New & Noteworthy

SGD Homology Data Now Available On New Homology Pages

March 25, 2021

SGD is excited to introduce our new Homology Pages! These pages can be accessed by clicking on the Homology tab in the header of SGD gene pages, as seen below.

The information displayed on the Homology Pages is divided into several sections:

• Homologs: Information about known homologs for the gene of interest, such as the species of the homolog, the corresponding Gene ID from the Alliance of Genome Resources, and the name of the homolog.
• Functional Complementation: Data about cross-species functional complementation between yeast and other species, curated by SGD and the Princeton Protein Orthology Database (P-POD).
• Fungal Homologs: Curated homolog information for 24 additional species of fungi. View the species of the fungal homolog, the database source of the entry, and the Gene ID of the homolog from that database.
• External Identifiers: A list of external identifiers for the protein from various database sources.

If you have any questions or feedback regarding our new Homology Pages, please do not hesitate to contact us at any time.

Categories: Data updates, Homologs, New Data, Yeast and Human Disease

Feed a Cold, Starve a Cancer?

March 02, 2017

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You may have heard the old wives’ tale of feed a cold, starve a fever. Turns out that this isn’t particularly good advice (although some studies do suggest that with a fever, you shouldn’t force-feed yourself). It also turns out to have probably originated in the 19th century and not from Chaucer in the 14th as many websites claim.

But while starving a fever is probably never a good idea, starving a cancer can be. Not by following the medical myth that since cancers use a lot of sugar, you can starve them by cutting down on sugar in your diet. Instead you can starve some cancers by denying them the amino acid asparagine (Asn).

On their way to becoming cancerous, acute lymphoblastic leukemia (ALL) cells lose their ability to make Asn. This means that unlike the cells around it, they need to pull Asn from the blood to make their proteins and to survive.

Doctors exploit this weakness by injecting L-asparaginase amidohydralase (L-ASNase) into patients which starves the cancer cell by depleting Asn levels in the blood. The cells around the cancer cells are fine because they can still make Asn.

Right now doctors use L-ASNase from two different bacterial sources: Escherichia coli and Erwinia chrysanthemi. But if a recent study by Costa and coworkers in Scientific Reports holds up, they might want to think about switching to using the Saccharomyces cerevisiae L-ASNase encoded by the ASP1 gene.

An older study had suggested that the yeast enzyme might be too weak to be useful. This new study finds that this is not the case.

The difference between the older study and this one was the purification protocol. The older study purified the native enzyme through multiple chromatography steps while this study used a single affinity chromatography step. The purified yeast and E. coli versions have comparable activity in this study.

They are also comparable in terms of being able to work with very low concentrations of Asn. This is important as Asn levels are very low in the blood.

What makes the yeast enzyme potentially better is that it is much worse at hydrolyzing a second amino acid, glutamine, than are the bacterial versions. This higher specificity for Asn is important because one of the major side effects of the current treatment is neurotoxicity caused by decreased levels of glutamine in the blood. Since the yeast version hydrolyzes glutamine at a lower rate, they predict patients may not suffer as badly from this side effect with the yeast version.

Of course this is all for naught if the yeast enzyme can’t kill cancer cells! Or if it kills cells indiscriminately.

The S. cerevisiae version was nearly as good as the E.coli version in tissue culture. After 72 hours of incubation, both versions had little effect on normal cells (HUVEC), and both were cytotoxic to the L-ASNase-sensitive cell line MOLT-4 with the E. coli version killing 95% of MOLT-4 cells and the yeast version killing 85% of them.

Taken together these results suggest that the S. cerevisiae version may be an alternative to the bacterial versions. It may be able to kill cancer cells with fewer side effects.

But the yeast version is not the only alternative in town. Another group is engineering the E. coli version to lessen its propensity for hydrolyzing glutamine. Either way it looks like certain leukemia patients may be getting an effective cancer treatment with fewer side effects.

Beer, wine, bread, chocolate, and now maybe a treatment for a nasty form of leukemia. Yeast may be humanity’s best friend. #APOYG!

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: Acute lymphocytic leukemia, cancer, Proteins, Recombinant protein therapy

Sign Up Now! Next SGD Webinar: December 14, 2016

December 12, 2016

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Looking for human disease-related information in SGD? There is so much to find! Active areas of curation at SGD include yeast-human homology, disease associations, alleles and phenotype variants, and functional complementation relationships.

Join our upcoming webinar on December 14th, 9:30 AM PST to learn about homology and disease data in SGD. In this quick 15 minute session, we will demonstrate the best ways to research this information on our website and provide a helpful tutorial on related SGD tools and features. Our webinars are always an excellent opportunity to connect with the SGD team–be sure to bring questions if you have them!

