New & Noteworthy

Chaperone Wars: The Last Co-chaperone

August 22, 2018


In Star Wars: The Force Awakens, the evil First Order rises up and threatens to eliminate the New Republic. The protagonists, who join forces of the Resistance, must find the reclusive Jedi Master Luke Skywalker so that he can take up arms against the First Order and save the galaxy.

(Warning: Star Wars spoilers below!)

One of the protagonists, Rey, keeps precious cargo with her as she searches for Luke: the lightsaber of Anakin Skywalker, Luke’s father. She must deliver the lightsaber to Luke in order to galvanize his joining the Resistance and to help motivate him to resume his role as a Jedi Master.

Just like how the galaxy would be doomed if Rey could not reach Luke, budding yeast cells would be doomed if a Hsp70 protein chaperone could not interact with another chaperone, Hsp90. But Hsp70 isn’t trying to deliver a lightsaber to Hsp90—instead, Hsp70 is trying to deliver “client” protein substrates, so that Hsp90 can help fold and mature these proteins.

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Like how R2-D2 helped Rey find Luke, Sti1p helps Hsp70 and Hsp90 interact. Image from http://starwars.wikia.com/wiki/Star_Wars:_Episode_VII_The_Force_Awakens.

In Star Wars, the trusty droid R2-D2 was ultimately there to help Rey find Luke with a holographic map to Luke’s location. But is our friend Saccharomyces cerevisiae just as fortunate? In fact, it just so happens that S. cerevisiae also has an “R2-D2” of its own: the co-chaperone Sti1p. Just like how R2-D2 helps Rey find Luke, Sti1p helps bring Hsp70 and Hsp90 together so that Hsp90 can receive its protein substrates and save the galaxy (or at least help fold proteins and save the yeast cell).

To give some background, Hsp90 (encoded by HSP82 and HSC82 in yeast) is a molecular chaperone that assists in the folding and maturation of specific protein substrates, or “clients”. It functions as a homodimer that undergoes ATP-regulated cycles of “opening” up to receive clients, closing, and then opening again. Many clients of Hsp90 first bind to a chaperone of the Hsp70 family, such as Ssa2p, which assists in the early stages of protein folding before interacting with Hsp90 and passing on the client.

The co-chaperone Sti1p comes into the picture by bridging Hsp70 and Hsp90 and helping them interact, so that the Hsp70-bound client can be delivered to Hsp90. Although this function of Sti1p has long been known, the exact mechanistic details have been obscure, and mutational studies have suggested that Sti1p does more than just bridge the two chaperones together.

Thanks to a recent GENETICS study by Reidy and coworkers, we now know more about how Sti1p helps save the galaxy: it not only helps Hsp90 interact with Hsp70, but also prepares Hsp90 to receive its client protein and advance in its reaction cycle.

To uncover the role of Sti1p in the Hsp90 cycle, the authors examined Hsp90 mutants that were dependent on Sti1p for viability. They mapped both previously-known and newly found Sti1-dependent mutants and found that all of the mutations clustered in just two sites. They designated the sites Sti1-dependent N and C-terminal domain proximal, or “SdN” and “SdC”, respectively.

Because previous studies showed that some Hsp90 SdN mutants don’t interact well with Hsp70, and that analogous SdC mutations in E. coli weaken interactions with Hsp90 client proteins, the authors hypothesized that Sti1p assists Hsp90 with these functions in particular.

To clarify how Sti1p and Hsp90 cooperate, the authors utilized a combination of mutational studies, pull downs with purified proteins, mass spectrometry, and more. They observed that SdN mutations in Hsp90 reduce interactions with Hsp70, while SdC mutations do not. Further, they found that the Sti1p dependency of SdN mutants could be cured through a novel suppressor mutation (E402R), which increases the interaction of Hsp90 with Hsp70. These results suggest that Hsp90 interacts with Hsp70 through the SdN region, and that Sti1p is needed to bring the two chaperones together if they aren’t able to do so well enough on their own.

