New & Noteworthy

Using Yeast to Find New Treatments for Huntington’s Disease

September 05, 2013

Huntington’s disease (HD) is a truly awful, inherited and ultimately fatal genetic disease.  People with this neurodegenerative disorder typically start having trouble with their coordination and displaying mild cognitive and psychiatric problems in mid-adulthood.  Their symptoms continue to worsen, with most of these folks passing away within 20 years of their diagnosis.  This disease strikes down adults in their prime.

Scientists have known for decades what causes HD—too many CAG repeats in the huntingtin (htt) gene.  What they haven’t been able to figure out is what to do about this misfolded protein.  To date, the treatment options are very limited.

A new study out by Mason and coworkers has a chance to change all of that.  Using an unbiased screen in Saccharomyces cerevisiae, these authors were able to identify a class of proteins, the glutathione peroxidases, that when overexpressed protected yeast from the harmful effects of the mutant htt protein.  They then followed up and showed that these proteins had a similar effect in fruit fly and mouse HD cell models as well as in a whole fruit fly model.  And this isn’t even the exciting part.

There are druggable small molecules that when added to cells (or whole animals) can upregulate the activity of glutathione peroxidases.  The authors used one of these molecules, ebselen, and showed that it mimicked the effects of overexpressing various glutathione peroxidases in cells and, more importantly, in whole fruit flies.  When these flies were fed ebselen, their neurons degenerated at a much slower rate.  Mason and coworkers have identified a small molecule that can mitigate the effects of the mutant htt protein in model systems.

While we shouldn’t get ahead of ourselves here, this is all pretty exciting news.  How cool would it be if one day people with HD lived longer, happier lives because of a drug identified using our favorite model organism?  (Pay attention NIH!)

Mason and coworkers looked in S. cerevisiae for open reading frames that, when overexpressed, would lower the toxicity of the mutant htt protein.  They identified 317 of these, and used a variety of bioinformatics tools to group them into different pathways and gene networks.

In the end, they decided to focus on two powerful suppressors, the glutathione peroxidases Gpx1p and Hyr1p (also known as Gpx3p), for a variety of different reasons. These proteins are powerful antioxidants, and oxidative stress is known to contribute to HD symptoms.  Also, these proteins aren’t already upregulated in patients with Huntington’s disease, suggesting that it might be possible to increase their activity using drug therapy.

Now of course yeast aren’t mammals, so Mason and coworkers needed to show that having extra glutathione peroxidase activity would help in mammalian cells too. And this is just what they did: adding a mouse version of glutathione peroxidase, mGPx1, suppressed cellular toxicity in mouse cells that overexpressed the mutant form of htt.

Next they tested whether activating glutathione peroxidases would have the same effect.  They focused specifically on a selenocysteine-containing molecule called ebselen because it is highly bioavailable, can cross the blood-brain barrier (critical for HD) and has been used in treating stroke and noise induced hearing loss. When added to the mouse HD model cell system, ebselen had very similar effects to overexpressing mGPx1.

So upregulating glutathione peroxidase activity by either overexpressing mGPx1 or adding the small molecule ebselen appears to help in a couple of different model cell systems.  But what about a whole animal?  Looks like it can help there too.

Mason and coworkers looked at HD in a fruit fly.  When they added mGPx1 to this model fly, various neurons in these flies were protected from the effects of HD.  And they got similar results when they fed these flies the molecule ebselen.

As a final experiment, the authors wanted to figure out whether glutathione peroxidases were really having their effect because of their antioxidant activity.  In one way it makes sense that this activity is why they are so effective at mitigating the effects of the mutant HD—scientists have known for a while that oxidative stress is a major contributor to symptoms of HD. But on the other hand no antioxidant therapies have worked to date for HD.  In fact, if anything they made matters worse.  So one thought was that there was something special about the antioxidant activity of these proteins.  For these experiments, they needed to go back to yeast.

The authors looked at a variety of antioxidant proteins, including superoxide dismutase, catalases, and glutathione reductases, and none protected the yeast from the effects of the mutant htt protein.  They then checked the effects of catalase and superoxide dismutase in the HD mouse cells, and again saw no effect.

