New & Noteworthy

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).

1011genomes_FigS1bPie_chart_PaperVersion-crop

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

Transcription Factors: Kings of the Genome Jungle

October 20, 2017


Imagine that Jane is in trouble on the edge of the jungle. She needs to be saved soon or she will be sent back to Europe (which she does not want).

june71

Tarzan got to Jane in time by swinging on vines. Just like Mig1p needs to “swing” on DNA to get to its binding sites in the yeast genome on time. (DisneyClips)

Tarzan knows he can’t get there in time by running along the jungle floor. But of course he has a trick up his sleeve—swinging from vine to vine!

He gets there in time, fends off her would-be kidnappers, and saves the day. All because he transferred from one strand of vine to another instead of “sliding” along the jungle floor.

A new study by Wollman and coworkers shows that at least two transcription factors in Saccharomyces cerevisiae, Mig1p and Msn2p, seem to share a lot in common with Tarzan. In a process termed intersegment transfer, they get to where they need to in the genome by “swinging” from DNA segment to DNA segment instead of just sliding along the DNA.

And transcription factors like these need to get to the right place in time to save the cell from outside threats. Just like Tarzan had to swing from vine to vine to save Jane.

This sort of approach would not necessarily work well with just a single transcription factor with a single DNA binding domain. It would sort of be like a one-armed Tarzan—it is hard to take advantage of swinging from vine to vine without at least two arms!

The authors argue that Mig1p gains its “extra arms” through joining together into a cluster. Now each Mig1p can bind DNA and drag the other transcription factors with them. A neat solution to the one arm problem.

Their basic approach is to use fluorescence microscopy to follow a GFP-Mig1p fusion protein in a single cell, something that has only become possible recently. What they found was that there were two populations of transcription factors—a diffuse set of smaller molecules and distinct, larger clusters made up of multiple Mig1p’s.

The clusters appeared to be the ones doing the work in the nucleus. Kinetic studies showed that they stayed in one place in the nucleus for over 100 seconds which is consistent with the clusters and not the monomers being bound to the DNA.

OK so this transcription factor tends to clump up into clusters and it looks like these clusters are the ones regulating gene expression. They also showed that a second transcription factor, Msn2p, did the same thing.

The authors next set out to see if this approach made sense for genetic regulation by running simulations of Mig1p finding its sites in the nucleus as either monomers or as clusters. It made sense to form clusters.

A lot of previous work has been done in S. cerevisiae in terms of the three dimensional map of the genome and where Mig1p DNA binding sites were located in this mesh of DNA. And in the course of their studies, Wollman and coworkers were able to estimate how many Mig1p molecules were in yeast cells and how many were in clusters.

MultiArmedMonster

This mythical beast is more like a cluster of Mig1p proteins with its multiple arms representing multiple DNA binding domains with which to grab strands of DNA. (Wikimedia Commons)

They now had all the information they needed to run their simulations. When they crunched the numbers, they found that clusters fit their data much better than monomers (R2 = 0.75 vs. R2 < 0).

The final step was to work out what part of Mig1p was involved in forming the clusters. To do this, the authors compared Mig1p and Msn2p, the second transcription factor they studied that also formed into clusters, and looked for structural regions they might have in common.

What they found was both proteins had a highly disordered region. For Mig1p, it was at the C-terminus and for Msn2p it was at the N-terminus.

The hypothesis is that these disordered regions, which are both at the opposite end of the protein from the DNA binding domain, interact and form ordered structures that enable clusters to form.  Wollman and coworkers used circular dichroism to show that when Mig1p was put in conditions that favor cluster formation, there was a transition consistent with unstructured protein becoming structured.

What we seem to have is a cluster of transcription factors connected in the middle with their DNA binding domains pointing out. This rolling cluster can more easily hop from DNA strand to DNA strand to find the right spots to bind.

Without this mechanism, Mig1p couldn’t get to where it needs to in time. It is as if Tarzan had 6-9 arms circling his body so he could get to Jane even more quickly.

With the wide range of tools available and our deep understanding of how yeast works and how a yeast cell is organized, our trusted ally S. cerevisiae again teaches us something fundamental about how our biology works.

Mig1p may be able to swing like Tarzan but it can’t yell like him. Or like Carol Burnett!

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Department of Genetics

Categories: Research Spotlight

Tags: free diffusion, MIG1, intersegment transfer, Saccharomyces cerevisiae, transcription factor

Of Medieval Market Townes and Wasp Guts

February 03, 2016


As market towns like this one were a place where isolated medieval Europeans could find partners to take back home, so to are a wasp’s gut for yeast. Image from Wikimedia Commons.

Back before trains, planes and automobiles, people didn’t get around as much. And for the people of medieval Europe, this could be a real problem genetically.

At this time there were a lot of small, isolated villages scattered across Europe. If people in these villages stayed put, inbreeding might have gotten as bad as the poor Spanish Hapsburgs. Their last king, Charles II, was infertile, riddled with genetic diseases and his royal line died out with him.

One reason (among many) that this didn’t happen to people all over Europe was market towns. These were centrally located places where villagers came to sell goods. And where they also found partners to bring home to freshen up the gene pool.

Turns out that out in the wild, our friend yeast is in an even worse predicament than medieval Europeans. Because they are all clones of each other, they exist in isolated colonies with almost no genetic diversity.

