New & Noteworthy

Polygamous DNA Replication

June 26, 2014

Once someone is married, there are lots of things that keep them from starting a second marriage at the same time.  Laws, fear of losing the first spouse, social mores and so on all create a situation where the vast majority of people have only a single spouse at any one time. 

As each of these inhibitions is lifted, people will be more or less inclined towards polygamy, depending on who they are and the culture they live in.  For example, if having multiple spouses becomes acceptable socially, then some people might dive right in while others might hold off.

It turns out that origins of replication are similar.  There are many layers of control that keep an origin from firing more than once during any cell cycle.  But just like people and polygamy, when a few inhibitory layers are removed, some origins are more likely to fire more than once in a cell cycle than are others.

In a new study out in PLOS Genetics, Richardson and Li have identified a DNA sequence that makes nearby origins of replication fire more than once during a cell cycle when certain regulatory mechanisms have been disabled.  The authors hypothesize that these reinitiation promoters (RIPs) may be important for promoting genetic diversity by causing genomic duplication of specific regions under certain circumstances. 

This lab had previously shown that the origin ARS317 reinitiates more frequently when global regulation is removed from some key players in initiation: Cdc6p, the Mcm2-7 complex, and the origin recognition complex (ORC).  They disabled the regulation of all three of these by mutating each to prevent their recognition by the master regulator cyclin-dependent kinase (CDK, whose catalytic subunit is Cdc28p). In this study, they identified a second origin, ARS1238, that also reinitiated more often under these conditions.  The authors next set out to identify why these origins reinitiated under these conditions.

The first thing they found was that chromosomal context didn’t matter a whole lot.  Both origins reinitiated at around the same rate when they were in their natural context or when moved to other chromosomes.  The ability to reinitiate must be contained in the sequence of the DNA that was moved.

They next showed through deletion and linker scanning analysis that the two origins both required an AT-rich, ~60 base pair sequence to reinitiate.  This sequence needed to be within around 35-75 base pairs of the origin to promote reinitiation. Not any old stretch of AT-rich DNA would do; a specific DNA sequence was necessary, suggesting that this DNA is not required for reinitiation just because it is more easily unwound. 

These authors have shed light on a key process in the life of a cell—the firing of an origin of replication once and only once during any cell cycle.  It is critical for a cell that origins do not routinely reinitiate to prevent widespread genomic duplications that left unchecked would be very dangerous to the cell.

Richardson and Li have shown that not all origins are created equally, in that some are more likely to reinitiate under certain conditions than are others. If similar regions in mammalian cells turn out to be hotspots for genetic changes in cancers, then scientists may be able to target them to prevent the cancer’s genetic progression.  We may be able to reintroduce laws to keep polygamy at bay.

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

Categories: Research Spotlight

Tags: DNA replication, replication origin, Saccharomyces cerevisiae

Like People, Prions Need Intimate Contact to Spread

June 19, 2014

In the Matrix Trilogy, the delicate balance of a virtual world is upset by a rogue computer program that goes by the name of Agent Smith.  This program finds and touches other agent programs, converting them into copies of itself.  Eventually, all the agent programs are copies of Agent Smith and only the hero Neo can save humanity in an epic battle within the virtual world of the Matrix.

A new study out in GENETICS by Li and Du provides additional evidence that prions in the yeast Saccharomyces cerevisiae work similarly to Agent Smith, in that they spread through a direct contact model.  These prions are proteins that have entered a rogue conformation, and they end up converting all copies of the same protein into a similar rogue conformation.  The proteins change from a hardworking Agent Smith trying to do its job into something that mucks up the working of a cell.  And the results, at least in humans, can be as catastrophic for the cell as Agent Smith was for the Matrix. 

Mad cow disease, for example, is caused by prions converting the prion protein (PrP) in the brain cells of people from a useful conformation to a dangerous one that spreads.  As the conformation spreads throughout the cell, these prions form amyloid fibrils that eventually kill the cell.  When enough brain cells are killed, the person dies.