It is well known that antioxidants negatively affect autophagy and that disrupting this process can make HD symptoms worse.  From this the authors reasoned that glutathione peroxidases were special because they were antioxidants that did not affect autophagy.  They provided support for their idea by showing that ebselen did not affect autophagy in yeast while a control antioxidant, N-acetylcysteine, did.

Once again, yeast shows why it is such an important tool in finding potential new treatments for human disease.  Without the unbiased screen, it’s difficult to imagine how scientists would have found this target. You can really only do this easily in a beast like yeast. 

 

Symptoms like these may one day be delayed because of the awesome power of yeast genetics.

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

Categories: Research Spotlight, Yeast and Human Disease

Anchors Aweigh for Peroxisomes

August 15, 2013

Suppose you had a fleet of rowboats that you wanted to split up evenly between two shores.  You probably wouldn’t just set them free and hope they drifted to the right places. A better way might be to run some ropes from the distant shore and pull half the rowboats across.  That way you’d be sure to get the distribution of boats that you wanted.

Dividing cells face a similar problem with their organelles.  When cells divide, they need to make sure mother and daughter receive the right number of organelles.  Otherwise one or both could die!

This is obviously too important to just leave to chance, which is why cells have devised ways to precisely control how many organelles end up in mother and daughter.  But not every organelle is divided in the same way.  In our boat analogy, some are pulled over with ropes, others with chains, some with winches and so on. 

We don’t know a lot about how some organelles are distributed between mother and daughter cells.  For example, the details have not been worked out for peroxisomes, the organelles that contain enzymes for beta-oxidation of fatty acids. Until now, that is…

In a recent study in The EMBO Journal, Knoblach and colleagues looked in great detail at how yeast cells distribute their peroxisomes. During budding, some peroxisomes stay tied up in the mother cell while others are transported into the bud and re-tied there.  

The authors found that the structure that ties up peroxisomes is like a rope with hooks at both ends. One hook attaches to the peroxisome, while the other hook attaches to the cortical endoplasmic reticulum (ER) near the cell wall. Surprisingly, the same protein, Pex3p, acts as the hook at both ends of the rope, connecting it to both ER and peroxisomes.

The authors already knew that some peroxisomes stayed anchored around the edges of the mother cell while others were “mobile” and moved to the daughter when yeast cells divided. They also knew that the protein Inp1p was important for anchoring peroxisomes. In the inp1 null mutant all the peroxisomes are mobile and end up in the bud, while overexpressing Inp1p causes all the peroxisomes to be anchored in the mother cell and stay there.

Knoblach and colleagues suspected that Inp1p might act as the rope that tethers peroxisomes. To test this, they fused Inp1p to a protein that sits in the mitochondrial outer membrane, Tom70p. Now peroxisomes in this strain were attached to mitochondria! This established that Inp1p is the tether.

Another major molecular player in this process is Pex3p. The pex3 null mutant phenotype looks a lot like the inp1 mutant phenotype: the mother cell loses all its peroxisomes and they end up in the bud. Pex3p is an integral membrane protein that is channeled through the ER on its way to the peroxisomal membrane – so it can be present in both places. The authors found that both the N terminus and C terminus of Inp1p bind to Pex3p. All this suggested that together, Inp1p and Pex3p might form a structure that links peroxisomes to the ER.

They were able to show that Inp1p and Pex3p interact directly both at the peroxisome and at the ER using a neat trick called bimolecular fluorescence complementation. This simply means that if the two halves of green fluorescent protein (GFP) are brought close to each other, they can fluoresce like the intact protein.  The basic idea is that they fused the first half of GFP with Inp1p and the second half with Pex3p and looked for green spots to turn up in the right place of the cell.  Of course this is easier said than done!

To pull this off, the authors had to first make two haploid strains of opposite mating types. The first had a pex3 mutation that caused all Pex3p to be stuck in the ER, anchoring all its peroxisomes there. It also carried a version of INP1 that was fused to half of GFP.

The second strain had a different pex3 mutation that set all peroxisomes adrift, and this mutant pex3 gene was fused to the other half of GFP. This strain also had an additional marker that made peroxisomes glow red.

When the cytoplasms of these two strains had a chance to mix after mating, the zygote had red peroxisomes with glowing green spots, showing that Inp1p-half GFP from the first strain was interacting with Pex3p-half GFP from the second strain.  Because the Inp1p-half GFP of the first strain was bound to ER-localized Pex3p, and the Pex3p-half GFP of the second strain was localized to peroxisomes, this result showed that Inp1p connects peroxisomes with the ER.