Yeast are also way less mobile than people. They do have spores but these don’t tend to travel very far without help.

And yet, looking at yeast DNA shows that yeast definitely get around. There are all sorts of signs of various DNA mixing over time. So where are all these yeast hooking up?

A new study by Stefanini and coworkers in PNAS suggests that yeasts’ market towns are in the guts of wasps. It is there that various yeasts can meet and mate before heading back to their “villages.”

This makes sense in a lot of ways. First off, as we described in an earlier blog, there is good evidence that yeast winter in wasp guts.

So there are definitely a variety of yeast hanging around for months, waiting for warmer weather. The gut is also the kind of harsh place where spore dissolution, the first step in yeast mating, can happen.

When the authors looked at the yeast isolates from a wasp’s gut they saw a lot more outbreeding compared to other sources. This suggests that a lot of mating is indeed going on there.

The next step was to directly test how much mating can actually happen in a wasp gut. Stefanini and coworkers tested this by having the wasps eat five different yeast strains and then analyzing the isolates genetically over time. They compared the results from this experiment to the amount of mating that happens in wine must and under ideal lab conditions.

What they found was a whole lot of mating going on.

After two months, around 1/3 of the yeast in the wasp’s gut were outcrossed. This is OK but pretty comparable to what is found in wine must.

It was a different story after four months. Now 90% of the yeast were outcrossed. This is an even better result than scientists typically get in the lab. Clearly the wasp gut is a great place for a yeast to find a partner.

The authors also found that the S. paradoxus strain had to mate to survive in the gut. The only time they found this strain in yeast isolates was in hybrids with S. cerevisiae.

The next steps will be to see if this kind of mating actually has a big effect on yeast diversity in the wild. And of course what, if anything, the wasp gets out of hosting these cavorting yeast.

A market town was great for both the town and the visitors. People met up, sold goods, found partners and the towns prospered from all of this traffic. I can’t wait to find out if the wasp/yeast situation is so mutually beneficial as well.

Jerry Lee Lewis has a whole lot of shakin’ going on, just like a wasp’s gut has a whole lot of matin’ going on.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: inbreeding, Saccharomyces cerevisiae, mating, Saccharomyces paradoxus, outcrossing

Clearing Customs in the Nucleus

January 06, 2016


Getting through the nuclear pore is like going through customs at the airport. And now we can see the mRNA make this journey in real time. Image from Wikimedia Commons.

Going through customs at the airport is a necessary evil. Once off the plane, you need to stand in line, scan for an open station, have various forms looked over and possibly stamped before you can pass through the airport doors and get into a new country.

And of course if there is anything wrong, you can be sent back to get your papers in order. A pain but it does help protect people.

Things work pretty similarly in the nucleus. The mRNA disembarks off the DNA, gathers up a set of proteins, and heads for the nuclear pore. There its proteins are checked and if everything is in order, it is allowed to proceed to the cytoplasm. And if there are problems, it is denied entry.

A couple of new studies out in the Journal of Cell Biology use imaging microscopy to give us a close up view of the bustling airport that is the nucleus of a yeast cell. It is utterly fascinating.

Both studies showed that mRNAs often hang out at the nuclear envelope, pausing at a nuclear pore and then sometimes moving to a new one. And that factors both in the nuclear pore and bound to the mRNA affect this scanning of the nuclear envelope.

The basic strategy with both studies is to fluorescently label specific mRNAs in a live yeast cell and follow its journey from the nucleus to the cytoplasm. To do this, they also needed to fluorescently label the nuclear pores, the custom stations in the nuclear envelope.

They labeled the mRNA using the bacteriophage PP7 RNA-labeling system. Basically, they load up the untranslated region (UTR) of a specific gene with sequences that form specific loops. Once transcribed, these loops are then bound by fluorescently labeled PP7 coat protein. Now they can track this labeled mRNA.

To more easily track mRNAs, they chose low expressing genes. That way they could follow a single mRNA more easily. They also needed to get rid of the yeast cell wall so they could see inside the cell better.

Overall they found that at least in yeast, the mRNA takes around 200 milliseconds to get exported to the cytoplasm. Very little of this time is spent in the nucleoplasm; the mRNA very quickly makes its way to a nuclear pore.

Once there things slow down. The mRNA stays at a nuclear pore or slides along the nuclear envelope to a different pore in a process the authors call scanning. Eventually the lucky successfully make it through the pore to the cytoplasm where they can seek out a ribosome for translation. Around 90% of the mRNAs they studied made it through.

They had a couple of different ideas about why the mRNA hangs around the nuclear envelope for so long. One is that the extended stay at the pore is to make sure everything is in order with the mRNA. It can’t pass through customs unless all of the right forms have been filled out properly.

Another possibility is that by scanning it is looking for a nuclear pore that is competent for exporting. It has to search for an available customs agent.

Now that the authors had established a system to look at mRNA export, they next set out to see which factors play important roles. As you might guess, mucking with parts of the nuclear pores or the proteins that bind the mRNA can throw a monkey wrench into the process.

In the first study, Smith and coworkers looked at what happens to the process when one of the key mRNA binding proteins, Mex67p is mutated. This protein is known to interact with the nuclear pore.

Losing the nuclear basket means mRNAs fall away from nuclear pores more easily. It is like getting to the head of the line and then having it close for lunch. Image from thornet on flickr.

