New & Noteworthy
Apply Now for the 2023 Yeast Genetics and Genomics Course
March 10, 2023
For over 50 years, the legendary Yeast Genetics & Genomics course has been taught each summer at Cold Spring Harbor Laboratory. (OK, the name didn’t include “Genomics” in the beginning…). The list of people who have taken the course reads like a Who’s Who of yeast research, including Nobel laureates and many of today’s leading scientists.
The application deadline is April 1st, so don’t miss your chance!
Find all the details and application form at the CSHL Meetings & Courses site. This year’s instructors – Grant Brown, Maitreya Dunham, Soni Lacefield, and Greg Lang – have designed a course (July 25 – August 15) that provides a comprehensive education in all things yeast, from classical genetics through up-to-the-minute genomics. Students will perform and interpret experiments, learning about things like:
- Finding and Analyzing Yeast Information Using SGD
- Transformation & genome engineering
- Microscopy
- Manipulating yeast
- Dissecting tetrads
- Isolating mutants
- Working with essential genes
- Synthetic genetic arrays
- Fluctuation assays
- Whole genome sequencing & analysis
- Deep mutational scanning
Techniques have been summarized in the accompanying course manual, published by CSHL Press.
Scientists who aren’t part of large, well-known yeast labs are especially encouraged to apply – for example, professors and instructors who want to incorporate yeast into their undergraduate genetics classrooms; scientists who want to transition from mathematical, computational, or engineering disciplines into bench science; and researchers from small labs or institutions where it would otherwise be difficult to learn the fundamentals of yeast genetics and genomics. Significant stipends (in the 30-50% range of total fees) are available to individuals expressing a need for financial support and who are selected into the course.
Besides its scientific content, the fun and camaraderie at the course is also legendary. In between all the hard work there are late-night chats at the bar and swimming at the beach. There’s a fierce competition between students at the various CSHL courses in the Plate Race, which is a relay in which teams have to carry stacks of 40 Petri dishes (used, of course). There’s also typically a sailboat trip, a microscopy contest, and a mysterious “Dr. Evil” lab!
The Yeast Genetics & Genomics Course is loads of fun – don’t miss out!
Categories: Announcements, Conferences
Apply Now for the 2023 Fungal Pathogen Genomics Course
February 02, 2023
Fungal Pathogen Genomics is an exciting several day long course that provides experimental biologists working on fungal organisms with hands-on experience in genomic-scale data analysis. Through a collaborative teaching effort between the web-based fungal data mining resources FungiDB, EnsemblFungi, PomBase, SGD, CGD, MycoCosm, and JGI, students will learn how to utilize the unique tools provided by each database, develop testable hypotheses, and analyze various ‘omics’ datasets across multiple databases.
Daily activities will include individual and group training exercises, supplementary lectures on bioinformatics techniques and tools used by various databases, and presentations by distinguished guest speakers covering the following topics:
- Comparative genomics, gene trees, whole-genome alignment
- Identification of orthologs and orthology-based inference
- Gene pages and genome browsers
- RNA-Seq analysis and visualization in VEuPathDB Galaxy
- Variant calling analysis and Ensembl Variant Effect Predictor (VEP) tool
- Development of advanced biologically relevant queries using FungiDB ‘search strategies’ and mining integrated datasets (proteomics, transcriptomics, phenotypes, etc.)
- Genetic interactions, virulence genes, secondary metabolites
- Overview of ontology structure, evidence, available tools, slimming and enrichment
- Introduction to manual genome annotation and curation using Apollo, a web-based platform for structural and functional genome annotation (MycoCosm, Ensembl Fungi, FungiDB)
The application deadline for the Fungal Pathogen Genomics workshop to be held May 9-13, 2023 is February 16, 2023.
Don’t miss out – apply now!
Categories: Announcements, Conferences
Happy Holidays from SGD!
December 19, 2022
Stanford University will be closed for two weeks starting Wednesday, December 21, and will reopen on Wednesday, January 4, 2023. SGD staff members will be taking time off, but the website will be up and running throughout the winter break, and we will resume responding to user requests and questions in the new year.
