Biology subjects

Patel, H. P.

Publications and source records attributed to Patel, H. P..

3 recordsLinked to original sources

bioRxiv2022

DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes

DNA supercoiling has emerged as a major contributor to gene regulation in bacteria. The impact of DNA supercoiling on transcription dynamics in eukaryotes is less clear. Here, using single-molecule dual-color RNA imaging in budding yeast, we show that transcriptional bursting of the divergent and tandem GAL genes is coupled. Upon topoisomerase degradation, supercoils that buildup from transcription inhibit subsequent transcription at neighboring genes, thereby reducing their simultaneous bursting. GAL gene transcription is inhibited more by negative than by positive supercoiling accumulation. Unlike bacteria, wildtype yeast has sufficient topoisomerase levels to minimize inhibition from supercoils at adjacent genes. Overall, we discover fundamental differences in supercoiling-mediated gene regulation between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes. One sentence summaryTranscription causes twisting of the DNA double helix, which can inhibit transcription of adjacent genes.

molecular biology↗

bioRxiv2021

Accurate inference of stochastic gene expression from nascent transcript heterogeneity

Transcriptional rates are often estimated by fitting the distribution of mature mRNA numbers measured using smFISH (single molecule fluorescence in situ 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. Whilst it is often presumed that post-transcriptional noise and gene copy number variation affect transcriptional parameter estimation, the size of the error introduced remains unclear. To address this issue, here we measure both mature and nascent mRNA distributions of GAL10 in yeast cells using smFISH and classify each cell according to its cell cycle phase. We infer transcriptional parameters from mature and nascent mRNA distributions, with and without accounting for cell cycle phase and compare the results to live-cell transcription measurements of the same gene. We find that: (i) correcting for cell cycle dynamics decreases the promoter switching rates and the initiation rate, and increases the fraction of time spent in the active state, as well as the burst size; (ii) additional correction for post-transcriptional noise leads to further increases in the burst size and to a large reduction in the errors in parameter estimation. Furthermore, we outline how to correctly adjust for measurement noise in smFISH due to uncertainty in transcription site localisation when introns cannot be labelled. Simulations with parameters estimated from nascent smFISH data, which is corrected for cell cycle phases and measurement noise, leads to autocorrelation functions that agree with those obtained from live-cell imaging.

systems biology↗

bioRxiv2021

Hda1C restricts the transcription initiation frequency to limit divergent non-coding RNA transcription

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 with an unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast. We identified the Hda1 histone deacetylase complex (Hda1C) as a repressor of DNC in high-throughput reverse genetic screens based on quantitative single-cell fluorescence measurements. Nascent transcription profiling showed a genome-wide role of Hda1C in DNC repression. Live-cell imaging of transcription revealed that Hda1C reduced the frequency of DNC transcription. Hda1C contributed to decreased acetylation of histone H3 in DNC regions, supporting DNC repression by histone deacetylation. Our data support the interpretation that DNC results as a consequence of the NDR-based architecture of eukaryotic promoters, but that it is governed by locus-specific repression to maintain genome fidelity.

genomics↗