It has also been proposed that Mex67p is important in making sure the trip through the pore is one way. Once the mRNA goes through, it releases Mex67p which makes the mRNA let go of the cytoplasmic side of the nuclear pore. The imaging studies here confirmed that Mex67p is indeed important for mRNA directionality.

Using a temperature sensitive mutant of Mex67p the researchers found that the mRNA they tracked stayed at the nuclear envelope about three times longer than in a wild type strain. The process was also much less efficient with only 32% making it to the cytoplasm instead of the 90% seen in the wild type strain. And of the 14 mRNAs which failed to make it through the pore, 7 headed back through the pore to the nucleus.

In the second study, Saroufim and coworkers concentrated on a part of the nuclear pore called the nuclear basket. This is the first part of the nuclear pore that the mRNP, the mRNA plus its proteins, encounters.

They found that deleting or mutating two key parts of the nuclear basket, MLP1 and MLP2, made the mRNA linger for a shorter time at the pore. The mRNA no longer scans the nuclear envelope.

But that didn’t mean the mRNA passed through to the cytoplasm more quickly. No, it just tended to fall back into the nucleoplasm and then have to reattach more often.

It is as if you had to deal with a customs agent who keeps sending you back into the airport. Or agents who keep putting up the “Out to Lunch” sign as soon as you get to the head of the line.

These two studies give researchers a way to study mRNA export in live cells in real time. As we piece together which proteins play what role, we will get a better handle on this important part of gene expression.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, mRNA export, nuclear pore

Pulsating Yeast

November 19, 2015


A glass of tepid water will do little for a sprained ankle. Just like adding activators and repressors to a gene will have little effect. Image from Wikimedia Commons

Sometimes when you get a minor injury, doctors will recommend alternating heat and cold as a therapy. The heat opens things up and the cold shuts them back down again.

Now obviously it would be pretty useless to apply both at the same time. Adding a bit of lukewarm water to an injury is not going to be very helpful at all.

The same thing holds true for many genes. If activators and repressors all turned on at the same time, there wouldn’t be much of an effect on the expression of a gene regulated by both. It is no way to respond to something in the environment!

Instead, if you want a gene to go up and then go back down again, you’d have the activator turn on first, followed by the repressor. Another way to put this is you’d have a pulse where all of the activators activate their genes at once and then stop working followed by a pulse where all of the repressors work at once.

This is exactly what Lin and colleagues found in their recent study in Nature. There they looked at the effect of certain external stimuli on the timing of when the activator Msn2p activated genes and when the repressor Mig1p repressed genes in our favorite yeast S. cerevisiae. These transcription factors coregulate many of the same genes.

The authors found that in the presence of either lowered glucose concentrations or 100 mM NaCl, most of the Msn2p in the cell turned on first followed closely by the Mig1p repressors. In the absence of either stimulus, there was no coordination.

So there does seem to be a carefully choreographed dance between these two transcriptional regulators with these signals. But of course gene regulation is a bit more complex than a sprained ankle.

There may be situations where a cell wants both regulators to do their jobs at the same time. Sometimes lukewarm water may be just what the doctor ordered.

And this is what Lin and colleagues found with 2.5% ethanol. Under this condition, the pulses of the two regulators overlapped—both were on at the same time. Apparently different stimuli call for different responses which means different timing of transcription factor pulses.

The authors next wanted to get at why Mig1p repression lagged behind Msn2p activation. Since both transcription factors can only enter the nucleus and do their job after they lose a few key phosphate groups, the authors reasoned that perhaps Mig1p dephosphorylation lagged behind that of Msn2p.

They decided to look at the PP1 phosphatase, Glc7p, as previous work had shown that it can indirectly regulate both Msn2p and Mig1p. And indeed, when the authors lowered the expression of GLC7, Msn2p and Mig1p no longer pulsed one after the other at lower glucose concentrations. It looks like Glc7p is a key player in controlling the pulsing of these two regulators.

Even though much of this work was done with synthetic promoters with Mig1p and Msn2p binding sites, the results were not restricted to these artificial constructs. Lin and colleagues found that around 30 endogenous targets also responded to lowered glucose concentrations in a coordinated way just like their synthetic construct. Yeast regulates genes by controlling when activators and repressors pulse.

Finally, all of these studies were done using fluorescent proteins and filming single cells in real time. (Is biology cool or what?) This makes sense because subtle signs of synchronization can be lost when averaged over a large population.

Just like a synchronized swim team, yeast regulates genes by controlling when activators and repressors can work. Image from Wikimedia Commons.

This also allowed the authors to investigate what happens in unstimulated cells. In other words, what happens when both regulators enter the nucleus at the same time? Or if a repressor gets in first?

The first thing they found was that even in the absence of stimulation, there were still pulses. So at seemingly random times, suddenly all of the Msn2p would swoop into the nucleus at the same time and then all leave a short time later. Or the same thing would happen with Mig1p.

If by chance the two entered the nucleus at the same time, both the synthetic reporter and an endogenous gene, GSY1, were not activated. But if Msn2p happens to get in there first, both were activated.

And if the repressor Mig1p managed to get into the nucleus at least 4-5 minutes before Msn2p, activation by Msn2p was muted. The presence of Mig1p beforehand seemed to keep Msn2p from activating coregulated genes to as high a level.

