New & Noteworthy

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

Saccharomyces cerevisiae, the Party Animal

June 26, 2013

Like a barfly before his liver gives out, the yeast S. cerevisiae can tolerate incredibly high levels of ethanol.  But unlike the town drunk, S. cerevisiae uses this skill to its own advantage.

In the wild, this yeast ferments sugars to flood the local environment with alcohol.  The end result is that it does just fine but any nearby microorganisms are killed.

Humans also take advantage of this unique property of S. cerevisiae.  Not only does it allow us to brew beer and ferment grapes into wine without worrying too much about bacterial contamination, but it also helps us generate ethanol as a biofuel.  What a cool and useful little beast!

Given how important this property is for both yeast and us, it is perhaps surprising how little we know about how S. cerevisiae pulls this off.  A new study out in PLOS Genetics by Pais and coworkers sets out to rectify this situation.

The study yielded a number of interesting findings.  It might seem obvious that an organism that can make a lot of ethanol should able to grow in the high-ethanol environment that it created. However, by looking at these characteristics in 68 different S. cerevisiae strains, the authors found that the ability to produce ethanol was at least partially separate at the genetic level from the ability to thrive in it. Pais and coworkers called the first process “high ethanol accumulation capacity” and the second “tolerance of cell proliferation to high ethanol levels.”

Second, they identified DNA differences in three different genes – ADE1, URA3, and KIN3 – that all work together to give certain strains of yeast their high ethanol accumulation capacity.  The most interesting of these three is KIN3.

Kin3p is a protein kinase that has a role in DNA repair.  Since ethanol is a known mutagen, it may be that the DNA differences in the KIN3 gene make its protein better at DNA repair, rescuing the cell from the DNA damage from high ethanol levels.

The authors found these genes as part of a larger study to identify at the genetic level why some strains of S. cerevisiae did better than others in ethanol.  They focused on two strains, CBS1585 and BY710.  CBS1585 is a sake yeast strain that can tolerate and grow in high levels of ethanol while BY710 is a laboratory strain that doesn’t do well with either (although still better than most any other beast out there in nature!).

They created diploids using these two strains, sporulated them into haploids, and then screened these haploids for their ability to deal with high levels of alcohol.  As would be predicted from their survey of the 68 strains, the haploids could be grouped into three distinct but overlapping pools: 

1)   High ethanol accumulation capacity in the absence of cell proliferation

2)   Cell proliferation at high ethanol levels

3)   Poor tolerance and growth in high ethanol

The authors then used pooled-segregant whole-genome sequence analysis to identify the DNA regions critical to the first two functions.  Basically this is just what it sounds like.  They isolated DNA from the three pools and looked for differences in pools 1 and 2 that weren’t in 3.  (OK that is dangerously simplified, but that is the gist of it.)

This is how they identified ADE1, URA3, and KIN3 as important in high ethanol accumulation capacity.  We will have to wait for them to pinpoint the important genes in the regions they identified for pool 2 to begin to understand why some strains can proliferate in the presence of high alcohol concentrations.

Once they have identified all of the genes that make certain yeast strains so good at dealing with alcohol, we may be able to engineer a yeast that can make more ethanol for less money.  We can make a great little alcohol producer even better!  

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

Categories: Research Spotlight

Tags: biofuel, brewing, ethanol tolerance, Saccharomyces cerevisiae

Flip-Flopping Yeasts

June 20, 2013

We all have certain things we can’t live without. But what’s essential to one person may be completely trivial to another. For example, a teenager who can’t live without his video games is fine without that antique tea set,  while the opposite may be true for grandma. 

In a new article in Genome Biology and Evolution, Ryan and coworkers find that the same holds true for different yeast species.  One gene that is essential in one yeast is dispensable in another.  And furthermore, they tend to be essential or nonessential in sets. Just like grandma’s tea set and the teenager’s games.

It’s been seen in S. cerevisiae that the genes that encode the proteins in a complex tend to be either mostly essential or mostly nonessential.  It is like the teapot and the cups and saucers all being essential to grandma or all being nonessential to the teenager.  This is called modular essentiality.

Ryan and coworkers found that if some protein complex is essential in one yeast, most or all of those genes will be essential in that species.  On the other hand, if that protein complex is dispensable in a different yeast, then most or all of those genes will be nonessential.  The genes encoding the entire complex flip together from essential to nonessential – again, just like the tea set.  It isn’t as if one of the tea cups happens to stay essential when the teenager gets ahold of the set! 