The authors chose to work in yeast because unlike in people, there are multiple examples of proteins in yeast that can go prion.  The list includes Sup35p, Ure2p, Rnq1p, Swi1p, Cyc8p, Mot3p, Sfp1p, Mod5p and Nup100p.  As you might guess from the sheer number of these prion-ready proteins, prions actually do more than kill a cell in yeast; they can serve useful functions. Scientists have yet to identify any useful functions for the prion form of PrP in people. 

Having multiple prions in a cell allowed Li and Du to perform some experiments to try to distinguish between two models of prion conformation spreading.  In the first, called the cross-seeding model, the prion acts very much like Agent Smith in that it needs to contact a “healthy” protein to convert it into a prion.  In the second model, the titration model, factors in the cell that prevent prion formation are titrated out when prions form.  As the factors are taken out of commission, prions are free to form.

The main evidence in this study that supports the cross-seeding model has to do with the localization of pre-existing prions during the de novo formation of a new prion.  Li and Du found that the prion [SWI+] localized to newly forming [PSI+] prions but not to already formed [PSI+] prions.  This is not the result we would expect if prion formation were due to titrating out of inhibitors of prion formation.  If that were the mechanism, then there would be no reason for [SWI+] to colocalize with newly forming [PSI+]. These experiments are like having a google map of the Matrix where we could see Smiths converting other agents by touch and then moving on and touching other agents.

Work like this is important for helping to find treatments for prion associated diseases and, perhaps, other amyloid fibril forming diseases like Huntington’s or Alzheimer’s.  Scientists need to focus on the amyloid fiber forming proteins themselves instead of trying, for example, to ramp up the activity of factors that inhibit formation.  Scientists probably need to eliminate Agent Smith to prevent the destruction of the Matrix and all of mankind.

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This is how prions turn other proteins into copies of themselves:

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: prions, Saccharomyces cerevisiae

Holding Back the Translation Torrent

June 12, 2014

We all know the story of the little Dutch boy who stuck his finger into a hole in a dike to keep his village from being flooded.  Now, a new study out in Molecular Cell by Pircher and coworkers has identified a novel regulatory mechanism involving a small 18-nucleotide RNA that behaves similarly to this boy.

A big difference in our story is that unlike the broken dike, the “water flow” in yeast cells is usually a good thing.  It is the continuous stream of protein translation that goes through the ribosome. 

When it’s under stress, though, yeast needs to slow down translation in order to make sure that it is making and folding each protein correctly. This gives it a better shot at surviving the stress.  Once the stress is gone, translation can ramp up again. This is where that 18-nt RNA comes in.

This group identified this 18-nt RNA as a ribosome binding RNA in a previous study. Because there are only a couple of known cases where a noncoding RNA (ncRNA) regulates the ribosome directly, Zywicki and colleagues had wanted to see whether this happens in yeast.  They found about 20 ncRNAs that bound to the ribosome, with the most abundant being an 18-nt fragment that corresponded to part of the coding sequence of the TRM10 gene that encodes a tRNA methyltransferase.

In the current study Pircher and coworkers reconfirmed that in yeast cells about 80% of this 18-nt RNA is associated with ribosomes. To verify whether it really bound to the ribosome rather than to the mRNA being translated, they broke apart polysomes with the chelating agent EDTA. This separated the large and small ribosomal subunits from each other and from mRNA.

All of the 18-mer stayed with the large subunit, showing that it really does interact with the ribosome. The researchers also found that under normal conditions it is bound to nontranslating ribosomes, while in stressed cells it shifts to actively translating polysomes.

The mutant phenotype of the trm10 null mutant suggested that the 18-mer might have a role in adapting to stress conditions. This mutant looks normal under standard conditions, but grows slower than wild type when under osmotic stress.

Pircher and colleagues used a clever strategy to find out whether this phenotype was due to the absence of Trm10p or to the absence of the 18-mer. First, they added a stop codon into the TRM10 gene, outside the region encoding the 18-mer. This mutation blocked production of Trm10p, but didn’t affect the 18-mer. The mutant looked just like wild type under osmotic stress conditions, showing that Trm10p isn’t involved in the stress response.

