Poster abstracts

POSTER SESSION 1, LAST NAME A-H

1. Direct mRNA sequencing of aged yeast reveals alterations in pseudouridylation, polyadenylation, and gene expression

Sara Alladin, University of Oregon - David Garcia Lab

Alladin, S.; Shaw, E.A.; Garcia, D.M.

Epitranscriptomics, Aging, Polyadenylation, Gene Expression

Aging is a complex process that involves many changes to cellular homeostasis, sometimes resulting from disruptions in transcriptional and post-transcriptional regulation. RNA modifications and RNA processing steps may contribute to age-associated transcriptome changes. We used Nanopore direct RNA sequencing (DRS) to profile pseudouridylation, polyadenylation, and differential gene expression in aging Saccharomyces cerevisiae. We compared transcriptomes from the mid-logarithmic phase, saturated, and aged yeast cells to measure whether these features changed. Our analysis identified 11 conserved pseudouridylated sites in the young and aged yeast mRNA with varying levels of pseudouridylation. One example was the mRNA TEF1 (encoding the eukaryotic translation elongation EF-Tu factor), which had reduced levels of an ORF pseudouridine site as yeast aged. Pseudouridylated sites unique to younger yeast were found in mRNAs related to cell growth, including amino acid biosynthesis, oxidative stress response, mitochondrial function, and translation initiation. We also identified pseudouridine sites found in saturated and aged but not young yeast. We observed a global shift in the polyadenylation landscape between young and old yeast, with poly(A) tails being longer in saturated and aged yeast (median = ~35nt and ~31nt, respectively) compared to the younger yeast (median = ~27nt). These changes occurred in the background of other changes to gene expression. This analysis revealed hundreds of genes up or downregulated in young vs. older yeast, including those encoding stationary phase survival components, DNA replication processes and glycolysis. Several snoRNAs were also downregulated in older yeast. In summary, our analysis of the aged yeast transcriptome revealed changes in pseudouridylation and poly(A) tail length, in addition to gene expression. These findings expand our understanding of whether RNA modifications and processing are affected in aging.

2. Exploring the Impact of H2A.Z Depletion on Nascent Transcription Regulation

Eully Ao, University of British Columbia - Michael Kobor Lab

Ao, E.*; Brewis, H.T.; Kobor, M.S.

Yeast, H2A.Z, nascent transcription, depletion system

H2A.Z is an evolutionarily conserved histone variant. In S. cerevisiae, it is incorporated into chromatin by the SWR1 chromatin-remodeling complex. While implicated in transcriptional regulation, among many other processes, H2AZ’s exact role in this process is still debated as gene expression does not appear to be correlated with its localization at gene promoters. The limited transcriptional changes observed may be due to experimental design. Previous studies utilized 1) H2A.Z knockout models; 2) total cellular RNA; and 3) nutrient-rich conditions. However, these may be limitations because these studies may be overlooking 1) compensatory mechanisms; 2) exclusive transcriptional activity; and 3) H2A.Z's potential role during cellular stress, particularly in priming genes for transcription in specific environmental conditions. To address these limitations, I propose an alternative approach to reveal the immediate effects of H2A.Z loss and uncover previously undetected changes in gene expression to explore H2A.Z’s role in transcriptional regulation. I will be 1) using the Anchor-away system to deplete H2A.Z; 2) measuring nascent transcription with SLAM-seq (thiol(SH)-linked alkylation for the metabolic sequencing of RNA); and 3) inducing cellular stress with caffeine. These analyses aim to identify the timing and extent of transcriptome alterations post-H2A.Z depletion, discern differences between bulk RNA and nascent RNA readouts, and analyse responses transcriptome-wide to induced stress upon H2A.Z depletion. Overall, this project will dissect the role of H2A.Z in nascent transcriptional regulation, particularly under stress conditions, contributing to our understanding of its impact on eukaryotic cellular processes and helping resolve the debate surrounding H2A.Z’s role in transcription regulation.

3. yEvo: Leveraging a high school teaching lab to study antifungal resistance mutations

Randi Avery, University of Washington - Maitreya Dunham Lab

Anderson, L.; Geck, R.; Armstrong, J.; Avery, R.; Schoch, J.; Wang, V.; Zeng, Z.; Gorjifard, S.; UW Genome Sciences Hackathon Team; Dunham, M’

Experimental evolution, education, mutation browser, antifungal resistance

Evolution is a topic that is particularly difficult for high school students to understand, with many common misconceptions continuing into the general population. With today’s political climate, it is increasingly important to properly teach high school students evolution in an accessible manner, as programs serving underrepresented students are likely to decrease. Toward this goal, the Dunham lab has developed a curriculum implementing a hands-on approach where students can see evolution in real-time using a yeast experimental evolution and genomics lab named “yEvo.” Briefly, students passage eight ancestral strains of Saccharomyces cerevisiae, which have been engineered to express various color phenotypes, in increasing concentrations of an antifungal drug. Since its inception, yEvo has been performed in dozens of classrooms and hundreds of students have studied both the azole and echinocandin classes of antifungal drugs. Evolved strains that show an antifungal-resistant phenotype compared to their ancestors are whole-genome sequenced by the Dunham lab. We then return data on the de novo mutations in the students’ evolved strains to the students via an original Mutation Browser website (https://yevo.org/mutation-browser/). The students can view the types of mutations found, which genes developed mutations, and the functions of those genes. We in the Dunham lab then leverage the data on recurring mutations to draw conclusions about antifungal resistance, as those mutations are robust to batch effects due to the various approaches the different high school classes take. Not only is yEvo beneficial for educating the next generation of adults, it also provides us with a high-throughput approach for elucidating antifungal resistance mutations.

4. Contribution of Adaptor Protein 1 (AP-1) Complex to Cryptococcal Disease

Kabir Bhalla, University of British Columbia - James Kronstad Lab

Leon-Hing, E.; Bhalla, K.*; Huang, Y-H.; French, V.; Beskrovnaya, P.; Lagace, M.; Neogi, A.; Foster, E.; Tocheva, E.; Johnson, P.; Kronstad, J.

