New & Noteworthy

Friends with Benefits

August 10, 2016

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A 2013 poll identified the top 20 modern necessities British people couldn’t live without. Some we can all relate to like smartphones, daily showers, and the internet, while others are more British-specific like a cup of tea or a full English breakfast.

Of course none of these are true necessities like food, water or air. We wouldn’t be as happy, nor as competitive, without some of these modern necessities, but we’d obviously still be alive. (But is life without the Internet really living?)

It turns out that the GCN5 gene is more like water or air for most eukaryotes—they can’t live without it. But our old friend Saccharomyces cerevisiae is different. This yeast isn’t as happy without GCN5, but it soldiers on nonetheless.

This nonessentiality, combined with its powerful genetics, makes yeast a great system for exploring what GCN5 does. And this is just what Petty and coworkers did with this important member of the histone acetyltransferase (HAT) family in a new study in GENETICS.

GCN5, like other HATs, transfers an acetyl group to histones, which results in increased activity of nearby genes. Consistent with this, previous work has shown that GCN5 acetylates histone H3 in the promoters of active genes.

HATs, along with their countervailing proteins histone deacetylases, as well as kinases, phosphatases, methyltransferases and so on, all work together to change gene expression on the fly in response to all sorts of different stimuli. These can include environmental signals, entering the cell cycle, or whatever.

Understanding how HATs work is critical for understanding how we (and other beasts) change gene expression in response to these signals. Which is what makes GCN5 in yeast such a great system. A strain deleted for GCN5 is sick, but alive, so we can study what happens when it is gone. And we can explore what we can do to fix its problems.

One of the many problems that yeast lacking GCN5 have is that they grow more poorly at high temperature than do yeast with GCN5. These researchers took advantage of this and looked for high copy suppressors of this temperature sensitivity. They found multiple genes, but the most common was RTS1, one of two regulatory subunits of the PP2A phosphatase complex.

Deleting GCN5 causes more problems than temperature-sensitivity, and overexpressing RTS1 restored some, but not all, of them. For example, RTS1 overexpression helped make the Δgcn5 strain less sensitive to DNA damage, less susceptible to microtubule disruption, better able to grow on nonfermentable carbon sources like glycerol or ethanol, and more able to progress into S phase during mitosis. But lots of PP2A^Rts1 could not rescue the abnormal buds nor the sporulation problems seen in a diploid lacking GCN5.

When the researchers deleted RTS1 or some of the genes that code for other critical components of the PP2A^Rts1 complex in a Δgcn5 strain, the strain died. The same was not true of a second regulatory subunit that can be part of the PP2A complex, CDC55—its deletion was not lethal, nor did it rescue the temperature sensitivity of the Δgcn5 strain.

Petty and coworkers provided evidence that the phosphatase activity of the PP2A^Rts1 complex was important by showing that okadaic acid, an inhibitor of this family of phosphatases, prevented Rts1p from rescuing the Δgcn5 strain’s temperature sensitivity. The easiest explanation is that the rescue happens because of the phosphatase activity of PP2A^Rts1, but it is also possible that a different member of the family might be providing the phosphatase activity.

So it looks like there is something important happening between PP2A and GCN5. Petty and coworkers next set out to find out what that might be.

First they showed that deleting GCN5 causes a decrease in the levels of core histones in the cell and that RTS1 overexpression fixed this problem. This happened at the transcription level as the RNA levels of the yeast histone genes (with the exception of HTA1) all showed reduced expression in the absence of GCN5, which again was restored when RTS1 was overexpressed.

Histone genes are normally turned on at the end of G1, then shut off at the end of S phase. This makes sense, as a cell needs to make more histones when it makes a new copy of its genome and this happens during S phase!

Consistent with the reduced histone gene expression seen earlier, the Δgcn5 strain failed to increase histone gene activity at the end of G1. Overexpressing RTS1 restored this induction. So it looks like GCN5 is involved in turning on all the histone genes, except maybe HTA1, at the right time, and that RTS1 can compensate if there is a lot of it around.

As a final set of experiments, the authors looked at what PP2A^Rts1 might be doing to rescue histone gene expression when GCN5 was deleted. They decided to look at histone modifications.

For this they used the SHIMA (Scanning HIstone Mutagenesis with Alanine) library, in which all key serine and threonine residues were individually mutagenized to alanine. Even though PP2A^Rts1 is thought to be primarily a serine/threonine phosphatase, they also looked at three tyrosine residues (Y40, Y43, and Y45) on H2B by mutating each individually to phenylalanine.

They found that two residues on histone H2B, Y40 and T91, were required for RTS1 to be able to rescue the temperature sensitivity of a Δgcn5 strain. And mimicking the permanent phosphorylation of T91, by mutating it to either aspartic or glutamic acid, slowed the growth of wild type yeast and killed the deletion strain.

This tells us a lot about what GCN5 is doing in yeast, and it might also help us better understand certain human cancers. Turns out that the residue equivalent to T91 in mammals is phosphorylated in these cancers.

Petty and coworkers were able to learn all of this because of yeast’s powerful genetic tools, and because GCN5 is not essential in yeast. Once again, the awesome power of yeast genetics (#APOYG!) can help us understand human cell biology.