Categories: Announcements
Global Biodata Coalition selects SGD in first set of Global Core Biodata Resources
December 15, 2022
We are proud that SGD has been included in the first list of Global Core Biodata Resources (GCBRs) announced by the Global Biodata Coalition (GBC)! This collection of 37 resources comprises deposition databases which archive and preserve primary research data, and knowledgebases, such as SGD, that add value to research data through expert curation and annotation. The list is meant to highlight those data resources whose long term funding and sustainability is critical to life science and biomedical research worldwide.
GCBRs represent the most crucial resources within the global life science data community. SGD’s selection as a key global data resource recognizes that SGD is essential to the global research endeavor.
For more information regarding the Global Biodata Coalition, including a link to the full list of selected core biodata resources, please see the full press release from the GBC.
Categories: Announcements
Regulation of yeast inducible promoters by SAGA and TFIID
November 15, 2022
Gene transcription — the elaborate process that our cells use to read genetic information stored in DNA – was long thought to be turned on only when certain regulatory factors traveled to specific DNA sequences. In a new study published in Genes & Development, Mittal et al., 2022, discovered that a subset of genes has their transcription regulatory factors and cofactors already in place, but in a latent state. With the appropriate signals, these “poised” genes become highly active.
The authors calculated the DNA-bound preinitiation complex/TBP-associated factor (PIC/TAF) ratio at all yeast genes and identified two major classes. The first, and largest, group provides basic “housekeeping” functions and are usually “on” at very low levels all the time (i.e., “constitutive”). The second class, the “inducible” genes, has a whole entourage of “poised” proteins assembled nearby which provides a guiding hand to transcription machinery when triggered by environmental signals, resulting in high levels of “induced” transcription.
Using CRISPR-Cas9 mediated protein depletion/degradation and gene knockout techniques, Mittal et al. removed parts of the SAGA and TFIID cofactors to systematically examine the role they play in regulating the above gene classes. They discovered that the constitutive class largely depended on TFIID, whereas the inducible genes required both SAGA and TFIID, suggesting an integrated pathway of PIC assembly at inducible genes. If true, such a possibility would be different from previous models where the two cofactors were thought to engage in somewhat distinct mechanisms of PIC assembly.
The authors further examined the above results and were surprised to find that at the inducible promoters, SAGA stabilized Taf1p (and thus TFIID) which helped in a rapid and robust transcription initiation of these genes upon acute environmental changes.
In addition, Mittal et al. also removed gene-specific transcription factors (TFs) to address how these factors recruited TBP upon sensing changes in environmental signals. They showed that TFs such as Hsf1p and associated cofactors were already bound to gene promoters prior to induction, instead of traveling to cognate sites upon induction. These cofactors, thus, lend a helping hand in recruiting TBP and the associated machinery at the induced genes.
Lastly, Mittal et al. removed Gcn5p and DUB subunits of SAGA to examine the effect on histone acetylation and ubiquitylation, respectively. They showed that global histone acetylation and ubiquitylation levels require active Gcn5 and the deubiquitination (DUB) activities, suggesting a SAGA independent moiety contributing towards maintaining total cellular acetylated and ubiquitylated histone pools.
Image credits: Image 1 – Mittal et al., 2022; Image 2 – B. Fanklin Pugh, Cornell University; Image 3 – Mittal et al., 2022.
Contributed by Chitvan Mittal.
Categories: Research Spotlight
2D RNA structures from RNAcentral
October 20, 2022
SGD has updated our RNA pages to add secondary structures provided by RNAcentral and generated by R2DT. Thumbnails and linkouts to RNAcentral via RNAcentral IDs are shown on the Summary and Sequence pages.
Interactive secondary structure viewers are available on the Sequence pages.
Take the pages for a spin! For more information about the structures, please see the Help page at RNAcentral.