To do this work, Ryan and coworkers started out by looking at S. cerevisiae.  As all of us here at SGD know, this was an excellent choice!  But not just because we work on it…

Because the S. cerevisiae genome sequence has been available for quite a while, we know which genes are essential to life, which genes interact genetically, and which proteins interact physically with each other. We also have a very good list of the protein complexes that exist in yeast and what their subunits are.

Ryan and coworkers used updated data to confirm that modular essentiality exists in S. cerevisiae.  Most of the proteins in an essential complex tend to come from essential genes and most of the proteins in nonessential complexes come from nonessential genes.  There is very little overlap…the tea set does not often contain a video game!

Next the authors asked whether this modular essentiality is found in other species too. At the moment, Schizosaccharomyces pombe, or fission yeast, is the only other eukaryote with complete data on the essentiality of genes. Although it’s also a single-celled yeast, S. pombe is about as far away from S. cerevisiae as you can get and still be a yeast. The two are thought to have diverged as much as 400 million years ago.

Even though it is so different, S. pombe also shows modular essentiality. And using an incomplete set of data from knockout mice, the authors see a similar pattern! So it looks like modular essentiality is at least conserved across fungi, and may be universal.

Next they asked if complexes that have flipped from essential to nonessential over time still maintain their modular essentiality.  Do all the tea cups become nonessential, or just some of them? 

Most (83%) of the genes that are present as one-to-one orthologs in both yeasts are either essential in both or nonessential in both. Ryan and coworkers focused on the other 17%, where a gene was essential in one species but not in the other.

In the cases where essentiality is “flipped” between the species, whole protein complexes tend to flip as a unit. The subunits of a complex that is nonessential in budding yeast are mostly nonessential, while the subunits of the analogous complex in fission yeast are mostly essential.

An example of this is the large subunit of the mitochondrial ribosome. Mitochondrial translation is optional for S. cerevisiae, but obligatory for S. pombe. In keeping with this, almost all the proteins that make up the S. cerevisiae large mitochondrial ribosomal subunit are nonessential. In S. pombe, the situation is flipped.

So the essentiality of a complex mirrors the lifestyle of its owner, just like the teenager and his grandmother.  The two yeasts, with their different lifestyles, place different importance on the mitochondrial ribosome. This wasn’t a big surprise, since this lifestyle difference was already known. But other complexes that are flipped between the species may point to things that we don’t yet know about their physiology.

These results support the idea that modular essentiality is universal, which would mean that in various organisms we can expect that mutants in subunits of a complex will share the same phenotype, disease association, and drug sensitivity. Obviously there are important implications here for antifungal drug design or for disease treatment: if you want to stop a complex from working, any of its subunits (or perhaps several at the same time) might prove to be good targets.

But another bigger point is how much we can learn from a deep understanding of an organism’s genome.  By teasing apart what is essential and what isn’t we can learn a lot about the beast we’re studying.  And someday, maybe a lot about ourselves.

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

Categories: Research Spotlight

Tags: essentiality, evolution, 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

Proteins Hate Crowds Too

May 29, 2013

A goldfish will swim faster through water than it will through gravy or Jell-O. And according to the results of a new study by Miermont and coworkers, it looks like the same may be true for proteins in a yeast cell.

Now obviously the authors didn’t replace the insides of a yeast cell with gravy or Jell-O. Instead, they used severe osmotic stress to remove water from the cell, making the interior more viscous and the proteins more crowded.

The movement of a wide range of proteins within the cell slowed to a crawl in these shrunken cells. In fact, if the cell was subjected to enough stress, the proteins in the cell essentially stopped moving at all. This lack of movement wasn’t because the high osmotic stress had killed the yeast…they recovered just fine when put back into a more osmotically friendly environment.

The first case the authors looked at was the pathway that helps yeast respond to osmotic stress, the HOG pathway. Once a cell is subjected to osmotic stress, Hog1p is phosphorylated and translocated into the nucleus where it can then turn on the genes needed to respond to this environmental insult.

Miermont and coworkers traced the movement of Hog1p using a Hog1p-GFP fusion protein. At 1 M sorbitol, which subjects the cell to mild osmotic stress, this fusion protein was phosphorylated within two minutes and had reached the nucleus within five.  There were already signs of cell shrinkage even under these mild conditions.

As sorbitol concentrations increased, the phosphorylation and nuclear localization took longer and longer to happen. At 1.8 M sorbitol, Hog1p-GFP didn’t reach maximum fluorescence in the nucleus until 55 minutes. At 2 M sorbitol and above, the cells were shrunken down to their minimal volume, which was 40% of their original size. Under those conditions, only 25% of cells had any nuclear fluorescence even after several hours.