Importantly, although the E402R suppressor mutation was able to “cure” SdN mutants of their Sti1p dependency, it was unable to do so in SdC mutants. This indicated that SdC mutants are defective in a function that Sti1p assists with, but one that’s separate from having Hsp70 interact with Hsp90.

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Luke needed a little motivation from R2-D2, as does Hsp90 from Sti1p. Image from http://starwars.wikia.com/wiki/Luke_Skywalker

To uncover why SdC mutants depend upon Sti1p for viability, the authors investigated suppressor mutations. The authors isolated multiple SdC suppressors and also found that a previously characterized mutation, A107N, is able to ameliorate the effects of SdC mutations. Previous studies on A107N show that this mutation promotes closure of the open-state Hsp90 heterodimer, which is an important step of the Hsp90 reaction cycle. The authors found that other SdC suppressor mutations were consistent with A107N and could relieve the Sti1p dependence of SdC but not SdN mutants. These results indicate that Sti1p not only promotes Hsp90-Hsp70 interaction, but also has an additional function in promoting Hsp90 heterodimer closure and progression of the Hsp90 cycle.

So it turns out that Sti1p is like R2-D2 in more ways than one. In its first role, Sti1p helps Hsp70 and Hsp90 interact, much like how R2-D2 helps Rey find Luke in The Force Awakens. In its second role, Sti1p helps Hsp90 accept its substrates, progress through its reaction cycle, and perform its function. This is similar to what R2-D2 does in Star Wars: The Last Jedi. In the movie, Luke initially shows reluctance to resume his role as a Jedi Master and help save the galaxy, despite being found by Rey. But thankfully, R2-D2 was there to motivate Luke to return to his heroic duties, much like how Sti1p is there to “motivate” Hsp90 to capture client proteins and do its job.

Thanks to the efforts of Reidy and coworkers, how Sti1p helps save the galaxy yeast cell is that much clearer. Not only does Sti1p help Hsp70 interact with Hsp90 and deliver lightsabers client proteins, but it also helps Hsp90 do its job as a Jedi Master chaperone by promoting progression of the Hsp90 reaction cycle!

by Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: chaperones

(Almost) All Hands on Deck for Calcineurin

July 31, 2018


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Multiplex flag of countries in the 2018 World Cup (from pixabay.com)

If there was a World Cup soccer championship for cellular proteins, it’s a pretty sure bet that calcineurin wouldn’t make the team. That’s because this protein is one of those players that just can’t help but use their hands! And as pretty much everyone knows, that’s a big no-no for soccer players (except goalies, of course).

Conserved across virtually all eukaryotic organisms — from plants and protozoa to fungi and humans — calcineurin is a very abundant calcium-binding protein. In fact, it’s so abundant that it makes up 1% of the total protein content in a cow’s brain!

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Moo! (from pngimg.com)

But what does this ubiquitous protein do? Well, it’s “merely” responsible for regulating many diverse, fundamental life processes… things such as fertilization, development, behavior, life span, responses to environmental cues, immune responses, cell death, and so on.

For eukaryotes, certain environmental and developmental cues, such as hormones or nutrient availability, can initially signal their presence by causing a change in the cell’s internal calcium levels. Calcineurin helps detect these calcium level changes and then passes the signal on in a chain of events.

Calcineurin is a calcium-regulated protein phosphatase, meaning that when it is activated by calcium level changes, it removes a phosphate group(s) from other proteins, particularly transcription factors. When these transcription factors have their phosphate group(s) chopped off by calcineurin, they travel into the cell’s nucleus and turn on a specific set of genes needed to make the cell respond appropriately to the original environmental or developmental cue.

Calcineurin is actually made up of two different proteins that bind together. One is a catalytic protein subunit (called CNA) and the other is a regulatory subunit (CNB). In our yeast friend Saccharomyces cerevisiae, the regulatory CNB protein subunit of the calcineurin complex is encoded by the CNB1 gene.