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

Categories: Research Spotlight, Yeast and Human Disease

Keep Only What You Need

July 13, 2016

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In the Lord of the Rings trilogy, the evacuees from Edoras are warned to take only what they need. Aragorn, Gimli and Legolas do the same thing when they chase down the orcs who kidnapped Merry and Pippin. And Sam and Frodo get rid of all nonessentials so they can make it to Mount Doom.

They all need to do this because if they are weighed down they won’t make it to their goal or maybe even die. If Sam and Frodo had kept all of their equipment, they would have died on the Plateau of Gorgoroth before reaching Mount Doom and saving Middle Earth.

In some ways, the hurly burly world of yeast is a bit like these characters in Middle Earth. If yeast cells are weighed down by slightly deleterious or even nonessential items, they will not survive. They will be outcompeted by their leaner, less burdened peers. The orcs would have made it to Isengard if Aragorn, Gimli, and Legolas had been slowed down by too much extra stuff.

One place where we can see this is with introns. Unlike many other eukaryotes, S. cerevisiae has gotten rid of almost all of its introns—only around 5% of its genes have them. This suggests that the ones that have stuck around are doing something important.

In a new study out in GENETICS, Hooks and coworkers explore the idea that at least some of the remaining introns have hung around because they play an important role as untranslated RNAs with specific secondary structures. In fact, they provide evidence that the secondary RNA structure of an intron in the GLC7 gene in critical for the cell’s ability to respond to salt stress.

The first step was to identify introns with a conserved secondary structure. They compared 36 fungal genomes using three different RNA structure prediction tools and found that all three programs were able to identify structures for 14 of the introns. They also found 3 introns that scored very well with at least two of the programs. With the exception of known snoRNAs, none of these matched any other noncoding RNAs.

Next, Hooks and coworkers used RT-PCR as well as re-analysis of deep sequencing data of total RNA to figure out which of these introns might actually be a real noncoding RNA. They found that six of the introns remained intact in the cell much longer than is typical for excised introns and that noncoding RNAs were further processed in two of them, an intron from GLC7 and one from RPL7B.

They set out to determine if the predicted secondary structure of the RNA of the intron in GLC7 really did anything important in the cell. GLC7 was a good choice as it has been previously reported that this intron is involved in a cell’s response to high salt. So if the structure is important, than if it is disrupted, the cell should not respond as well to high salt.

They used a couple of different mutants to get at this question. The first mutant, the GLC7 ncRNA deletion mutant, simply deleted the predicted noncoding RNA from the intron. The second mutant, the GLC7 ncRNA insertion mutant, inserted 139 base pairs in the middle of the predicted noncoding sequence. The researchers found that neither responded as well to a high salt concentration, 0.9 M NaCl, as did a wild type or a negative control deletion that removed part of the intron that did not overlap with the predicted noncoding RNA sequence.

They also found that this loss in response could not be rescued with the noncoding RNA being expressed in trans from a separate, constitutive promoter. The secondary structure of this intron plays an important role in dealing with the stress of high salt in cis.

While deletion of the predicted noncoding RNA had little effect on GLC7 expression at low salt, the same was not true at higher salt. At 0.9 M NaCl, GLC7 mRNA levels were about half of that of the wild type or the negative control deletion mutant. It looks like under high salt conditions, this intron is important for getting enough GLC7 made to deal with the stress.

So, like Gimli’s axe or Sam’s water bottle, yeast has maintained this intron because it plays an important role in survival. It will be interesting to see why other introns have been maintained and if they too play their roles as noncoding RNAs.

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

Categories: Research Spotlight

Tags: introns, ncRNA, RNA structure

Some Like it Hot

June 28, 2016

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According to an AFI poll, the best comedy of all time was the 1959 film “Some Like It Hot.” In this classic screwball comedy two men have to dress up as women to escape the mob and still make money as musicians. All sorts of hilarity ensues as one of them falls in love with a woman and a man falls in love with the other as a woman.

The key to this comedy is that the two actors, Tony Curtis and Jack Lemmon, have to play both the male and female parts. If they were played by separate actors and actresses, the movie would die at the box office. It would be a lethal mutation.

A new study by Solis and coworkers in Molecular Cell presents evidence that in yeast, the heat shock transcription factor Hsf1p is a bit like Tony Curtis and Jack Lemmon—it plays dual roles, both in maintaining basal levels of various heat shock proteins and in turning the appropriate genes up in response to a heat shock. This is different than in mammalian cells where HSF1 is only responsible for turning up heat shock genes in response to a spike in temperature. Something else maintains the levels of these proteins needed for survival.

So yeast is more like the comedy “Some Like it Hot,” or perhaps Tootsie, while mammalian cells are more conventional comedies where different actors play the male and female roles. Because Hsf1p plays a dual role in yeast, its deletion causes the cell to die. Mammalian cells can survive without HSF1 as long as it doesn’t encounter any temperature spikes.

Solis and coworkers started out by coming up with a way to dissociate the genes that Hsf1 regulates under normal conditions from those upregulated under heat shock conditions. For this they used the “Anchor-Away” approach to remove Hsf1p from the nucleus under normal conditions.