Second, to see directly whether the 18-mer is important, they mutated its sequence by changing some of the codons within it to other, synonymous codons encoding the same amino acid. So the Trm10p derived from this gene was wild-type, although the 18-mer sequence was different.

A couple of mutants of this type both showed the same phenotype of slow growth under osmotic stress. So production of the 18-mer is in fact important for maintaining growth rate under stress conditions. These mutant 18-mers also failed to bind to ribosomes. 

To find out what this little RNA actually does, they used electroporation to load up each cell with about 200,000 molecules of the 18-mer. This was about the same as the number of ribosomes per cell. Translation was almost completely inhibited. When they did the same experiment with an 18-mer with a scrambled sequence, it had no effect.

Further in vitro experiments confirmed the inhibitory effect of the 18-mer on translation, and showed that the inhibited step is translation initiation. It’s not completely clear why slowing down translation promotes cell growth during stress, but the authors speculate that it leads to more accurate translation and protein folding, which improves protein homeostasis and adaptation to stress. It also remains to be determined whether the 18-mer is created by processing of the TRM10 mRNA or is transcribed independently.

This regulatory mechanism is surprising and relatively novel: there are just a couple of known cases of ncRNAs regulating the ribosome directly. But it makes sense that regulating translation in this way allows the cell to react very quickly to changing environmental conditions, without needing to synthesize any new molecules.

Small ncRNAs like microRNAs or small interfering RNAs are emerging as big players in regulation in many organisms. However, miRNAs and siRNAs are not found in S. cerevisiae. But as this study shows, this does not mean that yeast doesn’t use small RNAs for regulation.  And one of the most surprising things about this story is that such a tiny scrap of RNA can regulate the ribosome, with its 5.5 kb of rRNA and 80 proteins. The little Dutch boy’s finger is immense by comparison!

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

Categories: Research Spotlight

Tags: ncRNA, osmotic stress, Saccharomyces cerevisiae, translation

Saddling Mom with the Burden of Old Age

June 05, 2014

In A World Out of Time by Larry Niven, people live forever by teleporting the unfolded protein aggregates associated with aging out of their cells.  Turns out that our very clever yeast Saccharomyces cerevisiae can do the same thing and it doesn’t need a machine.  

Instead of teleporting the aggregates away, yeast saddles the mother cell with them when it buds.  The daughter has now regained her youth and the mother is left to struggle with old age.

In a new study in Science, Hill and coworkers show that the yeast metacaspase gene MCA1 is critical in this process.  But it doesn’t look like it is involved in segregating these bundles to the mother cell.  Instead, it appears to help clear away many of the bundles left in the daughter.   If it were in Niven’s original story, Mca1p might be a little nanobot that chewed up any aggregates the teleporter missed.  

This all makes sense given caspases’ role in multicellular beasts.  There, these executioner proteases chew up cellular proteins during apoptosis, the process of programmed cell death that is a critically important part of development and growth. 

Although apoptosis has been observed in yeast and Mca1p is involved in the process, it has always been a bit of a mystery why a single-celled organism needs a mechanism for suicide.  This study now suggests that yeast’s only caspase, Mca1p, has a role as a healer as well as an executioner. It saves the daughter by degrading and proteolytically clearing away the aggregated bundles clogging up her cell. 

Scientists already knew that HSP104 was a key player in making sure that aggregates stayed with mom.  Hill and coworkers used this fact and performed a genetic interaction screen using HSP104 to identify MCA1 as required to keep protein aggregates out of the daughter in response to a heat shock.  Follow up work confirmed this result by showing that overexpressing MCA1 led to more efficient segregation of aggregates and that deleting it led to poor segregation of aggregates.

Digging deeper, these authors found that this poor segregation was because Mca1p was not eliminating aggregates in the daughter, as opposed to affecting the segregation itself.  They also showed that the protease activity of Mca1p was needed for this effect.

