As early as 1958, Francis Crick is thought to have coined the “central dogma” of molecular biology, which dictates that essential biological information only moves forward from genes to messages (through transcription) to protein (through translation), but never backward. Soon afterwards, reverse-transcriptases, a class of enzymes that could build DNA from RNA (as the name implies) were discovered, thereby violating Crick’s “central dogma”. While the underlying principle of the central dogma still holds true, where the flow of biological information goes from DNA to RNA to protein, it was nonetheless a sobering moment for us biologists to steer away from “dogmas” as they often proved fragile in the face of scientific inquisition.
The Hahn lab (Basic Sciences Division) is interested in understanding the molecular mechanisms underlying eukaryotic transcription. Transcriptional regulation plays crucial roles in controlling processes such as cell growth, differentiation, development, and cellular stress response. Deciphering these regulatory mechanisms can lead to understanding the molecular mechanisms underlying many types of human disease. Using yeast as a model organism, they have previously investigated how transcription coactivator complexes TFIID and SAGA regulate gene expression, thanks to recently developed techniques that allows the rapid depletion of essential proteins without the need for traditional heat-sensitive alleles.
Both TFIID and SAGA complexes are comprised of over ten different subunits, some of which are shared. Traditional loss-of-function techniques to permanently disable specific subunits of SAGA caused strong genome-wide defects in transcription and SAGA-mediated chromatin modifications. As such, these methods fell short of elucidating the early/immediate effects of transient SAGA loss. But is it possible to isolate early/immediate roles of SAGA in transcription activation from the well-established long-term effects? Dr. Rafał Donczew and Linda Warfield from the Hahn lab co-led a study to address this problem. They published their findings in a recent issue of eLife.
Dr. Donczew explained the importance of their study, “our work brings together many conflicting studies on coactivator dependence of yeast genes. Particularly, we identified two independent functions of the SAGA coactivator complex, which modifies chromatin at all active genes and, together with TFIID, coregulates a small subset of genes, likely acting as a TBP (TATA binding protein) loading factor. Based on our findings we classified yeast genes into two classes that differ in their coactivator requirements. This will be an important resource for the transcription community going forward and provides unexpected insights into how all RNA Pol II-transcribed genes are regulated.”
This study was only possible thanks to a technical breakthrough. The authors used a recently developed technology that allowed rapid depletion of SAGA followed by measurement of newly-synthesized RNA, thereby enabling them to accurately measure SAGA-dependent transcription. Dr. Donczew elaborates, “rapid depletion has very different effects on transcription compared with strains having deletions of SAGA subunits. By focusing on newly-synthesized RNA labeled with 4-thio uracil, and not steady state mRNA, we are able to uncouple changes in transcription from alterations in chromatin modifications that change slowly after rapid SAGA depletion and from secondary effects on RNA stability.”
Using this loss-of-function approach, the authors showed that rapid depletion of SAGA exhibited only modest transcription defects at about 13% of protein-coding genes, most of which were also modestly sensitive to TFIID depletion thereby suggesting functional redundancy between TFIID and SAGA at this gene set. The remaining 87% of genes were sensitive to rapid TFIID depletion but their immediate/early transcription was SAGA independent. Confirming their hypothesis of some genes redundantly regulated by TFIID and SAGA, they found that rapid depletion of subunits in both coactivators strongly reduced transcription of all RNA PolII-transcribed genes. The authors next performed a genome-wide mapping of SAGA and TFIID occupancy and found that binding of both factors at many genes to be independent of gene class. By conducting promoter analysis for different SAGA/TFIID subunits, they concluded that the distinction between the gene classes cannot be ascribed to any single regulatory factor but rather to multiple components.
Dr. Donczew reflected on their findings, “Available data does not provide an explanation why the coactivator-redundant genes require both SAGA and TFIID for proper function. Similarly, it is unclear why the majority of genes are insensitive to rapid SAGA depletion. Our future studies will focus on exploring the mechanisms underlying this behavior.”
This work was funded by the National Institutes of Health.
UW/Fred Hutch Cancer Consortium member Steven Hahn contributed to this work.
Donczew R, Warfield L, Pacheco D, Erijman A, Hahn S. 2020. Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA. eLife.