Trainee talk abstracts

Session 1 

1. TATA box binding protein core domain dictates its evolutionarily conserved function

Hazel Cui, University of British Columbia - Sheila Teves Lab

Cui, J.H.*; Teves, S..

The TATA box-binding protein (TBP) is a highly conserved basal transcription factor essential for the assembly of the pre-initiation complex across all three eukaryotic RNA polymerases (Pol I, II, and III). TBPs and their paralogs share a conserved C-terminal DNA-binding domain and possess divergent N-terminal domains (NTDs), whose functional roles remain incompletely understood. To dissect the contributions of these domains, we investigated the functionality of chimeric yeast TBPs containing varying NTD lengths, as well as murine TBP and its paralogs, in complementing TBP depletion in Saccharomyces cerevisiae. Despite strong conservation in the DNA-binding domain, none of the homologs fully restored viability in TBP-deficient yeast, correlating with their inability to sustain transcription by RNA Pol II and III. Moreover, chimeric yeast TBPs differentially support stress-induced transcription reprogramming, implicating the NTD in modulating transcriptional adaptability. Collectively, our findings highlight the dual necessity of structural conservation and domain-specific diversity in TBP function, emphasizing the balance between essential transcriptional roles and regulatory flexibility among eukaryotic TBP homologs.

2. A high-throughput platform for transcription factor characterization in yeast

Omar Tariq, University of British Columbia - Carl de Boer Lab

Tariq, O.*; Cheney, W.; de Boer, C.

Transcription factors (TFs) are DNA-binding proteins which act as the central regulators of gene expression. TFs function by recognizing a short sequence motif (a group of similar sequences) and binding it to activate or repress the expression of nearby target genes. Over the years, several methods have been developed for characterizing TF binding genome-wide (ex. ChIP-seq) or against a large pool of synthetic sequences (ex. HT-SELEX). However, these methods are sub-optimal for profiling many TFs in a single experiment. We have developed the TF-GPRA (Transcription Factor Gigantically Parallel Reporter Assay) to learn binding for many TFs in parallel. The TF-GPRA involves exogenously expressing eukaryotic TFs in yeast, along with a library of randomized promoter sequences controlling the expression of a fluorescent reporter. By sorting cells according to reporter expression, we can develop a tripartite TF-Sequence-Expression dataset for training machine learning models. These models can then learn motifs for both yeast and human TFs. This approach could be expanded to studying TF variants, isoforms or TFs from a poorly studied genome.

3. How natural variation in transcript properties modulates mRNA and protein levels in budding yeast

Nadine Tietz, Western Washington University - Dan Pollard Lab

Tietz, N.*; Pollard, D.

Natural variation in gene expression drives trait differences in organisms, yet the genetic mechanisms underlying this variation remain poorly understood. Polymorphisms that modify codon bias, mRNA folding strength, transcription elongation rate, and amino acid properties such as charge and weight influence gene expression, with their effects potentially depending on their position within the transcript. Despite their potential role in shaping natural trait variation, systematic studies on how these transcript properties relate to gene expression in Saccharomyces cerevisiae are limited. Our study utilizes genomic, transcriptomic, and proteomic data for 22 S. cerevisiae isolates for 1,447 genes. For each gene, we calculated across-isolate changes for the five transcript properties and estimated mRNA transcript abundance, protein abundance, and translational efficiency. We then built linear mixed-effects models associating allelic variation in these transcript properties with allelic variation for our 3 expression levels.  We found that mRNA folding strength, codon bias, and AA weight have strong effects and act on all three levels of expression. Codon bias and AA weight have consistent effects across expression levels, however, mRNA folding strength negatively influences transcript abundance while positively influencing translational efficiency and protein abundance. Transcription elongation rate and AA charge have weaker effects and only associate with transcript abundance. Codon bias, AA charge, and AA weight showed independent and synergistic effects with mRNA folding strength, particularly on translational efficiency. The impact of polymorphism location varied: codon bias acted mainly through domain-encoding and 3′ coding regions, while mRNA folding strength effects were concentrated around start/stop codons and within coding regions. Our results provide a comprehensive view of how transcript-level polymorphisms influence gene expression in yeast. These insights have important implications for pharmaceutical companies developing mRNA vaccines, as optimizing mRNA to enhance protein production could improve vaccine efficacy.