The authors used fluorescence recovery after photobleaching (FRAP) to confirm that these effects were due to slowed protein movement. Basically they zapped a small portion of a cell to burn out the fluorescence of the Hog1p-GFP in that region and then timed how long it took fluorescing Hog1p-GFP from other parts of the cytoplasm to diffuse into that area. To prevent the complication of nuclear translocation in these experiments, they used a pbs2 mutant strain, in which the HOG pathway is blocked and Hog1p stays in the cytoplasm.

They found that fluorescence recovery took less than one second in normal media, about five seconds in 1 M sorbitol, and never happened at 2 M sorbitol. Clearly the protein’s movement was slowed with increasing osmotic pressure. But the effects weren’t permanent: once the cells were put back into normal media, the protein returned to its original kinetics.

The authors expanded their studies to look at proteins that shuttle between the nucleus and cytoplasm in other pathways, including Msn2p, Yap1p, Crz1p, and Mig1p, and obtained similar results. All four proteins took longer to reach the nucleus after being exposed to high osmotic stress. They also looked at other cellular processes where protein movement is critical, such as endocytosis, and again found that increased osmotic stress led to slower moving proteins.

So it looks like molecular crowding can definitely slow down protein movement and affect how a cell functions. For example, the time it takes to respond to environmental stimuli could be significantly slowed, affecting the cell’s chances for survival.  Since yeast cells in the wild encounter high-sugar environments (like rotting grapes), their protein density must be regulated so that they can get through stresses like this. They are ultimately much better adapted than that goldfish in the bowl of Jell-O.

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

Categories: Research Spotlight

Tags: osmotic stress, protein crowding, Saccharomyces cerevisiae

A Radical Discovery About a Well-Known Enzyme

May 22, 2013

Living your life puts a lot of wear and tear on you. A big reason is that as your cells go about their business, they churn out lots of damaging chemicals. 

One of the worst offenders is the free radical superoxide, O₂^–. Cells can’t help producing this powerful oxidant during normal metabolism, but it’s so toxic that it can destroy proteins and damage DNA.

Cells have come up with a two-step process to deal with this toxic waste. In the first step, they use the enzyme superoxide dismutase (Sod1p is the cytosolic form in yeast) to convert superoxide into the less harmful hydrogen peroxide (H₂O₂) and water. The cells then use catalases to take care of the H₂O₂, converting it to water and molecular oxygen. 

We’ve known about the first enzyme, superoxide dismutase, for decades. It has always been thought to have a simple role, sitting in the cytoplasm and detoxifying O₂^–. But new research shows that its job is considerably more interesting than that: it also has a role in a regulatory process known as the Crabtree effect.

The Crabtree effect is named after the scientist who first described it way back in 1929. Some types of cells are able to produce energy by either fermentation or respiration in the presence of oxygen. Since these two processes have different metabolic costs and consequences, which one to use is a critically important choice.

If lots of glucose is around, yeast cells choose fermentation. They prevent respiration by repressing production of the necessary enzymes, and this glucose-dependent repression is the Crabtree effect. It happens not only in yeast, but also in some types of proliferating cancer cells.

A new study by Reddi and Culotta shows that Sod1p is actually a key player in the Crabtree effect. In response to oxygen, glucose, and superoxide levels, it stabilizes two key kinases that are involved in glucose repression.

It was recently found that the sod1 null mutant can’t repress respiration when glucose is around.  This is different from the wild type, which is subject to the Crabtree effect. 

Reddi and Culotta started by investigating this observation and found that SOD1 is part of the glucose repression pathway that also involves the two homologous protein kinases Yck1p and Yck2p. They found that Sod1p binds to Yck1p, which wasn’t totally unexpected since this interaction had been seen before in a large-scale screen. The unexpected part was that Sod1p binding actually stabilizes Yck1p and Yck2p.  These stabilized kinases can now phosphorylate targets that propagate the glucose signal down the pathway and ultimately repress respiration.

Now the question is why does Sod1p binding stabilize the kinases? It turns out that its enzymatic activity is crucial for stabilization. One idea is that the hydrogen peroxide that Sod1p makes in the neighborhood of the kinases could inactivate ubiquitin ligases that would target them for degradation. Ubiquitin ligases are rich in cysteine residues, and so could be especially sensitive to oxidation by H₂O₂.

This regulation might also feed into other pathways: these kinases are also involved in response to amino acid levels, and the sod1 null mutant was seen to affect the amino acid sensing pathway in this study.

Most excitingly, this mechanism is not just a peculiarity of yeast Sod1p. The authors mixed and matched yeast, worm, and mammalian superoxide dismutases and casein kinase gamma (the mammalian equivalent of Yck1p/Yck2p), and found that binding and stabilization works in the same way across all these species.