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Talk to the (EF) hand! …named for Helices E and F (image from Wikimedia Commons)

The Cnb1 protein contains a set of four almost-identical short amino acid domains that are conserved in the CNB proteins of all other organisms. These motifs are called “EF hand” domains because each of them looks like a spread thumb and forefinger of a human hand. And crucially, each “EF hand” can grab and hold onto calcium ions (Ca2+). The four EF hands of the Cnb1 protein are called EF1, EF2, EF3 and EF4, respectively.

But besides holding onto calcium ions, what else do these hands do? Why are there 4 of them? Are each of the hands doing the same thing? Does the right hand know what the left (or the middle, or the other middle) hand is doing?

Well, in the July issue of GENETICS, Connolly and co-workers describe experiments they’ve performed that help figure out some of the answers to these questions for the yeast Cnb1 protein.

The authors made a series of 4 different mutant CNB1 genes, each one having a disabling mutation in one of the 4 EF hands so that the hand can’t grab calcium ions any more. They put these mutant-handed CNB1 genes into yeast cells that had their normal CNB1 gene completely removed. The yeast cell thus ends up depending on a Cnb1 protein with one mutant hand and three functional hands.  In a way, they are making Cnb1p have one of its 4 hands tied behind its back, and then seeing how well it can do its job!

How did they test how well each of the mutant-handed proteins works? Remember that the Cnb1 protein is the regulatory subunit of calcineurin, and calcineurin activates a transcription factor by dephosphorylating it. In yeast, the transcription factor regulated by calcineurin is encoded by the CRZ1 gene. The Crz1 protein recognizes and binds a certain DNA sequence (called CDRE) located just upstream of each one of its target genes and turns these genes on, ultimately changing the yeast cell’s behavior during calcium signaling. The authors put a special reporter gene into their yeast strains; this reporter gene has the special CDRE DNA sequence fused to an often-used bacterial gene called lacZ. The amount of lacZ protein produced by the yeast cells (which positively correlates with calcineurin function) can be sensitively monitored by an easy test tube assay.

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Totally tubular! (from pixabay.com)

Using this test tube assay, Connolly and co-workers tested how well each of the EF hand mutants worked. First they tested how well each could turn on the CRZ1-regulated genes, and also how well the mutant proteins detected calcium. Then they also tested how well each of the mutant-handed Cnb1 proteins worked in high salt environments, during the mating response, under oxidative stress conditions, and even in the presence of immunosuppressive drugs! Why the latter? Calcineurin is a target of immunosuppressive drugs, which are used when people get organ transplants to stop their own bodies from attacking the “foreign” organ. Yeast calcineurin is so similar to human calcineurin that it too is affected by these drugs!

The results were clear (well, actually yellow in the assay)—in all cases, the Cnb1 protein was able to have its EF4 hand disabled and still function perfectly or almost as well as the intact Cnb1 protein! But whenever one of the other EF hands was disabled, the function of calcineurin suffered, and this was true for each of the many ways it was tested, from salt to mating to immunosuppressive drugs.

It appears that when any of the useful hands (EF1, 2 or 3) were mutated, it causes Cnb1p to improperly change its shape in response to calcium, and this misshapen protein can’t do its job of activating Crz1p and ultimately getting the cell to respond to calcium-mediated signals properly.

And (#APOYG alert!) these yeast genetic results for the 4 EF hands match very closely to what’s been seen for mammalian calcineurin EF hand mutants in test tube (“in vitro“) experiments, giving an even stronger confirmation to these mammalian results. Maybe yeast will help develop new strategies for calcineurin-related diseases!

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You’ve gotta “hand” it to calcineurin! (from Wikimedia Commons and Pixabay)

So now back to soccer… As we’ve found out, calcineurin HAS to use its hands, so it’s not a good pick for a regular soccer position player. But maybe it could be a super-awesome goalie since it has 4 hands! It even seems that you can tie its EF4 hand behind its back and Cnb1p can still guard the goal just fine with its remaining 3 hands – the EF4 hand seems to be a totally useless appendage! But if you disable any of the other hands, then it causes the Cnb1 protein to bend awkwardly and not do its job anymore.