4. Regulation of chromatin structure and transcription by yeast general regulatory factors

Anuradha Venkatramani, Fred Hutchinson Cancer Centre - Steve Hahn Lab

Venkatramani, A.*, Mahendrawada, L. and Hahn, S.

Yeast general regulatory factors (GRFs; Abf1, Rap1, Reb1, Cbf1) are pioneer-like transcription factors that can invade nucleosomal DNA, leading to the formation and maintenance of nucleosome-depleted regions. A recent study (Mahendrawada et al) mapped the genome-wide binding and regulatory targets for most yeast transcription factors, revealing an unexpectedly low overlap between transcription factor binding and target gene regulation. Because GRFs are expected to directly bind gene regulatory regions, we investigated the overlap between GRF-regulated genes, genes with detectable GRF-DNA binding, and genes where nucleosome positioning is dependent on the GRFs. GRFs were rapidly depleted in yeast using the auxin-inducible degron system, and nucleosome position and occupancy was mapped genome-wide using H3 ChIP-seq and MNase-seq. We found that only a subset of GRF-regulated genes (9-30%) respond to rapid depletion of each GRF by increasing nucleosome occupancy at the promoter region. Most of these genes are highly downregulated following GRF depletion, suggesting that GRFs regulate these genes through organization of chromatin structure.  Surprisingly, ~50% of this gene set is not detectably bound by the respective GRFs. Possible explanations include GRFs acting at some genes from low occupancy sites or functional binding sites lying outside the linked regulatory regions. Additionally, many genes bind GRFs but show no apparent changes in chromatin structure or transcription upon GRF depletion. This may suggest redundant function of the GRFs or other chromatin remodelers at these genes. Finally, another set of GRF-regulated genes do not show obvious chromatin changes upon GRF depletion suggesting that GRFs regulate these genes by other mechanisms. Future studies aim to understand mechanisms used by the GRFs to shape chromatin structure and transcription. The findings of this study will enhance our understanding of the role of pioneer-like factors in gene regulation.

Session 3

5. Experimental evolution reveals new drivers of freeze-thaw tolerance in Saccharomyces cerevisiae

Leah Anderson, University of Washington - Maitreya Dunham Lab

Anderson, L.*; Miksovsky, L.; Dunham, M.

Although freezing and thawing is commonplace for Saccharomyces cerevisiae in both laboratory and natural environments, the cellular mechanisms underlying freeze-thaw tolerance remain understudied. The goal of my project is to identify genetic factors that contribute to freeze-thaw tolerance and functionally characterize the variation at these loci. To investigate this, I performed experimental evolution by subjecting initially clonal yeast populations to repeated cycles of freezing and thawing. After 20 cycles of freeze-thaw-regrowth, increased freeze-thaw tolerance was observed in 34 independent populations. Whole-genome sequencing of the evolved populations revealed SNPs, copy number variations, and transposon insertions that differ from the ancestral genome. Several likely loss-of-function mutations were identified in the serine proteases KEX1 and KEX2. Mutations also emerged in the sphingolipid biosynthesis gene ELO3 and the proton pump gene PMA1, which likely cause altered gene functions. Supporting this, PMA1 is essential and unlikely to tolerate complete loss-of-function mutations, and deletion of ELO3 in the ancestral background does not confer increased freeze-thaw tolerance. In contrast, deletion of KEX1 or KEX2 in the reference strain S288C was sufficient to increase freeze-thaw tolerance, validating their role in the phenotype. These findings reveal new genetic determinants of freeze-thaw tolerance including genes not previously associated with this stress. Interestingly, prior research has identified genetic interactions among KEX2, PMA1, and ELO3, suggesting the possible role of a gene network in regulating the freeze-thaw response. Building on these insights, my next steps are to clarify how these genes interact functionally and whether they act in coordination to shape the cellular response to freeze-thaw stress. 