In the final set of experiments we’ll discuss, the authors looked to see what effect MCA1 has on the life span of a yeast cell.  They saw little effect of deleting MCA1 unless a second gene was also deleted: YDJ1, which encodes an HSP40 co-chaperone.  The double deletion mutant yeast were able to divide fewer times before petering out.  Consistent with this, overexpressing MCA1 led to increased life span and this effect was enhanced in the absence of YDJ1.

Finally, cells lived for a shorter time if just the active site of the Mca1p protease was compromised in a ydj1 deletion background.  This again confirms that proteolysis is key to MCA1’s effects on aging. 

So yeast attains eternal youth by both dumping its age-related aggregates on its mother and by using Mca1p to destroy any aggregates that managed to get into the daughter.  The daughter gets a reset until she builds up too many aggregates, in which case she gets saddled with them.

Yeast may be showing us another way to live a longer life.  If we can specifically degrade our aggregates without causing our cells to commit mass suicide, maybe we can extend our lives.  And we don’t even need fancy teleporting machinery; we just need to adapt the molecular machinery yeast is born with.  Feel free to use this idea for a new science fiction story!

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

Categories: Research Spotlight

Tags: ageing, caspase, protein aggregates, Saccharomyces cerevisiae

Slipping Through Haldane’s Sieve

May 22, 2014

Imagine you are panning for gold in a river and there are two kinds of nuggets.  One type is naked gold while the other is gold hidden inside of normal rock.  Pretty easy to figure out which nuggets you’ll gather first!

Now imagine instead that the process of panning is a rough one that knocks the shell off of the second type of nugget revealing the gold inside.  Now there won’t be any difference between the two.  You will be just as likely to keep both types of nuggets.

The same sort of situation applies to new beneficial mutations in a changing environment.  Back in 1927, J. B. S. Haldane predicted that the more dominant a mutation, the more likely it was to help a diploid beast adapt to a new environment.  The naked gold was more likely to be taken over the covered gold.

Gerstein and coworkers show in a new study that at least in the yeast Saccharomyces cerevisiae, Haldane’s sieve (as it is called) may not always apply.  The process of adapting to a new environment can strip away the dominant older allele, revealing the recessive one.  Loss of heterozygosity (LOH) uncovers the hidden gold of the recessive phenotype.

The authors had previously identified haploid mutants that were able to survive in the presence of the fungicide nystatin.  They mated these mutants to create either heterozygotes or homozygous recessive mutants and compared these to wild-type diploids growing either in the presence or absence of nystatin. 

Gerstein and coworkers found a wide range of effects of these mutations in the absence of nystatin.  Sometimes heterozygotes grew better than either homozygote, sometimes homozygous recessive strains did best, and sometimes wild type grew best.   Phenotypes were all over the map.

The story was very different in the presence of nystatin where only the homozygous recessives managed to grow.  This appears to contradict Haldane’s sieve.  Here there were no dominant mutations that allowed for survival.

Gerstein and coworkers found that some heterozygote replicates started to grow after a prolonged lag period.  A closer look at the heterozygotes that grew showed that they had lost the dominant allele so that they could now show the recessive phenotype and survive.  LOH had broken Haldane’s sieve. 

The authors found that the lower the nystatin levels, the more likely a population was to break through Haldane’s sieve.  They postulate that the populations survive longer at lower levels of nystatin, which increases the chances that a LOH will happen.  It is a race between survival and eliminating the dominant allele that keeps them from growing. 

The next step was to determine if LOH was common enough that populations with a small percentage of heterozygotes could survive.  They found that even in populations where only 2% were heterozygotes, around 5% of the 96 replicate populations managed to lose an allele and grow.  So even at low levels, a recessive mutation can give a population the advantage it needs to adapt and survive.

Combining the awesome power of yeast genetics with cheap sequencing is allowing scientists to test fundamental models of genetics that will unearth how populations adapt and survive in new environments.  We are finding those nuggets of scientific knowledge that have remained hidden.

