Research

New: Postdoctoral position available

Open position for a postoctoral fellow to investigate mechanisms of transcriptional regulation using yeast as a model system.  Examples of possible projects include: studies on the genome-wide specificity and mechanisms of coactivators including Mediator, SAGA, NuA4 and/or TFIID; investigating mechanisms and specificity of transcription activators; probing the roles of transcription factors in orchestrating genome-wide expression. A variety of approaches such as molecular genetics, biochemistry, genomics, and computational biology will be utilized. The precise topic of the research project will be determined jointly with the candidate and candidates that have a demonstrated interest in gene regulation and that display initiative and creativity will be given priority.   Qualifications and link to application are here.


Overview

Eukaryotic RNA polymerases are components of large protein machines that integrate numerous regulatory signals to precisely control gene expression. Key regulatory factors include gene-specific transcription factors that either activate or repress transcription in response to various signaling pathways. These factors often work via recruitment of transcription coactivators to gene regulatory regions. Coactivators are large protein complexes that interface with the basal transcription machinery and/or modify nucleosome positioning or covalent nucleosome modifications. Research in our laboratory aims to understand the function of the gene-specific transcription factors, how they interface with coactivator complexes and how these factors modulate early steps in the transcription pathway.

The lab uses a multi-disciplinary approach including molecular genetics, genomics, computational biology, biochemistry, structural, and biophysical methods to uncover new fundamental mechanisms used in gene regulation. We use S. cerevisiae (budding yeast) as our experimental system because of the powerful mix of genomics, molecular genetics and biochemical methods that can readily be used in 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.
 

Mechanisms of Eukaryotic transcriptional regulation

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 of the gene-specific transcription factors (TFs), coactivator 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, coactivators, 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. The work in our laboratory is focused on two problems central to eukaryotic transcriptional regulation: a) the function of gene-specific transcription factors, and b) genome-wide specificity and mechanisms of transcription coactivators. These two problems are highly interrelated. For example, TFs, chromatin architecture, and promoter sequences play a critical role in determining coactivator requirements and the assembly pathway for the Pol II transcription machinery at particular genes. Examples for some of our current work is given below. In future work, we aim to use a wide variety of approaches to address mechanisms of TFs including novel transcription activation domains, TF cooperativity, and coactivator function and specificity.


Function and genome-wide specificity of the coactivators TFIID and SAGA

Often recruited by transcription activators, coactivators play roles in processes including promoter recognition, PIC formation, chromatin modifications, and nucleosome positioning. Much is known about the structure, enzymatic functions, and protein interactions among coactivators such as TFIID, SAGA, NuA4, Mediator, etc., but relatively little is known about mechanisms that determine their genome-wide specificity and the molecular mechanisms of how they promote activated transcription. Over the past five years, we’ve combined genomics, genetics and biochemistry to address coactivator specificity and mechanisms.

Our goal of investigating transcription initiation mechanisms from non-TATA promoters led us to examine the functions and specificity of the conserved coactivators TFIID and SAGA, two TBP (TATA binding protein) loading factors that play important roles in Pol II preinitiation complex (PIC) assembly. Long-term ablation of yeast SAGA function (e.g., a deletion of the gene coding for Spt3- a key SAGA subunit) showed that SAGA has a strong genome-wide role in all Pol II transcription, mediated in large part by its histone H3 HAT subunit, Gcn5 (Fig 1). However, rapid depletion of TFIID or SAGA using the auxin degron system identified two gene classes that differ in their requirements for SAGA and TFIID, likely caused by differences in TBP loading mechanisms. The coactivator redundant (CR) genes (~13% of yeast genes) can utilize either TFIID or SAGA to promote transcription; rapid depletion of both coactivators is necessary for strong defects in transcription (e.g., Taf13/Spt3 and Taf13/Spt7-degrons; Fig 1A). The TFIID-dependent genes (TFIID genes; ~87% of genes) are sensitive only to rapid depletion of TFIID. Promoter analysis suggests that no single feature determines promoter class; we found no TF motif or promoter element exclusive to either the TFIID or CR genes. Our results differ from early pioneering work where gene transcription was proposed to be dominated by either TFIID or SAGA with many of the highest expressed genes seeming TFIID-independent. Our new findings are an important start for understanding differences in gene regulation and PIC architecture at cellular genes.

Two yeast promoter classes: coactivator redundant (CR) and TFIID dependent.

Fig 1A. Two yeast promoter classes: coactivator redundant (CR) and TFIID dependent. Heat map showing changes in newly synthesized mRNA upon degron depletion (deg) or gene deletion of selected coactivator subunits. SAGA subunits: Spt3, Spt7; TFIID subunit: Taf13. Genes are clustered into the two classes as indicated.

Genome-wide functions of TFIID and SAGA.