Superoxide dismutases may have been drafted into this regulatory role during evolution because they are the only molecules that sense superoxide, whose levels reflect both glucose and oxygen conditions. A radical idea indeed!

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

Categories: Research Spotlight

Tags: fermentation, regulation, respiration, Saccharomyces cerevisiae

Codependent Genes

May 16, 2013

When a gene is duplicated, one copy usually dies. It is battered by harmful mutations until it eventually just fades into background DNA.

But this isn’t the fate of all duplicated genes.  Sometimes they can survive by gaining new, useful functions.  The genes responsible for snake venom proteins are a great example of this.

Another way for a duplicated gene to live on is when both copies get different mutations that confer different functions, so that a cell needs both to survive.  Two examples of this type of codependent gene survival are highlighted in a new study by Marshall and coworkers.  They compared various fungal species and identified cases where two functions were carried out by either one gene or by two separate genes.  Surprisingly, these cases involve alternative mRNA splicing, which is a rare process in fungi.

The first gene pair they focused on was SKI7 and HBS1 from Saccharomyces cerevisiae.  In this yeast these two genes exist as separate entities, but in other yeasts like Lachancea kluyveri they exist as a single gene which the authors have called SKI7/HBS1. 

The SKI7/HBS1 gene makes two differently spliced mRNAs, each of which encodes a protein that matches up with either Ski7p or Hbs1p.  In addition, the SKI7/HBS1 gene can rescue a S. cerevisiae strain missing either or both the SKI7 and HBS1 genes.  Taken together, this is compelling evidence that SKI7 and HBS1 existed as a single gene in the ancestor of these two fungal species. In S. cerevisiae, after this gene was duplicated each copy lost the ability to produce one spliced form.

The second gene Marshall and coworkers looked at experienced the reverse situation during evolution.  PTC7 exists as a single gene that makes two mRNA isoforms in S. cerevisiae: an unspliced form that generates a nuclear-localized protein, and a spliced form that produces a mitochondrial protein. 

But in Tetrapisispara blattae, these two forms exist as separate genes.  The PTC7a gene is similar to the unspliced form in S. cerevisiae and the protein ends up in the nucleus, while the PTC7b gene is similar to the spliced S. cerevisiae version and its product is mitochondrial.  

Because an ancestor of S. cerevisiae had every one of its genes duplicated about 100 million years ago, yeasts have been a great system to study the fate of duplicated genes.  This study shows that even though gene duplication is widespread in fungi and alternative splicing is rare, these mechanisms are actually interrelated and each can increase the diversity of the proteins produced by a species.

Fun fact: 544 genes survived duplication in S. cerevisiae.  That is around 10%.

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

Categories: Research Spotlight

Tags: alternative splicing, evolution, gene duplication, Saccharomyces cerevisiae

Keeping the Noise Down

May 08, 2013

When you get down to a single cell, things can get really noisy. Instead of the nice, smoothed over data that you see in populations, you see some variation from cell to cell. This is even if all the cells are identical genetically.

Of course this makes perfect sense if you think about it. Part of the variation comes from slightly different environments. Conditions at the bottom of the flask are bound to be different from those at the top! This goes by the name of extrinsic noise.

Another source of variation has to do with levels of reactants within the cell and the chances that they encounter each other so they can react. These effects can be especially pronounced when there aren’t a lot of reactants around. This goes by the name intrinsic noise.

One process with a lot of noise is gene regulation. It is often affected by minor fluctuations in the environment and there are usually just one or two copies of the gene itself. This is the perfect recipe for noise.

The noisiness of gene expression can be split into two steps. One, called burst frequency, reflects how often RNA polymerase sits down and starts transcribing a gene. The second, burst size, has to do with how many proteins are produced each time a gene is turned on.

Of these two processes, the most sensitive to noise is usually burst frequency. A transcription factor (TF) has to find the promoter of the gene it is supposed to turn on and then bring the polymerase over to that gene. This is dependent on the amount of TF in a cell and the number of TF binding sites on the DNA. What this means is that most of the time, genes with low levels of expression tend to be very noisy.

There are some situations, though, where it is very important to have low expression and low noise: for example, where a cell needs at least a few copies of a protein, but can’t tolerate too many. For most promoters, low levels of expression mean high noise, which in turn means there will be some cells that lack this key protein entirely. But a new study out in PLOS Biology shows one way that a promoter can have the best of both worlds.

In this study, Carey and coworkers examined the noisiness of sixteen different naturally occurring promoters in the yeast S. cerevisiae, controlled by the TF Zap1p. This is a great system because the activity of Zap1p is determined by the concentration of zinc in the medium. This means the authors were able to look at the noisiness of these promoters under a broad range of gene activities.

