New & Noteworthy

Budding Yeast Diversifies its Phosphatase Portfolio

March 10, 2016

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You’ve probably heard the old saying, “Don’t put all your eggs in one basket.” The idea of course is that the wise thing to do is to spread out your possessions so when something happens to one set, you still have the rest. (See what Homer and Marge Simpson think of this saying.)

If it really is wise to follow this saying, then according to the results of a new study just published in GENETICS by Kennedy and coworkers, the budding yeast S. cerevisiae is wiser than the fission yeast S. pombe. Well, at least as far as for one part of entry into mitosis.

To enter mitosis, every eukaryote tested so far needs to increase the activity of cyclin dependent kinase 1 (Cdk1). Dephosphorylation of a key tyrosine residue in Cdk1 is an important part of this increased activity.

One of the big players in this dephosphorylation is the phosphatase Cdc25 in S. pombe or Mih1p in S. cerevisiae. In fact, it is so important in S. pombe, that deleting it is lethal. These poor cells arrest in G2 and eventually die.

The same is not true for S. cerevisiae. Deleting MIH1 has only mild effects—a slight delay in entering mitosis and starting anaphase. The phosphorylation on the key tyrosine on Cdk1p, Y19, remains for a longer period of time in this strain, but does eventually clear, explaining the delayed mitotic entry.

One interpretation of this result is that S. cerevisiae has spread its Cdk1 phosphatase activity over multiple proteins. Knocking out MIH1 still leaves enough Cdk1 activity to allow the cell to enter mitosis, albeit more slowly.

One likely suspect in S. cerevisiae is Ptp1p. Previous work had shown that in S. pombe, Pyp3, the homologue of Ptp1p, can also dephosphorylate Cdk1-Y19.

Kennedy and coworkers found that deleting both MIH1 and PTP1 in S. cerevisiae had a more severe effect on mitotic entry and exit from anaphase compared to deleting only MIH1. In addition, the level of Y19 phosphorylation on Cdk1p remained for an even longer period in the mih1 ptp1 deletion strain. But it was still not lethal and the cells did eventually manage to pass through mitosis.

These results suggest there is still another player involved. The next suspect these researchers focused on was protein phosphatase 2A (PP2A). Previous work had shown that mutation of the B-regulatory subunits of PP2A, Cdc55p and Rts1p, both affect Cdk1p phosphorylation.

Because of the multiple routes by which PP2A can affect entry into mitosis, the authors designed an in vivo phosphatase assay to accurately measure the level of phosphorylation of Y19 of Cdk1p. The results of this assay suggested that PP2A^Rts1 and not PP2A^Cdc55 affected the phosphorylation state of Y19.

Kennedy and coworkers finally managed to kill off their yeast by deleting MIH1, PTP1, and PP2A^Rts1! They had finally found enough of this yeast’s phosphatase activity to mimic the effects of just Cdc25 in the fission yeast S. pombe.

Using immunopurified protein complexes, Kennedy and coworkers were able to show that both Mih1p and Ptp1p could dephosphorylate Y19 of Cdk1. They could not, however, see dephosphorylation by PP2A^Rts1. It could be that their in vitro assay did not detect it for this protein or that PP2A^Rts1 works on a different phosphatase that affects Cdk1.

Bottom line is that the budding yeast has evolved such that the phosphatase activity needed to enter mitosis is spread out over multiple proteins. The fission yeast evolved in a way that kept all of its phosphatase eggs in the same basket, Cdc25. We’ll let you decide which yeast you think is the wiser.

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

Categories: Research Spotlight

Tags: Cdk1, mitosis, phosphatase, PP2A, redundant regulation

The Benefits of Sex

March 03, 2016

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While you doggedly swipe right and left or wait night after night at that club, you may be wondering whether it is all worth it. Biologists have been wondering something similar.

Now they haven’t been wondering about the value of sex…since everything from amoebas to zebras has sex, it must be pretty important. No, the hard part has been figuring out why it is so beneficial.

On balance it can seem that the minuses of disease risk and passing on only half of your DNA outweighs the benefits of the combining two individual sets of DNA for some brand new combination. A new study by McDonald and coworkers in Nature using our old friend S. cerevisiae provides compelling evidence for a couple of ways that sex is good for a species.