Fig 1B. Genome-wide functions of TFIID and SAGA. The drawing shows the role of TFIID and SAGA at the two different gene classes.  SAGA and TFIID have partially redundant roles at the CR genes while the TFIID gene class is insensitive to rapid SAGA depletion. SAGA also has a role in genome-wide transcription via its Gcn5 HAT subunit that is revealed by long-term SAGA depletion.


Mechanisms of transcription activator function

Transcription activators are TFs that stimulate transcription via their activation domains (ADs). In many cases, ADs function in part by recruitment of coactivators that regulate processes such as PIC assembly, post-initiation steps, covalent chromatin modifications, and nucleosome positioning. Pioneering early work characterized unusual properties of ADs such as intrinsic disorder and biased low complexity sequences. Combined, early studies suggested that activator function does not involve precise molecular complementarity with their protein targets but left open the important question of how their unusual properties translate into molecular a mechanism for function. Our breakthrough in this field resulted partly from a collaboration with NMR spectroscopist Rachel Klevit to identify dynamic mechanisms of AD-coactivator binding and specificity. Exemplified by the yeast TF Gcn4 that contains strong tandem ADs binding to the Mediator subunit Med15, we found that the AD-Med15 interface is highly dynamic and “fuzzy”, with no unique protein-protein interface (Fig 2). With the recent recognition of condensates at some enhancer regions, the dynamic AD-ABD binding we uncovered neatly fits with how small numbers of DNA-bound TFs can lead to large dynamic phase shifted complexes at enhancers. At the same time, we collaborated with computational biologist Johannes Söding to develop a deep learning predictor for acidic AD function termed ADpred. Working backwards from ADpred allowed us to identify sequence features that specify AD function in natural transcription factors and, importantly, to relate these properties to a molecular mechanism for acidic AD function. Proteome analysis with ADpred suggests that acidic ADs account for less than half of metazoan ADs. Our combined work is an important advance in understanding the molecular basis for acidic AD function and opens a pathway to investigate an important set of questions about other types of ADs and how TFs cooperate to regulate cellular transcription.

Structure coordinates in pymol format

Fig 2A. "Fuzzy" complex of the Gcn4 central AD and Mediator subunit Med15 (Gal11) activator binding domain 1. Download structure coordinates in pymol format

transcriptions

Fig 2B. Atomic details for one of the many possible Gcn4-Med15 binding states. See Brzovic et al (2011) and Tuttle et al (2018) for more details.


Genome-wide functions of yeast bromodomain-containing BET proteins

Bromodomains are protein domains that allow targeting to chromatin via interactions with acetylated histones.  One class of double bromodomain-containing proteins, the human BET factors, have wide-ranging importance for transcription in some cancer cells and are promising targets for the therapy of cancer and immunoinflammatory diseases.  However, their mechanisms of action and functional redundancies are poorly understood.  Yeast has homologs of the BET factors, and these were thought to function via the coactivator TFIID by helping to target it to promoter regions with acetylated nucleosomes. Using rapid depletion of the yeast BET proteins (Bdf1 and Bdf2) and measuring changes in nascent transcription, we found that the Bdfs have a critical role in genome-wide transcription for most genes. At many genes, the transcription defect upon Bdf depletion is greater than is found upon TFIID depletion.  We found that the Bdfs modulate preinitiation complex formation through recruitment of TFIID, Mediator and basal transcription factors to chromatin. Surprisingly, we discovered that hypersensitivity of genes to Bdf1/2 depletion results from combined defects in transcription initiation and early elongation, a striking functional similarity to human BET proteins, most notably Brd4. Our results have established Bdf1/2 as critical for yeast transcription and provide important mechanistic insights into the function of BET proteins in all eukaryotes.


Related Recent Publications from the Hahn Laboratory

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 Nat Commun. 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 9, e50109.  doi: 10.7554/eLife.50109. PMID: 31913117

Donczew, R., and Hahn, S. (2018). Mechanistic Differences in Transcription Initiation at TATA-Less and TATA-Containing Promoters. Molecular and Cellular Biology Dec 13;38(1). pii: e00448-17. doi: 10.1128/MCB.00448-17. PMID: 29038161.

Pacheco, D*., Warfield, L*., Brajcich, M., Robbins, H., Luo, J., Ranish, J., and Hahn, S. (2018). Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator. Molecular and Cellular Biology Apr 30;38(10). pii: e00038-18. doi: 10.1128/MCB.00038-18. PMID: 29507182

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.

Warfield, L., Tuttle, L.M., Pacheco, D., Klevit, R.E., and Hahn, S. (2014). A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface. Proceedings of the National Academy of Sciences 111, E3506-13.

Brzovic, P.S., Heikaus, C.C., Kisselev, L., Vernon, R., Herbig, E., Pacheco, D., Warfield, L., Littlefield, P., Baker, D., Klevit, R.E., and S. Hahn. (2011). The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex. Molecular Cell 44, 942–953.