The authors studied the kinetics of this process in a lot more detail and even tracked the wanderings of individual peroxisomes. The model that comes from all this work is that at start of budding, some peroxisomes are bound to Inp1p and others aren’t. Those that aren’t bound to Inp1p move to the bud, and the others stay anchored to the mother cell’s ER. Meanwhile, during budding Pex3p passes through the ER and re-emerges at the ER membrane at the bud cortex. It can then bind Inp1p, which in turn binds to the Pex3p on the surface of the migrating peroxisomes to dock them in the bud.

Not only is this cool just from a basic biology perspective, but it may also help us deal with some human peroxisome biogenesis disorders.  For example, Zellweger syndrome and infantile Refsum disease are associated with specific mutations in the human ortholog of PEX3. Once again, little S. cerevisiae is helping us navigate the inner workings of eukaryotic cells.  

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight, Yeast and Human Disease

Tags: peroxisomes, Saccharomyces cerevisiae, yeast model for human disease

Hanging on by a Thread

July 02, 2013

When Nik Wallenda recently made his incredible tightrope walk over a 1500 foot-deep gorge, the attachment of the cable he walked on was critical. If that had failed, it would have been a very unhappy ending for Nik.

Something equally dramatic can happen in a cell.  If  the attachment of spindle microtubules to chromosomes during cell division fails, then the chromosomes don’t end up in the right place. When this happens, the cell can end up dead, or even worse, cancerous.  This is as bad as falling off a tightrope without a net!

In a cell, the chromosome is attached to the spindle with something called the kinetochore. It is like the spike driven into the side of the gorge the tightrope walker is going over. One end is attached to the chromosome (the side of the gorge) and the other is attached to the spindle (the rope that is tied to the spike).

This is where the analogy ends though…a kinetochore is way more complicated than a metal spike. It is a huge, multi-protein complex with lots of specialized parts. The way in which the whole complex assembles still isn’t completely understood.

In a new paper in GENETICS, Akiyoshi and coworkers unraveled a bit of the mystery behind it.  They found that phosphorylation by a highly conserved protein kinase known as Aurora B (Ipl1p in S. cerevisiae) of one kinetochore subunit, Dsn1p, provides some of the glue that holds the structure together.  More specifically, they found that phosphorylated Dsn1p does a better job at keeping inner kinetochore proteins attached to the complex.  It drives the spike deeper into the gorge.

The researchers mutated two residues in Dsn1p that are sites for Ipl1p phosphorylation.  They mutated one or both to alanine, which prevents phosphorylation, or to aspartic acid, which mimics the phosphorylated state.  They found that preventing phosphorylation of these sites loosened the complex and keeping them “phosphorylated” tightened it. 

First, to try to look at what happens when Dsn1p isn’t phosphorylated by Ipl1p, they mutated the two sites to alanine. Either site could be mutated with no apparent effects, but mutating both was lethal. Clearly these sites are doing something!

The researchers got around this lethality issue by mutating a third site in Dsn1p. This site is a target for phosphorylation by a different kinase, Cdk kinase (Cdc28p). The idea is that preventing phosphorylation by Ipl1p makes Dsn1p unstable, but then preventing phosphorylation by Cdc28p can stabilize the mutant protein.

Now that they had a living yeast strain in which Dsn1p wasn’t phosphorylated by Ipl1p, they could look to see what was different about the kinetochore in this mutant. When they pulled down the mutant Dsn1p using antibody and a Flag-tag, it brought down normal levels of outer kinetochore proteins but reduced levels of inner kinetochore proteins. So this suggested that Ipl1p phosphorylation promotes interactions between Dsn1p and inner kinetochore proteins.

Supporting this idea, an ipl1 mutant that phosphorylated Dsn1p to a lower extent showed lower-than-wild-type levels of inner kinetochore proteins associated with Dsn1p.  And, when they looked at a mutant where those Dsn1p residues were changed to aspartic acid, mimicking constant phosphorylation, higher levels of inner kinetochore proteins were pulled down. All of this evidence, and more, points to Ipl1p phosphorylation of Dsn1p as critical for attachment of inner kinetochore proteins to the kinetochore complex.