First it is a way of combining individual beneficial mutations into a single individual. Now rather than having a couple of well adapted individuals battling for supremacy, the mutations can merge into one super beast that can outcompete everyone else.

This benefit, recombination speeds adaptation by eliminating competition among beneficial mutations, had been predicted and goes by the name of the Fisher-Muller effect. But this is the first time scientists have actually seen it playing out at the DNA level.

The second big benefit of sex is freeing good mutations from a bad genetic background. Now the beneficial mutation is not weighed down by other negative mutations. It’s like finally getting rid of that concrete block tied around your ankle.

Yeast is an ideal system for studying the benefits of sex because it can happily exist as a sexual or asexual creature. This means that researchers can directly compare the two in the same experiment. Which is just what McDonald and coworkers did.

They followed 6 sexual and 12 asexual populations through about 1000 generations of adaptation. The only difference between the asexual and sexual populations was, as you might have guessed, sex.

The sexual populations included 11 bouts of sex. In other words, every 90 generations or so, an ‘alpha’ cell would swipe left and find an ‘a’ cell to hook up with.

As expected and has been seen before, the sexual populations were much better adapted to their environment than were the asexual populations. Sex is clearly a good thing! The next step was to tally up the mutations in each population to try to figure out why.

What McDonald and coworkers found was that there wasn’t a lot of difference in the mutations that crop up in each. Over time, both groups had about the same number and ratio of intergenic, synonymous, and nonsynonymous mutations.

The big difference between the asexual and the sexual populations was in the mutations that became fixed. In the sexual group, most mutations were weeded out over time. In their experiment, 78% of mutations became fixed in the asexual population while only 16% hung around in the sexual population.

Sheer numbers wasn’t the only difference between the two either. The kinds of mutations that became fixed differed significantly in both as well.

In the asexual population, each of the three kinds of mutations fixed at around the same rate. Around 75-80% of intergenic, synonymous and nonsynonymous mutations became established in this population.

It was a different story in the sexual population. Here, 22% of the nonsynonymous, 11% of the intergenic and none of the synonymous mutations became fixed. It seems like only mutations that make a difference end up getting selected for.

Further analysis revealed two big reasons why the two populations differed. First, good mutations ended up getting stuck with other bad mutations in the asexual population. This blunted the positive effects of the beneficial mutation.

And second, the various good mutations tended to be spread out among different groups in the asexual population. The end result was that instead of working together, these groups battled each other for supremacy resulting in some beneficial mutations being lost.

So no need to wonder anymore about the benefits of sex to a species. It is a strong purifier, weeding out unimportant or damaging mutations and a powerful aggregator, squirrelling all the good ones into one group. No wonder most every beast does it!

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

Categories: Research Spotlight

Tags: Fisher-Muller effect, mutation, nonsynonymous, selection, synonymous

Not Quite The Same

February 24, 2016

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Imagine a world where you either make your own bread from scratch or have it delivered to your doorstep. Not much of a difference, right? Either way you’re eating bread.

Except of course that the two are pretty different. Having your bread delivered frees up time to do other things.

It turns out that something similar may be going on in our old friend, Saccharomyces cerevisiae. According to a new study in Nature Microbiology by Alam and coworkers, a yeast able to make its own amino acids or nucleobases works very differently than one that can’t but is supplied all the nutrients it can use.

This is important for yeast studies because these sorts of auxotrophic markers are used all the time. It means researchers need to be very careful about comparing a wild type yeast strain with a yeast strain deleted for, say, URA3, but grown in the presence of plenty of uracil. The two are not equivalent.

And the study may even have implications for other folks as well. For example, cancer cells have many mutations, some of which can be in metabolic genes. These mutations may affect how these cancers respond to drug treatment.

This all might not matter much if the effects were small. But they weren’t in this study. The changes were profound.

Alam and coworkers compared 16 different strains that were identical except that four different metabolic genes were deleted in various combinations. These genes included HIS3, LEU2, URA3 and MET15 (also known as MET17).

Using mRNA sequencing, they found that 5,011 out of 5,923 transcripts were affected in one strain or the other. This is 85% of the coding genome of yeast!