Cryptococcosis, fungi, immune response, adaptor proteins, AP-1 complex, macrophages

Cryptococcus neoformans, one of the highest-ranked ‘critical’ fungal pathogens on the World Health Organization’s (WHO) Fungal Priority Pathogens List (FPPL), primarily impacts individuals with compromised immune systems. Due to its high antifungal resistance and mortality rates ranging from 41% to 61%, there is a growing need for improved therapies and interventions. With its unique arsenal of complex virulence factors such as the polysaccharide capsule and melanin production, this fungal pathogen continues to remain the most common cause of fungal meningitis. Adaptor protein (AP) complexes have crucial roles in endocytic and secretory pathways, being involved in vesicular trafficking to maintain the physiological functions of eukaryotic cells. We have shown that the AP-1 complex is required for the elaboration of virulence factors, including the production of melanin and urease, and for virulence in the murine inhalational model of cryptococcosis. Cells deficient in AP-1 complex subunits exhibit abnormal cellular morphology, particularly at elevated temperatures and in the macrophage phagolysosome, and have an altered cell surface architecture. Moreover, we have highlighted the role of these proteins in the trafficking of polyphosphate (polyP).

5. Investigating a prion form of a yeast mRNA capping enzyme

Preeti Bhattacharjee, University of Oregon - David Garcia Lab

Bhattacharjee, P.*, Capage, M., Evarts, J., Garcia, D.M.

Prion, ABD1, mRNA, capping, epigenetic

Prions are altered conformations of proteins that can affect physiological function and are inherited through a self-templating mechanism, constituting a unique form of epigenetic inheritance that is widespread across all domains of life, including mammals, plants, fungi, bacteria, and viruses. Prions in budding yeast can confer adaptive advantages in environmental stress. Of particular interest to our lab are prions that may regulate RNA. Through a large phenotypic screen in budding yeast, we discovered prion-like states of several RNA-modifying enzymes. Among these were ABD1 and CET1, which are novel examples of prion-like forms of essential genes required for 5 ́ capping of mRNA, a function that is conserved across all eukaryotes. The prion-like states of Abd1 and Cet1 enable resistance to translation inhibition by cycloheximide. This trait lasts through hundreds of generations of yeast outgrowth, shows dominant inheritance through mitosis and meiosis, and is dependent on the chaperone Hsp70. We excluded genetic effects on prion-based phenotypes through whole genome sequencing. Data on chaperone dependence suggest that prion-like Abd1 and Cet1 do not form amyloids like the majority of known prions. This raises questions about the biophysical state of these prions and the consequence for their canonical function. The Abd1 prion confers resistance to growth inhibition by a catalytic inhibitor, suggesting that the protein might have an altered structure in its prion-like state, and/or altered catalytic activity. Intrinsic disorder is a hallmark of prions – however, the disordered N-terminus of Abd1 is dispensable for its prion phenotype. In ongoing work, we explore additional sequence features that are important for the prion phenotypes, and are using microscopy to visualize subcellular localization of the Abd1 in naïve and prion strains. Using whole cell quantitative proteomics, we hope to gain a molecular and systems-level understanding of how the Abd1 prion leads to the observed growth phenotypes.

6. Experimental Techniques for Quantifying mRNA Folding Strength’s Impacts on Translation in Saccharomyces cerevisiae

Max Cannata, Western Washington University - Dan Pollard Lab

Cannata, M* Geels, A* Pollard, D

mRNA, transcripts, translation, ribosomes, secondary structure, protein, synthesis

Messenger RNA’s function in the cell is to deliver protein information from nuclear DNA to cytoplasmic ribosomes, where codons are translated into their corresponding amino acids during protein synthesis. Free mRNAs fold  on themselves and undergo intramolecular base pairing to achieve a secondary structure which is more thermodynamically stable than when they are linearized. During translation, these secondary structures must be unwound by ribosomes traversing mRNA transcripts. It is therefore hypothesized that the secondary structure of mRNAs can impact protein synthesis. Biophysical experiments indicate that higher levels of structure in mRNA transcripts slows down ribosomes; however, comparative studies of secondary structure and protein abundance across genes and alleles suggest a positive correlation between the two. One hypothesis explaining the mechanism of this effect is that a higher secondary structure results in higher ribosome density, which reduces opportunities for transcripts to refold between ribosomes. This “refolding avoidance” would result in less secondary structure in polysomes, which in turn increases translational efficiency. Experimental measurements of mRNA folding strength (mF)  both before and after translation starts are not available. This project proposes a method to collect measurements of mF before and during ribosomal association, which will allow experimental investigation of the refolding avoidance model. More broadly, these data will help clarify the relationship between mRNA structure and protein synthesis, with implications for understanding translational regulation. Measurements of mF will be assessed via the psoralen-TEG azide crosslinking of RNA molecules in Saccharomyces cerevisiae to maintain their secondary structures. By preserving mRNA regions with structure, chimeric transcripts indicating the location of RNA-RNA hybridization can be created and sequenced. Through computational methods, these experimentally collected hybridization sites can be mapped to the transcripts sequencing during structure prediction.

7. Quantifying effects of natural genetic variation on lifespan of Saccharomyces cerevisiae isolates

Aidan Corbin, Western Washingotn University - Dan Pollard Lab

Corbin, A.*; Wolin, K.; Boggan, L.