Basically, they co-expressed HSF1 fused to FRB, the FKBP rapamycin-binding domain, and a ribosomal protein L13A-FKBP12 fusion. When they add rapamycin to this strain, the two proteins heterodimerize and Hsf1p is dragged out of the nucleus. They confirmed that Hsf1p was gone from the nucleus within a few minutes.

Next, they used native elongating transcript sequencing (NET-seq) 15, 30, and 60 minutes after rapamycin addition to see which genes were affected when Hsf1p left the nucleus. They found that only 25 genes were repressed and five were induced at these time points. Using RNA-seq and ChIP of Hsf1p they showed that Hsf1p was probably responsible for the expression of 18 of the 25 repressed genes and none of the induced ones.

So yeast Hsf1p is involved in the basal expression of a number of chaperone genes. In a set of experiments that I don’t have time to go over here, they also showed that most of the heat shock response was independent of Hsf1p in yeast. Their data suggests that Msn2/4p may be the key player instead.

They next did a similar set of experiments in mammalian cells but with a couple of differences. First off, these cells can survive HSF1 deletion, meaning they didn’t need to do anything fancy—they just used CRISPR/Cas9 to delete the gene in mouse embryonic stem cells and mouse embryonic fibroblasts.

Under normal conditions they found that the deletion of this gene caused two genes to go up in expression and two to go down. This is what you might expect by chance suggesting that in mammalian cells, HSF1 isn’t involved in basal expression of any genes.

They next used RNA-seq to compare gene expression of these cells and their undeleted counterparts under normal and heat shock conditions. They found a set of nine genes that were induced in both wild type cells and repressed in the HSF1-deleted cells under heat shock conditions. Eight out of nine of these are involved in chaperone pathways and they overlap surprisingly well with the yeast genes that Hsf1p controls under basal conditions.

Taken together these experiments paint an interesting picture. In yeast, HSF1 is mostly responsible for the basal expression of chaperone genes, and in mouse cells it is a key player in the heat shock response of a similar set of genes. This suggests that deletion of HSF1 is lethal in yeast because the decreased expression of one or more of the genes it regulates under normal conditions.

They tested this by expressing 15 of the 18 genes (three are redundant to some of the others) on four different plasmids and saw that a yeast strain that is deleted for HSF1 now survives. So one or more of these genes is responsible for yeast death in the absence of HSF1.

Through a process of elimination, Solis and coworkers found that the key genes were SSA2, a member of the HSP70 family, and HSC82, an HSP90 family member. The decrease in expression of these two genes cause by the deletion of HSF1 results in a dead yeast cell.

These experiments are so cool. In yeast, HSF1 makes sure there is enough of these chaperones around in good times to fold proteins properly and has a minor role in the heat shock response, while in mouse cells, the same gene plays no real role in basal levels of expression of chaperone genes and instead is critical for responding to heat shock. The protein regulates similar genes, just under different conditions.

These neat science experiments can tell us more about diseases, like cancer too. Turns out that some cancer cells may be more like yeast cells in that deletion of HSF1 stops them from growing and causes an increase in poisonous protein aggregates which may give us a new way to target HSF1-dependent cancers. For example, it may be that targeting Hsp70 or Hsp90 could be useful for treating HSF1-dependent cancers.

In cancer cells then, HSF1, like Dustin Hoffman in Tootsie, Milton Berle in the Milton Berle Show, or Bugs Bunny in many different cartoon shorts, takes on dual roles in the cell. And as we learned from yeast, this could be these cancers’ Achilles heel.

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

Categories: Research Spotlight, Yeast and Human Disease

Look Before You Leap…Into DNA Repair

June 09, 2016

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Sometimes in an emergency, it can be useful to take a pause before trying to fix a problem. If the ambulance is on the way you may not want to start removing someone’s appendix right then and there. Yes you may save them, but the ambulance and EMTs will do less damage to the patient once they get there.

The same sort of logic applies to cells too. For example, a double stranded break is a deadly emergency that can wreak havoc with a cell’s genome.

The cell can deal with this in a few ways; the big two being non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the simplest in that the broken ends are simply stuck back together. However, it often results in a bit of DNA damage as a few bases are added or lost at the ligation point.

The other option, homologous recombination (HR), involves using a homologous DNA region as a template to essentially resynthesize the broken area. There are two possible ways this can happen in a cell: gene conversion and break induced replication (BIR).

If both ends are available without too much of a gap and in the right orientation, the cell opts for gene conversion, the safer of the two options. Only a small section of DNA is resynthesized, keeping most of the original DNA intact.

It is a different story for BIR, the second approach. Here, a large swath of DNA gets resynthesized, meaning a big loss of heterozygosity (LOH)—two sections of DNA are now identical over a large region. BIR only happens when there is only one end available or when there is a big gap of missing DNA.

Of the two HR possibilities, gene conversion is preferable over BIR. This is why the cell wisely waits to make sure there is no other option before starting down this path. This pause goes by the name of recombination execution checkpoint (REC).