While not all of these changes were huge, 573 of them differed by 2-fold or more. In other words, around 10% of the genome is significantly affected when a yeast cell is provided a nutrient instead of having to make it itself. Not surprisingly, the affected genes were enriched for those involved in metabolic activity and enzymatic function.

The authors next looked at some publically available gene expression experiments that used auxotrophs in the same BY4741 background. These studies primarily looked at the how the knocking out of a specific gene affected global gene expression. The vast majority of deleted genes were not metabolic.

Alam and coworkers found that a sizeable minority of changes overlapped with the ones they saw with deleting HIS3, LEU2, URA3 or MET15. In other words, on average, at least 18% of the changes in the genes identified in these studies were not due to the gene deletion they were studying. They were instead due to the deletion of a “housekeeping” metabolic gene.

This all might be less of a big deal if the affected genes were always the same. Then you could just be on the lookout for these genes when using a specific auxotroph.

Unfortunately, it isn’t so easy. Different combinations of deleted metabolic genes yield different changes in gene expression patterns with very little overlap.

So, for example, when the authors compared a his3 deletion strain to one deleted for both HIS3 and URA3, a very different set of genes was affected. And these were different from a strain deleted for HIS3 and MET15, and so on. Looking at all the possible combinations confirmed that universal gene targets were rare.

The bottom line from these experiments is that researchers need to be very careful about the strains they compare because they may not be as equivalent as they think. Just because the older Star Trek films and the newer ones have a Spock, that doesn’t mean the half Vulcan is the same in both. Just ask Uhura.

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

Categories: Research Spotlight

Tags: auxotrophic markers, auxotrophy, gene expression, metabolic background, supplementation

Unlocking Chromatin

February 10, 2016

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In Die Hard, Hans Gruber and associates need to break through seven locks in the right order on a safe to get to bearer bonds worth 640 million dollars. Of course the hero John McClane foils the plot and beats the villains.

Nothing so exciting in yeast, but some genes are nearly as hard to turn on as that safe was to open. One of the most stubborn is the HO gene. It requires three locks or gates be opened in the right order to start making the HO endonuclease.

A new study in GENETICS by Yarrington and coworkers shows that the second lock for HO is a set of nucleosomes that blocks the binding of the transcription activator SBF. When they rejiggered this promoter so that these nucleosomes were removed, the HO gene needed fewer steps to get activated.

It is as if Hans Gruber and his gang only had five or six locks to get through to open their safe. And the 7th, hardest one was removed.

The HO gene is usually turned on in three sequential steps. First the Swi5p activator binds to a region called URS1, which recruits coactivators that then remodel the chromatin at the left half of URS2 (URS2-L). This allows SBF to bind its previously hidden binding sites which then remodels the chromatin again. Now a second set of SBF sites is revealed in the right half of URS2 (URS2-R).

These authors set out to provide direct proof that nucleosome positioning over URS2-L is the key to the second lock. They did this by making a set of chimeric promoters between HO and CLN2.

Both of these promoters are activated by SBF. A key difference between the two is that the CLN2 promoter, like 95% of yeast promoters, is in a nucleosome depleted region (NDR).

The idea then is to make an HO promoter in which the usual URS2-L is replaced with the NDR region of CLN2. If the nucleosomes matter over URS2-L, then this construct should be activated in two instead of three steps.

Or, to put it another way, Swi5p binding to URS1, the first lock, will no longer be needed to open the second lock. HO activation will now be Swi5p independent. This is what the authors found.

When they looked at their chimeric protein that lacked nucleosomes over URS2-L, they found that using a strain deleted for SWI5 had very little effect on activity. There was only around a 2-fold difference in activity with this construct in the wild type and SWI5-deleted strains. This is very different than the wild type HO promoter where there was around a 15-fold difference between the two strains.

The authors then did an additional experiment where they took their chimeric reporter and mutated the nucleosome depleted region such that nucleosomes could bind there. This construct was now more Swi5p-dependent: there was around a 5-fold difference in activity between the wild type and SWI5 deletion strains. They had at least partially rebuilt that second lock.

Yarrington and coworkers continued with ChIP experiments to confirm that their chimeric construct was indeed depleted for nucleosomes, as well as other experiments to tease out more subtle details about the regulation.

Given that it switches a yeast cell’s mating type, it isn’t surprising that the HO gene is under such lock and key. The yeast cell wants to make sure it only turns on when needed. Just as the Nakatomi Corporation wanted to make sure only the right people could get to that fortune in bearer bonds.