Aging

Understanding the genetic basis of natural variation in aging remains a central challenge in biology. This study aims to characterize the genetic networks underlying variation in replicative lifespan (RLS) in Saccharomyces cerevisiae. A major obstacle is that aged mother cells constitute only a small fraction of a growing culture, complicating large-scale genetic analysis. To overcome this, we utilize miniature aging chemostat devices (MADs), which magnetically retain mother cells and continuously remove daughter cells, enabling long-term growth and analysis of aging populations. Preliminary experiments using MADs to characterize RLS in the S288c and YJM145 strain backgrounds demonstrate that viable and nonviable aged cells can be clearly distinguished by flow cytometry. These assays reveal strain-specific differences in the proportion of viable aged cells, supporting the presence of underlying genetic variation in aging phenotypes. Building on these results, we are employing a QTL mapping approach known as FACS-BSAseq, which uses fluorescent markers to track aging-related traits in large segregant populations. By integrating high-throughput phenotyping with bulk segregant sequencing, this work aims to identify genomic regions and regulatory networks associated with both RLS and aberrant gene expression. These insights will advance our understanding of the molecular determinants of longevity and help define genetic targets for future aging research.

8. Leveraging mutational screening to uncover dominant REV3 alleles as a novel cancer therapeutic

Cathy Cozma, University of British Columbia - Peter Stirling Lab

Cozma, E.*, Stirling, P.

Synthetic lethal (SL) therapies can exploit genome instability present in rapidly dividing cancer cells by targeting key DNA damage repair pathways. Clinically successful SL therapies, such as PARP inhibitors, act by ‘trapping’ the PARP protein at DNA damage sites, creating persistent cytotoxic protein-DNA lesions and limiting accessibility by alternative repair mechanisms. While PARP trapping is an effective SL therapeutic approach, it is difficult to identify de novo inhibitors with trapping properties. Cancer cells rely on error-prone mechanisms like translesion synthesis (TLS) for chemoresistance, making TLS polymerases, such as polymerase Z, high-value cancer therapeutic targets. We have engineered a high- throughput mutagenesis pipeline to identify missense variants in DNA polymerase Z (REV3) that cause dominant negative growth phenotypes. Dominant rev3 variants in the Rev7 and Pol31 interaction interfaces and N-terminal Rev3 region have been identified and characterized for altered DNA binding kinetics and protein-protein interaction strength. Dominant rev3 variants show increased replication stress phenotypes and decreased fitness in a variety of DNA damage repair deficient genetic backgrounds. Overall, the identification of druggable dominant hotspots in Rev3 may help to guide rational inhibitor development to selectively kill cancer cells dependent on TLS.

9. Investigating the molecular mechanism of the beneficial yeast prion [BIG+]

Emily Dennis, University of Oregon - David Garcia Lab

Dennis, E.*; Alladin, S.; Shaw, E.; Vaaler, A.; Bhattacharjee, P.; Garcia, D.

Prion, RNA-modifying enzyme, Translation, Non-amyloid

Prions are a specialized class of proteins that can fold into alternative conformations, potentially altering protein function. They can self-template by converting copies with the naïve (non-prion) conformation to adopt the prion conformation, modulating cellular activities epigenetically based on protein fold rather than genetic code. Historically, prions have been viewed predominantly as deadly disease agents. But prions can also have adaptive roles, sometimes benefiting organisms by responding to environmental change. One recently discovered prion in budding yeast, named [BIG+] for “better-in-growth,” is that of a highly conserved RNA-modifying enzyme, Pus4. Pus4 is a pseudouridine synthase that catalyzes a ubiquitous tRNA modification of uridine to pseudouridine at position 55 and can also modify some mRNAs. [BIG+] cells can grow faster and larger under some conditions but have shorter lifespans. They also have increased protein synthesis—albeit with reduced translation fidelity—compared to cells with naïve Pus4. This leads to a fitness advantage among fast growing cells in nutrient-rich environments. Our initial approach sought to determine if the catalytic activity of Pus4 was altered. Direct RNA sequencing of both tRNAs and mRNAs indicated no differences in pseudouridine modification between naïve and [BIG+] strains. We then wondered if other features of Pus4 were altered, such as an ability to aggregate/oligomerize. Through several biochemical approaches, including sucrose gradient density ultracentrifugation, we show that Pus4, both in the naïve and the prion state, does not form a higher-order assembly. These data suggest that [BIG+] could represent a biophysical state that is distinct from that of previously characterized amyloid-forming prions. Whole proteome mass spectrometry revealed several proteins involved in glucose-sensing whose levels were reduced in [BIG+] cells compared to naïve cells, leading to a model for how epigenetic-based alteration of metabolism can lead to altered growth and lifespan in a eukaryote.

11. Investigating the effect of long-term freezing on Saccharomyces cerevisiae Bar-seq collections

Zanny Ham, University of Washington - Maitreya Dunham Lab

Ham, Z.*, Armstrong, J., Anderson, L, Dunham, M.

Freeze-thaw, Saccharomyces cerevisiae, bar-seq

This study investigates the long-term viability and genetic stability of Saccharomyces cerevisiae stored in frozen conditions for over a decade. The primary objective was to determine whether yeast can be revived after 10 years in the freezer and to identify genotypes affected by prolonged freeze-thaw stress. The experimental plan involved comparing barcode frequencies of yeast samples from 2016 and 2025, using identical chemostat growth conditions as was done in 2016. Results indicated no significant change in barcode frequency for most samples, suggesting no obvious sensitivity or tolerance to freeze-thaw stress. However, certain genotypes exhibited higher or lower frequencies, indicating varying levels of sensitivity. Gene ontology analysis revealed that genes related to DNA repair, sporulation, cell cycle progression, osmotic stress response, and transcription initiation were affected. The study concludes that while most yeast strains remain viable after long-term freezing, specific genotypes show differential responses, warranting further investigation into the underlying mechanisms. Future steps include replicating the experiment using other yeast labs' archival collections and exploring the functions of genes with significantly different frequencies in the population after freezing for 10 years.

12. High-throughput antimicrobial peptide expression screening in Komagataella phaffii by peptide barcoding

Oliver Hong, University of British Columbia - Thibault Mayor Lab

Hong, O.*, Mayor, T.