6. Profiling the fitness of wild & domestic Saccharomyces cerevisiae strains in response to cytotoxic compounds via CRISPR-Cas9 barcoding

Jackson Moore, University of British Columbia - Vivien Measday Lab

Moore, J.*; Barazendeh, M.; Nislow, C.; Measday, V.

The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is a powerful model system for eukaryotic biology. Experimental data is often restricted however to a limited set of domesticated laboratory strains, typically derived from S288c. S288c is a phenotypic outlier when compared to the species as a whole and is not biologically representative of industrial or wild strains. Here we describe a genetically diverse collection of 265 barcoded S. cerevisiae strains, representing multiple phylogenetic clades, that enable pooled competitive fitness assays. Using this collection, we examined strain fitness along evolutionary and phylogenetic lines in response to 33 cytotoxic drugs and identified novel genetic associations with drug resistance and sensitivity. The collection included strains we isolated from British Columbian and Californian wine regions, representing three distinct clades: domesticated Wine/European, wild Transpacific Oak, and admixed Pacific West Coast Wine. Marker-less genetic barcodes were introduced into the genomes of strains by targeting the HO endonuclease locus via CRISPR-Cas9 and replacing the gene with single-stranded oligonucleotide donor DNA (ssODN) consisting of a 20-nucleotide barcode and two short 40-nucleotide homology arms. High-throughput drug screening was then performed using pooled bar-seq assays in response to antineoplastic and antifungal compounds targeting multiple biological pathways. Genome-Wide Association analysis identified 625 non-S288c alleles linked to compound resistance or sensitivity, including missense mutations in Pol3 (DNA polymerase delta, mechlorethamine resistance), and Yrr1 (multi-drug resistance transcription factor, nocodazole resistance). This study highlights the power of natural variation in uncovering genetic mechanisms of drug resistance and sensitivity, with implications for synthetic lethality strategies and resistance pathways.

7. Mining yeast diversity for recombinant production goals

Ryan Wong, University of British Columbia - Thibault Mayor Lab

Wong, R.W.K.*; Foo, M.; Lay, J.R.S.; Wai, T.L.T.; Moore, J.; Dutreux, F.; Molzahn, C.; Nislow, C.; Measday, V.; Schacherer, J.; Mayor, T.

The budding yeast Saccharomyces cerevisiae is a widely utilized host cell for recombinant protein production due to its well studied and annotated genome, its ability to secrete large and post-translationally modified proteins, fast growth and cost-effective culturing. However, recombinant protein yields from S. cerevisiae often fall behind that of other host systems. To address this, we developed a high-throughput screen of wild, industrial and laboratory S. cerevisiae isolates to identify strains with a natural propensity for greater recombinant protein production, specifically focussing on laccase multicopper oxidases from the fungi Trametes trogii and Myceliophthora thermophila. Using this method, we identified 20 non-laboratory strains with higher capacity to produce active laccase. Interestingly, lower levels of laccase mRNA were measured in most cases, indicating that the drivers of elevated protein production capacity lie beyond the regulation of recombinant gene expression. We characterized the identified strains using complementary genomic and proteomic approaches to reveal several potential pathways driving the improved expression phenotype. Gene ontology analysis suggests broad changes in cellular metabolism, specifically in genes/proteins involved in carbohydrate catabolism, thiamine biosynthesis, transmembrane transport and vacuolar degradation. Targeted deletions of the hexose transporter HXT11 and the Coat protein complex II interacting paralogs PRM8 and 9, involved in ER to Golgi transport, resulted in significantly improved laccase production from the S288C laboratory strain. Whereas the deletion of the Hsp110 SSE1 gene, guided by our proteomic analysis, also led to higher laccase activity, we did not observe major changes of the protein homeostasis network within the strains with higher laccase activity. This study opens new avenues to leverage the vast diversity of Saccharomyces cerevisiae for recombinant protein production, as well as offers new strategies and insights to enhance recombinant protein yields of current strains.