Categories: New Data, Website changes
Tags: RNA structure
Role of Sumoylation in Regulating Replication
September 09, 2022
There are ~400 origins of replication in yeast, each of which can be “licensed” by the binding of the conserved origin recognition complex (ORC) and then the MCM replicative helicase complex, all of which happens in G1 phase. During the subsequent S phase, origins are then “activated” by binding of several other replication factors, leading to unwinding and then nascent strand synthesis.
The regulation of origin licensing and activation is a complex, multi-level process, for which numerous aspects of the picture remain unclear. As yeast has the most tools for studying this process, including a full map of origins and numerous options for genetic and biochemical modulations, it presents the ideal model in which to ask probing questions. A recent study by Regan-Mochrie et al. in Genes & Development has revealed the key role of sumoylation in regulation of genome replication.
The origin recognition complex comprises six subunits, encoded by ORC1 to ORC6. The authors built on previous results showing sumoylation of these proteins during DNA damage to ask about their modification status under normal growth. They were able to assess sumoylation status for four of the six subunits, observing various degrees of sumoylation of Orc1p, Orc2p, Orc4p, and Orc5p. They then created a construct to hypersumoylate Orc2p to ask how this affects cells, and found it led to cell lethality. This lethal effect was specific to the Orc protein, as other non-Orc hypersumoylated proteins were tolerated.
The lethality could be rescued by reducing the levels of a SUMO-conjugating enzyme, further indicating the specificity of the effect. The authors identified a subset of early origins that were preferentially inhibited upon hypersumoylation and, upon study, determined that the extra sumoylation interfered with loading of the MCM complex.
After identifying the residues becoming sumoylated on Orc2p, the authors were able to generate mutants to ask about the converse, i.e. hyposumoylation. Indeed, as might be hypothesized, lack of sumoylation caused DNA replication defects via abnormal increased firing of early origins.
Thus, by close study in yeast, the role of sumoylation in genomic stability becomes more clear, where sumoylation of ORC subunits affects loading of the MCM complex, which is itself the substrate for loading of activation factors. Modulated sumoylation status appears to provide a key level of regulatory control.
Categories: Research Spotlight
Tags: cell cycle control, DNA replication, ORC complex, origin recognition complex, regulation of replication, Saccharomyces cerevisiae, sumoylation
Humanizing Glycolysis in Yeast
September 02, 2022
While glycolysis is highly conserved between unicellular yeast and multicellular humans, it appears that not only the glycolytic functions but also the secondary (aka “moonlighting”) functions of the relevant proteins remain largely consistent. This consistency presents unique opportunities for better understanding human glycolysis in muscle tissue and elsewhere.
The technical challenges of studying glycolytic enzymes are not trivial—largely due to genetic redundancy for such critical components—but a recent report by Boonekamp et al. in Cell Reports explains how the authors built on previous work to overcome the obstacles in yeast. Employing a minimized set of glycolytic genes that were also relocated to a single chromosome (strain SwYG) made it possible to swap in human orthologs with relative ease.
The authors first looked at direct complementation of yeast genes by 25 human glycolytic enzymes. Remarkably, 22 of 25 human genes readily complemented their yeast counterpart. The three exceptions were hexokinases 1, 2, and 3 (HsHK1, HsHK2, and HsHK3), which are roughly twice the size of their yeast orthologs and have lower sequence conservation.
As glycolysis cannot take place without the hexokinases, the authors first looked more closely at HsHK1 and HsHK2 to ask why they fail to function in yeast. Upon exposure to glucose, for which the native human proteins could not support growth, they created conditions to select for systematic mutations that conferred improved glycolysis. The mutant proteins proved to be less sensitive to inhibition by glucose-6-phosphate (G6P), a potent allosteric inhibitor of hexokinase activity.
With this understanding, the authors set out to complement the entire pathway in yeast with human proteins. They created two different strains, one with HsHK2 because it is considered the main isoenzyme in human muscle (strain HsGly-HK2), and one with HsHK4 because it shows less inhibition by G6P (strain HsGly-HK4). The HsGly-HK2 strain could not grow well on glucose until after a long lag phase in which mutations were selected near the G6P-binding site of HsHK2.