Komagataella phaffii, Pichia pastoris, Antimicrobial peptide, peptide barcoding, expression screening, protein production

As antimicrobial resistance, a major global health threat identified by the World Health Organization continues to increase worldwide, so does the need for new treatment modalities and mechanisms of action. Antimicrobial peptides (AMPs) represent a diverse family of nature-proven bioactive molecules found in nearly all multicellular organisms, and display reduced susceptibility to resistance compared to conventional antibiotics. More than 5000 natural AMPs have currently been identified, providing a rich reservoir of antimicrobial potential. We investigate the expression of AMPs in the industrial yeast Komagataella phaffii as an alternative strategy to more costly solid phase peptide synthesis. We find AMP secretion levels to vary considerably amongst different AMPs, expression clones, and expression scale, highlighting the need for an accurate high-throughput screening method which can facilitate iterative improvement of AMP production. We establish a method to generate strain libraries expressing different AMPs and expression in a pooled culture simulating bioreactor conditions. Relative expression of different AMPs was quantified through detection of uniquely assigned tryptic peptide “barcodes” by mass spectrometry as an alternative to the direct detection of AMPs which we found to be non-quantitative. We further optimized this workflow for larger libraries by scaling down sample requirements and expanding the barcode library.

13. Regulation of Meiotic DNA Double-strand Break Formation in S. pombe

Randy Hyppa, Fred Hutchinson Cancer Center - Gerald Smith Lab

Hyppa, R.*, Smith, G.R.

Meiotic recombination, DNA double-strand breaks, Tel1 (ATM), MRN (Mre11-Rad50-Nbs1), meiotic cohesin, interference

Meiotic recombination is initiated by DNA double-strand breaks (DSBs). Factors that control the frequency of DSBs are important for successful generation of viable progeny. In many organisms the ATM (Tel1) protein kinase prevents excessive meiotic DSBs, especially nearby DSBs on the same chromatid. Normally, two close DSBs are made less often than expected from independence, a feature known as DSB interference, which is lost in a tel1 mutant. In the fission yeast Schizosaccharomyces pombe, high-level DSB formation depends on the linear element (LinE) and meiotic cohesin complexes; when either complex is impaired, Tel1 significantly represses DSB formation. In tel1 mutants, two coincident DSBs on the same DNA chromatid increase dramatically and manifest high negative interference (more double events than expected from independence) when either cohesin or LinEs are absent. Formation of crossovers and gene convertants also shows corresponding negative interference in these same mutants, evidence that DSB interference leads to genetic interference in S. pombe. The conserved MRN (Mre11-Rad50-Nbs1) complex is required for DSB repair and ATM (Tel1) activity during the mitotic DNA damage response. We found this is also true for meiotic DSBs: mre11 and rad50 mutants also lose meiotic DSB interference, like tel1, and display highly negative DSB interference in strains lacking cohesin. Additionally, we found that protein phosphatase Pph3 (PP4) antagonizes Tel1 kinase and is required for high-level DSB formation and recombination. Thus, phosphorylation by Tel1 is regulated to both promote and restrict meiotic DSB formation and, consequently, genetic recombination.

POSTER SESSION 2, LAST NAME I-X

14. Organelle-specific adaptors share an overlapping, but not identical, interface with the yeast Vps13 VAB domain

Kevin Jeffers, University of British Columbia - Elizabeth Conibear Lab

Jeffers, K.R.* , Davey, M. , Conibear, E.

Bridge-like lipid transport, membrane contact sites, protein-protein interactions

Bridge-like lipid transport proteins, including vacuolar protein sorting 13 (Vps13), directly transport lipids between organelles at membrane contact sites (MCSs). Yeast Vps13 acts at multiple sites, where organelle-specific adaptor proteins compete for Vps13 availability. Adaptors at MCSs have a conserved proline-X-proline (PxP) motif that interacts with the Vps13 adaptor-binding (VAB) domain. A structural study revealed the binding interface between VAB and the mitochondrial PxP adaptor, Mcp1, in Chaetomium thermophilum (Adlakha et al. (2022)). However, it was unclear how much of this interface was shared with the vacuole/endosome adaptor Ypt35 or the prospore membrane adaptor Spo71. We found that though the three adaptors have an overlapping core VAB-PxP interface, the VAB-Spo71 interface extends slightly farther. We demonstrate that mutations at the core interface disrupt interactions with all PxP adaptors, whereas a mutation at the unique extension between Vps13 and Spo71 reduces only their colocalization. We further demonstrate these same PxP-disrupting mutations do not necessarily affect another Vps13 function, carboxypeptidase-Y (CPY) sorting. CPY sorting is not impaired when the VAB-PxP interface is disrupted, whereas mutations at other sites in the VAB domain cause significant CPY secretion. This suggests that while several Vps13 functions are dependent on the VAB-PxP interface, others may require a different interface that still requires a functional VAB domain.

15. Moonlighting to Create the RNA Goldilocks Zone: Cbf5’s novel role in DNA Double Strand Break repair

Andrew Loe, University of Victoria - Jennifer Cobb Lab

Loe, A*. Mojumdar, A. Lunke, M. Cobb, J.

Cbf5, DNA double strand repair, DNA:RNA hybrid.

Pseudouridine (Ψ) is the most abundant RNA modification and is known to enhance transcript stability. Pseudouridine synthases, such as Cbf5 (dyskerin in humans), convert uridine to Ψ. In humans, mutations in dyskerin (DKC1) cause Dyskeratosis Congenita (DC), a premature aging disorder associated with shortened telomeres and cancer. Notably, 140+ mutations in DKC1 have been identified in sporadic cancers, many of which are not linked to DC, suggesting broader roles in genome stability. Moreover, DKC1 mutations lead to increased γ-H2AX foci and persistent DNA damage in both patient fibroblasts and mouse models, implicating dyskerin in double-strand break (DSB) repair. Here, we define a direct, telomerase-independent function for Cbf5 in DSB repair in Saccharomyces cerevisiae. Using an HO endonuclease induced DSB near the MAT1 locus, we show that Cbf5 is dynamically recruited to DSBs through the MRX complex, ongoing RNA polymerase II transcription, and intact nascent RNA. Cbf5 occupancy increases over time, peaking ~3 hours post-induction in wild-type cells; however, recruitment is accelerated in nej1Δ mutants and abolished in mre11Δ, rpb1-1, and RNase A/H–treated conditions. The catalytically inactive mutant, Cbf5R35K, fails to localize to breaks, resulting in diminished in RNA:DNA hybrids accumulation relative to wild-type near the DSB in rnh201Δ (~2-fold) and are further elevated in the cbf5R35K mutant (~2.5-fold), with an additive ~4-fold increase in the cbf5R35K rnh201Δ double mutant. Together, these findings support a model in which catalytically active Cbf5 is recruited to DSBs through a transcription-coupled, MRX-dependent mechanism. Our study begins to characterize a previously unrecognized role for Cbf5 in maintaining genome stability and identifies RNA as a key regulatory axis linking transcriptional context to the DNA damage response.