8. Engineering stress-resistant yeast with tardigrade damage suppressor gene (Dsup): genomics and large-scale phenotyping to elucidate function

Hamid Kian Gaikani, University of British Columbia - Corey Nislow Lab

Gaikani, H.K.*; Barazandeh, M.; Campbel, E.; Hitchens, L.; Giaever, G.; Nislow, C.

Tardigrades are well-known for their extraordinary resilience. They are capable of surviving desiccation, extreme temperatures, osmotic stress, and massive doses of radiation. Several proteins have been implicated in tardigrade stress resistance, including the Damage suppressor (Dsup) protein, which has been shown to offer protection against DNA damage when expressed in human cells. The mechanism that underlies this protection is, however, not well established, and in fact, exogenous expression of Dsup appears to be deleterious to certain normal cells. Here, we investigate the function of Dsup in Saccharomyces cerevisiae. We designed and tested multiple Dsup expression strategies, including episomal plasmids and chromosomal integrations with diverse constitutive and inducible promoters. We demonstrate that Dsup enhances resistance to oxidative stress in yeast, although its expression is accompanied by an evident fitness cost. Using a DNA damage response marker (RNR3), we show that Dsup expression reduces the DNA damage response following exposure to the alkylating agent MMS. To ask how exogenous Dsup interacts with the yeast genome to confer stress resistance, we used SGA technology to introduce Dsup into ˜4000 haploid deletion mutants and performed pooled Bar-seq screens. To probe how Dsup might act at the level of the genome and transcriptome, we used MNase-seq to investigate chromatin architecture and RNA-seq to examine transcriptomic alterations. In parallel, we are expressing additional tardigrade candidate genes (e.g. Tdr1) using this approach. This work establishes yeast as a functional platform for expressing and dissecting stress-resistance genes from extremophiles, enabling the study of gene–environment (G´E) interactions beyond native biological contexts.

Session 5

9. Using yeast to bring research into the teaching lab: A yeast CRISPR CURE for biochemistry, geroscience, and other fields

Kristen Mittl, Western University of Health Sciences - Brian Wasko Lab

Mittl, K.*; Wasko, B.

Research experiences are associated with numerous positive outcomes for students, yet traditional teaching labs often rely on predetermined experiments that lack authentic inquiry. A key challenge in expanding access to research is scalability, as there aren’t enough spots in individual faculty labs to accommodate all interested students. Course-based Undergraduate Research Experiences (CUREs) address these issues by embedding genuine research into laboratory course curriculum. We initially aimed to develop a cost-effective CURE that uses CRISPR, maintains a connection to human health, and is well-suited for a biochemistry laboratory setting. Building on this initial yeast CRISPR CURE framework, we are designing a related project focused on the biology of aging (Geroscience). This project centers on a drug rapamycin that is known to extend longevity from yeast to primates by binding to FKBP12/Fpr1 and inhibiting mTOR, a protein kinase that functions as a nutrient responsive regulator of cell growth. Recognizing that genotype-dependent responses may influence the efficacy of rapamycin, our CURE is designed to have students identify candidate human missense mutations in FKBP12 and mTOR using publicly available databases. In a proof-of-principle experiment, we selected human single nucleotide polymorphisms (SNPs) that are within residues conserved in yeast, located within 5 Å of rapamycin in crystal structure data, and are predicted to be deleterious by the machine learning algorithm AlphaMissense. Our results show that incorporating an FKBP12 allele from a human SNP database into the yeast homolog (Fpr1-F43S) confers strong resistance to rapamycin-induced growth inhibition, demonstrating support that human SNPs can modulate the activity of rapamycin. This project also highlights that the yeast CRISPR CURE developed is a dynamic and modular platform that can be adapted to promote various fields, such as Geroscience, to enhance the research training pipeline.