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

Categories: Research Spotlight

Tags: chromatin remodeling, HO endonuclease, nucleosomes, transcription regulation

Of Medieval Market Townes and Wasp Guts

February 03, 2016

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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, mating, outcrossing, Saccharomyces cerevisiae, Saccharomyces paradoxus

Unfrying An Egg

January 20, 2016

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Eggs start out as slimy and awful, but can end up warm, firm and wonderful. All it takes is some heat to denature the egg proteins and voilà, a tasty breakfast.

Not that anyone would want to do it, but of course it is impossible to do the reverse. You can’t take a fried egg and turn it back into a raw one. The denaturation is pretty much permanent.

When a cell is hit with high temperatures, its proteins start to denature as well. And scientists thought that most of the denaturation of many of these proteins was as irreversible as the eggs. The thought was that many or most of these denatured proteins were “eaten” through proteolytic degradation. Although cellular chaperones are capable of disaggregating and refolding some heat-denatured proteins, it wasn’t known which aggregated proteins met which fate in a living cell.

A new study out in Cell by Wallace and colleagues shows that at least in yeast, most eggs get unfried. After a heat shock, aggregated proteins in the cell return to their unaggregated form and get back to work.

Now those earlier scientists weren’t crazy or anything. The proteins they looked at did indeed clump up and get broken down by the cell after a heat shock. But these were proteins introduced to the cell.

In the current study, Wallace and colleagues looked at normal yeast proteins being made at their normal levels. And now what happens after a brief heat shock is an entirely different story.

The first experiment they did looked at which endogenous yeast proteins aggregated after they were shifted from their normal 30 to 46 degrees Celsius for 2, 4, or 8 minutes. The researchers detected aggregation using ultracentrifugation—those proteins that shifted from the supernatant to the pellet after a spin in the centrifuge were said to have aggregated.

Using stable isotope labeling and liquid chromatography coupled to tandem mass spectroscopy (LC-MS/MS), they were able to detect 982 yeast proteins easily. Of these, 177 went from the supernatant to the pellet after the temperature shift. (And 4 did the reverse and went from the pellet to the supernatant!)

After doing some important work investigating these aggregated proteins, the researchers next set out to see what happened to them when the cells are returned to 30 degrees Celsius. Are they chewed up and recycled, or nursed back to health and returned to the wild?

To figure this out they did an experiment where proteins are labeled at two different times using two different labels. The researchers first grew the yeast cells at 30 degrees Celsius in the presence of arginine and lysine with a “light” label. This labels all of the proteins in the cell that have an arginine and/or lysine.

Then the cells are washed and a new media is added that contains “heavy” labeled arginine and lysine. The cells are shifted to 42 degrees Celsius for 10 minutes and then allowed to recover for 0, 20, or 60 minutes.

After 60 minutes of recovery, the ratio of light to heavy aggregated proteins looked the same as proteins that hadn’t aggregated. In other words, aggregation did not cause proteins to turn over more quickly.

It looks as if aggregated proteins are untangled and allowed to go about their business. So after a heat shock the cell doesn’t throw its hands in the air and simply start things over.

Other experiments done by Wallace and coworkers in this study, that we do not have the space to tackle here, suggest that the cell has an orderly process for dealing with heat stress. After a heat shock, certain proteins aggregate with chaperones in specific areas of the cell. Once the temperature returns to normal, these stress granules disassemble and the aggregated proteins are released intact.

None of this will help us unfry an egg — a denatured egg protein is obviously significantly different than an aggregated protein protected by chaperones in a stress granule. But this study does help us better understand how our cells work. And that’s a good thing.

Unlike Mr. Bill’s dog, most aggregated yeast proteins can return from a heat shock.

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

Categories: Research Spotlight

Tags: chaperones, heat shock, protein aggregates, protein aggregation

Clearing Customs in the Nucleus

January 06, 2016

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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.

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: mRNA export, nuclear pore, Saccharomyces cerevisiae

Yeast on Their Best Behavior

December 10, 2015

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As the holidays approach, many of us are getting ready to crowd around the table for a big family dinner. Some of us may behave differently around family than we might with friends or coworkers.