16. The effects of near identical H2A and H2B genes on Ty1 retrotransposon insertion

Jonah Lu, University of British Columbia - Vivien Measday Lab

Lu, J*. Howe, L. Measday, V.

Yeast, Retrotransposon, Ty1, Histone, H2A, H2B

Transposable elements are mobile genetic elements that can move from one region of the genome to another. The Ty1 retrotransposon is a long terminal repeat retrotransposon in Saccharomyces cerevisiae that utilizes reverse transcription of its messenger RNA (mRNA) to complementary DNA (cDNA) and Ty1-integrase to reintegrate into new regions of the genome. The structure and function of the Ty1 retrotransposon are like that of retroviruses, making Ty1 a useful model for retroviral and retroelement integration studies. Retroelements and retroviruses can target integration of their cDNA into nucleosome-occupied regions. In eukaryotes, nucleosomes contain four heterodimers of the histone proteins H2A, H2B, H3, and H4 wrapped by 1.7 turns of DNA. Ty1 integration, targeted upstream of RNA polymerase III transcribed genes, is correlated with where the DNA interfaces with the H2A-H2B dimer. Each H2A and H2B protein is produced by two different genetic loci each containing bidirectional promoters and code for slightly different histone subtypes. It is not fully understood how the H2A and H2B subtypes affect Ty1 insertion. I examined the effects of the different H2A-H2B gene loci on Ty1 integration in S. cerevisiae. Histone H2A and H2B levels were lowered by deleting both genomic copies of the H2A-H2B loci and adding a plasmid containing either the HTB1-HTA1 locus or the HTA2-HTB2 locus. Strains containing decreased histone levels stall in G2/M phase despite the lack of changes in genome-wide nucleosome phasing. Ty1 integration upstream of tGLY genes decreased in strains with a lower copy number of H2A-H2B indicating a change in Ty1 target frequency. Genome-wide Ty1 mobility increased 8.4 to 9.4-fold in strains lacking the HTB1-HTA1 locus while strains lacking the HTA2-HTB2 locus had a 2 to 3.7-fold increase in Ty1 mobility. Further work is needed to determine whether the total histone amounts, or the histone subtypes themselves influence Ty1 integration.

17. From Oak to Wine: Evolution, Genomics, and Fermentation Potential of Saccharomyces cerevisiae in North America

Alex Marr, University of British Columbia - Vivien Measday Lab

Marr, R. A.*; Moore, J.; Monpetit, R.; Monpetit, B.; Measday, V.

S. cerevisiae, genome wide association studies, high-throughput phenotyping, fermentation

Saccharomyces cerevisiae (S. cerevisiae), the yeast species commonly associated with baked goods and alcoholic beverages, exists in wild and domesticated lineages, including oak and wine-associated populations. Here, we investigate the evolutionary origins and phenotypic diversity of North American (NA) S. cerevisiae strains collected from uninoculated wine fermentations and natural oak habitats. The Measday lab’s unique strain collection includes 172 isolates from the Okanagan Valley (British Columbia) and California that were the focus of this work, including 161 strains from uninoculated wine fermentations and 11 strains from California oak trees (Quercus agrifolia). Phenotypic profiling of 272 strains (172 from this study and 100 global strains) across 86 central metabolism and environmental stress conditions identified 35 conditions that distinguished strains from the Wine/European, Pacific west coast wine and Transpacific oak clades. Fermentation trials in 1.5mL synthetic grape must showed that 248 of 272 strains fermented to dryness (<4 g/L residual sugar), as confirmed by metabolic analysis via high-performance liquid chromatography, measuring glucose, fructose, ethanol, glycerol, and organic acids. Whole genome sequencing of 97 strains in this study, combined with 175 previously sequenced strains, enabled Genome-wide association studies (GWAS). GWAS using FastLMM, analyzing 75,354 genomic variants—including 68,039 SNPs/indels, 7,103 copy number variations (CNVs), and 212 pangenomic open reading frames (ORFs)—identified 507 significant genetic associations across 93 conditions. These findings underscore the evolutionary complexity and winemaking potential of NA S. cerevisiae strains, providing valuable insights into their adaptive traits and applications in the fermentation industry. Future work will employ CRISPR-Cas9 allele swapping to functionally validate GWAS hits associated with phenotypic diversity and fermentation traits in NA strains.

18. Deciphering Transcription Factor Clustering in Yeast Using Proximity-Dependent Biotin Labeling

Saket Mishra, Fred Hutchinson Cancer Center - Steve Hahn Lab 

Mishra, S.*; Mahendrawada, L.; Hahn, S

Transcription factors (TFs), Proximity-dependent biotin labeling (TurboID), Gene regulation