A new study by Jain and coworkers out in GENETICS has identified two of the key players involved in this checkpoint—SGS1 and MPH1. Both are highly conserved 3’ to 5’ helicases.

These researchers found that when both are deleted, the REC disappears. And that isn’t all. In situations where the wild type cell would choose to repair its DNA by BIR, the double deletion strain no longer does. Instead, it now uses a process that is more similar to gene conversion.

Jain and coworkers found this using a reporter that contained an HO endonuclease site in the middle of the LEU2 gene. Once the HO endonuclease is activated, it makes a double strand DNA break, cutting the LEU2 gene in half.

The cell was provided with a variety of templates to be used in HR. One of these only provides the part of the LEU2 gene to the right of the HO endonuclease site. As only one of the ends has homology to the cleaved LEU2 gene, with this template, the cell can only use BIR to repair the break.

The researchers saw a huge increase in how fast the DNA was repaired using the BIR-only template in the strain deleted for both sgs1 and mph1 compared to the wild type strain. The double mutant repaired the DNA using BIR nearly as quickly as a wild type cell could using gene conversion. The pause before repair was essentially lost in the double mutant.

The double deletion strain wasn’t just faster with the BIR-specific template either. It repaired DNA via this pathway about 4 times better than the wild type strain did.

There are at least a couple of different reasons why the double mutant could be so much better at repairing DNA in BIR situations. In the first, the cell is just better at BIR—it can initiate the BIR pathway much more quickly than wild type. The second possibility is that this double mutant isn’t using the BIR pathway anymore and is using something closer to gene conversion.

Jain and coworkers found that the second option is the more likely of the two. The way they figured this out was to make a mutation in the POL30 gene, a gene required for DNA repair by BIR but not gene conversion. So, they tested what happens when POL30 is mutated in the wild type and the sgs1 mph1 double deletion strains.

They found that while a dominant negative mutant of pol30 had the predicted effect of severely compromising repair in wild type cells using their BIR-specific reporter, it had no effect in the double deletion mutant. Since Pol30p is needed for the BIR pathway, the strain deleted for sgs1 and mph1 must be using a different pathway to fix the DNA damage. So the pause is eliminated because the cell isn’t really using the BIR pathway anymore.

We don’t have time to go into it here, but there is a lot more research in this paper that looks at why the REC might be lost and that probes the differences in how MPH1 and SGS1 influence the BIR pathway that I encourage you to read. For example, deleting SGS1 is not identical to deleting MPH1 in terms of the BIR DNA repair pathway. And each single mutant still uses the BIR DNA pathway as the pol30 mutation severely compromises the ability of each to repair the BIR-specific reporter.

There is a lot more fascinating stuff like this in the study. The bottom line, though, is that MPH1 and SGS1 are the level-headed people urging folks not to panic and to wait for the EMTs to get to the accident scene to help. When MPH1 and SGS1 are gone, the cell dives right in and starts repairing breaks without waiting for the safest option—gene conversion. Who knows what havoc is wreaked in their absence!

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

Categories: Research Spotlight

Sometimes You Need Half a Trillion

June 01, 2016

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In the Foundation series by Isaac Asimov, Hari Seldon invented a field of study called psychohistory which was able to “make general predictions” about human history. It only worked because the population of the Galactic Empire was in the quadrillions. You need huge numbers to get useful predictions.

Sometimes real life biologists need lots of individuals to see what they are looking for too. In the case of a recent paper in PNAS by Lee and Stevens, they needed to sift through half a trillion yeast to find what they were looking for. And boy was it worth it!

They saw an intron jump from one gene to another. Twice. In 500 billion tries.

This is the first example where we have caught an intron in the act of moving from one place to another. This has important implications for the study of evolution where over time introns spread or recede. It might even help us better understand cancer where intron loss can play a role.

Seeing something as rare as this means making a very specific, complicated reporter. You don’t want to manually sort those 500 billion colonies…or at least I wouldn’t want to.

Here is a “simplified” schematic of the reporter they came up with:

It is as complicated as it looks but it got the job done! Let me walk you through what everything is and how it works.

The GU and AG sequences are splice site junctions. Sequences between a GU and an AG can be spliced out.

The red gene is the S. pombe his5+ gene that has a promoter driving its expression shown with the red arrow. It is in the reverse orientation compared to the eGFP gene which is under the control of the promoter represented by the blue arrow.

The S. pombe his5+ gene works fine in S. cerevisiae his3 mutants to make up for histidine auxotrophy. But it won’t work from this construct.

See, the his5+ gene has an intron that keeps it from being translated correctly unless the intron is spliced out. But this intron can’t be spliced out when the gene is transcribed using the red promoter because the splice junctions GU2 and AG1 are in the wrong orientation. Any transcripts from the red promoter will not work because the intron cannot be spliced out.

You also can’t get His5 from the blue promoter. Even with splicing (which will work in that orientation), the gene can’t be translated because it is in the wrong orientation.

To get any his5+ transcript, the yeast needs to take the spliced RNA from between GU1 and AG2 and get it into DNA. It also needs to get rid of the intron in the middle of his5+ gene before it happens.