Taken together these results confirm that just like a synchronized swim team, yeast regulates genes by controlling when activators and repressors can work. First there is a pulse where the all of the molecules of a certain activator are primed to do their job and then, after a short time, they all stop doing their job. This can then be followed later by a pulse of repressors shutting it all down.

And this isn’t just in yeast either. For example, these kinds of pulses are important in neuroscience as well.

This work suggests that in dissecting regulatory pathways, researchers may need to pay more attention to the timing of pulses. Then they can see that hot followed by cold makes much more sense than both together.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, transcription regulation

Yeast, the Spam Filter

November 11, 2015


If you don’t have a good spam filter for your email, you may be overwhelmed—just as the sheer number of variants of human genes can be overwhelming. Luckily, yeast can help us filter out the variants that matter. Image by Jean Pierre Gallot via Flickr

Imagine what our email inboxes would look like if we didn’t have spam filters! To find the meaningful emails, we’d have to wade through hundreds of messages about winning lottery tickets, discount medications, and other things that don’t interest us.

When it comes to sorting out meaningful mutations from meaningless variation in human genes, it turns out that our friend S. cerevisiae makes a pretty good spam filter. And as more and more human genomic sequence data are becoming available every day, this is becoming more and more important.

For example, when you look at the sequence of a gene from, say, a cancer cell, you may see many differences from the wild-type gene. How can you tell which changes are significant and which are not?

SuperBud to the rescue! Because many human proteins can work in yeast, simple phenotypes like viability or growth rate can be assayed to test whether variations in human genes affect the function of their gene products. This may be one answer to the increasingly thorny problem of variants of uncertain significance—those dreaded VUS’s.

In a new paper in GENETICS, Hamza and colleagues systematically screened for human genes that can replace their yeast equivalents, and went on to test the function of tumor-specific variants in several selected genes that maintain chromosome stability in S. cerevisiae. This work extends the growing catalog of human genes that can replace yeast genes.

More importantly, it also provides compelling evidence that yeast can help us tell which mutations in a cancer cell are driver mutations, the ones that are involved in tumorigenesis, and which are the passenger mutations, those that are just the consequence of a seriously messed up cell. Talk about a useful filter!

The researchers started by testing systematically for human genes that could complement yeast mutations. Other groups have done similar large-scale screens, but this study had a couple of different twists.

Previous work from the Hieter lab had identified genes in yeast that, when mutated, made chromosomes unstable: the CIN (Chromosome INstability) phenotype. Reduction-of-function alleles of a significant fraction (29%) of essential genes confer a CIN phenotype. The human orthologs of these genes could be important in cancer, since tumor cells often show chromosome rearrangements or loss. 

So in one experiment, Hamza and colleagues focused specifically on the set of CIN genes, starting with a set of 322 pairs of yeast CIN genes and their human homologs. They tested functional complementation by transforming plasmids expressing the human cDNAs into diploid yeast strains that were heterozygous null mutant for the corresponding CIN genes. Since all of the CIN genes were essential, sporulating those diploids would generate inviable spores—unless the human gene could step in and provide the missing function.

In addition to this one-to-one test, the researchers cast a wider net by doing a pool-to-pool transformation. They mixed cultures of diploid heterozygous null mutants in 621 essential yeast genes, and transformed the pooled strains with a mixture of 1010 human cDNAs. This unbiased strategy could identify unrecognized orthologs, or demonstrate complementation between non-orthologous genes.

In combination, these two screens found 65 human cDNAs that complemented null mutations in 58 essential yeast genes. Twenty of these yeast-human gene pairs were previously undiscovered.

The investigators looked at this group of “replaceable” yeast genes as a whole to see whether they shared any characteristics. Most of their gene products localized to the cytoplasm or cytoplasmic organelles rather than to the nucleus. They also tended to have enzymatic activity rather than, for example, regulatory roles. And they had relatively few physical interactions.

So yeast could “receive messages” from human genes, allowing us to see their function in yeast. But could it filter out the meaningful messages—variations that actually affect function—from the spam? 

The authors chose three CIN genes that were functionally complemented by their human orthologs and screened 35 missense mutations that are found in those orthologs in colorectal cancer cells. Four of the human missense variants failed to support the life of the corresponding yeast null mutant, pointing to these mutations as potentially the most significant of the set.

Despite the fact that these mutations block the function of the human proteins, a mutation in one of the yeast orthologs that is analogous to one of these mutations, changing the same conserved residue, doesn’t destroy the yeast protein’s function. This underscores that whenever possible, testing mutations in the context of the entire human protein is preferable to creating disease-analogous mutations in the yeast ortholog.

Another 19 of the missense mutations allowed the yeast mutants to grow, but at a different rate from the wild-type human gene. (Eighteen conferred slower growth, but one actually made the yeast grow faster!)

For those 19 human variants that did support life for the yeast mutants, Hamza and colleagues tested the sensitivity of the complemented strains to MMS and HU, two agents that cause DNA damage. Most of the alleles altered resistance to these chemicals, making the yeast either more or less resistant than did the wild-type human gene. This is consistent with the idea that the cancer-associated mutations in these human CIN gene orthologs affect chromosome dynamics.