Thanks to the efforts of Connolly and coworkers, we now know that it’s not quite “all hands on deck” for Cnb1p, but rather “EF 1, 2, and 3 hands on deck” in order to carry out its “goal” of regulating cellular responses to calcium!

by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: signal transduction, protein structure

New and Improved Gene/Sequence Resources

July 12, 2018


Gene/Sequence Resources (GSR) is a great way to figure out which tools at SGD can help you analyze the yeast genome. Given a gene name, chromosomal region, or raw DNA/protein sequence, GSR retrieves a list of sequence analysis tools, options for accessing biological information, and table/map displays.

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Pick up to 11 yeast strains in the new Gene/Sequence Resources

We’ve recently updated GSR to expand its search capabilities and provide more options for downloading, analyzing, and comparing sequences from different S. cerevisiae strains. New features in Gene/Sequence Resources include:

  • Options to select up to 11 different yeast strains for gene searches and sequence downloading
  • Ability to search for multiple genes at a time
  • Sequence alignment/variation tools such as Variant Viewer
  • Convenient, automatic loading of your sequence/gene of interest into other tools at SGD

Be sure to check out the new and improved Gene/Sequence Resources, accessible via the Sequence section in the top purple toolbar, and let us know if you have any feedback.

Categories: Website changes

Too Much of a Good Thing Can Be Hazardous to Your Health

July 02, 2018


When someone says they can “read you like a book”, they probably aren’t saying that they know your entire genome sequence. (…or for you Westworld fans, you certainly hope they aren’t saying they’ve got access to the Delos Incorporated “library”!).

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Can someone read you (or your genome) like an open book?
(from maxpixel.net)

But in fact everyone’s genome CAN be thought of as a book, sort of like a giant cookbook. Within your genome are many “recipes” for gene products — DNA sequences that each give instructions on how, when, and where to make a protein or RNA molecule. The recipes of the genome are used to “cook up” a person!

When a parent cell divides and creates another cell, it makes a copy of its genome “cookbook” and passes it down to the new cell. It’s extremely important to make sure that there aren’t any errors in the newly copied text, as mistakes in recipes for crucial enzymes can have disastrous results for the cell. Indeed, our cells (as well as yeast cells, and in fact, all organisms) have enzymes that are dedicated to scrupulously inspecting the new copies of our genome cookbooks that are made during cell division, and then helping to correct any errors.

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No typos here!
(from pixabay.com)

A certain class of these enzymes are known as DNA mismatch repair (MMR) proteins. These enzymes carefully proofread the newly made copy of the genome, and if something doesn’t match the original version, they will help fix the error.

You might think that having more of these MMR proteins would be a good thing, because there would be more thorough checking and repairing of the new copy of the genome. But the situation isn’t so simple, especially when it comes to human cancer.

Previous studies show that some cancers and cancer predisposition syndromes have less of the MMR proteins. This makes sense because cancers almost always arise due to genome mutations, and it is known that if a cell has less of the error-checking MMR proteins, there are more genome mutations. However, other studies report a puzzling discrepancy: in some cancers, there are in fact MORE of the MMR proteins rather than less!

In their recent GENETICS study, Chakraborty and coworkers decided to investigate this discrepancy further. They analyzed data from two databases of human cancer genome information, The Cancer Genome Atlas (TCGA) and the cBioPortal for Cancer Genomics, specifically looking at how much the genes encoding MMR proteins were turned on or off in cancer.

They observed that many types of human cancer cells make more of two particular MMR proteins: MSH2 and MSH6. It turns out (as it often does!) that our good buddy Saccharomyces cerevisiae has MMR proteins very similar to the human ones, also encoded by genes called MSH2 and MSH6. So Chakraborty and coworkers made yeast cells that overexpress the yeast genes MSH2 and MSH6 and used the awesome power of yeast genetics (and genomics) (#APOYG!) to investigate whether this might lead to cellular and genome changes like those seen in human cancers.

