Overview

Our laboratory studies a wide range of mechanisms that regulate the transcription of eukaryotic protein coding genes. mRNA synthesis is regulated by many signaling pathways that control processes such as development, growth, and the response to environmental conditions such as stress. Even genes that are constitutively transcribed appear to be regulated by complex mechanisms that ensure mRNA synthesis rates are stable during different environmental conditions and cell cycle phases. Misregulation of transcription is a major cause of human disease and our work addresses the molecular basis for some of these defects. The lab uses a wide variety of experimental technologies to explore these topics such as molecular genetics, genomics, high throughput screening, computational biology, biochemistry, structural biology, and biophysics. We use . cerevisiae (budding yeast) as our experimental system because of the powerful mix of genomics, molecular genetics and biochemical methods that can readily be applied to this model organism. Because the transcription machinery and its regulatory factors are well-conserved throughout evolution, fundamental gene regulatory mechanisms in yeast are nearly always conserved in metazoans.

Mechanistic studies on eukaryotic transcription began over 50 years ago with the identification of the three forms of RNA Polymerase (Pol) and the discovery in the 1980’s of eukaryotic basal transcription factors. Since then, great progress has been made in identifying important principles of transcription and its regulation. These include: (i) the discovery of nearly all the regulatory transcription factors (TFs), cofactor complexes, and components of the basal transcription machinery, (ii) recognition of the important roles that chromatin, chromatin modifications, and higher order chromosome architecture plays in gene regulation, and (iii) fundamental insights into the molecular mechanisms of gene-specific TFs, cofactors, and RNA polymerases. With this background, and the availability of important new technologies, this is an excellent time to tackle the next level of fundamental problems in gene regulation. Outlines of our current projects are given below.

The function of DNA sequence-specific transcription factors

A major focus of the lab is to investigate the function of regulatory TFs, many of which bind DNA in a sequence-specific manner. Yeast contain ~150 TFs, 10-fold less than mammalian cells, which make yeast a great model system to examine genome-wide functions and interactions between the complete set of TFs. In our initial studies, we mapped the genome-wide binding locations for nearly all the TFs and found three distinct sets of genes that differ in the number and types of TFs that bind to their gene regulatory regions. To explore the functions of TFs, we have rapidly depleted individual factors to identify the gene regulatory targets for each TF. These approaches have led to many surprising results. For example, we found that many TF binding locations do not obviously contribute to expression from the nearby gene and that depletion of individual TFs often affects expression of genes that do not contain an obvious nearby binding site. These results have opened many new research directions. For example, we will be exploring why certain TF binding sites are functional vs nonfunctional, how individual TFs contribute to transcription and chromatin architecture, and how TF and cofactor activities are integrated to control genome-wide transcription. Another major surprise is that only a small percentage of TFs are strongly biased toward either transcription activation or repression. Instead, most yeast TFs have dual functions. For example, when many TFs are rapidly depleted, roughly equal numbers of genes are either increased or decreased in expression. Exploring the mechanisms of dual function TFs is another area of interest.

The molecular basis for cofactor function and gene specificity

Another major interest is investigating the mechanisms of three conserved transcription cofactors, Mediator, SAGA and TFIID, that integrate regulatory signals and that are links between regulatory transcription factors (TFs) and the core transcription machinery. By combining genetics, genomics, and biochemical approaches, we are investigating the gene-specificity of these cofactors and how they interact with TFs and the core transcription machinery. For example, in a recent study we found that, in contrast to earlier models for Mediator function, the principal role of Mediator at many genes is not to function as a direct signaling target of transcription activators, but rather as an essential component of the transcription machinery at core promoters. In another example, we recently found that yeast contain three distinct types of protein-coding genes, each regulated by a specific set of cofactors. Finally, we are using Massively Parallel Reporter Assays (MRPAs) to investigate enhancer-promoter specificity, testing the function and cofactor specificity of nearly all yeast enhancers driving expression from the three classes of yeast promoters.

Transcription Bursting

Prior work from many laboratories has shown that gene transcription can occur in bursts with genes in alternating on and off states. Changes in transcription can result from regulating the length of the on or off states, the amount of transcription occurring during the on state (bursting) and/or the fraction of cells that are in the on vs off state. Most earlier studies used single cell microscopy to investigate these parameters, but the disadvantage of this approach is that transcription from only one gene at a time can be examined in detail. By metabolically labeling mRNA for different times, combined with single cell RNA-seq, we have estimated the on/off fraction and relative bursting for thousands of genes in a single experiment. We are extending this work to examine the effects of gene activation and the roles of several transcription cofactors on the kinetics of transcription for many genes.

Recent Publications from the Hahn Laboratory

Schofield, JA and S. Hahn. (2024) Transcriptional noise, gene activation, and roles of SAGA and Mediator Tail measured using nucleotide recoding single cell RNA-seq. Cell Reports, Aug 27; 43:114593. doi: 10.1016/j.celrep.2024.114593

Mahendrawada, L., Warfield, L., Donczew, R., and S. Hahn (2023). Surprising connections between DNA binding and function for the near-complete set of yeast transcription factors.  Biorxiv   https://doi.org/10.1101/2024.03.08.584165

Schofield, JA and S. Hahn (2023) Broad compatibility between yeast UAS elements and core promoters and identification of promoter elements that determine cofactor specificity.  Cell Reports Apr 12;42(4):112387. doi: 10.1016/j.celrep.2023.112387

Warfield, L.*, Donczew, R.*, Mahendrawada, L., and S. Hahn (2022) Yeast Mediator facilitates transcription initiation at most promoters via a Tail-independent mechanism.  Mol Cell, Nov 3;82(21):4033-4048.e7. doi: 10.1016/j.molcel.2022.09.016. Epub 2022 Oct 7.

Donczew, R and S. Hahn (2021). BET family members Bdf1/2 modulate global transcription initiation and elongation in Saccharomyces cerevisiae.  Elife. 2021 Jun 17;10:e69619. doi: 10.7554/eLife.69619. Online ahead of print.PMID: 34137374

Tuttle,  L.M., Pacheco, D., Warfield, L., Wilburn, D.L., Hahn, S., and Klevit, R.E. (2021). Mediator subunit Med15 dictates the conserved “fuzzy” binding mechanism of yeast transcription activators Gal4 and Gcn4. Nature Comm. Apr 13;12(1):2220. doi: 10.1038/s41467-021-22441-4..

Erijman, A., Kozlowski, L., Sohrabi-Jahromi, S., Fishburn, J., Warfield, L., Schreiber, J., Noble, W., Söding, J., and Hahn, S. (2020). A high-throughput screen for transcription activation domains reveals their sequence characteristics and permits reliable prediction by deep learning. Mol Cell May 12:S1097-2765(20)30262-8. doi: 10.1016/j.molcel.2020.04.020..

Donczew, R*., Warfield, L.*, Pacheco, D., Erijman, A., and Hahn, S. (2020). Two roles for the yeast transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA. Elife. 2020 Jan 8;9. pii: e50109. doi: 10.7554/eLife.50109. PMID: 31913117 

Tuttle, L.M., Pacheco, D., Warfield, L., Luo, J., Ranish, J., Hahn, S., and Klevit, R.E. (2018). Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex. Cell Rep 22, 3251–3264. https://doi.org/10.1016/j.celrep.2018.02.097

Hahn S. (2018). Phase Separation, Protein Disorder, and Enhancer Function. Cell 175:1723-1725. doi: 10.1016/j.cell.2018.11.034.