All are welcome to this event. If you are interested attending, please register here: http://bit.ly/SGDwebinar6

This is the sixth episode in the SGD Webinar Series. For more information on the SGD Webinar Series, please visit our wiki page: SGD Webinar Series.

Categories: Announcements, Homologs, Tutorial, Yeast and Human Disease

Too Much of a Good Thing

November 09, 2016

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Like a ruined cookie with too much salt, a cell can go haywire when it has too many copies of certain genes. And of course, cells can deal perfectly well with too many copies of other genes. Just like adding too many chocolate chips to your cookies might make an even better cookie!

Finding out which genes are like salt and which ones are like chocolate chips is of more than just general biological interest. It might help us to explain why cancer happens and to possibly find better treatments.

As you probably know, cancer cells are pretty messed up genetically. Their DNA is littered with mutations, rearrangements and somatic copy number amplifications (SCNAs).

A big reason for this genetic jumble is early DNA changes that increase the rate of mutations in a cell. This “mutator” trait makes a cell more likely to stumble on the mutations it needs to grow out of control or refuse to die.

In their new study in GENETICS, Ang and coworkers set out to find genes that can cause a mutator phenotype when they are part of a SCNA. In other words, which genes lead to an increased mutation rate when expressed at a higher level.

This is important because there are so many SCNAs in a typical cancer cell that it can be hard to figure out which ones matter and which ones don’t (or to put it into cancer parlance, to tell the drivers from the passengers). And despite all of the CRISPR hoopla and other mammalian resources, it would still be a very long process to find “dosage mutator” genes in cell culture and/or living animals.

Which is why Ang and coworkers used our favorite workhorse, the yeast Saccharomyces cerevisiae, to find genes that may cause an increased mutation rate when overexpressed.

The assay is conceptually simple. Yeast that have a functioning CAN1 gene do not survive in the presence of the drug canavanine. So these researchers looked for cells that did better in the presence canavanine when overexpressing a single gene. Presumably, they are surviving because that extra gene resulted in the CAN1 gene being mutated more often because of an increased mutation rate.

They found 37 genes that fit the bill, 18 of which that were involved in biological pathways known to affect genome stability. Combining this with previous studies that looked at gene deletions, this brings the grand total of suspected yeast mutator genes to 210.

Most of these 210 were identified because of mutations that made them stop working which can make figuring out why they cause the mutator phenotype relatively simple. For example, if a mutation kills a gene responsible for fixing DNA mistakes, then you are going to get more DNA mistakes in that cell. It is a little trickier to understand how extra copies of a gene might cause an increased mutation rate.

Ang and coworkers focused on trying to figure out the mechanism behind their top 5 dosage mutator genes: PIF1, MPH1, UBP12, RRM3, and DNA2. Since 4/5 of these code for helicases, they first checked to see if just being a helicase is enough to be a dosage mutator gene. It isn’t.

They retested 48 DNA helicases in their assay and found that none of them caused an increased mutation rate when mutated. There is more to a dosage mutator than being a helicase!

In the next set of experiments, they wanted to determine if the five strains, each overexpressing one of these five genes, had a higher mutation rate by the same mechanism. They tested this by determining the sensitivity of these 5 strains to 3 different DNA damaging agents. The idea is that if they share the same mechanism, they should have the same sensitivity profiles to each of these agents. They did not.

For example, overexpressing MPH1 resulted in a higher sensitivity to all three agents while overexpressing UBP12 only increased sensitivity to two of them. So each strain probably has an increased mutation rate for a different reason.

They next wanted to see if the increased mutation rate was due to a loss or gain of function. They did this by comparing the profiles of strains either deleted for or overexpressing the dosage mutator genes. The idea is that if overexpression leads to a loss of function, then deleting and overexpressing the genes should have the same profile. The three they could test like this did not.

The authors conclude from this that the increased mutation rate for MPH1, UBP12, and RRM3 is most likely due to the gain of an inappropriate function as opposed to a loss of function. In a final set of experiments, Ang and coworkers focused on what that new function might be in their strongest mutant, MPH1.

First they showed that of the three activities associated with Mph1p, only DNA binding and not its ATPase or helicase activities were important for it causing an increased mutation rate when overexpressed. From this they reasoned that perhaps Mph1p was displacing some other important DNA binding protein and that it was this displacement that was causing the increased mutation rate.