Using various yeast genetic assays to measure rates of genome alterations such as homologous recombination, mutations, and loss of large chromosome regions, Chakraborty et al. observed increased rates for all of these measures of genome instability in cells when both MSH2 and MSH6 were overexpressed, but not for overexpression of either one alone (or of other MMR proteins). So even though there are more of the “good guy” MMR proteins in these cells, this actually ends up making the genome of the cell MORE likely to get damaging mutations of the same types seen in cancer cells.

So why is too much of a good thing such a bad thing in this case? The authors hypothesize that both Msh2p and Msh6p act together as a joined pair to go to the spot where the chromosomal DNA is actively being copied (“replicated”). When they are both overexpressed, too many of the Msh2p-Msh6p pairs can go to the replication spot and actually interfere with the copying process.

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Medieval scribe and author Jean Miélot at his desk, late 15th century (from Wikimedia Commons)

It’s as if you were a medieval scribe carefully copying an illuminated manuscript of a genome cookbook, and instead of one supervisor occasionally checking your work, there are a bunch of people constantly looking over your shoulder and maybe even bumping into your arm, causing you to make mistakes in your writing. They may even make you drop the book you’re copying, scattering pages so that you might leave some out or put them back in the wrong order, seriously messing up your work!

Here’s hoping our cells don’t overdo it with their MMR proteins, so that they can be careful with their cookbook copying job and do it “write”!

by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: MSH6, DNA replication, DNA mismatch repair, MSH2, cancer

Mixing Mitochondria Makes Magic

May 31, 2018


In the Harry Potter universe, there are two materials that make up a wand: the wood, which comes from trees like cedar and holly, and the core, which is a magical substance such as the feather of a phoenix or a unicorn hair.

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A variety of wizarding wands. From Flickr.

Every wand has unique properties that depend mainly on the combination of its wood and core. Different wood-core pairings give different characteristics that can either antagonize or synergize with the wizard using it. When a wand meets up with its ideal owner, it will begin to learn from and teach its human partner. Such auspicious pairings can continuously improve the wizard’s spell-casting, helping the wizard perform better and better under ever more varied circumstances. However, a poor pairing between a wand and a wizard can be devastating, enfeebling the wizard’s magic or even causing it to backfire.

And just like wizards and wands, it turns out that mitochondrial DNA and nuclear DNA in a cell need to be properly paired to perform the “magic” of running a cell in the most efficient way.

Mitochondria are dynamic structures inside eukaryotic cells that provide much of the energy to keep a cell humming along.

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A mitochondrion. These sub-cellular organelles produce much of the energy for a cell in the form of ATP. From Wikimedia Commons.

Mitochondria contain their own DNA, encoding genes necessary for the organelle to do its work. Although mitochondrial DNA is physically separate from nuclear DNA, it turns out that the two need to work together if the cell is to make functioning mitochondria.

Like the wand-wizard pairing in Harry Potter’s world, the combination of a specific mitochondrial genome (the wand) with a particular nuclear genome (the wizard) is important for making a healthy mitochondrion. Some mitochondrial-nuclear combinations work well and others not so much, but not a lot is known about where different mitochondrial DNAs come from and how they end up paired with their favored nuclear genomes.

Knowing more about this may help us understand how mitochondrial genomes evolve during interspecific hybridizations, such as in lager beer yeast and certain other fermentation yeasts.

A new study in GENETICS from Wolters et al. shows that when S. cerevisiae yeast cells go through the mating process, there is often mixing of mitochondrial genomes to give new combinations of mitochondrial genes — almost as if lots of new wood-core combinations of wands were being created.

How do these new mitochondrial combinations arise? When two haploid yeast cells mate, they merge to form a single diploid cell that contains mitochondria from both of its parents. Sometimes, these mitochondria exchange pieces of DNA, mixing-and-matching genes in a process known as mitochondrial recombination.

