Delineating transcriptional regulatory processes in yeast

From the Hahn Lab, Basic Sciences Division

Yeast is a single cell eukaryotic organism, that incites in this Irish scientist an intense memory of the smell of brewing Guinness, wafting down the River Liffey on a drizzly Dublin day as I sit at my desk a long way from home. Fortunately for you reader, this article will not be an ‘Ode to Guinness’ as yeast is not merely a nostalgia inducing tool for the homesick, but rather more importantly, it is a key model for understanding complex transcriptional behavior in eukaryotes. This is something appreciated by Dr. Jeremy Schofield, a postdoctoral research fellow in Dr. Steven Hahn’s lab in Fred Hutch’s Basic Sciences Division. Gene regulation is complex, involving the interaction between different regulatory mechanisms that include numerous core promoters and co-activators. “In yeast, transcription of most genes depends on the cofactor TFIID” explained Dr. Schofield. “Transcription from a subset of genes (coactivator redundant, or CR) additionally depend on SAGA and often Mediator-Tail (the transcription activator-binding domain of Mediator).  The CR class genes tend to contain TATA boxes in their core promoters and are typically highly regulated by stress or other signaling pathways.”

What remains unknown however, is how the expression of these distinct yeast gene classes is regulated and what roles are played by upstream activator sequences (UAS) and the aforementioned core promoters. In their recent study, published in Cell Reports, Dr. Schofield and Dr. Hahn, sought to address “whether UAS sequences (and likely the factors binding these sequences) display specificity for the core promoters they regulate, and whether cofactor specificity can be explained by UAS, core promoters, or a combination of both”. They utilized yeast to understand essential transcriptional process in this study and “because the yeast genome is highly tractable,” added Dr. Schofield. “We readily combined a high throughput screen with yeast engineered for rapid depletion of essential regulatory factors. This approach provided unique insights into how eukaryotic protein coding genes respond to regulatory signals and transcription cofactors.  The cofactors we studied in this work are highly conserved between yeast and other eukaryotes, therefore insights gained in the yeast model can be directly related to regulatory mechanisms in higher eukaryotes, including humans,” he continued.

Upstream activator sequences matched with core promoters from the same yeast gene class result in high transcriptional activity.
Upstream activator sequences matched with core promoters from the same yeast gene class result in high transcriptional activity. Figure provided by Dr. Schofield.

The authors began their study by designing a large-scale reporter assay that allowed them to study the transcriptional activity of tens of thousands of UAS and promoter combinations. When comparing the UAS to non-UAS control sequences, they were able to determine that UAS can broadly activate core promoters, irrespective of matching gene class, and that genomic characteristics such as binding motifs for specific regulatory factors that were linked to high transcriptional activity were also present irrespective of gene regulatory class. They then asked the question whether UAS can activate transcription at an even greater frequency if matched promoters and gene classes were analyzed. They observed that UAS from TFIID genes activate TFIID core promoters to a greater extent, a finding that was consistent for UAS from CR genes and CR core promoters. These data suggest that “higher levels of transcription are often achieved through matching of UASs and cores from the same gene class,” said Dr. Schofield.  Although, activation by unmatched promoters can still occur. Next, they sought to address cofactor dependence associated with UAS, the core promoter, or both. To achieve this, the authors rapidly depleted core elements Taf13 (TFIID), Spt7 (SAGA) or Med15 (MED Tail) and assessed changes in transcriptional activity via RNA-sequencing. They noted that “specificity for SAGA and Mediator-Tail [cofactors] depends on a combination of UAS and core promoter, whereas TFIID [cofactor] specificity is dependent on the core promoter alone.” These data “underscore the differences in regulation at the separate classes of yeast genes, and supports work in higher eukaryotes, where there is an increasing body of evidence demonstrating the functional importance of enhancer/promoter compatibility,” said Dr. Schofield.

Next steps for the authors include “examining the mechanistic differences between the gene classes of yeast and how this relates to compatibility between UASs and core promoters. Eukaryotic genes can be roughly divided into constitutive and inducible genes. Prior studies have suggested that at least some genes are transcribed in short bursts interspersed with periods where gene expression is turned off. We are investigating how widespread this bursting behavior is and whether it is confined to any particular class of protein coding genes,” said Dr. Schofield. Further describing their experimental plan, he continued by saying “to investigate this question and to identify gene features associated with transcriptional variation, we are measuring bursting behavior across the yeast genome using time-resolved single-cell RNA-seq. This approach, paired with rapid depletion of transcriptional cofactors, will further our understanding of the transcriptional mechanisms associated with constitutive vs. inducible gene expression and how genes are regulated under a wide variety of conditions and genetic backgrounds.”

Notably, according to Dr. Schofield, the insights into yeast transcriptional machinery gleaned from this study, “will be important for understanding how genes are regulated both in normal and cancer cells.”

This work was supported by funding from the National Institutes of Health and a Cancer Center Support Grant to Fred Hutch Genomics.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Steven Hahn contributed to this work.

Schofield JA, Hahn S. 2023. Broad compatibility between yeast UAS elements and core promoters and identification of promoter elements that determine cofactor specificity. Cell Rep. 42(4):112387.  Available online ahead of print.