As researchers are inundated by a tsunami of genomic data, they may be able to turn to yeast to help discover the mutations that matter for human disease. They can help us separate those emails touting the virtues of Viagra from those not-to-be-missed kitten videos. And when we know which mutations are likely to be important for disease, we’re one step closer to finding ways to alleviate their effects. 

by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD

Categories: Research Spotlight, Yeast and Human Disease

Tags: chromosome instability, yeast model for human disease, functional complementation, Saccharomyces cerevisiae

Life Needs to be More Like a 1950’s Chevy

October 21, 2015


Stripped of modern bells and whistles, cars last a lot longer. The same may be true of life. It may last longer when some extra, nonessential genes are removed. Image via Wikimedia Commons

In the old days, a car came with the bare minimum of features to get from point A to point B. The windows rolled down with a crank and it usually had a radio. That was about it.

As the car has evolved, it has gained a huge number of bells and whistles. There are power windows and power brakes, a baffling number of computer-based bonus features, personal wifi hotspots, and so on. All of these have undoubtedly made cars more fun and comfortable to drive. But they have come at a cost. Many cars simply do not last as long as their predecessors because these extras break easily.

Turns out life may be like a modern car. It has lots of nice features that help it to do better in the world. But a lot of these features may shorten its life span.

This point was reinforced in a recent study by McCormick and coworkers. They painstakingly searched through a library of 4,698 single gene deletion strains in S. cerevisiae and found that 238 of these strains were able to produce significantly more buds over their lifetime. Many nonessential genes seem to shorten a yeast’s life.

And boy was it painstaking! Believe it or not, they manually dissected over 2.2 million individual yeast daughter cells to generate these results. Luckily it was worth it, as they found so many interesting things.

First off, many of the genes they found fall into a set of five pathways that includes cytosolic and mitochondrial translation, the SAGA complex, protein mannosylation, the TCA cycle, and proteasomal activity. So there are certain pathways we can target to extend the lifespan of our friend yeast. And even better, yeast may not be the only beneficiary of these studies.

Two of the pathways, cytosolic and mitochondrial translation and the TCA cycle, have also been found to be significant in extending the life of the roundworm C. elegans. These pathways are also shared with humans.

And just because the authors found no overlap with the other three pathways in other beasts doesn’t mean they may not be targets for life extension in them too. It could be that previous screens in C. elegans simply missed genes from these pathways.

It could also be that what is found in yeast may turn out to be important in people but not in C. elegans. For example, the authors failed to find any equivalent to the SAGA complex in C. elegans. Either the roundworm lost this complex during evolution, or the homologs between yeast and C. elegans are so different that they’re unrecognizable. In any event, humans at least do have an equivalent to SAGA, called STAGA.

All of this suggests that there may be common ways to make organisms, including people, live longer, healthier lives. Here’s hoping!

And these five pathways are certainly not the whole story. The majority of the genes McCormick and coworkers identified were not in these five, which means there are probably lots of other ways to get at living longer.

One fascinating example that the authors decided to look at in depth was LOS1. Deleting it had one of the biggest effects on a yeast’s reproductive life span.

At first this seems a little weird, as Los1p exports tRNAs out of the nucleus. As expected, deleting LOS1 led to a buildup in tRNAs in the nucleus. The authors confirmed that this buildup is important by showing that overexpressing MTR10, a gene involved in transporting tRNAs from the cytoplasm to the nucleus, led to a longer lived yeast with a buildup of tRNAs in its nucleus.

The next step was to figure out why having a lot of tRNA in the nucleus makes yeast live longer. It was known previously that Los1p is kept out of the nucleus under glucose starvation conditions. The authors confirmed this result.

Most everyone knows that restricting calorie intake (also called dietary restriction or DR) can extend the lives of most every beast tested so far, including yeast. The authors found that growing a los1 deletion strain at low glucose did not increase the lifespan of this strain any further. It thus appears that an important consequence of DR is keeping Los1p out of the nucleus and thereby increasing the amount of tRNA in the nucleus.

While we don’t know yet exactly why keeping tRNAs in the nucleus helps yeast live longer, it is interesting that the increased lifespan associated with the loss of LOS1 is linked to caloric restriction. Finding a way to inhibit Los1p has to be better than starving yourself!

This study has identified 238 genes to follow up on for future studies. And of course there is a whole class of genes that haven’t yet been investigated—the essential genes! Many of these may be important for extending life too. 

Stripping life down to its bare essentials may help individuals live longer at the expense of being the most fit in terms of survival in the hurly burly world of nature. After all, those “nonessential” genes undoubtedly have a function in helping yeast outcompete their less well-endowed yeast neighbors. Just like those power sliding doors are way better than the manual ones on a minivan.

But if you want a long-lived minivan, get the one with the manual doors. And if you want a long-lived yeast (or person), get rid of some of those nonessential genes that cause you to break down.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: aging, Saccharomyces cerevisiae, lifespan

You Can Take Yeast Off of the Grapevine, But…

October 14, 2015


All over the world and through the ages, people have moved from the country into the big city to look for a better life. These folks often find that even though they can adapt to city life and city ways, they still hang on to their core country values. As the old saying goes, “You can take the boy out of the country, but you can’t take the country out of the boy.”

Even when he goes to the city, the country mouse hangs on to his country ways. The same is true for S. cerevisiae—even though it entered the lab, it still clung to genes that were most useful out in the vineyard. Illustration by Arthur Rackham (1912) via Wikimedia Commons

Our friend Saccharomyces cerevisiae didn’t migrate voluntarily into the lab. But it ended up there, and has been as lonely as a new migrant in a big city. 