Their research yields a treasure trove of information about the noisiness of these promoters at varying levels of expression. As we might predict, noise decreased at most (11/13) of the reporter genes as more active Zap1p was around. This makes sense, as cell to cell variability will decrease as genes are turned on more often. Higher burst frequency means less noise.

The opposite was true for most (2/3) of the reporters repressed by Zap1p. As more Zap1p was around, transcription of the reporter gene became less frequent, which meant that the noise effects became more prominent.

One of the more interesting findings in this study focused on an exception to this rule. The ZRT2 promoter showed a bimodal expression pattern, as it was activated at low levels of zinc and repressed at high levels. What makes it so interesting is that its noise level stays fairly constant.

As the zinc concentration increases and activity goes up, the noise goes down. This is what we would expect. But when zinc levels get high enough so that the gene is repressed, the noise levels do not increase. They stay similar to the levels seen with the activated gene.

The authors show that this promoter is repressed differently than the other two repressed promoters, ADH1 and ADH3. These promoters are repressed by decreasing the burst frequency: they fire less often when repressed. In contrast, the ZRT2 promoter fires at the same activated rate when repressed, but yields less protein with each firing: repression decreases burst size.

So this is how a cell can manage to get a gene turned on at low levels more or less uniformly through a cell’s population. If it can create a situation where the gene fires a lot but very little protein is made with each firing, then the cell will have relatively constant but low levels of that protein.

This study also provides a new tool for dissecting how a TF affects the expression of a gene. If a repressor decreases expression without an increase in noise, then it is probably affecting burst size. If on the other hand the noise goes up as expression goes down, then the repressor is affecting burst frequency.

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

Categories: Research Spotlight

Tags: cellular noise, RNA polymerase II, Saccharomyces cerevisiae, transcription

Breaking Up is Hard to Do

May 01, 2013

When a cell goes cancerous, its chromosomes get seriously messed up. Pieces get deleted, duplicated, mixed and matched. One of the worst things that can happen, in terms of a cell keeping its chromosomes together, is that a chromosome ends up with two centromeres.

A centromere is the part of a chromosome that gets attached to the spindle so it can be moved to the right place during cell division. When there are two centromeres, both get attached and something has to give if the chromosome is pulled in two different directions. Often this means that the DNA of the chromosome breaks between the two centromeres.

This isn’t as simple as the rope breaking during a tug-of-war, though. A chromosome can withstand around 480 piconewtons of force before breaking, but the force exerted by the spindle that breaks the chromosome between the centromeres is just one piconewton or less. Clearly something else is going on to create those breaks!

In a new study out in GENETICS, Song and coworkers looked more closely at what happens when a dicentric chromosome breaks. They used a diploid strain of S. cerevisiae to show that where the DNA breaks is not random. In their experiments, the break tended to happen within 10 kilobases (kb) of the “foreign” centromere.

They used a previously described system where a conditional centromere was placed 50 kb from the normal centromere on chromosome III. This conditional centromere is only turned on in the absence of galactose. They then mated this strain to an unrelated one, resulting in a diploid with a high degree of heterozygosity. In other words, the chromosomes from each strain were different at lots of different places.

Song and coworkers streaked diploids from isolated colonies to a plate lacking galactose and then investigated how the yeast managed to resolve its double centromere issue. Two key ways that the yeast could eliminate the additional centromere involve crossing over between sister chromatids or break-induced repair. They focused on these as it is relatively easy to identify the DNA breakpoint. Because the two chromosomes in each pair are so different, they just needed to look for a loss of heterozygosity. In other words, where did the chromosomes become the same?

When they looked through 27 colonies, they found that the breaks weren’t randomly spread between the centromeres. Surprisingly, about half of them happened very near the conditional centromere. To make sure that there wasn’t something special about these sequences, they looked at two different strains with the conditional centromere located in different places on chromosome V instead of III. They obtained similar results.

Since the force exerted by the spindle isn’t enough to break the chromosome, there must be enzymes involved in creating the DNA breaks. But why do they prefer the region near the conditional centromere? One possibility is that the DNA there is stretched and is more open to enzymes. As the chromosome is being pulled apart, an enzyme gets into this region and manages to cut the DNA.

Although we don’t have time to go into it here, the paper also has a lot to say about the variety of ways that a diploid cell resolves its extra centromere in a way that allows it to survive. And that will inform the study of chromosome dynamics in all kinds of cells.

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

Categories: Research Spotlight

Tags: cancer, DNA repair, Saccharomyces cerevisiae

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

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