The authors found that a surprising proportion of mated yeast cells (~40%) had recombinant mitochondrial DNA. And in many cases, the recombined mitochondrial genomes work even better with the nuclear genome to make a super healthy cell. Often these optimal pairings allowed the cells to develop new powers, tolerating higher temperatures and more oxygen-stressed conditions than the original parent cells — in other words, the cell has found its optimal “wand”!

But other pairing combinations were inauspicious, giving sickly or dead mitochondria that can harm the cell, especially when it is growing under stressful conditions. For instance, when the authors swapped the mitochondrial-nuclear pairing for two different but very fit cell types, these new pairings gave unhealthy cells, meaning that the original fit cells had already found their perfect “wand”.

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Harry Potter first meeting his perfectly-paired wand. From harrypotter.wikia.com.

So just like when Harry Potter was in Ollivander’s wand shop and finally found his holly-phoenix feather wand and felt unified with its amazing magic, yeast cells can acquire new super powers when their nuclear and mitochondrial genomes are perfectly paired!

by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.

…and we wish a fond farewell to Barry Starr, Ph.D. who has left the Stanford Department of Genetics for new horizons. We miss you Barry and wish you well!

Categories: Research Spotlight

Tags: recombination, mitochondria, environmental stress

SGD April 2018 Newsletter

May 01, 2018

SGD periodically sends out its newsletter to colleagues designated as contacts in SGD. This April 2018 newsletter is also available on the community wiki.

Categories: Newsletter

Out of China: Changing our Views on the Origins of Budding Yeast

April 17, 2018

1,011. That’s the number of different Saccharomyces cerevisiae yeast strains that were whole-genome sequenced and phenotyped by a team of researchers jointly led by Joseph Schacherer and Gianni Liti, published this week in Nature (Peter et al., 2018; data at: http://bit.ly/1011genomes-DataAtSGD).

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Ecological origins of the 1,011 isolates (from Peter et al., 2018; Creative Commons license)

Scrupulously gathering isolates of S. cerevisiae from as many diverse geographical locations and ecological niches as possible, the authors and their collaborators plucked yeast cells not only from the familiar wine, beer and bread sources, but also from rotting bananas, sea water, human blood, sewage, termite mounds, and more. The authors then surveyed the evolutionary relationships among the strains to describe the worldwide population distribution of this species and deduce its historical spread.

They found that the greatest amount of genome sequence diversity existed among the S. cerevisiae strains collected from Taiwan, mainland China, and other regions of East Asia. This means that in all likelihood the geographic origin of S. cerevisiae lies somewhere in East Asia. According to the authors, our budding yeast friend began spreading around the globe about 15,000 years ago, undergoing several independent domestication events during its worldwide journey. For example, it turns out that wine yeast and sake yeast were domesticated from different ancestors, thousands of years apart from each other. Whereas genomic markers of domestication appeared about 4,000 years ago in sake yeast, such markers appeared in wine yeast only 1,500 years ago.

Additionally — and similar to the situation where human interspecific hybridization with Neanderthals occurred only after humans migrated out of Africa — it appears that S. cerevisiae has inter-bred very frequently with other Saccharomyces species, especially S. paradoxus, but that most of these interspecific hybridization events occurred after the out-of-China dispersal.

There are many more gems to be found among the treasure trove of information in this paper. Some notable conclusions from the authors include: diploids are the most fit ploidy; copy number variation (CNV) is the most prevalent type of variation; most single nucleotide polymorphisms (SNPs) are very rare alleles in the population; extensive loss of heterozygosity is observed among many strains. There are also phenotype results (fitness values) for 971 strains across 36 different growth conditions.

As is often the case for yeast, the ability to sequence and analyze whole genomes at very deep coverage has yielded broad insights on eukaryotic genome evolution. The team’s work highlights this by presenting a comprehensive view of genome evolution on many different levels (e.g., differences in ploidy, aneuploidy, genetic variants, hybridization, and introgressions) that is difficult to obtain at the same scale and accuracy for other eukaryotic organisms.

