DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes.

DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how DNA supercoiling impacts transcription dynamics in eukaryotes is unclear. Here, 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.

Transcription factor clusters enable target search but do not contribute to target gene activation.

Many transcription factors (TFs) localize in nuclear clusters of locally increased concentrations, but how TF clustering is regulated and how it influences gene expression is not well understood. Here, we use quantitative microscopy in living cells to study the regulation and function of clustering of the budding yeast TF Gal4 in its endogenous context. Our results show that Gal4 forms clusters that overlap with the GAL loci.

Quantifying how post-transcriptional noise and gene copy number variation bias transcriptional parameter inference from mRNA distributions.

Transcriptional rates are often estimated by fitting the distribution of mature mRNA numbers measured using smFISH (single molecule fluorescence hybridization) with the distribution predicted by the telegraph model of gene expression, which defines two promoter states of activity and inactivity. However, fluctuations in mature mRNA numbers are strongly affected by processes downstream of transcription. In addition, the telegraph model assumes one gene copy but in experiments, cells may have two gene copies as cells replicate their genome during the cell cycle.

Following the tracks: How transcription factor binding dynamics control transcription.

Transcription, the process of copying genetic information from DNA to messenger RNA, is regulated by sequence-specific DNA-binding proteins known as transcription factors (TFs). Recent advances in single-molecule tracking (SMT) technologies have enabled visualization of individual TF molecules as they diffuse and interact with the DNA in the context of living cells. These SMT studies have uncovered multiple populations of DNA-binding events characterized by their distinctive DNA residence times.

The Hda1 histone deacetylase limits divergent non-coding transcription and restricts transcription initiation frequency.

Nucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA and a divergent non-coding (DNC) transcript of unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast.

Optimized protocol for single-molecule RNA FISH to visualize gene expression in .

Single-molecule RNA fluorescence hybridization (smFISH) allows subcellular visualization, localization, and quantification of endogenous RNA molecules in fixed cells. The spatial and intensity information of each RNA can be used to distinguish mature from nascent transcripts inside each cell, revealing both past and instantaneous transcriptional activity. Here, we describe an optimized protocol for smFISH in with optimized lyticase digestion time and hybrization steps for more homogenous results.

Single-Molecule Fluorescence Imaging in Living Cells.

This protocol describes how to image fluorescently tagged proteins, RNA, or DNA inside living cells at the single-molecule level. Imaging inside living cells, as opposed to fixed materials, gives access to real-time kinetic information. Although various single-molecule imaging applications are discussed, we focus on imaging of gene transcription at the single-RNA level.

Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting.

Transcription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of and single-molecule imaging approaches, we directly correlated binding of the Gal4 transcription factor with the transcriptional bursting kinetics of the Gal4 target genes and in living yeast cells. We find that Gal4 dwell time sets the transcriptional burst size.