So what they are looking for are yeast that glow green and can survive in the absence of histidine. There are a couple of ways this can happen.

The more common way results in the his5+ gene recombining back into the plasmid such that the intron is missing from its middle. Basically the RNA between GU2 and AG1 is spliced out and the resulting RNA is reverse transcribed into DNA. This DNA then undergoes homologous recombination with the plasmid DNA resulting in a working his5+ gene. They got over 10,000 of these in their experiment.

The much less common way involves the intron being inserted into a gene within the genome. This way uses some of the same steps with one extra one—a reverse splicing event.

As I said, they got two of these with one ending up in the RPL8B gene and the other in the ADH2 gene.

Here’s how they think this happened…

The spliced out RNA from the plasmid was left for a brief time in the spliceosome (or what remained of it after the splicing reaction). During this time, a second mRNA arrived for splicing while the intron from the reporter was still there.

Then something called reverse splicing happened which basically replaced the native intron of the RPL8B or the ADH2 gene with the spliced out intron from the reporter. Next this was turned into DNA with reverse transcriptases and then this construct ended up in the genome through homologous recombination.

No wonder this was so rare! Reverse splicing is thought to be really uncommon in vivo as is reverse transcription of DNA. Add in a loitering intron on the remnants of a spliceosome and you can see why this was a 1 in 250 billion shot.

So there you have it, one way a transposon can hop. It is no transposon, but occasionally an intron can move to a new gene.

And of course we turned to the awesome power of yeast genetics to help us figure this out. Only with yeast can we sift through half a trillion cells to find the two that show us how intronogenesis, the introduction of an intron to a new site, might happen. #APOYG!

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

Categories: Research Spotlight

Tags: mobile intron, reverse splicing

Control (Apple) F for Genetics

May 18, 2016

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A long time ago, through the mists of time, finding a phrase or a passage in a document was hard. If you didn’t have the foresight to highlight it, you were stuck.

You could reread the whole document but that might take way too much time. Or you could narrow down where to look by remembering the chapter it was in and rereading just that.

Nowadays of course, finding that phrase or passage is trivial in a Google or Word doc. You just use the find function!

Finding the genetic variant responsible for a given trait is still, in many ways, in the age of the typewriter. A genetic association study can find a part of the DNA responsible for that trait but homing in on the exact variant is terribly time consuming and labor-intensive.

Enter that wonder of a gene editing tool, CRISPR.

In a new study in Science, Sadhu and coworkers have essentially turned CRISPR into a find function for genetic association studies. They use CRISPR’s double stranded DNA cutting ability and mitotic recombination in a heterozygote yeast strain to blanket a region of DNA with crossover events.

Sifting through the results let them find the actual amino acid change responsible for manganese sensitivity with much less effort than would have been required with the old method. They only needed to use 358 lines instead of the 7,500 or so they predict they would have had to go through without good old CRISPR.

And think how much time might be saved with a more complicated beast! Given its short generation time and relatively small genome, yeast is like a magazine article. A human is more like War and Peace. This new system might make linking human traits/diseases to the causative SNP much simpler.

The usual way to link a trait to the DNA that causes it is to see what known traits tend to get passed down with it. If you know trait A is at position X on chromosome Y and the trait you are interested in, trait B, is usually passed down with A, then trait A and trait B are close to each other (this is called linkage mapping). This is the easy part.

The hard part is getting from A to B. To get finer mapping, you need to rely on the recombination that happens during meiosis. This is the step where this new study can help.

Instead of relying on the slow, natural process of meiotic recombination, these authors use the CRISPR-Cas9 system to jump start mitotic recombination.

They start off with a heterozygote in which one of the parent strains has a trait and the second does not. They chose the lab strain, BY, and the vineyard strain, RM, as the parents.

Next they designed 95 guide RNAs that would direct Cas9 to 95 different spots along the left arm of chromosome 7. Sadhu and coworkers targeted sites that were heterozygous in the two strains.

Once Cas9 cuts the DNA, the cell uses homologous recombination to repair the cut using the DNA on the other chromosome in the pair as the template. This results in a loss of heterozygosity (LOH) which can reveal recessive traits.

They picked 384 lines, approximately 4 from each cutting event and found that 95% of these had undergone LOH with hardly any off-target effects.

They next measured the growth of these strains in 12 different conditions and found that one trait, growth on 10 mM manganese sulfate could work for their purposes. Using the more traditional approach with 768 segregants obtained through meiotic recombination, they were able to narrow it down to an overlapping piece of DNA of 3,900 bases. However, using CRISPR-Cas9, they were able to narrow down the location of the variant to a 2,900 base pair stretch of DNA. Score one for the new method!

Next they did finer mapping by targeting the 2,900 bases they had identified with CRISPR-Cas9. Even though they only used three guide RNAs that targeted three locations, they were able to look at many more places on the DNA than this because the length of DNA repaired around the cut varies a bit between strains.

They isolated 358 lines of which 46 or 13.1% had a recombination event in the 2,900 bases they were interested in. Only 0.7% of segregants obtained the old way were in the right place. Score another one for the new method!