Session 6

10. Concurrent detection of chemically modified bases in yeast mitochondrial tRNAs by Nanopore direct RNA sequencing

Julia Reinsch, University of Oregon - David Garcia Lab

Reinsch, J.L.*; Garcia, D.M.

The mitochondrial genome of Saccharomyces cerevisiae encodes 24 transfer RNAs (tRNAs) which are essential for mitochondrial translation and respiration. Mitochondrial tRNAs (mt-tRNAs) are chemically modified by nuclear-encoded enzymes that are imported into the organelle; these modifications are important for tRNA structure, stability, and decoding during translation. Some mutations in enzymes that modify mt-tRNAs are associated with severe mitochondrial diseases in humans. Saccharomyces cerevisiae has been used to model the impact of these mutations due to enzyme conservation and relative ease of phenotyping mitochondrial defects. Yet the mature sequences of all yeast mt-tRNAs, including their myriad of chemically modified bases, have not been sequenced in their entirety. We used Nanopore direct RNA-sequencing (DRS) combined with mitochondrial enrichment and enzyme knockouts to map base modifications across 24 mt-tRNA isoacceptors. Analysis of wild-type yeast revealed modification signals that were coincident with known modification sites at annotated positions, as well as at unknown, previously undescribed sites. We found that the loss of certain modifications led to an increase in putative modification levels at other sites, providing new evidence for interactions between these chemical modifications and other modification enzymes. The loss of the modification pseudouridine at U28—catalyzed by Pus2—in  one mt-tRNA led to a putative increase in modification levels in the anticodon, which may directly impact decoding. Mutations that reduce catalytic activity of Pus2’s human ortholog, PUS1, are associated with the disease Mitochondrial Myopathy and Sideroblastic Anemia (MLASA). These results suggest that disruption of individual mt-tRNA modification enzymes can lead to pleiotropic consequences, causing further dysregulation of subsequently added modifications that may influence the disease mechanism. Overall, we demonstrated that DRS is a sensitive method for profiling most mt-tRNA modifications, we expanded the list of known modifications present in yeast mt-tRNA, and we discovered new interactions between mt-tRNA modification sites.

11. Uncovering the functional impact of missense mutations proteome-wide using mistranslation and mass spectrometry

Matthew Berg, University of Washington - Judith Villen Lab

Berg, M.D.*; Chang, A.; Hess, K.; Rodriguez-Mias, R.A.; Villen, J.

DNA sequencing has identified millions of natural genetic variants that alter protein sequence. However, determining the functional impact of these variants remains challenging. Traditional mutagenesis approaches are not scalable for millions of variants and high-throughput approaches such as deep mutational scanning are limited to investigating one protein per experiment. Recently, our lab established Miro – a high-throughput proteomic approach to functionally annotate the impact of missense mutations across entire proteomes. In this approach, stochastic errors in protein synthesis are induced to create amino acid substitutions throughout all expressed proteins within a cell. Biochemical selections that probe general protein properties like solubility, thermal stability, ligand binding, protein-protein interactions and post-translational modifications are then applied to the collection of protein variants. After selection, variants are quantified by mass spectrometry to determine the functional impact of each mutation on the measured property. Here, we harness a collection of tRNAs that we engineered to mis-incorporate alanine at non-alanine codons and determine the impact of over 30,000 alanine substitutions on the thermal stability of more than 1500 yeast proteins. Using this data, we uncover functionally important residues and protein regions, including those involved in ligand binding and protein-protein interactions. Our work represents the first proteome-wide alanine scan and provides insight into various aspects of protein biology including the structural and functional context underlying mutational sensitivity.