In yeast there is just one Aurora kinase, and Dsn1p is just one of its substrates. In human cells there are multiple versions of Aurora, and they are implicated in cancer development. Clearly, yeast will be a helpful model in understanding all the details of how Aurora influences kinetochore structure and chromosome segregation. And that will be a much more impressive and useful feat than a tightrope walk!

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight, Yeast and Human Disease

Tags: Aurora kinase, kinetochore, Saccharomyces cerevisiae

Alternative Ways to Increase a Cell’s Shelf Life

June 05, 2013

Like milk or eggs, most cells with linear chromosomes have a shelf life. Each time these cells divide, they lose a little off the end of their chromosomes. Eventually, too much is lost and the cells crap out. Or, to use a more scientific term, they become senescent.

But this is not the fate of every cell. Some cells, like those that go on to become sperm or eggs, use a reverse transcriptase called telomerase to extend their telomeres as part of their normal life cycle. And they aren’t the only ones. Around 85% of cancers hijack the telomerase and use it for their own nefarious ends.

The other 15% of cancers use a variety of different mechanisms to keep their telomeres from getting too short (Cesare and Reddel, 2010). All these different ways are lumped together in a single category called alternative lengthening of telomeres or ALT. The telomeres are lengthened in these cells by recombination with other telomeres, either those on other chromosomes or those that exist as shed, extrachromasomal bits. 

While telomere extension may keep cells alive, it can sometimes be a double-edged sword. A double stranded DNA break is usually recognized as DNA damage. However, if the break happens near a telomere seed (a sequence that looks like a telomere), then the DNA damage response can be suppressed and the end can be extended into a new telomere, in a process called chromosome healing. But now the cell could be in trouble, with new, partial chromosomes being created and getting pulled this way and that.

In a new study out in GENETICS, Lai and Heierhorst decided to investigate whether chromosome healing happens in yeast cells that have stayed alive because of ALT.  What they found was that chromosome healing at telomere seeds was suppressed in these post-senescence survivors.

They created these ALT dependent, post-senescence survivors from an est2 mutant strain that lacked the catalytic subunit of telomerase.  Without telomerase, the only way for these cells to survive is by using ALT. 

In the first experiment, they looked at whether the post-senescence survivors could create a new telomere by chromosome healing.  The authors used a galactose inducible HO endonuclease to create a double stranded break near an 81 base pair sequence known to be a telomere seed sequence in wild type. 

Broken DNA usually signals cells to pause the cell cycle until the damage is repaired. This is known as the DNA damage checkpoint. During chromosome healing in wild type, this checkpoint is suppressed so the chromosome break isn’t recognized as DNA damage.

In the post-senescence survivors, even after 21 hours there was no evidence of a telomere forming.  They didn’t suppress the DNA damage checkpoint either.

Lai and Heierhorst determined that these ALT-dependent cells could still repair a different break that was not near a telomere seed sequence. They just couldn’t repair the break at the telomere seed. And this wasn’t because the DNA damage checkpoint was active. When they prevented the checkpoint by using a rad53 mutant, the telomere still wasn’t repaired.

Instead, the post-senescence survivors eventually repaired the break by some other mechanism, generating lots of differing products in the process. When they repaired breaks at sites that were not telomere seeds, they were able to use homologous recombination. But homologous recombination was suppressed at the telomere seed site.

Since ALT is used in cancer cells, and happens most often in some of the least-curable types of cancer, whatever we can learn about the process in yeast is valuable. It may give us clues on how to change the expiration date of those cancer cells to “ASAP”.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: cancer, DNA damage checkpoint, Saccharomyces cerevisiae, telomere

When Half a Loaf is Too Much

April 18, 2013

One of the ways you can tell a human cell is cancerous is by taking a peek at its genome. Instead of the orderly 23 pairs of chromosomes seen in a normal cell, the cancerous one has a jumbled mess of a genome. There are extra chunks sticking here and there, chunks missing, and lots of other oddities.

Besides looking untidy, this sort of chaos also causes something called copy number variation (CNV). In CNV, there are either more or less than the usual two copies of some genes. Having the wrong number of copies of certain genes can definitely cause problems.