For example, with your relatives, you might bite your tongue if your political views vary greatly from theirs. Where we are and with whom we interact can sometimes affect what we do.

Turns out that yeast growing in a colony can be the same way. Though of course they aren’t keeping their opinions to themselves. (Well, we don’t think they are…)

A yeast cell can end up acting differently depending on where it is in a colony. For example, only a narrow band of cells gets to sporulate while all the others are left to plod through mitosis.

A new study out in GENETICS by Piccirillo and coworkers shows that these cells sporulate because nearby cells “encourage” them to. They are being influenced to sporulate because of the cells around them. Just like your relatives might influence you to change your behavior at the dinner table.

The first step in showing that one set of cells signals a second set to sporulate was to find the genes involved in setting up this pattern. Since the authors were looking at Saccharomyces cerevisiae, it was pretty easy to get mutants to study. They just had to open their freezer and pull out their yeast homozygous diploid deletion library.

Initially, they looked for strains where the usual pattern of sporulating cells was disrupted. They then took these candidates and looked for those that could still sporulate normally in suspension. They wanted mutants that could sporulate but couldn’t do it in the right place.

They found seven strains that fit the bill. Three of the deleted genes, MPK1/SLT2, BCK1, and SMI1, were in the cell-wall integrity pathway (CWI). They also showed that mutation of three other genes in the pathway, SLG1/WSC1,TUS1 and RLM1, all impacted colony sporulation as well.

Further work showed that the transcription factor RLM1 was induced 1-2 days before the master regulator IME1 was turned on. IME1 is a key player in getting meiosis started so that yeast cells can sporulate.

So the story seemed to be that RLM1 is turned up which then turns on IME1, which kick starts meiosis. Makes sense except it is unlikely that Rlm1p is directly activating IME1. There is no obvious Rlm1p site in the IME1 promoter.

A close look at the colonies showed that RLM1 is upregulated in a layer of cells just under the ones where IME1 is upregulated. Deletions in the CWI pathway seemed to have disrupted a group of “feeder” cells whose job it is to get nearby cells to sporulate.

To show this, the authors used a chimeric colony assay that consisted of two strains. The first strain, which had functional Rlm1p, had a reporter, either RFP or lacZ, under the control of the IME1 promoter. The second strain was either wild type or deleted for the transcription factor RLM1.

They created colonies with equal amounts of each strain and looked at IME activation. The idea is that if RLM1 is important in the cells that sporulate, then the second strain shouldn’t matter. You should get the same number of cells in which the IME1 promoter is activated whether or not adjacent cells express RLM1.

But if it is important for RLM1 to be expressed in nearby cells, then there should be a falloff in activation if adjacent cells are deleted for RLM1. This is just what the authors found.

And it wasn’t just the artificial reporter system that was affected either. There was also a drop off in the number of cells that sporulated in the case where some of the cells lacked RLM1.

In a further set of experiments, Piccirillo and coworkers showed that these feeder cells became more osmosensitive compared to the ones that go on to sporulate. While they did not find the signal that prompted the meiosis of nearby cells, this change in osmosensitivity is consistent with the cells preparing to release something into the environment.

So it looks like activating the CWI pathway in one set of cells causes a second set to start down the road of sporulation. And if the CWI pathway is disabled in these cells, then the second set of cells no longer changes their behavior and begin to go through meiosis.

This all seems weird at first until you realize that the cells in a colony usually all share the same DNA. What is good for one set of cells is good for the survival of the DNA even if it is at the expense of other cells in the colony.

Yeast cells tend to sporulate when food grows scarce. But sporulating takes a lot of energy. Colonies may get around this paradox by having some of the cells in the colony give up nutrients or energy to a few cells that go on to sporulate. The feeder cells deprive themselves so that other cells have a better shot at survival.

Now the DNA, shared by all the cells, can live on for the next round of holiday dinners….

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

Categories: Research Spotlight

Tags: cell wall integrity pathway, IME1, RLM1, sporulation

Keeping Gen(i)e Drives in Their Lamps

December 02, 2015

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Everyone knows about genies. They have almost infinite power, can grant you three wishes, and are kept under control by the owner of their lamp.