The Cofactor Redundant (CR) class of genes in S. cerevisiae (yeast) represents a subset (13%) of highly regulated genes that can utilize either TFIID or SAGA to promote transcription 1. CR gene promoters interact with many transcription factors (TFs) with a median of 67 TFs detected per promoter 2. Interestingly, many TFs bind to these promoters in the absence of their DNA binding motif, raising the question of how TFs are targeted to their binding sites on chromatin. One model that can explain our observations is the so called “wolfpack model” 3 where it’s proposed that groups of TFs form dynamic clusters mediated by intrinsically disordered regions (IDRs). By this model, TF recruitment may be mediated by only a few of the TFs in the cluster that directly interact with DNA. To experimentally test whether TFs cluster in cells, we used proximity-dependent biotin labeling (TurboID) to identify protein interaction networks for TFs enriched at CR genes. In a pilot experiment, Cyc8, a broadly acting TF that regulates nearly half the protein-coding genes, was fused to TurboID. Proximity labeling identified both known and novel Cyc8-associated factors, including Tup1, general regulatory factor TFs (Reb1, Cbf1), chromatin regulators (Isw2, Bre1), initiation factor (Taf4), and elongation/termination factors (Ctk1, Rat1). These findings suggest enrichment of regulatory factors with Cyc8, which may help Cyc8 modulate many steps in the transcription process (chromatin, initiation, elongation, termination), explaining its broad role in gene regulation. Additional ongoing experiments are extending this approach to Gal4 and a curated set of 18 dual-acting TFs to map in vivo TF-TF interactions, identify key TF "scaffolds" versus "clients" in the cluster, and assess the functional consequences of disrupting TF clustering on gene regulation. Together, this study aims to uncover fundamental principles of how TFs locate their chromosomal binding sites.

19. ER-PM contact sites reinforce nuclear membrane structure and regulate mitotic nuclear division

Aleksa Nenadic, Simon Fraser University - Christopher Beh Lab

Nenadic, A.; Gurung Jason, R.B., Beh, C.T.

ESCRT III, ER-PM membrane contacts sites, closed mitosis, membrane tether proteins, CDC5, nuclear envelope ruptures, nuclear segregation, karyokinesis, nuclear ER, spindle position checkpoint (SPOC)

The endoplasmic reticulum (ER) radiates from the nuclear envelope into the cytoplasm reaching the cell cortex, where it attaches to the plasma membrane (PM) through ER-PM membrane contact sites (MCSs). As a contiguous extension of the nuclear ER, we hypothesized that cortical ER attachments to the PM at MCSs contribute to the morphology, positioning and integrity of nuclei. In yeast ∆-super-tether (∆-s-tether) cells, which lack 7 ER-PM tethering factors, cortical ER is detached from the PM. In these cells, the nuclear ER membrane is misshapen and often ruptures, requiring repair by the nuclear ESCRT-III (Endosomal Sorting Complex Required for Transport-III) complex. Without ER-PM MCSs, mitotic nuclear division and segregation is also impaired, triggering a Spindle Position Checkpoint (SPOC)-dependent cell-cycle delay and disrupting the spatiotemporal regulation of the polo-like kinase Cdc5p. Defects in nuclear membrane morphology, integrity, and mitotic nuclear segregation caused by the removal of ER-PM tethers are rescued by two independent mechanisms: restorating phospholipid metabolism or non-specific re-establishing ER-PM contact. Our findings show that ER-PM MCSs stabilize the nuclear envelope/ER membrane by maintaining its physical structure and lipid composition, which promotes Cdc5p-dependent coordination of cell and nuclear ER membrane division.

20. One CEN to rule them all: Characterization of S. cerevisiae and S. uvarum centromeric sequences to engineer the Saccharomyces panCEN

Rachel Powell, University of Washington - Maitreya Dunham  Lab

Powell, R.L.; Sanchez, M.R.; Ollodart, A.R.; Liachko, I.; Dunham, M.J.

centromere, evolution, technology development, plasmid loss rate, fiber-seq, accessibility, chromatin

Saccharomyces cerevisiae is one of the most important experimental systems for modern genomics research and biological engineering. Many applications of S. cerevisiae rely on the use of plasmids or yeast artificial chromosomes (YACs), which both require a centromere in order to promote segregation. Budding yeasts utilize “point centromeres” composed of three highly conserved centromere DNA elements in a relatively small chromosomal region. This feature makes them easy to clone onto plasmids and YACs; the most commonly used centromeric sequence for yeast expression vectors is a 125 bp fragment of S. cerevisiae CEN6, denoted ScCEN6. However, it is uncertain if ScCEN6 is the optimal sequence for high fidelity plasmid segregation, particularly when used across diverse yeast strains and species. For example, previous work demonstrates that ScCEN6 cannot effectively function in S. uvarum; ScCEN6 has a ~20% loss per generation when measured through a classic plasmid loss rate assay. Interestingly, a larger CEN fragment improves this rate to ~5% loss per generation, indicating that S. uvarum CENs may require additional flanking sequences for full functionality. To achieve the most optimized CEN across all Saccharomyces strains and species, a panCEN sequence will be engineered based on data from two major aims: 1. Determine optimal CEN sequences for S. cerevisiae and S. uvarum; and 2. Characterize chromatin accessibility for CEN sequences across different Saccharomyces to understand how CEN sequences behave in situ.

21. Endoplasmic Reticulum Function Contributes to Caspofungin Tolerance in Cryptococcus neoformans

Sabrina Xianya Qu, University of British Columbia - James Kronstad Lab

Qu, X., Hu, G. Horianopoulos, L.C., Kronstad, J.

Antifungal, Endoplasmic Reticulum (ER) stress, caspofungin tolerance, cell wall, chitin