There is some debate out there about whether CNV causes a cell to go cancerous or if it is just an effect of the cancer. In a new study, de Clare and coworkers provide strong evidence that for many genes in the yeast Saccharomyces cerevisiae, having just one copy in a diploid background leads to faster growth, poor cell cycle control, and an aversion to apoptosis (programmed cell death). This argues strongly that CNV can actually cause a cell to go cancerous. This suggestion is strengthened further by the fact that many of the genes they identified are orthologs of human genes that exist as single copies in certain cancers.

Earlier studies from this group looked at the growth rates of over 5,800 heterozygous diploid yeast mutants, each missing one copy of a particular gene, and found around 600 that actually grew faster than wild type. You might not expect such a high number at first blush, since it seems like a single celled organism would have evolved to grow as fast as it can. The authors hypothesized that there must be a strong selective advantage to having these genes, outweighing the fact that they slow down growth.

Looking more closely, they found that the genes in this set were significantly more likely than the average gene to have functions that keep the genome stable, such as DNA damage repair. They were also highly conserved across the Ascomycete fungi, confirming their importance.

The next step was to see whether there might be any connection to human cancer. They took a subset of these genes – 30 genes involved in DNA repair and sister chromatid segregation – and compared them to human genes. Nineteen of the yeast genes had a human ortholog, and 17 of those human genes exist as a single copy in many cancers, suggesting that having only one copy of these genes may contribute to a cell’s cancer phenotype.

If copy number variation of those genes contributes to cancer in human cells, does it confer a cancer-like phenotype on yeast? The researchers found that the heterozygous yeast mutants showed characteristics of cancer cells such as altered cell cycle, a decrease in apoptosis, and lowered sensitivity to anti-cancer drugs. So the increased growth conferred by the mutations comes with a high cost: increased genome instability and cancer-like symptoms.

Because this cancer-like phenotype occurs in yeast, it will be an excellent model to study exactly how particular genes contribute to it. But these findings could also have a more immediate impact on cancer treatment. Certain experimental cancer treatments work by decreasing the activity of the proteins produced by some of these genes. If a treatment only partly knocks down the activity, then it may actually encourage cancer growth. It would mimic the effects of having a single copy of a gene. The authors actually show that this is the case in yeast for some of the drugs they tested.

And this isn’t a worry just for the drug targets themselves. The drugs aren’t completely specific…they can affect other genes too, again mimicking the effects of having a single copy of one of these other genes. Add to this the fact that each genomically jumbled cancer cell may have different proportions of genes, and you have quite a mess. As usual, yeast can swoop in and save the day.

Scientists may be able to use this and other yeast libraries to quickly screen varying amounts of potential new drugs for their effects on growth. Not only that, they’ll be able to identify what pathways these drugs are hitting in addition to the one(s) that are targeted. This should make the process of drug optimization move ahead much more quickly. Thanks yeast!

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

Categories: Research Spotlight, Yeast and Human Disease

Hate the CIN, Love the CINner

March 21, 2013

At first our favorite small eukaryote, S. cerevisiae, might not seem like a great model for cancer studies. After all, budding yeast can’t tell us anything about some of the pathways that go wrong in cancer, like growth factor signaling. And it clearly can’t help explain what happens in specific tissues of the human body. But in other ways, it actually turns out to be a great model.

For example, all the details of cell cycle control were originally worked out in yeast.  And now a whole new batch of genes has been found that influence a phenomenon, chromosome instability (CIN), that is important in both yeast and cancer cells.

As the name implies, chromosomes are unstable in cells suffering from CIN. Big chunks of DNA are lost, or break off and fuse to different chromosomes, turning the genome into an aneuploid mess. And this mess has consequences.

CIN can cause new mutations or make old ones have a stronger effect.  Eventually these mutations can affect genes that are important for keeping a cell’s growth in line.  Once these are compromised, a tumor cell is born. 

Since CIN is pretty common in yeast, we might be able to better understand it in cancer cells by studying it in yeast. The Hieter lab at the University of British Columbia has come up with a powerful screen to get yeast to confess why it CINs. 

A previous study from the group set the stage by finding a large group of mutants that have CIN phenotypes, implying that those genes are involved in keeping chromosome structure stable. In a new paper in G3: Genes, Genomes, Genetics, van Pel et al. uncovered the network of interactions among the genes in this set, using synthetic genetic array (SGA) technology. And they confirmed that the human homologs of some of these genes interact in the same way as in yeast, making them potential targets for cancer therapies.