SGD is happy to announce that in conjunction with the authors and publishers, we are hosting the datasets from the paper at this SGD download site. These datasets include: the actual genome sequences of the 1,011 isolates; the list of 4,940 common “core” ORFs plus 2,856 ORFs that are variable within the population (together these make up the “pangenome”); copy number variation (CNV) data; phenotyping data for 36 conditions; SNPs and indels relative to the S288C genome; and much more. We hope that the easy availability of these large datasets will be useful to many yeast (and non-yeast) researchers, and as the authors say, will help to “guide future population genomics and genotype–phenotype studies in this classic model system.”

Categories: Announcements, New Data

Tags: strains, evolution, genome wide association study, Saccharomyces cerevisiae

In Memoriam: André Goffeau

April 12, 2018

It was with great sadness that we learned that André Goffeau, renowned yeast researcher and Professor at the Université Catholique de Louvain in Belgium, passed away on April 2, 2018.

Prof. Goffeau worked on yeast transporter genes and multidrug resistance for much of his scientific career, and made many contributions to this field. But he will forever be remembered for his visionary idea to sequence the entire genome of Saccharomyces cerevisiae, ultimately leading to the coordination of a world-wide collaborative effort during the late 1980s and early 1990s by researchers from 19 countries working in 94 laboratories. The sequencing project, which represented the first completely sequenced eukaryotic genome, culminated in the landmark publication “Life with 6000 Genes” (Goffeau et al. 1996). But of course this was only the beginning of a cascading myriad of discoveries, methods, resources and careers built upon the existence of the yeast genome sequence.

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The collaborative nature of the yeast community’s effort was nicely summed up in the 1996 Goffeau et al. paper: “Whether they worked in large centers or small laboratories, most of the 600 or so scientists involved in sequencing the yeast genome share the feeling that the worldwide ties created by this venture are of inestimable value to the future of yeast research” and indeed this has proved true. Prof. Goffeau was recognized with many awards and honors over his career, including the 2002 Beadle Medal of the Genetics Society of America for his work in having “initiated and successfully led the yeast genome sequencing project”. After the completion of the S. cerevisiae genome he continued to sequence whole genomes of other microbes and also worked on novel anti-cancer agents. Prof. Goffeau was a highly praised mentor and published hundreds of scientific papers of which many resulted from large collaborations; he also served on journal editorial boards, organized meetings, and performed many other valuable services to the scientific community over his career. He was an active and treasured part of the yeast community and we will miss him greatly.

Categories: Announcements

Trouble with Triplets

April 06, 2018


This Research Spotlight also comes as a video animation. Be sure to check it out!

In the Star Wars movie Attack of the Clones, Padme and Anakin end up fighting Genosians on an automated assembly line. Padme manages to survive because the assembly line is set up in an ordered and predictable way. She knows when to jump, when to pause and so on to survive. If there was more chaos in the line, she might have been killed and then there’d be no Luke or Leia!

If the Genosian assembly line weren’t predictable, Padme might’ve ended up a pancake.

The same kind of thing can happen when cells read genes. An enzyme called RNA Polymerase II (Pol II) slides along the gene, spewing out a long string of messenger RNA behind it.

Just like the assembly line on Genosis, the gene is set up in an ordered and predictable way. Nucleosomes (barrel-shaped clusters of proteins) are spaced along the gene’s DNA to help keep it from getting tangled up. But when Pol II comes hustling along, the nucleosomes need to be carefully removed in front and then replaced after it passes.

Similar to the plight of poor Padme getting hurt if the assembly line were not predictable, genes can likewise get damaged if the nucleosome removal/replacement process goes haywire. In Star Wars, the result of a defective assembly line is damaged droids and TIE fighters. In cells, the result is a changed assembly line—the gene can end up longer! This change can be harmful for both the gene and the cell.

In a new study in GENETICS, Koch and coworkers use our favorite beast, the yeast Saccharomyces cerevisiae, to show that the ISW1 gene is a key player in making sure that the nucleosomes get back on DNA after being taken off. When yeast lack the ISW1 gene, nucleosomes don’t always return after being removed to make way for Pol II. And for some genes, this spells even more trouble.