Echinocandin drugs are antifungal agents that target the cell wall by inhibiting β-1,3-glucan synthesis. Widely used in clinical settings, echinocandins like caspofungin, micafungin, and anidulafungin are effective against Aspergillus and Candida species. However, they are not effective against Cryptococcus neoformans, the fungus responsible for life-threatening meningoencephalitis in immunocompromised individuals. Caspofungin does inhibit C. neoformans β-1,3-glucan synthase at high concentrations, but these levels are not achievable in a clinical setting. The mechanisms leading to echinocandin tolerance in C. neoformans are emerging and are associated with calcineurin signaling, and cell wall and membrane composition. The synergistic effects of caspofungin have been studied in combination with various inhibitors in C. neoformans including FK506 (calcineurin inhibitor), manumycin (to disrupt Ras pathway signaling), clorgyline (a monoamine oxidase and ABC transporter inhibitor), 2BP (a palmitoyltransferase inhibitor), and SDS (a challenge to plasma membrane integrity). One hypothesis suggests that the polysaccharide capsule or cell wall components may block permeability to caspofungin, preventing it from reaching its target enzyme. To begin to test this hypothesis, we examined the impact of other inhibitors that influence capsule.  For example, previous studies revealed that wild-type C. neoformans strains exhibit reduced capsule formation when exposed to tunicamycin, a protein glycosylation inhibitor. Our experiments revealed a synergistic effect between caspofungin and tunicamycin in sensitivity tests on wild-type strains, with a significant reduction in both capsule and chitin levels when the drugs were combined. We propose that tunicamycin disrupts capsule formation to enable caspofungin to penetrate the polysaccharide structure, target the cell wall, and inhibit growth. Our analysis offers further evidence of the endoplasmic reticulum's role in capsule formation and provides new insights into the connection between caspofungin treatment and ER function in C. neoformans.

22. Transcriptional Squelching by Overexpression of a Conserved Dimerization Motif 

Julie Ray, University of Washington - Jennifer Nemhauser Lab

Ray, J., Leydon, A., Nemhauser, J.

Transcription, Transcriptional Regulation, Synthetic Biology 

Transcriptional squelching is a general term for the redistribution of cofactors away from a promoter, causing a decrease in transcription. Squelching was initially identified in yeast when overexpression of an isolated activation domain from the Gal4 transcription factor (Gal4-AD) caused a decrease in reporter transcription (Gill and Ptashne 1988). Further analysis revealed that Gal4-AD repressed transcription by sequestering the Mediator complex away from promoters. We recently identified a protein-protein interaction motif from a plant corepressor that can interact with conserved coactivators in plant, yeast and human cells. We hypothesize that, like Gal4-AD, overexpression of this motif might squelch transcription. To test this hypothesis, we have constructed a synthetic circuit where squelching can be assayed with flow cytometry. We have found that our original dimerization motif, as well as another homologous sequence, were able to significantly squelch transcription, and we are currently testing whether this effect is via sequestering of general transcription factors or other coactivators. Inducible squelching has potential benefits for design of synthetic circuits, as well as in design of novel therapies to fight cancer or infections.

23. Using dominant genetics to understand dominant states of cohesin

Elizabeth Stephens, University of British Columbia - Phil Hieter and Peter Stirling Labs

Stephens, E.*, O’Neil, N., Stirling, P., Hieter, P.

mutational mapping, cohesin, gain of function

Targeting synthetic lethal (SL) interactions is an approach to selectively kill tumor cells by exploiting cancer-specific mutations. While synthetic lethality has been studied extensively, few examples have translated to the clinic, possibly due to the limitations of gene knockout models to accurately represent in vivo protein inhibition. To address this, we deployed a dominant SL screening method, focused on dominant gain-of-function genetics. The goal is to model proteoforms with missense mutations, to guide small molecule inhibitor design. Here, we investigate the cohesin complex, involved in sister chromatid cohesion and DNA repair, as a potential SL drug target. We screened a random mutagenesis library in S. cerevisiae to identify dominant lethal Smc1 mutations in a cohesin-compromised background. Key residues identified map to the C-terminal ATPase domain. Ectopic expression of these proteoforms cause cell cycle disruptions, sensitize cells to DNA damaging agents and are dominantly SL with cancer-like backgrounds, such as those with mitotic defects and cohesion dysregulation. Structural mapping revealed key residues are conserved in humans and localized to ATP-interacting residues, suggesting a druggable domain. These findings can inform in silico docking studies to design small molecules that can be tested for phenocopying of the dominant state for better clinical translation.

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

Nadine Tietz, Western Washington University - Dan Pollard Lab

Tietz, N*.; Ichwan, G.; Pollard, D

natural allelic variation, transcript properties, gene expression outcomes 

Natural variation in gene expression drives trait differences, but the genetic mechanisms behind this variation are not well understood. Polymorphisms affecting codon bias, mRNA folding strength, transcription elongation rate, and amino acid (AA) properties (charge and weight) can influence gene expression, depending in part on their position within the transcript. Despite their potential role in trait diversity, systematic studies of these factors in Saccharomyces cerevisiae remain limited. We analyzed genomic, transcriptomic, and proteomic data for 1,447 genes across 22 S. cerevisiae isolates. For each gene, we calculated across-isolate variation in five transcript properties and estimated mRNA abundance, protein abundance, and translational efficiency. Using linear mixed-effects models, we examined how allelic variation in these properties correlates with variation in expression levels. We also investigated positional effects, interactions among transcript properties, and whether gene-level characteristics modulate these relationships. We found that mRNA folding strength, codon bias, and AA weight significantly impact all three expression levels. Codon bias and AA weight had consistent effects across expression levels, while mRNA folding strength reduced transcript abundance but increased translational efficiency and protein levels. Transcription elongation rate and AA charge had weaker effects, mainly on transcript abundance. We also observed independent and synergistic effects, particularly between codon bias, AA charge/weight, and mRNA folding strength, most notably on translational efficiency. Positional analyses showed codon bias effects concentrated in domain-encoding and 3′ coding regions, while mRNA folding strength effects were strongest near start/stop codons and within coding regions. Together, our findings provide a comprehensive view of how transcript-level polymorphisms shape gene expression in yeast. These insights may help guide mRNA vaccine design by informing strategies to optimize protein production through transcript sequence engineering.

