding dynamics and transcriptional output in the same cell. In combination with other in vitro and in vitro single-molecule imaging approaches, we were able to correlate the binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We found that Gal4 dwell time sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Our data support a model in which multiple RNA polymerases initiate transcription during one burst as long as the transcription factor is bound to DNA, and bursts terminate upon transcription factor dissociation..
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Simultanenous imaging of GAL10 transcription (green) and the binding dynamics of single Gal4 transcription factor molecules (red) in the same cell.

Non-coding RNA transcription

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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Cell switches from antisense transcription (green) to sense transcription (red). Cells are shown from the top and two sides.

Cell transcribing antisense (green) and sense (red) RNA simultaneously at the same locus. Cells are shown from the top and two sides.