Which is of course how we need it to be. One of the basic tenets of classical microbiology is that you can’t begin to study an organism until you’ve isolated it in a pure culture.

And studying pure S. cerevisiae has yielded a huge body of knowledge about molecular biology, cell biology, and genetics. But by not studying yeast in the context of its old country home, we may have missed a few things.

In a new article in PLOS ONE, Rossouw and colleagues uncover one of them. S. cerevisiae has a family of FLO genes that promote flocculation, the adherence of yeast cells to each other. It has always been a bit puzzling why a whole family of genes that are pretty much redundant with each other would be maintained through evolution.

When the researchers took S. cerevisiae out of its lab isolation by mixing it with other yeast species, they found that the different flocculation genes actually determine which species it can co-flocculate with. Different Flo proteins prefer different partners. 

This discovery helps us understand the evolution of this gene family and also opens the door to further study of inter-species interactions in the vineyard. And since flocculation is an important property in winemaking and brewing, there could even be tasty practical applications of this knowledge.

The researchers started by surveying 18 non-Saccharomyces yeast strains that are found in vineyards. They looked at the ability of the yeasts to flocculate both as pure strains and when mixed with either of two S. cerevisiae wine strains.

Intriguingly, certain species showed a synergistic effect when mixed with S. cerevisiae, flocculating more than either species on its own. Rossouw and colleagues used microscopy to confirm that the “flocs” did indeed contain both yeast species—a simple observation, since the cells of different species have slightly different shapes.

To test the effects of different FLO genes on co-flocculation, the authors assayed the co-flocculation ability of flo1, flo10, and flo11 deletion mutants as well as Flo1, Flo5, and Flo11 overproducers in individual combinations with six of the non-Saccharomyces yeasts. 

The results showed that Flo1 has general effects on flocculation. Overproduction increased co-flocculation across the board with all the species tested, while deletion of FLO1 consistently decreased it. In contrast, deletion of FLO10 didn’t have much effect on co-flocculation.

It was a different story for Flo5 and Flo11, though. Overproduction of each of these not only affected co-flocculation, but had species-specific or even strain-specific effects. Flo5 overproduction caused a relative increase in co-flocculation with Metchnikowia fructicola and a substantial decrease in co-flocculation with two different strains of Hanseniaspora opuntiae. Flo11 overproduction reduced co-flocculation with one of the Hanseniaspora opuntiae strains but not with the other. 

All of these experiments were done on mixtures of two species at a time. To get S. cerevisiae even further out of the lab, Rossouw and colleagues created a “consortium” of wine yeasts, a mixture of six species that are found in wine must (freshly pressed grapes) at the start of fermentation. They then added the FLO overproducer strains individually to the consortium, to see their effects in a more natural situation.

They let the yeast consortium flocculate, extracted total DNA from the flocculated or supernatant parts of the culture, and then used automated ribosomal intergenic spacer analysis (ARISA) to see which strains had co-flocculated. This technique can determine the relative abundance of different yeast species in a sample by sequencing a particular region of ribosomal DNA.

In this experiment, overexpression of each of the three FLO genes had significant effects on at least one of the species in the consortium. The species composition of the flocculated yeasts was uniquely different, depending on which gene was overexpressed.

The discovery that the flocculation genes have individual effects on association with other species goes a long way towards explaining why S. cerevisiae has maintained this gene family with so many members that apparently have the same function—at least, when you study a pure culture. Differential regulation of the FLO genes could affect the spectrum of other species that our favorite yeast interacts with. 

So, our friend S. cerevisiae didn’t actually get out of the lab in these experiments, but at least it got to rub shoulders with some of its old friends (buds?) from the vineyard. These experiments are a good reminder for researchers to think outside the lab.

And when S. cerevisiae and its friends get together outside the lab, beautiful things can happen. We’ll drink a toast to that!

by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD

If yeast could sing about its forced migration to the lab, it might sound like this.

Categories: Research Spotlight

Tags: vineyard, Saccharomyces cerevisiae, flocculation

Unleashing the Awesome Power of Yeast Transcription

October 07, 2015


With the right mutations, yeast can activate transcription over long distances—just as with the right social media, distance is meaningless for finding and organizing like-minded people. Image via Pixabay.com

In the old days, before the internet, planes or even mass publishing, it was hard to spark a quick, worldwide movement. You simply couldn’t reach out to likeminded people who lived far away.

Nowadays things are very different. With the advent of social media, it is now trivially easy to spread the word. Using Twitter and Facebook, organizers can easily and effectively organize people who live on the other side of the world.

In terms of transcription activation, our friend Saccharomyces cerevisiae seems to be stuck in the old world. Its transcription factors can only turn up nearby genes. This is different from most other eukaryotic beasts, where activation at a distance is routine.

Except maybe yeast isn’t as backward as we think. It may be that yeast has the potential to activate transcription at a distance, but keeps that potential locked away.

This idea is supported in a new study out in GENETICS by Reavey and coworkers. These authors found that they could mutate away yeast’s inability to activate transcription at a distance.

In other words, this ability is there but is just prevented from being used. Yeast is keeping social media out of the hands of its genes.

Getting around social media/internet controls is not easy. Pressure might need to be applied at multiple points before people power is finally released through social media. And even when it does happen, it can sometimes be hard to figure out exactly why certain events tipped the balance.