Through a set of experiments we don’t have time to go into here, they provided evidence that Mph1p was outcompeting the flap endonuclease Rad27p for DNA binding. This makes some sense as previous work had shown that deleting RAD27 causes mutation rates to go way up. So too much Mph1p keeps Rad27p from getting to where it needs to be with the end result being an increased mutation rate.

All this MPH1 work may have important implications in some human cancers. Nonsense or missense mutations in FANCM, the human homolog of MPH1, are known to make people more likely to get cancer. And there are examples of cancers where FANCM is overexpressed. Perhaps that overexpression results in an increased mutation rate in these cancers.

Yet again yeast is giving researchers new targets for, and new ways to think about, human disease. Thanks, yeast, for finding all of these mutator genes for us to investigate further! #APOYG!

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: cancer, forward mutation, genome-wide, MPH1, mutator, overexpression

New SGD Help Video: Finding Human Homology & Disease Information

October 24, 2016

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Looking for human disease-related information in SGD? There is so much to find! Active areas of curation at SGD include yeast-human homology, alleles and phenotype variants, functional complementation relationships, and disease associations. There are plenty of ways to find this information on our website, and it takes just 90 seconds to learn how – what are you waiting for?

For more SGD Help Videos, visit our YouTube channel, and be sure to subscribe so you don’t miss anything!

Categories: Announcements, Homologs, Tutorial, Yeast and Human Disease

Winter is Coming (for Cancer)

October 17, 2016

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I may be a little late to the game, but over the last few weeks I have started consuming episodes of Game of Thrones voraciously. It is such a fun show to watch! And this isn’t the only HBO show I enjoy. Veep, Silicon Valley, and Last Week Tonight with John Oliver all have my attention as well.

You might say that I need HBO, because without it I can’t get my fill of these shows. (Well, there are other routes, but HBO or HBO GO are the easiest). My over watching of these shows has made me dependent on HBO.

Something similar can happen in cancers. Sometimes a key player in keeping a cell cancerous is an overexpressed gene. And just like my binging of Game of Thrones makes me dependent on HBO, so this overexpressed gene (the TV show) makes the cancer cell dependent on another gene (HBO).

A real life example might make this clearer. Most folks have heard of BRCA1 and BRCA2 especially since the Angelina Jolie story. When either of these genes is damaged, you can end up with cancer.

Both of these genes are involved in DNA repair and damaging them means the cell builds up mutations. Making lots of DNA mistakes is a good thing for cancers but only up to a point. Too much damage and the cancer cell dies.

What this means is that these cancer cells are now more dependent on other DNA repair genes. Which means these other DNA repair genes are now targets to go after to selectively kill the cancer cells.

For cancer cells lacking BRCA1 or BRCA2 function, research has shown that these cells are now dependent on a second gene, PARP1. If PARP1 expression is turned down, normal cells survive but BRCA1/BRCA2-dependent cancers die. So, we can kill cancer cells, or end their TV show watching, by going after PARP1, their HBO.

Finding these sorts of genes is not easy unless, of course, you turn to our favorite lab workhorse, the yeast Saccharomyces cerevisiae. Given all of the genetic techniques and tools available with this yeast, it is possible to quickly do a synthetic dosage lethality assay – to look for genes that are lethal only in combination with deleting your gene of interest.

This is just what Reid and coworkers did in a new study just out in GENETICS for CKS1B, a gene that is amplified and overexpressed in many cases of breast, lung, and liver cancers. And they found a more “druggable” target to go after, the kinase PLK1 (the human homolog of yeast CDC5). PLK1 even comes with its own kinase inhibitor, Volasertib.

Reid and coworkers transformed a low copy plasmid containing the CKS1 gene, the yeast homolog of CKS1B, under the control of the galactose promoter into two different yeast strain libraries. The first screen used 9600 yeast deletion strains, each with a single gene deleted in either a MATa or MATα strain. The second screen used strains with temperature sensitive mutants of essential genes. They now looked to see which yeast strains did poorly or couldn’t survive when they were overexpressing CSK1 in the presence of galactose.

In the end they came up with 44 different genes that, when deleted or weakened, had a severe effect on the growth of yeast that overexpressed CKS1. Given that CKS1 plays an important role in cell cycle progression, they focused on the 15 genes that affect mitotic progression. Eventually, through a set of experiments that I don’t have time to go into here, they settled in on CDC5, a polo-like kinase involved in both mitotic entry and exit.

The next step was to see if what they learned about in yeast has any bearing on cancer. It did.