Now of course, not every diploid is as numerous or as genetically flexible as yeast.  Cows, chickens, lizards, and people may all still be slaves to Haldane’s sieve.  We will need more studies to see if our recessive treasures can be uncovered in time to save us. 

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

Categories: Research Spotlight

Tags: antifungal resistance, evolution, Saccharomyces cerevisiae

Yeast: A One Man Band for Finding New Drug Leads

May 15, 2014

Imagine you are in a band and the only instruments you have are guitars.  Yes, you can play some beautiful music, but there will be a whole lot of music that your band won’t be able to play. 

In some ways, finding chemical leads to develop into drugs is similar to an all guitar band.  The compounds in available libraries all tend to have a lot in common.  They are like a vast array of subtly different guitars.

In a new study, Klein and coworkers use synthetic biology to have the yeast Saccharomyces cerevisiae make more varied libraries on its own.  As an added bonus, the authors also use the yeast to assay the new leads.  Not only have they expanded the range of instruments available to your band, but they’ve also made it so you can play all the instruments.  You are now a one man band!

The first step in all of this is to have an assay that can easily pick out the important leads.  Klein and coworkers use a galactose inducible Brome Mosaic Virus (BMV) system they had previously developed.

In this system, if one of the viral genes is on, then it produces a fusion protein that includes the Ura3 protein.  When the URA3 gene is expressed, yeast die in the presence of 5-fluoroorotic acid (5-FOA).  So any yeast that can make a compound that can inhibit viral expression will survive in 5-FOA.

The next step in creating these in vivo libraries was to randomly assemble various biochemical pathways into yeast artificial chromosomes (YACs) and to transform them into yeast.  These pathways were chosen because they have yielded important compounds before or because they come from medically important beasts.  This work was described in detail in a previous paper.

Specifically, Klein and coworkers randomly combined cDNA genes from eight biochemical pathways into YACs and transformed them into the BMV replication yeast strain.  They found 74 compounds that allowed the yeast to survive in the presence of 5-FOA.  Of these, 28 had activity in a secondary BMV assay. 

A close look at the 74 compounds showed that by and large, most had characteristics that put them in the right ballpark to be useful leads.  They had low molecular weight and the right hydrophobicity, and were chemically complex. In addition, many could easily be improved chemically (this last point is called optimizability).  Most importantly, they were pretty unique from a drug lead point of view. 

Over 75% of the compounds resembled nothing in known libraries.  And the compounds were not similar to one another.  Klein and coworkers had created a wide range of instruments other than guitars.

Of course, keeping a yeast strain alive is hardly reason to look for a new drug.  But that isn’t all these compounds can do.  At least some of these leads show excellent activity against two viruses related to BMV, Dengue and hepatitis C, and one looks particularly promising. 

With a random combination of genes from a variety of biochemical pathways, yeast has been coaxed into synthesizing chemical leads that can target two medically relevant viruses.  Scientists should be able to use a similar approach to tackle other diseases.  All they need is a yeast strain with the right assay.

Yeast can make our bread rise, get us drunk, and now maybe cure us of disease.  Is there anything yeast can’t do?  Well, they still can’t play a guitar. 

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: drug discovery, Saccharomyces cerevisiae

The Curious Case of the Proteasome in the Nucleus

May 07, 2014

In the Sherlock Holmes mystery story “The Adventure of the Crooked Man,” a man and his wife are heard having an argument behind closed doors. There is a crash, and the doors are opened to reveal that the man is dead in a pool of blood and his wife has fainted. No one else is nearby, and it seems beyond any doubt that the wife has murdered her husband…of course, until Sherlock Holmes delves into the details and uncovers the real story.

Something similar is going on in the nucleus behind those nuclear pores. The proteasome is a huge molecular machine that recognizes and degrades ubiquitinated proteins. Scientists have seen key parts of the proteasome in the nucleus, and nuclear proteins are degraded by this complex.  Seems like an open and shut case that the proteasome degrades nuclear proteins in the nucleus.  But like our Sherlock Holmes story, things aren’t always as they appear. 