Turns out that both of these are also true for long range transcription activation in yeast. Mutating a single gene was not enough—it took mutations in multiple genes to see any significant effect.

As you can imagine, it would be very tricky to hit all of the right mutations for a polygenic trait in one fell swoop. This is why Reavey and coworkers started their mutant hunt with a strain that could already weakly activate transcription from a distance, a strain in which the SIN4 gene was deleted. Now they just needed additional mutations to make the effect stronger.

In their screen they used a reporter in which the GAL4 upstream activating sequence (UAS) was placed 799 base pairs upstream of the HIS3 reporter. This reporter gives very low levels of activity in a wild type strain. They included a second reporter, the URA3 gene under control of the same upstream sequences, because Reavey and Winston had discovered in previous work using a single reporter that cis acting mutations and chromosomal rearrangements were a frequent source of false positive results.

The researchers put the reporter strain through multiple rounds of mutation with UV light and selection with increasing levels of 3AT, a competitive inhibitor of His3. After each round of selection, they measured mRNA levels for HIS3 and URA3 and chose strains that not only had higher 3AT resistance but also showed more transcription of the reporter genes.  In the end they found three strains that survived in the presence of galactose (to turn on the activator) and 10 mM 3AT.

As expected, each strain had multiple mutations. One strain had acquired mutations in the GRR1 and MOT3 genes. To confirm that these were the most important mutations, Reavey and colleagues engineered a fresh strain with just the original sin4 null mutation and the selected grr1-1 and mot3-1 mutations. The fresh strain completely recapitulated the selected strain, showing that these three mutations could unlock yeast’s potential for long-range transcriptional activation.

It makes sense that a grr1 mutation could affect transcriptional activation. Grr1 is a ubiquitin ligase that destabilizes Med3 (also known as Pdg1), a key component of the Mediator complex involved in transcription activation. The researchers provided evidence that this is how the grr1-1 mutation affects the process, by showing that mutating MED3 mimicked the effects of mutating GRR1.

It’s also not too hard to imagine how a mutation in Mot3, a sequence-specific transcriptional activator, could affect transcriptional activation, presumably by changing the expression of a gene under its control.

If you stay away from social media, you’ll lose opportunities for impromptu pillow fights. Who knows what yeast is missing without long-range transcription activation? Image via Wikimedia Commons

The results were not so clear-cut for two other strains that were selected. They arose from the same lineage, and each had acquired the ptr3-1 mutation on top of the original sin4 null mutation. One strain went on to further pick up the mit1-1 mutation, while the other got an msn2-1 mutation.

Again it isn’t too surprising that mutations in genes that encode sequence-specific transcriptional activators like Mit1 and Msn2 arose in these strains. But the selection of the ptr3 mutation in these lineages is something of a mystery.

It is hard to imagine how the usual job of Ptr3 in nutrient sensing and transport would be involved in keeping long range transcription activation down. Perhaps the researchers have uncovered a novel function for this gene.

And re-creating these two strains only partially restored the levels of transcription activation at a distance that were seen in the original strains. A little genetic detective work showed that a big reason for this was that both of the selected strains had acquired an extra copy of the chromosome that had the HIS3 reporter, chromosome III.

Reavey and colleagues deleted each of their identified genes to see which ones caused their effect through a loss of function mutation. Deleting GRR1, PTR3, and MSN2 all had the same effect as the original isolated mutations.

The same was not true for MOT3 and MIT1. Deleting either gene actually weakens long range transcription activation, suggesting that these two had their effect through gain of function mutations.

Finally, the researchers showed that the increase in long-distance transcriptional activation was not simply due to a general increase in transcription activation in the selected strains, by showing that their mutants did not have increased activity of a reporter with the GAL4 UAS placed 280 base pairs upstream of HIS3. In fact, if anything, the strains showed decreased activation with this reporter.

So this experimental strategy allowed Reavey and coworkers to identify some of the key genes involved in keeping transcription activation at a distance under control in yeast. In particular, they found compelling evidence that the Mediator complex is an important player. But there is still plenty of work to do. For example, which of the genes regulated by Mit1, Msn2, and Mot3 are important in long range activation? And what on Earth is Ptr3 doing in all of this?

The success of this approach also confirms that doing repeated rounds of selection in yeast is a viable way to select multiple mutants and study polygenic traits. This strategy may prove a boon for studying the many human diseases that are the result of polygenic traits.

Not only can we use yeast to uncover its activation potential, but we can also now potentially use it to uncover new treatments for human disease. Unleashing another awesome yeast power… 

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: transcription, Saccharomyces cerevisiae, polygenic trait

The Latest Buzz on Stressed-Out Mitochondria

September 30, 2015


Stinging wasps get our attention, and with good reason—getting stung hurts a lot! If you see wasps going into a nest within the walls of your house, you’ll likely try to block their access.

Just like wasps who can’t get into their nest, excess mitochondrial precursor proteins that can’t be imported into mitochondria are bad news for the cell—but it’s developed ways to deal with them. Image via Pixabay.com

But this could backfire: instead of being able to peacefully go into their nest, a swarm of angry wasps could be buzzing around looking for trouble. It might get so bad that you’ll need to call in an exterminator to take care of the problem.