The idea behind SGA studies is that if two proteins are involved in the same process, then a strain carrying mutations in both of their genes will be much worse off than a strain carrying either single mutation. In the worst case, the double mutant will be dead. This is known as a synthetic lethal interaction.

Yeast is a great model for doing these sorts of studies on a very large scale.  We can construct networks showing how lots of different genes interact, and most importantly, find the genes that are central to many interactions.   These “hubs” are likely to be the key players in those processes.

The researchers looked specifically for interactions between genes that are involved with CIN in yeast and are also similar to human cancer-related genes. They came up with various interaction hubs that will be interesting research subjects for a long time to come. In this study, they focused on one of these: genes involved with the DNA replication fork.

One of these in particular, CTF4, is a hub for both physical and genetic interactions. Unfortunately, Ctf4p doesn’t look like a good target for chemotherapy. It’s thought to act as a scaffold, and lacks any known activity that could potentially be inhibited by a drug. However, the interaction network around CTF4 that van Pel et al. uncovered suggests another way to target this hub. If a gene that interacts with CTF4 itself has a synthetic lethal interaction with another gene, and we could re-create the synthetic lethal phenotype in a cancer cell, we might be able to knock out the whole process. And that is just what they found in human cells.

First the authors identified a couple of human genes that were predicted from the yeast screen to be close to human CTF4 in the interaction network and to have a synthetic lethal interaction with each other. They then lowered the expression of one using small interfering RNA (siRNA), and reduced the activity of the other with a known inhibitor. Neither treatment alone had much effect, but combining them significantly reduced cell viability.

Since cancer cells frequently carry mutations in CIN genes, it should be possible to create a synthetic lethal interaction, guided by the yeast interaction network, where one partner is mutated in cancer cells (equivalent to using siRNA in this study) and the other partner is inhibited with a drug. Since it relies on a cancer-specific mutation, this approach has the potential to selectively target cancer cells while not disturbing normal cells, the ultimate goal for chemotherapy.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight, Yeast and Human Disease

Tags: cancer, DNA replication, Saccharomyces cerevisiae, yeast model for human disease

Cancerous Avalanche

March 05, 2013

Cancer often gets going with chromosome instability.  Basically a cell gets a mutation that causes its chromosomes to mutate at a higher rate.  Now it and any cells that come from it build mutations faster and faster until they hit on the right combination to make the cell cancerous.  An accelerating avalanche of mutations has led to cancer.

There are plenty of obvious candidates for the genes that start these avalanches: genes like those involved in segregating chromosomes and repairing DNA, for example.  But there are undoubtedly sleeper genes that no one has really thought of.  In a new study out in GENETICS, Minaker and coworkers have used the yeast S. cerevisiae to identify three of these genes — GPN1 (previously named NPA3), GPN2, and GPN3.

A mutation in any one of these genes leads to chromosomal problems.  For example, mutations in GPN1 and GPN2 cause defects in sister chromatid cohesion and mutations in GPN3 confer a visible chromosome transmission defect.  All of the mutants also show increased sensitivity to hydroxyurea and ultraviolet light, two potent mutagens.  And if two of the genes are mutated at once, these defects become more severe.  Clearly, mutating GPN1, GPN2, and/or GPN3 leads to an increased risk for even more mutations!

What makes this surprising is what these genes actually do in a cell.  They are responsible for getting RNA polymerase II (RNAPII) and RNA polymerase III (RNAPIII) into the nucleus and assembled properly.  This was known before for GPN1, but here the authors show that in gpn2 and gpn3 mutants, RNAPII and RNAPIII subunits also fail to get into the nucleus. Genetic and physical interactions between all three GPN proteins suggest that they work together in overlapping ways to get enough RNAPII and RNAPIII chugging away in the nucleus.