After measuring how well these 46 lines grew in manganese sulfate, the researchers were able to narrow the search to a single polymorphism that resulted in an amino acid change in the PMR1 gene. A perfectly reasonable result, given that Pmr1p is a manganese transporter. They then showed that this variant, pmr1-F548L, did indeed make a strain sensitive to manganese all on its own.

By targeting recombination events to regions of interest, these authors have provided proof of principle for a technique that should make connecting traits (phenotype) with DNA variants (genotype) much easier than it is currently. Behold yet again the awesome power of yeast genetics (#APOYG)!

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

Categories: Research Spotlight

Tags: CRISPR/Cas9, genotype, mapping, phenotype

Can’t Get There Like That

May 04, 2016

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As HBO’s Silicon Valley scathingly relates, the mapping app from Apple was truly terrible when it was first launched. There are all kinds of funny (scary?) stories in which people following the directions ended up in the wrong place. (Click here for a few more of the epic fails.)

And sometimes it would show impossible ways to get from one location to the other. For example, to get to a certain place, my iPhone would recommend two different routes. After choosing the seemingly easiest route I quickly realized that it would take me through a building. Sometimes, even though it seems there are a couple of different ways to get somewhere, there is actually only one.

It turns out that this can be true in gene expression too. While you might increase expression by either mutating the promoter or duplicating the whole gene, sometimes only duplication is enough. There is just one route from here to there.

This point is driven home in a new study out in GENETICS by Rich and coworkers. Here they show that, under sulfate-limiting conditions, the only way that yeast can boost the expression of the SUL1 high affinity sulfate transporter enough to thrive is by duplicating it.

In many previous experiments, whenever yeast is starved for sulfate, after 100 generations or so the population almost always ends up selecting for a SUL1 duplication rather than increasing expression with a promoter mutation. This duplication results in a fitness advantage of 35% or more, which makes sense – when there isn’t much sulfate available, those cells that can get more of what’s there will outcompete their neighbors.

There are a couple of possible reasons things go down like this. It could be that the duplication is simply the most likely way to increase expression enough to survive, meaning that promoter mutations are possible but rare. Alternatively there may be no way to mutate the promoter enough to adequately increase the activity in such a short window of time. You simply can’t get to enough increased fitness by this route.

The first step in figuring out how to get somewhere is to map the roads in the area. This is also what Rich and coworkers needed to do – they needed to figure out how SUL1 is regulated. They did this in the standard way by nibbling away bits of the sequences upstream of the gene until there was a significant impact of gene expression. This is how they identified the 493 base pairs upstream of SUL1 as its promoter.

In the meantime, they also managed to develop a broadly applicable methodology for investigating any promoter – saturation mutagenesis, chemostat selection, and DNA sequencing to track variants.

The next step was to generate a library of mostly single point mutations in this promoter using error-prone PCR. As might be expected, most of the mutants had no effect or decreased gene expression but a few did increase activity. However, none of these last set increased expression as much as duplicating the gene.

They found 8 mutants that gave a 5% or better increase in fitness with the best being a 9.4% increase. Even when they combined these point mutations they could not increase the fitness much beyond 11%, not even half of the increase in fitness that an extra SUL1 gene gives. It seems that to get the most bang for its buck, yeast needs to duplicate SUL1. At least in the time frame of the experiment, that is.

Using what is essentially the scanning mutagenesis of this promoter, they were able to identify three sites that were important for SUL1 regulation. One site at -465 to -448 corresponded to a Cbf1-Met28-Met4 regulatory site, the second site at around -407 was most similar to either a Met31 or Met32 site and the third site at around -350 matched a Met32 site.

Mutations that resulted in increased activity tended to bring one of these three sites closer to the consensus transcription factor binding site. For example, the strongest point mutation, -353T>G, did this with the Met32 site at -350.

Even with these stronger consensus sequences for sulfate-regulatory transcription factors, none of these point mutations could get the yeast to where it needed to be in the fitness landscape in order to be able to thrive under sulfur limitation. Point mutations just can’t hold a candle to simply duplicating the SUL1 gene. Sometimes there really is just one way to get from point A to point B.

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

Categories: Research Spotlight

Tags: adaptation, chemostat selection, fitness, gene duplication, saturation mutagenesis

Chocolate and Coffee Too?

April 20, 2016

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Most of us know about yeast’s big part in making bread and booze. But those aren’t yeast’s only wonderful gifts. It also plays a big role in chocolate and coffee too. Is there anything this marvelous microorganism can’t do?

A new study by Ludlow and coworkers in Current Biology set out to look at the strains involved in cacao and coffee fermentation. Unlike the extensively studied wine strains, these have mostly been ignored up until now.

These researchers found that cacao, coffee and wine strains were very much different from one another. And they also found that unlike wine yeasts, which are pretty much the same most everywhere in the world, coffee and cacao strains are different depending on where they come from.

But the cacao and coffee strains did have one thing in common. Each had a lot in common with the other strains in its country and even on its continent.

So, for example, coffee strains from all over South America are very similar to each other but different from the coffee strains from Africa. And the opposite is true as well. African coffee strains are all pretty similar to each other but different from the South American strains. The same sort of thing is true for cacao strains as well.