Mitochondrial proteins might seem a lot less scary than wasps, but it turns out that they can also cause trouble if they can’t get into mitochondria. In two new letters to Nature, Wrobel and colleagues and Wang and Chen used complementary approaches to ask what happens when dysfunctional mitochondria aren’t able to import all of the proteins that are waiting to get in. 

What they both found was that these piled up proteins cause real problems for the cells. In desperation, the cells slow down protein synthesis to reduce the excess, and also turn to their own exterminator, the proteasome, to keep these proteins under control.

This is a paradigm shift in thinking about how poorly functioning mitochondria cause disease. In the past, almost everyone focused on how damaged mitochondria couldn’t make enough energy for a cell. Now it looks like there are other ways for a nonworking mitochondria to do a cell in. And new targets for scientists to go after in treating mitochondrial disease.

As we all know, mitochondria are the powerhouses of the cell, where energy is generated, and they’re also the site of many other essential biochemical reactions. They’re composed of about 1,000 proteins, and nearly all of those are synthesized in the cytoplasm and then imported into mitochondria by an intricate system of transporters.

In order to find out what happens when these 1,000 proteins don’t get into mitochondria as efficiently as they should, both groups created strains whose mitochondrial import was impaired.

Wrobel and colleagues used the temperature-sensitive mia40-4int mutation, affecting an essential component of the mitochondrial import system. Wang and Chen started with the aac2-A128P mutation in PET9 (which is also known as AAC2), an ADP/ATP carrier of the mitochondrial inner membrane. Overexpression of the aac2-A128P allele causes mitochondrial dysfunction and eventual cell death.

Wrobel and colleagues decided to get a comprehensive look at what happens in the mia40-4int mutant by assaying its transcriptome and proteome, using RNA-seq and stable isotope labelling by amino acids in cell culture (SILAC), respectively. Surprisingly, one of the biggest differences from wild type that they saw in the import-defective mutant was a decrease in cytoplasmic translation. Whether they looked at the mRNAs encoding ribosomal proteins, the proteins themselves, or the polysome content and translational activity of the cells, everything pointed to down-regulation of translation. And at the same time, the proteasome—the molecular machine that breaks down unwanted proteins—was activated.

To verify that what they were seeing wasn’t peculiar to the mia40-4int mutant, Wrobel and colleagues slowed down mitochondrial import in several other ways: using different mia40 mutant alleles, other import mutants, or treatment with a chemical that destroys the mitochondrial membrane potential required for import. Under these different conditions causing the accumulation of mitochondrial precursor proteins in the cytoplasm, they still saw decreased cytoplasmic translation and increased proteasome activity.

Quick, Henry, the Flit!” When mitochondrial precursors start to swarm around the cytoplasm, the cell keeps them under control by activating the proteasome. Upper image, Flit insecticide, by Bullenwächter, lower image, structure of the yeast 26S proteasome by FridoFoe; both via Wikimedia Commons

Wang and Chen took a different approach, looking to see whether over-expression of any other genes could compensate for the lethality of overexpressing the aac2-A128P allele. The researchers transformed the mutant with a library of yeast genes on a multicopy plasmid, and found 40 genes whose expression could keep it alive.

The suppressor genes found by Wang and Chen were all involved in some aspect of synthesis or degradation of cytoplasmic proteins, just like the genes found by Wrobel and colleagues whose expression was altered in the mia40 mutant. And Wang and Chen also verified that these suppressors weren’t specific to the aac2-A128P mutation: they suppressed a variety of other mutations that decreased import.

Both groups observed precursors of mitochondrial proteins accumulating in the cytosol of the mutant strains they studied. Wang and Chen saw a couple other very interesting proteins increase in abundance: Gis2 and Nog2. These proteins are involved in regulating ribosome function, and the researchers speculate that their stabilization during this stress response contributes to the translational down-regulation. Intriguingly, their human orthologs are implicated in neuromuscular degenerative disease.

So, using orthogonal approaches, the two groups converged on the same model: a newly discovered cellular pathway that regulates cytosolic translation and protein degradation in order to deal with the stress of inefficient mitochondrial import. Wrobel and colleagues have named it UPRam, for Unfolded Protein Response activated by mistargeting of proteins, while Wang and Chen call it mPOS, mitochondrial Precursor Over-accumulation Stress.

Before this work, it was unknown whether cytosolic pathways were even affected by mitochondrial dysfunction. Now we know that the cell has a specific response when mitochondrial precursor proteins begin swarming in the cytosol, unable to get into their home: it slows down the production of those proteins and calls in the proteasome exterminator to take care of them.

We usually think of mitochondrial disease symptoms as being caused by the reduced energy generation of sick mitochondria, or by the lack of other key events that happen in mitochondria—for example, the synthesis of the iron-sulfur clusters that some vitally important enzymes need. Now, these findings raise the possibility that proteostatic stress on the cell caused by the accumulation of mitochondrial precursors could also lead to impaired cell function and disease.

Perhaps drugs that inhibited cytoplasmic translation, or activated the proteasome exterminator, would be helpful in reducing the buzzing swarm of mitochondrial precursor proteins. Wouldn’t it be wonderful if this knowledge suggested new avenues of treatment to take some of the sting out of human mitochondrial disease?

by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD

Categories: Research Spotlight

Tags: proteostatic stress, mitochondria, Saccharomyces cerevisiae, proteasome

Next