So it looks like having too little RNAPII and RNAPIII in the nucleus causes chromosome instability. This is consistent with previous work that shows that mutations in many of the RNAPII subunits have similar effects.  Still, these genes would not be the first ones most scientists would look at when trying to find causes of chromosomal instability. Score another point for unbiased screens in yeast leading to a better understanding of human disease.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: cancer, chromosome instability, RNA polymerase II, RNA polymerase III, Saccharomyces cerevisiae

Crowdsourcing Genetic Disease

February 28, 2013

Remember when sequencing the human genome was going to help us better understand and treat complex diseases like Type 2 diabetes or Parkinson’s? Well, ten years later, we’re still waiting.

Sure we’ve made some progress. Using genome wide association studies (GWAS), scientists have uncovered markers here and there that explain a bit about how a genetic disease is inherited. But despite a seemingly never-ending stream of these assays, scientists simply can’t explain all of the genetics behind most of these diseases.

So now scientists need to try to explain this missing heritability. If they can find out why they aren’t getting the answers they need from GWAS, then maybe they can restructure these assays to give better results.

As usual, when things get dicey genetically, scientists turn to the yeast Saccharomyces cerevisiae to help sort things out. And in a new study out in Nature, Bloom and coworkers have done just that.

In this study, they mated a laboratory and a wine strain of yeast to get 1,008 test subjects from their progeny. They extensively genotyped each of these 1008 and came up with a colony size assay that allowed them to determine how well each strain grew under various conditions. They settled on 46 different traits to study genetically.

What they found was that none of these traits was determined by a single gene. In fact, they found that each of the 46 different traits had between 5 and 29 different loci associated with it, with a median of 12 loci. This tells us that at least in yeast, many genetic loci each contribute a bit to the final phenotype. And if this is true in people, it could be a major factor behind the missing heritability in GWAS.

If a trait is dependent on many genetic loci that each have a small effect, then researchers need large populations in order to tease them out. In fact, when Bloom and coworkers restricted their population to 100 strains, they could only detect a subset of the genetic loci. For example, the number of loci went from 16 to 2 when they looked at growth in E6 berbamine.

So it may be that scientists are missing loci in GWAS because there are simply too few participants in their assays. If true, then the obvious answer is to increase the size of the populations being studied. Thank goodness DNA technologies get cheaper every year!

Of course as the authors themselves remind us, we do need to keep in mind that humans are a bit more complex than yeast. There may be other reasons that we aren’t turning up the genetic loci involved in various traits. It may be that we can’t as accurately measure the phenotypes in humans or that human traits are more complicated than the yeast ones studied. Another possibility is that in humans, there are more rare alleles that can contribute to a given trait. These would be very hard to find in any population studies like GWAS.

Still, this study at the very least tells us that larger populations will undoubtedly uncover more loci involved in human disease. Thank you again yeast.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: GWAS, model organism, Saccharomyces cerevisiae

Giving the Keys Back to the Cell

February 06, 2013

When someone has a bit too much to drink, it is a good idea to take away their car keys. This keeps them safe until they can drive again. But the next morning, that hung over person needs to get their keys back so they can get to work.

Cells sometimes face a similar situation. Instead of being drunk though, cells have something go wrong while they are growing and dividing. When this happens, the cell stops the cell cycle at the next checkpoint, fixes what is wrong, and then starts the cell cycle back up again where it left off.

Scientists have learned a lot about how the keys are taken from cells, but not a whole lot about how they get them back. Fong and coworkers help to rectify this situation in a new study out in GENETICS. There they identified proteins key to releasing a yeast cell from its S-phase checkpoint.

If a cell’s DNA is damaged while it is growing and dividing, replication is slowed at the S-phase checkpoint. This gives the cell a chance to fix the DNA before it is copied. The authors found that in the absence of the DIA2 gene, yeast cells had trouble getting replication up and running again. This implies that this gene is required for yeast to overcome the S-phase checkpoint. The cell needs DIA2 to get its keys back.

Dia2p is an F-box protein involved in identifying certain proteins for destruction. It is one of several interchangeable subunits that provide specificity to the SCF ubiquitin ligase complex. The idea would be that Dia2p is important for degrading the “keeper of the keys,” the protein responsible for stopping the cell cycle in the S-phase.

To test whether Dia2p is important for checkpoint recovery, Fong and coworkers first activated the S-phase checkpoint by adding the DNA damaging agent MMS. Then they removed the MMS and measured how long it took the cells to finish copying their DNA. The dia2Δ mutant was significantly slower than wild type.

