Biology subjects

Schofield, J. A.

Publications and source records attributed to Schofield, J. A..

3 recordsLinked to original sources

bioRxiv2024

Transcriptional bursting, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single cell RNA-seq

A time resolved nascent single-cell RNA-seq approach was developed to dissect gene-specific transcriptional bursting and the roles of SAGA and Mediator Tail (the activator-binding module). Most yeast genes show near-constitutive behavior while only a subset of genes show high mRNA variance suggestive of transcription bursting. Bursting behavior is highest in the coactivator redundant (CR) gene class (dependent on both SAGA and TFIID) and is strongest in TATA-containing CR genes. Applying this approach to analyze gene activation, we found that basal histone gene transcription is in a low level, low-noise constitutive mode while the activated state unexpectedly shows an increase in both the fraction of active promoters and a switch to a noisy and bursty transcription mode. Rapid depletion of either SAGA or Mediator Tail suggests that both factors play an important role in stimulating the fraction of active promoters at CR genes, with a variable gene-specific role in transcriptional bursting.

genomics↗

bioRxiv2023

Improving the study of RNA dynamics through advances in RNA-seq with metabolic labeling and nucleotide-recoding chemistry

RNA metabolic labeling using 4-thiouridine (s4U) captures the dynamics of RNA synthesis and decay. The power of this approach is dependent on appropriate quantification of labeled and unlabeled sequencing reads, which can be compromised by the apparent loss of s4U-labeled reads in a process we refer to as dropout. Here we show that s4U-containing transcripts can be selectively lost when RNA samples are handled under sub-optimal conditions, but that this loss can be minimized using an optimized protocol. We demonstrate a second cause of dropout in nucleotide recoding and RNA sequencing (NR-seq) experiments that is computational and downstream of library preparation. NR-seq experiments involve chemically converting s4U from a uridine analog to a cytidine analog and using the apparent T-to-C mutations to identify the populations of newly synthesized RNA. We show that high levels of T-to-C mutations can prevent read alignment with some computational pipelines, but that this bias can be overcome using improved alignment pipelines. Importantly, kinetic parameter estimates are affected by dropout independent of the NR chemistry employed, and all chemistries are practically indistinguishable in bulk, short-read RNA-seq experiments. Dropout is an avoidable problem that can be identified by including unlabeled controls, and mitigated through improved sample handing and read alignment that together improve the robustness and reproducibility of NR-seq experiments.

biochemistry↗

bioRxiv2022

Broad compatibility between yeast UAS elements and core promoters and identification of promoter elements that determine cofactor specificity

Three general classes of yeast protein-coding genes are distinguished by their dependence on the transcription cofactors TFIID, SAGA and Mediator (MED) Tail, but little is known about whether this dependence is determined by the core promoter, Upstream activation sites (UASs), or other gene features. It is also unclear whether UASs can broadly activate transcription from the different promoter classes or whether efficient transcription requires matching UASs and promoters of similar gene class. Here we measure transcription and cofactor specificity for tens of thousands of UAS-core promoter combinations. We find that <5% of UASs display strong core promoter specificity while most UASs can broadly activate promoters regardless of regulatory class. However, we find that matching UASs and promoters from the same gene class is generally important for optimal expression. From examining the cofactor dependence of this large UAS-promoter set, we find that sensitivity to rapid depletion of MED Tail or SAGA is dependent on the identity of both UAS and promoter while dependence on TFIID localizes to only the core promoter. Our results explain why transcription factor-mediated MED recruitment to the UAS does not always result in Tail-dependent transcription and highlight the role of TATA and TATA-like promoter sequences in MED Tail function.

molecular biology↗