25. Engineering Yeast with Open Tools

Emi Tong, Open Science Network Society - Scott Pownall

Tong, E.*; Caven, I.; Rae, M.; Fan, E.; Pownall, S.

open-source, Open Yeast Collection

To solve global issues, bioinnovators need to access open tools and resources. Open technologies, like the Open Yeast Collection (OYC), empower innovators across disciplines driving creative and collaborative problem-solving. Originally designed for S. cerevisiae and K. phaffii, OYC is an extensible, modular Golden Gate assembly library for engineering yeast that is released under the Open Materials Transfer Agreement (OpenMTA). The OpenMTA is a material transfer agreement that supports distribution that is free of royalties or fees, other than appropriate and nominal fees for preparation and distribution, attribution, and promotes reuse, redistribution and importantly non-discrimination in who can use these materials. The OpenMTA permits sharing amongst academia and not-for-profit but also, unlike most traditional MTAs, with for-profit startups and established companies. Synthesis of the core OYC was funded by the — now defunct — BioBricks Foundation's FreeGenes program. OYC permits the assembly of tools from reusable and redistributable DNA parts. Through both FreeGenes and the iGEMs annual Distribution the OYC has been distributed to over 50 countries. More recently, OYC was extended through the creation of the Yeast Protein Expression Toolkit (yPET), a collection of DNA parts which permits addition of a wide range of fusion tags including secretion signals, purification tags, and cleavage sites to either the N-terminus or the C-terminus of a CDS. Synthesis of yPET was funded through a University of Cambridge EPSRC Impact grant obtained by Dr. Jenny Molloy from the Open Bioeconomy Lab, UK. The OpenMTA also applies to strain organisms like the recently released K. phaffii NRRL YB-4290 Hoc1tr chassis.

26. Exploring the multifaceted roles of an RNA modification enzyme

Abby Vaaler, University of Oregon - David Garcia Lab

Vaaler, A.*; Alladin, S.; Barry, M.; Shaw, E. and Garcia, D.

RNA, RNA modification, tRNA, translation, pseudouridylation, pseudouridine, Pus4, catalysis, mutants, Nanopore, paromomycin, RNA sequencing, mistranslation, gene expression, RNA binding

Many types of RNA molecules are post-transcriptionally modified by a large and conserved class of enzymes, which are curiously often dispensable for life. We are using a combination of genetics, cell phenotyping, and direct RNA sequencing to investigate several of these enzymes and their molecular roles in budding yeast. One example is the pseudouridine synthase Pus4, that catalyzes pseudouridine-55 in the vast majority of tRNAs, throughout nature. Growth assays revealed a Pus4 catalytic mutant has increased sensitivity to paromomycin, an antibiotic that interferes with translation. In contrast, a strain deleted for the protein entirely does not exhibit such sensitivity. Conversely, cells deleted for Pus4 show increased mistranslation as measured with a reporter construct, while the catalytic mutant does not have increased mistranslation relative to wild-type. These results indicate Pus4 contains additional non-catalytic activities affecting translation. mRNA sequencing of these strains showed the Pus4 catalytic mutant lead to increased mRNA levels for several stress-response genes, including several encoding heat shock proteins, which was not the case from cells lacking the PUS4 gene entirely. While we and others have established Pus4’s role in modifying 64/66 yeast tRNA isoacceptors, our Nanopore direct RNA sequencing results show that in the absence of Pus4, some pseudouridylation of tRNAs at position 55 still occurs, suggesting another modification enzyme modifies U55 in vivo, not previously known. Ongoing work is establishing the identity of this other enzyme(s). We also created a Pus4 reduced-binding mutant enabling us to investigate Pus4 beyond its pseudouridylation role. Our results show this mutant does not affect tRNA modification but does affect mRNA modification, and leads to altered growth phenotypes under stress distinct from those by a catalytic mutant. In summary, our work demonstrates Pus4 has impacts beyond catalysis and it activities affects different aspects of gene expression in budding yeast.

27. Osh proteins Regulate PI(4,5)P2 Distribution To Promote Polarized Exocytosis

Matthew Volpiana, Simon Fraser University - Christopher Beh Lab

Volpiana, M.*; Nenadic, A.; Beh, C.T.

Oxysterol binding proteins, Osh, Phosphoinositide, PI4P, PI(4,5)P2, polarized exocytosis, exocyst complex, vesicles

Membrane attachments for both vesicle targeting and organelle membrane contact sites (MCSs) are mediated by the assembly of tethering proteins, which are regulated by phosphoinositide lipids. The yeast oxysterol-binding protein (OSBP) homologue (OSH) family represents a group of phosphoinositide-binding proteins involved in both polarized exocytosis and MCS assembly. Osh proteins, along with the exocyst tethering complex and SNARE membrane fusion proteins, regulate the plasma membrane (PM) distribution and synthesis of PI4P and PI(4,5)P2, the two primary phosphoinositides at the plasma membrane (PM). Through the regulation of PI4P and PI(4,5)P2 on joined membranes, We propose that Osh proteins play a central role in promoting and assembling membrane tethering complexes by regulating PI4P and PI(4,5)P2 on opposing membranes. We are determining the Osh-dependent regulatory mechanisms that controls PI4P and PI(4,5)P2 polarization during vesicle targeting to the PM and at endoplasmic reticulum (ER)-PM MCSs.

28. Conservation and Divergence of TBP and its Homologs Throughout Evolution

Henry Young, University of British Columbia - Sheila Teves Lab

Young, H.*; Teves, S.

Transcription is a highly conserved process essential for gene expression across all life forms, yet it has evolved unique adaptations to these mechanisms and associated machinery, sometimes even challenging the requirements of the original function. One such protein is the general transcription factor TATA-box-binding protein (TBP) and its homologs. While in budding yeast, TBP is required for all transcription and is the only TBP protein, multiple TBP homologs can be found in other species, such as the mouse TRF2 and TRF3 that are highly similar to TBP but serve independent roles in the mouse system. These phenomena raise the question of how these homologs maintain conserved yet divergent functions. This study aims and proposes to explore the distinctions between TBP and its homologs from multiple species using a conditional yeast TBP system, utilizing growth assays and chromatin profiling to assess their ability to support transcription. Additionally, the role of co-evolution between TBP homologs and promoter sequences will be investigated through GFP reporter assays to quantify binding to different RNA polymerase promoters. Results will shed light on the evolutionary balance between conservation and functional diversification in transcriptional regulation across species.