Given that Dia2p is involved in ubiquitin-mediated degradation, the authors reasoned that it may help a cell get out of S-phase arrest by degrading a protein that was keeping it there. To find this “keeper of the keys,” Fong and coworkers looked for mutations that rescued dia2Δ cells in the presence of high levels of MMS. The idea is that if they knock out the gene that is keeping the dia2Δ cells arrested, then the cells could overcome the block caused by the MMS.

One of the genes that came up in the screen was MRC1. To confirm that Dia2p and Mrc1p work together in releasing a yeast cell from the S-phase checkpoint, the authors constructed a double mutant carrying dia2Δ and a mutant version of MRC1, mrc1AQ, that they knew was checkpoint defective. Indeed, the double mutant behaved like wild type in their checkpoint recovery assay. Since the mutant Mrc1-AQp could not keep cells at the checkpoint, there was no need for Dia2p to target it for degradation. The double mutant cell never let go of its keys.

The simplest model to explain what happens in wild type is that when its DNA is damaged, a cell is prevented from progressing through S-phase by Mrc1p. Then when the DNA is repaired, Dia2p, providing specificity to the SCF ubiquitin ligase complex, targets Mrc1p for degradation. The cell is now released, allowing the cell cycle to continue.

The authors did a lot more work that we won’t go into here, but suffice it to say that Dia2p and Mrc1p are not the only players involved in releasing a cell from the S-phase checkpoint. There were other genes, both identified and unidentified, that came up in their screen. These will need to be studied as well.

And this isn’t all just interesting from a scientific standpoint. Many cancer treatments work by damaging the cancer cell’s DNA while it is growing and dividing. A better understanding of how cells are arrested and released may lead to better cancer treatments.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: DNA replication checkpoint, Saccharomyces cerevisiae

Ghosts of Centromeres Past

January 28, 2013

Every cell needs to correctly divvy up its chromosomes when it divides.  Otherwise one cell would end up with too many chromosomes, the other with too few and they’d both probably die.

Cells have developed elaborate machinery to make sure each daughter gets the right chromosomes.  One key part of the machinery is the centromere.  This is the part of the chromosome that attaches to the mitotic spindle so the chromosome gets dragged to the right place. 

Given how precise this dance is, it is surprising how sloppy the underlying centromeric DNA tends to be in most eukaryotes.  It is very long with lots of repeated sequences which make it very tricky to figure out which DNA sequences really matter.  An exception to this is the centromeres found in some budding yeasts like Saccharomyces cerevisiae.  These centromeres are around 125 base pairs long with easily identifiable important DNA sequences.

The current thought is that budding yeast used to have the usual diffuse, regional centromeres but that over time, they evolved these newer, more compact centromeres.  Work in a new study published in PLOS Genetics by Lefrançois and coworkers lends support to this idea.

These authors found that when they overexpressed a key centromeric protein, Cse4p (or CenH3 in humans), new centromere complexes formed on DNA sequences near the true centromeres. The authors termed these sequences CLR’s or Centromere-Like Regions.  And they showed that these complexes are functional.

When Lefrançois and coworkers kept the true centromere from functioning on chromosome 3 in cells overexpressing Cse4p, 82% of the cells were able to properly segregate chromosome 3.  This compares to the 62% of cells that pull this off with normal levels of Cse4p.  The advantage disappeared when the CLR on chromosome 3 was deleted.

A close look at the CLRs showed that they had a lot in common with both types of centromeres.  They had an AT-rich 90 base pair sequence that looked an awful lot like the kind of sequence that Cse4p prefers to bind and a lot like the repeats found within more traditional centromeres.  They also tended to be in areas of open chromatin and close to true centromeres. The obvious conclusion is that these are remnants of the regional centromeres budding yeast used to have. 

The hope is that the yeast CLRs might make studying regional centromeres easier.  They are so long and complicated that it is very difficult to pick out which sequences matter and which don’t, but the yeast CLRs could be a simpler model system.  Even better, the CLRs might shed some light on the process of neocentromerization – the formation of new centromeres outside of centromeric regions, which happens a lot in cancer cells. Once again, simple little S. cerevisiae may hold the key to understanding what’s going on in much larger organisms.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: centromeres, evolution, Saccharomyces cerevisiae, yeast model for human disease

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