First Reid and coworkers looked at a variety of cancer cells in The Cancer Genome Atlas (TCGA) and found that it was very rare for both PLK1 and CKS1B to be overexpressed in the same cancer at the same time. Next they looked at a data set of short hairpin RNA (shRNA) knockdowns of ~16,000 human genes and found that knocking down PLK1 had negative effects on cancers overexpressing CKS1B. These are consistent with the two genes having a synthetic lethal relationship.

They then took eight breast cancer lines where the shRNA against PLK1 had a negative effect on growth and tested the effects of targeting PLK1 on apoptosis. Did decreasing expression of PLK1 in cells that overexpress CKS1B cause an increase in apoptosis in their hands?

The short answer is yes. They repeated the experiments with the shRNA and also tested the PLK1-specific kinase inhibitor Volasertib and found that both treatments increased apoptosis in CKS1B overexpressing cancer cells. It looks like they may have uncovered a way to go after a subset of cancers using yeast!

Which shouldn’t surprise us. Yeast and other model organisms have been teaching us about cancer at least since the days when Hartwell, Hunt and Nurse first identified cyclins and CDKs (for which they got the 2001 Nobel Prize in Physiology or Medicine), and will continue to school us for years to come.

Hopefully researchers will continue to turn to yeast to continue to better understand and find new treatments for cancer. Yeast has so much more to teach us! #APOYG!

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: CKS1, cyclin-dependent kinase, polo-like kinase, synthetic dosage lethal

A Nobel Prize for Work in Yeast. Again!

October 03, 2016

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Dr. Yoshinori Ohsumi has won the 2016 Nobel Prize in Physiology or Medicine for his groundbreaking work on autophagy in yeast. This is the process whereby cells recycle their worn out parts or where a cell, like Mobius, the snake eating its own tail, eats less essential bits of itself to stay alive during times of starvation. Think Scarlett O’Hara using her drapes as a dress in Gone With the Wind (or Carol Burnett’s hilarious parody).

Like many, many Nobel Prizes in the past, Ohsumi’s work uncovered basic biological properties using a model organism. In this case he used our favorite lab workhorse, the yeast Saccharomyces cerevisiae, to piece together the steps involved in the recycling of a cell’s own internal structures.

And like many other basic biological studies, this one has important medical applications. In this case the two most obvious are chemotherapy resistance and amyloid-β aggregation in Alzheimer’s disease, but it isn’t restricted to just these two. For example, a specialized form of autophagy that targets damaged mitochondria, mitophagy, may not be working well in people with Parkinson’s disease.

The key to Ohsumi’s work was finding a way to disrupt this process in yeast so that he could find the important genes underlying autophagy using the awesome power of yeast genetics (#APOYG!). It turns out that this is trickier than it might seem because yeast and their autophagosomes, the little vesicles that surround and encase the bits to be degraded, are very small and so hard to see. In fact, they are so small that there was some question about whether yeast even had this process!

If yeast did, then it would take place in the vacuole, the recycling center in yeast. The equivalent organelle in people is the lysosome.

To see if autophagy happens in yeast, Ohsumi starved yeast that had vacuoles but couldn’t digest anything. The idea was that there would be a buildup of autophagosomes in the vacuole because the yeast would be desperately trying to eat itself but had no way to digest what it ate. He indeed saw that these poor yeast developed huge vacuoles bloated with autophagosomes.

Dr. Yoshinori Ohsumi now had the makings of a yeast screen! “All” he had to do was to look for mutants that didn’t form giant vacuoles under these conditions with the logic being that if you knocked out autophagy, you wouldn’t get a buildup of autophagosomes.

The rest, as they say, is history. Ohsumi and his lab managed to tease out the subtleties of this vital cellular process using good old baker’s yeast. What other nuggets of knowledge about ourselves will we pry out of this most useful of eukaryotes? I can’t wait to see what it reveals about us next!

Other Nobel Prizes have been awarded in recent years for work in yeast:

• Leland H. Hartwell, R. Timothy (Tim) Hunt, and Paul M. Nurse won the 2001 Nobel Prize in Physiology or Medicine for their discoveries of “key regulators of the cell cycle”.
• Roger D. Kornberg won the 2006 Nobel Prize in Chemistry “for his studies of the molecular basis of eukaryotic transcription”.
• Elizabeth H. Blackburn, Carol W. Greider, and Jack W. Szostak won the 2009 Nobel Prize in Physiology or Medicine “for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase”.
• James E. Rothman, Randy W. Schekman, and Thomas C. Südhof won the 2013 Nobel Prize in Physiology or Medicine “for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells”.