You can think of coffee and cacao strains as the large, flightless birds of the yeast universe. Like a rhea, emu, or ostrich, they stay on their own continent. Wine yeasts are more like those chickens that are basically the same worldwide because humans have taken them along with them in their migrations.

The first step in their analysis was for Ludlow and coworkers was to get a hold of a bunch of different samples of cacao and coffee yeast strains from all over the world. They managed to get 78 cacao strains from 13 different countries and 67 coffee strains from 14 countries. The countries were from Central and South America, Africa, Indonesia and the Middle East.

The next step was to compare the genomes of these strains with each other and with the wine strains. They decided to use a technique called restriction site-associated DNA sequencing, or RAD-seq, that would give them an in depth look at around 3% of the yeast genome. As there was already a database with 35 wine strains that used the same method, Ludlow and coworkers only needed to generate data for their newly isolated strains.

These data revealed that coffee and cacao yeast strains were very different from one another. It also showed that the closer two coffee or cacao strains were to each other geographically, the closer they were together genetically. Using just these strains they could accurately predict the country of origin for a coffee yeast strain 79% of the time and 86% of the time for those associated with cacao.

The researchers next expanded the number of species they compared by using two additional databases. One included RAD-seq data from 262 strains from a wide variety of different places while the other contained another 57 strains.

This analysis generated 12 distinct groups of yeast, many of which had been identified before. Their new strains formed four new groups which they called South America Cacao, Africa Cacao, South America Coffee and Africa Coffee.

By comparing the four groups to the eight older ones, they were able to see that the new groups were not novel but instead were made up of mixtures of some of the other known groups. And who they shared alleles with depended on where the yeast strain was located. So, for example, the two South America groups shared alleles with the North American Oak group while the two African groups shared alleles with the Asian and European groups.

It looks like that, unlike with wine where there are a limited set of best yeast strains that most people use, the yeast strains involved with making chocolate and coffee are continent and even country specific. This suggests that people either took their wine yeast with them when they moved or that cross contamination throughout the world resulted in homogenization of wine yeasts. Sort of like the chickens Pacific Islanders brought with them whenever they settled on a new island.

It is a very different story for cacao and coffee yeast strains. While there was cross contamination within countries and even continents, there was no worldwide contamination. The rhea stayed in South America and the ostrich in Africa.

Wherever these strains come from, they work hard to make our chocolate, coffee, bread, wine, and beer. What an amazing bounty! Make some room, Dog, good ol’ Saccharomyces cerevisiae is joining you as man’s best friend.

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

Categories: Research Spotlight

Tags: admixture, migration, population diversity

Lessons from Yeast: Poisoning Cancer

April 06, 2016

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In the book Dune, the mentat Thufir Hawat is captured by the evil Harkonnens and given a residual poison. He can only stay alive by getting a constant dose of the antidote. Once it is withdrawn, he will die.

A new study in the journal GENETICS by Dodgson and coworkers shows that the same sort of thing can happen to yeast that carry an extra chromosome. In this case, certain genes on the extra chromosome turn out to be like the residual poison. And a second gene turns out to be the antidote.

Once that second gene is deleted, the yeast cell dies. It has been deprived of its antidote.

This synthetic lethal phenotype isn’t just a cool finding in yeast either. Cancer cells invariably have extra and missing chromosomes. If scientists could find similar “antidote genes” in specific types of cancers and target them, then the cancer cell would die. And this would happen without damaging the other cells of the body that have a typical number of chromosomes.

The first thing these researchers did was to make separate yeast strains each with an extra chromosome I, V, VIII, IX, XI, XII, or XVI. The next step was to see what happens when every gene was deleted individually, one at a time, from each strain.

As expected, these yeast did pretty well when a gene on the extra chromosome was deleted. So, for example, a strain with an extra chromosome I tolerated a gene deleted from chromosome I. This makes sense as this just brings that gene back to its normal copy number.

But this was not the case with chromosomes VIII and XI. Here deleting genes on the extra chromosome often had a negative effect. This suggested that the screen probably had a high number of false positives and these researchers later confirmed this.

Likely reasons for the high number of false positives include the strain with the extra chromosome being W303 and the deletion strain being S288C, errors in the deletion collection itself, and what they refer to as neighboring gene effects. Basically this last one is the effect that deleting a gene has on nearby genes.

Once Dodgson and coworkers corrected for these problems, they found two broad sets of phenotypes – general and chromosome specific.

The general ones were the ones shared by most or all of the strains. These were deletions that affected the yeast no matter which chromosome they had an extra copy of.

For the most part, these genes were enriched for the Gene Ontology (GO) term vesicle-mediated transport, indicating that they have something to do with the transportation of substances in membrane-bounded vesicles. For example, deletion of MNN10, HOC1, and MNN11, genes all involved in protein transport and membrane-related processes, had a negative effect on many of the yeast strains with an extra chromosome. Consistent with this, brefeldin A, a drug that targets protein trafficking, negatively affected most of the strains.

Another gene that affected many of these strains when deleted was TPS1. This gene encodes a subunit of trehalose-6-phosphate synthase, a key enzyme for making trehalose, a molecule that helps yeast deal with stress. Perhaps not surprisingly, having an extra chromosome is stressful!