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“CAG” starts to spell “trouble” if genes get too many of them. from Wikimedia Commons.

The genes that have the most problems are those that have something called triplet repeats. Basically, this means the same 3 bases (e.g. CAG) are repeated many times in a row in the gene.

Using PCR, Koch and coworkers showed that a gene with triplet repeats ended up with more of them if the ISW1 protein (Isw1p) wasn’t there to shepherd the nucleosomes properly. And too many repeats in a gene can be harmful–not just to the yeast, but to us as well.

In fact, Triplet Repeat Expansion Disorders happen when the number of repeats increases from one generation to the next.  These diseases are some of the most devastating ones around.

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Woody Guthrie succumbed to a triplet repeat disorder, Huntington’s Disease. from Wikipedia.

They mostly cause slow but steady degeneration of nerve and brain cells, and the cruel symptoms (loss of body movement control, dementia) often only show up in mid-life. The most well-known is probably Huntington’s disease, which killed folk-singer Woody Guthrie.

So understanding how Isw1p helps keep the Pol II assembly line running smoothly in yeast might help us understand how triplet repeat expansion happens in humans, and may eventually give us ideas how to keep it from happening in the first place. This is especially likely because humans have proteins that are similar to Isw1p and probably do something similar.

To determine how Isw1p regulates nucleosome reassembly, Koch and coworkers used Southern blots to show that yeast that lacked Isw1p couldn’t replace their nucleosomes after Pol II had passed as efficiently as yeast that had the protein. This means that when cells are missing the ISW1 gene, a long stretch of the DNA is left bare after Pol II has passed by.

This stretch of nucleosome-free DNA can end up forming new structures called hairpins that can cause cells to send in DNA repair machinery to deal with it. Unfortunately this machinery isn’t always that great at fixing the DNA. Like the Three Stooges adding more pipe to try to fix a leak, the cell can end up adding more DNA to deal with the hairpin.

Like adding extra DNA, throwing extra pipes at a plumbing leak is a great way to make a bad problem worse.

But for the cell, the results are not as hilarious as they were for Moe, Larry, and Curly. That extra DNA can lead to deadly genetic diseases.

A yeast cell needs Isw1p to keep the cell from bringing in the Three Stooges to mess up its DNA and potentially cause devastating genetic diseases. If it turns out the same is true in people, then once again yeast will have shown us how human genetic diseases might happen. And perhaps provide a target for us to go after to prevent these diseases from happening. #APOYG!

by Barry Starr, Ph.D., Barbara Dunn, Ph.D., and Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: DNA repair, ISW1, RNA polymerase II, nucleosome, chromatin remodeling

Apply Now for the 2018 Yeast Genetics and Genomics Course

March 16, 2018

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For almost 50 years, the legendary Yeast Genetics and Genomics course has been taught each summer at Cold Spring Harbor Laboratory.

For almost 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 15th, so don’t miss your chance! Find all the details and application form here. This year’s instructors – Grant Brown, Greg Lang, and Elçin Ünal – have designed a course (July 24 – August 13) 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
  • Isolation and Characterization of Mutants
  • Yeast Transformation, Gene Replacement by PCR, and Construction and Analysis of Gene Fusions
  • Tetrad Analysis
  • Genome-scale Screens Using Synthetic Genetic Array (SGA) Methodology    
  • Deep Sequencing Applied to SNP Mapping and Deep Mutational Scanning
  • Exploring Synthetic Biology with CRISPR/Cas9-directed Engineering of Biosynthetic Pathways
  • Computational Methods for Data Analysis
  • Modern Cytological Approaches Including Epitope Tagging and Imaging Yeast Cells Using Fluorescence Microscopy

Techniques have been summarized in a completely updated course manual, which was recently published by CSHL Press.

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There’s fierce competition between students at CSHL courses in the Plate Race, a relay in which teams carry stacks of 40 Petri dishes (used, of course).

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 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!

 

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