12. Understanding the mechanisms of protein sequestration to the Intranuclear Quality Control site in Saccharomyces cerevisiae

Ryan Campbell, University of British Columbia - Peter Stirling Lab

Campbell, R.*; Kumar, A.; Stirling, P.

The fitness of a cell is best reflected by the state of the proteome, as the proteins are the workforce of the cell. When there are outside stressors that disrupt the proteins in the cell, they must enlist a multifaceted response to control the damage as otherwise they risk cell death. When proteins get disrupted, they can misfold and accumulate into protein aggregates, which are known to be involved in common diseases such as Alzheimer's and Parkinson's. Protein Quality Control (PQC) pathways are important in this regard, as they help remodel the proteome by refolding, disaggregating, and degrading misfolded proteins. One aspect of PQC that is underexplored is sequestering of the misfolded proteins to various cellular inclusions. Interestingly, yeast cells under various forms of stress will form a structure within the nucleus called the INtranuclear Quality control site (INQ) which also hosts various endogenous PQC machinery that is involved in various forms of DNA damage repair. Sequestration to INQ is promoted by molecular chaperones Hsp42 and Btn2 under DNA damage. Our preliminary data has also shown that yeast strains with deletions of either Hsp42 or Btn2 cannot form INQ under stress conditions. My goal with this project is to characterize the pathways that Hsp42 and Btn2 act upon, as well as identifying and understanding the PQC machinery that is sequestered to INQ. I hypothesize that nuclear protein sequestration is an adaptive mechanism to maintain cellular fitness during DNA damage across species. Additionally, I aim to identify the analogous pathway for INQ sequestration in human cells, as little is known about nuclear sequestration sites in humans. Further insight into these mechanisms could clarify the questions surrounding these debilitating diseases.

13. A Rab5-GEF and a regulatory site in the GAP adaptor BLOC coordinate isoform-specific Rab5 inactivation

Mia Frier, University of British Columbia - Elizabeth Conibear Lab

Frier, M.*; Shortill, S.; Davey, M.; Conibear, E.

Rab5-family GTPases establish endosomal membrane identity and function, and their subsequent inactivation is necessary for endosome maturation and vacuolar fusion. Both yeast and humans express multiple Rab5 family proteins and diverse VPS9-family GEFs that may couple Rab5 activation to different endosomal processes. For example, the GEF VARP associates with human retromer to promote protein recycling from endosomes. However, it is not known if each Rab5-family GTPase is regulated differently during endosome maturation, or what roles VPS9 GEFs may play in differential control of Rab5 isoforms. We recently described VINE, a VARP homolog in S. cerevisiae which is mutated and absent from commonly studied laboratory strains. Surprisingly given its role as a VPS9-GEF complex, we found that VINE suppresses the function of the yeast Rab5 homolog Vps21 but not the related Rab5-like GTPase Ypt52. This negative regulation did not require VINE’s GEF activity and was instead mediated by a site on its ankyrin repeat-containing domain (AnkRD). Using a genome-wide screen comparing proximity interactors of wild-type and mutant VINE, together with AlphaFold modeling, we identified yeast protein phosphatase 1 (PP1) as a potential binding partner for the VINE AnkRD. Because VINE specifically inactivated Vps21 and not the other yeast Rab5 homologs, we hypothesized that the VINE-PP1 complex might act on the Vps21-specific GAP Msb3 or its adaptor, BLOC. Indeed, we identified a disordered region in BLOC that mediated VINE’s effect on Vps21. Our data suggest that dephosphorylation of this regulatory site allows it to interact with Msb3, promoting GAP activity at endosomes which inactivates Vps21. Our data are consistent with a model in which selective Rab inactivation during membrane maturation reinforces the boundary between Rab5- and Rab7-marked membrane domains. These findings reveal a novel pathway for endosomal activation of a Rab5-GAP and highlight distinct regulatory mechanisms that control specific Rab5-family GTPases.