Comparing the uptake and output of the two humanized yeast strains to the native yeast strain revealed a number of intriguing differences, especially around the different enzyme activities between human and yeast. These differences led to different behaviors on different carbon sources and overall slower growth and glycolytic flux for the humanized strains versus native yeast.
Three glycolytic yeast enzymes have secondary “moonlighting” roles beyond their function in glycolysis. Hexokinases are involved in glucose repression of genes such as invertase (SUC2), and, indeed, it appears that human hexokinases can also at least partially complement this secondary function. Yeast aldolase (FBA1) plays a secondary role in vacuolar function that is required for growth at alkaline pH. This function is likewise complemented by the human orthologs, where the humanized strains can grow at pH 7.5. The third moonlighter is enolase, where yeast ENO2 is required for mitochondrial import of tRNALys, thereby allowing growth at higher temperatures and on non-fermentable carbon sources. All three human enolases can at least partially complement this growth defect, and thus appear to have the same secondary function.
Despite the high conservation of functions, the humanized yeast strains have a slow growth phenotype. The authors used this phenotype to employ adaptive laboratory evolution to see which genomic changes restore growth. Interestingly, the mutations that restored growth were mostly not in the glycolytic enzymes themselves, but in associated factors that regulate enzyme abundance and activity. The identity of these regulators and the potential for targeting them in human muscle have already rewarded the successful transfer of skeletal muscle glycolysis into yeast.
Categories: Research Spotlight
Tags: glycolysis, humanizing yeast, muscle glycolysis, Saccharomyces cerevisiae, yeast model for glycolysis
Link Between Aging and Iron
August 25, 2022
Perturbations in iron homeostasis affect aging, but how this happens has remained a bit of a black box. A new study by Patnaik et al. in Cell Reports illuminates this box by looking more closely at the transcription factors that are first to respond when iron becomes limiting.
Key among these are Atf1p and Atf2p, which activate the full suite of iron-mobilization genes, among which is TIS11/CTH2, which encodes an RNA-binding protein that targets specific messages for decay.
The targeted messages flagged for decay encode mitochondrial proteins, as these use iron but are not the most essential in the set. The most essential Fe-requiring enzymes are those involved in DNA synthesis and repair, such that slowing/shutting mitochondrial function is a response to iron deficiency. Intriguingly, mitochondrial function also happens to decline with age.
To find the specific mechanisms linking iron with aging, the authors used an unbiased analysis of genes involved in iron homeostasis to see which showed connection with aging. The strain with a tis11Δ mutation lived longer than any others, with a lifespan extended by 51.1%. In a broader sense, they found that genes involved in different aspects of response to iron deficiency also had different effects on fitness and aging.
Delving more deeply into the role of Tis11p/Cth2p in aging, the authors used RNA-seq and Ribo-seq to look at temporal changes in transcription versus translation in aging cells. They showed how, overall, aging leads to inhibition of translation—except for certain genes which are upregulated instead. Interestingly, most of the upregulated genes are in the Fe regulon that gets activated by the first responder Atf1p.
While the expression of TIS11/CTH2 increases both with aging and with iron deficiency, the deletion of the gene extends lifespan. Thus, multiple lines of evidence suggest Tis11p/Cth2p is a negative regulator of longevity. The key connection appears to be mitochondrial translation, where the function of Tis11p/Cth2p to inhibit translation of mitochondrial transcripts for repressing non-essential Fe-requiring enzymes serves to simultaneously repress overall mitochondrial respiration, which speeds aging.
As not all genes translationally upregulated in the tis11Δ mutant contained appropriate binding sites in the 3’ UTR, the authors looked further and found binding sequences for Puf3p, a protein known to bind and inhibit translation of mRNAs coding for mitochondrial ribosome proteins. Thus, Puf3p appears to be a critical partner for Tis11p/Cth2p in mediating downregulation of mitochondrial function. Further, they questioned the relationship with the Hap4p transcription factor, which regulates numerous components of the electron transport chain and whose overexpression extends lifespan. As the combination of a tis11Δ deletion with HAP4 overexpression had no additive effect in an epistasis experiment, they concluded that Tis11p-dependent repression acts through Hap4p.