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

Categories: News and Views, Yeast and Human Disease

When Running Out of Steam is a Good Thing

August 24, 2016

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Usain Bolt sprinting is a thing of beauty. It is just amazing how he can kick in the afterburners at the end of a race and just dominate the thing. I am sure Justin Gatlin of the U.S. would love for Bolt to lose this extra burst of speed so Gatlin could beat him at the Olympics.

Turns out that transcription elongation has an afterburner a bit like Bolt’s too. It goes by the name SPT4 in yeast and SUPT4H1 in you and me. The protein from this gene is needed to push through long transcripts.

A new study in Science by Kramer and coworkers suggests that like Gatlin, some people would like to see their cells lose the burst of speed that SUPT4H1 gives their polymerases. But instead of helping these folks win a race, this loss might help them deal with their amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).

ALS is a progressive neurodegenerative disease that is always fatal. It was first made famous by Lou Gehrig and later with the bucket challenge. After Alzheimer’s, FTD is the second most common form of dementia.

Back in 2011 two groups found that a significant number of cases of FTD and ALS were associated with a gene called C9orf72 (chromosome 9 open reading frame 72). These people had hundreds or even thousands of copies of the hexanucleotide repeat GGGGCC in the first intron of their gene instead of the 30 or so that is more typical.

Later studies showed that these repeats caused two very specific problems in cells. First, the RNA (and antisense RNA) from this allele tended to build up in small bundles called foci. Some researchers think that these foci trap some of the important RNA binding proteins that the cell needs.

The second phenotype is a strange one. These RNAs get translated by a process called repeat-associated non-ATG, or RAN, which causes a buildup of dipeptide repeat proteins. Apparently there is something about the secondary structure of the RNA that allows it to get translated without a typical AUG start codon.

The idea is that these foci and weird dipeptide proteins are at least part of the reason why these folks have their ALS symptoms. Ideally you’d want to get at all three issues (the sense and antisense RNA-laden foci, and those newly translated proteins) with a single approach.

Kramer and coworkers reasoned that they might get such a result if they could get the cell to make a whole lot less of SUPT4H1 (or Spt4p in yeast). They reasoned correctly.

Previous research had shown that its deletion didn’t affect too many genes except for those involved in diseases like Huntington’s – those with long CAG repeats. Perhaps, then, deleting it might also just affect the copies of the C9orf72 gene with those hexanucleotide repeats without affecting too many other genes.

When they forced yeast, nematode, fruit fly and human cells to make less Spt4p or SUPT4H1, the number of RNA foci went down or even disappeared in all of these different cells. There was also much less of those dipeptide repeat proteins lurking about the cell as well.

They first set out to do some experiments in everyone’s favorite workhorse, Saccharomyces cerevisiae. They found that expressing either the sense or antisense RNA with the 66 hexanucleotide repeats caused both the RNA foci and the dipeptide repeat proteins seen in the cells of ALS patients to form in yeast too. Neither happened with the sense or antisense 2 repeat constructs.

Next they showed that deleting SPT4 greatly reduced the level of 66 repeat RNA but had little effect on the 2 repeat RNA. These researchers also saw no RNA foci and much less dipeptide repeat proteins in the deletion strain expressing the 66 hexanucleotide repeats. All without much affecting any other genes.

This yeast work suggests that targeting SUPT4H1 might reduce the effects of the ALS version of the C9orf72 gene without affecting the more typical version. Now Kramer and coworkers were ready to see what happens in bigger beasts.

When they expressed the 66 repeat in Caenorhabditis elegans neurons, these nematodes lived for a shorter time and their neurons had RNA foci and the dipeptide repeat proteins. Expressing human SUPT4H1 in these worms’ neurons worsened their condition while feeding them RNAi against nematode SPT4 helped.

The RNAi let these worms live longer and it decreased the number of RNAi foci and the amount of dipeptide repeat proteins. They saw similar results with a Drosophila system.

Finally they moved to the main stage—human cells from ALS patients who had the C9orf72 protein with too many hexanucleotide repeats. RNAi against either SUPT4H1 or its partner in crime, SUPT5H, reduced the number of RNA foci and reduced the amount of dipeptide repeat proteins with no “overt toxicity.” RNA-seq showed that only a small subset of genes was affected with the RNAi treatment.

So it looks like targeting SUPT4H1 may be a good strategy for dealing with ALS if the RNA foci and dipeptide repeat proteins are a big part of the problem. This is a big if.

But if it all does work out, we can thank yeast yet again (#APOYG!) for showing us the way to a new treatment for a devastating disease. Of course, though, yeast can’t do everything. It is unlikely to show sprinters the best way to beat Usain Bolt in a race!

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

Categories: Research Spotlight, Yeast and Human Disease

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

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

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