In addition to the genes that affect many strains with an extra chromosome, there were also genes that were chromosome specific. The best characterized of these was the EDE1 gene in the strain with an extra chromosome IX. Deleting EDE1 in this strain increased its doubling time by more than 80 minutes while only causing an increase of 5 minutes in the doubling time of wild type yeast. This was a severe phenotype in their assay.

They next tried to find which gene on chromosome IX might be responsible for the severe effect of deleting EDE1. Since EDE1 is known to be involved in endocytosis, they looked for genes involved in the same process. And they found one – PRK1.

The strain with a deleted EDE1 gene and an extra chromosome IX was rescued by deleting one copy of the PRK1 gene. The extra PRK1 gene was the poison and the EDE1 gene was the antidote.

If a similar pair could be found in cancers that often have the same set of extra chromosomes, then perhaps scientists could develop drugs that target an antidote gene. Now the cancer cells would die and the “normal” cells would be fine. Thanks again, yeast, for pointing us toward new ways to treat human disease.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: aneuploidy, cancer, synthetic lethal

Sometimes Simple is Better

March 23, 2016

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Diagnosing why something has gone wrong in a complicated system can be difficult. There are so many bells and whistles that you can easily get lost.

That’s why it can sometimes help to turn to a simple system and then apply what you have learned to the more complicated one. This will, of course, sound familiar to any scientists studying that marvel of a model organism, Saccharomyces cerevisiae.

For example, it is amazing what you can glean from this yeast about human brain and blood diseases. Even though, of course, baker’s yeast has neither blood nor a brain!

This becomes very clear in a study out in PLOS Genetics by Fernandez-Murray and coworkers. In this study they use yeast to help figure out why mutations in the SLC25A38 gene in people leads to something called congenital sideroblastic anemia. And even better, their work hints at a possible treatment.

People with sideroblastic anemia make too little hemoglobin in their red blood cells and have too much iron in the mitochondria close to the nucleus (perinuclear mitochondria). The current treatment for this condition is not ideal, involving lots of transfusions and iron chelation.

Sometimes people are born with this anemia and sometimes people get it later in life. One subset of the inherited version happens because a gene with an unknown function, SLC25A38, isn’t working correctly. This group of patients is the focus of this study.

Fernandez-Murray and coworkers started out by using yeast to figure out what the yeast homolog, HEM25, does in a yeast cell. When the gene was deleted, the cells made about 50% less heme than wild type yeast and adding back the human gene, SLC25A38, to this deletion strain restored heme levels. Looks like they had made a yeast model of this inherited anemia.

Previous work had suggested that SLC25A38 might be a glycine or serine transporter and the next set of experiments confirmed it as a glycine transporter in a couple of ways. In both, they took advantage of cases in which yeast can use glycine as their sole nitrogen source if the glycine can make it into the mitochondria.

In the first case, they showed that yeast cells deleted for HEM25 grew poorly on plates where glycine was available as the only nitrogen source. In the second case, they showed that cells deleted for both SER1 and HEM25 grew poorly on plates where again glycine was the only nitrogen source. This last result confirms HEM25 as a glycine transporter since yeast deleted for the SER1 phosphoserine aminotransferase can only grow in the absence of serine if they can get glycine into their mitochondria. (There isn’t space to go into it here, but they also showed that HEM25 was not a serine transporter.)

OK so now they had created a yeast cell that mimicked the effect of sideroblastic anemia and figured out why people with a mutated SLC25A38 gene had the condition. Now it was time to find a treatment.

The researchers came up with three possibilities. The first treatment was just to give the yeast extra glycine, the second was to drive glycine synthesis within the cell by adding a lot of serine, and the third was to add a downstream precursor of heme synthesis, 5-aminolevulinic acid (5-Ala).

They tested each scenario on yeast cells deleted for HEM25 and found that both glycine and 5-Ala worked to restore heme synthesis, but that added serine had no effect. Both glycine and 5-Ala returned heme levels to that seen in wild type.

Of course we aren’t yeast, so they next tested their treatment on something a bit more complicated — zebrafish. By using morpholino technology to knock down both copies of the zebrafish SLC25A38 homolog, SLC25A38a and SLC25A38b, the researchers managed to lower a zebrafish’s heme levels to about 50% of normal.

When they gave these zebrafish extra glycine or 5-Ala, their heme levels did not improve. They were still anemic!

After a bit of thought, the researchers realized that folate might be what the zebrafish were missing. In work that we didn’t have time to go over before, the researchers had shown that a folate dependent pathway was critical for getting heme levels up to normal.

Yeast could get away without added folate in these experiments because they make their own. However, zebrafish, like people, do not.

So the final step was to try to add both glycine and folate to these fish. Now the zebrafish’s heme levels returned to about 80% of normal.

These results suggest a better treatment for some people with sideroblastic anemia — added folate and glycine. And it all came from studying the problem in the simpler, bloodless S. cerevisiae. Nice work again yeast.

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

Categories: Research Spotlight

Tags: anemia, human disease, model organism, zebrafish

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