The role of phosphorylation of Tis11p/Cth2p was examined by mutating N-terminal serine residues, which impairs degradation of the protein. Consistent with the converse result of extended lifespan in null mutants, the nondegradable version of the protein shortens lifespan.
Thus, the ease of the yeast model once more illuminates intricate connections between critical proteins, facilitating potential drug discovery around several new aging factors.
Categories: Research Spotlight
Tags: aging, cell aging, iron homeostasis, Saccharomyces cerevisiae, yeast model for aging
Sen1p Is the Traffic Cop for RNA Polymerase III
August 12, 2022
One way to imagine DNA is as a busy road with a lot of competing traffic. Say, a small village in southern Italy…where someone must mediate conflicts between competing vehicles to avoid disaster.
It turns out that the “someone” in yeast cells is Sen1p. Two recent papers from associated groups describe the intriguing detail of how the Sen1p helicase plays this role for RNA polymerase III transcription. The paper by Aiello et al. in Molecular Cell shows how Sen1p regulates transcription-driven conflicts between the several machineries all engaged with DNA. In the related paper by Xie et al. in Science Advances, the authors show how the Sen1p helicase mediates “fail-safe” methods of transcription termination for RNA Pol III, thereby promoting efficiency and avoiding conflict with other pieces of machinery.
The key conflict preventing RNA Pol III from transcribing noncoding genes is with RNA Pol II, which is busy transcribing coding genes. Aiello et al. show how Sen1p has two strategies for mediating these conflicts, both of which involve interactions between Sen1p and the replisome. One involves temporary release of RNA Pol II from DNA while the other resolves genotoxic R-loops in nascent RNA. Both are critical for preventing genome instability.
In the related paper by Xie et al., the authors focus on how termination of transcription of noncoding genes by RNA Pol III is achieved, and the role that Sen1p plays in termination. They show how Sen1p can interact with all three polymerases and also with the other two subunits (Nrd1p and Nab3p) of the NRD1 snoRNA termination (NNS) complex. More specifically, they show by mutation and co-immunoprecipitation that it is the N-terminal domain (NTD) of Sen1p that interacts with RNA Pol III and the replisome.
The authors use metagene analysis of RNA Pol II distribution at mRNA-coding genes to show how Sen1p can promote the release of RNA Pol II to resolve transcription-replication conflicts (TRCs). They further show how the association of Sen1p with the replisome is required for limiting TRCs at the ribosomal replication fork barrier, and how this action appears redundant with that of RNases H. The cooperation and redundancy in this role are key means to protect genome stability.
Not only is Sen1p required for termination of RNA Pol III transcription, but the authors show how this function is independent of the NNS complex. Unlike resolution of conflicts between RNA Pol II and RNA Pol III, the termination function of Sen1p does not require the replisome.
They asked the question of whether Sen1p acts via the primary termination site for RNA Pol III or, rather, a backup secondary termination that catches errors (i.e., when RNA Pol III reads through a weak termination site). Termination for RNA Pol III employs a tract of T nucleotides (T-tract) in the nontemplate strand and these T-tracts can be relatively weak or strong. When T-tracts prove insufficient to stop the polymerase, Sen1p plays a role by means of secondary structures in nascent RNAs, which act as auxiliary cis-acting elements. This backup method is termed the “fail-safe transcription termination pathway.” The RNA secondary structures are not absolutely required for RNAPIII termination, but can function as auxiliary elements that bypass weak or defective termination signals.
Once more, it is the power of the yeast model that has allowed investigation to such exquisite molecular detail. That cells preserve genomic stability and avoid pile-ups amid so much traffic along DNA remains truly remarkable–even when we know more of how it works.
Categories: Research Spotlight
Tags: replisome, RNA polymerase II, RNA polymerase III, Saccharomyces cerevisiae, transcription, transcription conflicts