And as we saw in Disney’s Aladdin, it is a good thing that the lamp is around! When the evil sorcerer Jafar was given the powers of a genie, he began to take over the world. Until, that is, Aladdin forced him back into his lamp where he could be kept under control.

In the last few years, scientists have come up with their own genies. While not as powerful as the “real” ones, these gene drives can still pack quite a punch. And maybe even grant us a few wishes.

Gene drives can force genes to spread quickly through a population whether those genes are good for a species or not. This means we might be able, for example, to force a “bad” gene to spread through the mosquitoes that transmit malaria. By causing the mosquito population to crash, our wish to save hundreds of thousands of lives each year would be granted!

But just like a genie, we need to keep gene drives under control. We do not want something that overrides natural selection to escape and wreak havoc with ecosystems.

Which is where, as usual, our friend yeast can help! In a new study out in Nature Biotechnology, DiCarlo and colleagues use yeast to test two different strategies to make gene drives safe enough to use. And, they argue, safe enough to research.

Gene drives are based on the idea of homing endonucleases. Basically, if a gene associated with a gene drive is on just one of the two chromosomes in a pair, the gene drive will copy and insert the gene into the other chromosome through a precise DNA cut.

Now both chromosomes end up with a copy of the gene. Which of course means all of the offspring will get the altered gene too. This copying will happen generation after generation until the new gene has swept through the population.

The idea for gene drives has been around since 2003 but really only became practical with the discovery of the CRISPR/Cas9 system. This genome editing tool, which is ludicrously simple to program to target most any DNA sequence, allows scientists to create most any gene drive they want.

The CRISPR/Cas9 system has two parts. One part is the guide RNA which leads the second part, the endonuclease Cas9, to the right spot in the genome to cut. What makes the system so powerful is that you just need to make a different guide RNA to target different sequences in the genome.

One easy way to help control a gene drive is to keep these two parts separate. Do not have the guide RNA and the Cas9 on the same piece of DNA. Then, if one part were to escape, it couldn’t do anything on its own.

This is of course easy to do in yeast. Just integrate one part into a chromosome and keep the second part on a plasmid.

This is just what DiCarlo and coworkers did. And they showed that this separation can be very effective.

They integrated a guide RNA into the ADE2 gene of a haploid yeast to create a gene drive designed to disrupt ADE2. As expected, this strain produced red colonies on adenine limiting media.

They next mated this strain to a wild type haploid. All of the resulting diploids were cream colored. This is what would be expected as both copies of ADE2 need to be disrupted to see red colonies in a diploid.

When these diploids were sporulated, the researchers got the expected 2:2 ratio of red to cream colored haploids. This all changed when they introduced a Cas9 containing plasmid into the experiment.

In the presence of Cas9, more than 99% of the resulting diploids were red. And when sporulated, these diploids produced all red haploid colonies.

The two parts of CRISPR/Cas9 together drove the disrupted ADE2 through the population. But importantly, just having the guide RNA integrated into ADE2 had no effect on how the two alleles were passed down. Once one part is removed, the gene drive stalls out.

The same system also worked when the ADE2 gene drive included the URA3 gene so that URA3 spread through the population as well. It also worked when the essential gene ABD1 was targeted.

And genetic background did not significantly affect how well this ADE2 gene drive worked. When they mated their haploid to six different strains of yeast they saw no loss in efficiency.

So separating the two parts of the gene drive is a pretty good failsafe. But of course nothing is perfect.

Ideally we need some way to shut the system down if all of our safety features fail. We want to be able to get rid of Jafar and the lamp entirely if possible.

DiCarlo and coworkers showed that they could create a gene drive that could overwrite and correct the ADE2 they had disrupted with the guide RNA. This new gene drive targeted a synthetic sequence in the original gene which means that it would only affect altered yeast. So even if things go awry, we may be able to erase the changes we made.

These two strategies should help keep gene drives in check both in the wild and the lab. But of course, again, it is important to keep in mind that nothing is foolproof.

At the end of Aladdin, they buried Jafar and his lamp deep in the desert to keep him from causing any more trouble. But his lamp was found and Jafar reemerged to wreak havoc in the second Aladdin movie, reminding us that we must be very careful when unleashing powerful forces.

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

Categories: Research Spotlight

Tags: CRISPR/Cas9, gene drives

Pulsating Yeast

November 19, 2015

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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.

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

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