Chen and Madura applied some detective work to the details of our nuclear protein degradation mystery in a new study published in GENETICS and found that it probably doesn’t work this way at all.  Nuclear proteins need to be exported out of the nucleus to be degraded by the proteasome.  

The researchers first confirmed earlier work showing that the Sts1 protein is responsible for escorting proteasomes to the nucleus. In the temperature-sensitive sts1-2 mutant at the restrictive temperature of 37 degrees, two proteasome subunits from different subcomplexes of the proteasome (Rpn11p from the regulatory particle and Pup1p from the catalytic particle) didn’t make it into the nucleus.

They then looked at two nuclear proteins, Rad4p and Pol1p (also known as Cdc17p) that are substrates of proteasomal degradation. When the proteasome subunits Rpn11p and Pup1p didn’t make it into the nucleus because of the sts1-2 mutation, Rad4p and Pol1p were not degraded.

So the proteasome needs to get into the nucleus in order for nuclear proteins to get degraded. Sounds like the proteasome is guilty of degrading proteins in the nucleus. But like a good mystery novel, the story takes an interesting twist here.

Chen and Madura found that when they raised the temperature of the sts1-2 mutant cells, Rad4p and Pol1p were stabilized immediately. This didn’t really make sense though. Even if the temperature-sensitive mutation blocked import of proteasomes into the nucleus as soon as the temperature increased, the proteasomes already inside the nucleus should have been able to continue degrading their substrates.

Wondering whether the substrates might be exported from the nucleus to be degraded elsewhere, they tested what happened to Rad4p and Pol1p when nuclear export of proteins was blocked. Using a few different ways to prevent nuclear export (combinations of mutations, chemicals, and temperature), they showed that if Rad4p and Pol1p could not get out of the nucleus, they were not degraded by the proteasome.

So it’s clear that nuclear export is part of the degradation process for at least these two nuclear proteins. Chen and Madura also detected a general increase in multi-ubiquitinated proteins (tagged for proteasomal degradation) in the nucleus under conditions where export was blocked, suggesting that this mechanism may apply to other proteins as well. And it’s been shown in human cells that several specific proteins, including the tumor suppressor p53, need to get out of the nucleus to be degraded.

There are still a lot of details to be filled in about where degradation is really happening. An intriguing clue comes from the fact that Sts1p is distantly related to the Schizosaccharomyces pombe protein Cut8, which is a nuclear envelope protein that tethers the proteasome to the nuclear membrane. Might nuclear proteasomes work on the outside of the nuclear envelope?

More detective work is needed to answer this question. But it’s clearly a very important one. Nuclear export and protein degradation are highly conserved processes, and both are currently under study as potential targets of cancer treatment.

Let this be a warning to all of us not to take everything at face value.  Just because someone is holding the bloody knife, that doesn’t mean he is the murderer.  And just because subunits and subcomplexes of the proteasome machinery look to be in the nucleus, that doesn’t mean nuclear proteins are degraded there.  It is elementary, my dear Watson.  

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

Categories: Research Spotlight

Tags: nuclear export, proteasome, Saccharomyces cerevisiae

Modifications? Heterochromatin Don’t Need No Stinking Modifications (for Activation)

May 01, 2014

When a character is asked to show his badge in the movie The Treasure of the Sierra Madre, he famously says something along the lines of, “Badges? We don’t need no stinkin’ badges!*” If histones in yeast heterochromatin could talk they might say something similar, except instead of badges they’d bring up modifications. Maybe something along the lines of, “Modifications? We don’t need no stinkin’ modifications for activation!” At least, they’d say this if a new study by Zhang and coworkers holds up.

In this study, the authors show that two different genes in the yeast S. cerevisiae are activated in heterochromatin in the absence of any significant changes to the surrounding chromatin.  This result is surprising because most researchers think activation and changes in chromatin always go hand in hand.  Apparently, in at least some situations they do not. 

This isn’t to say that chromatin didn’t do anything here…it most certainly did.  It served as a general damper on transcription.  But in this study chromatin was by no means the major player; it had a relatively small influence on the levels of basal and activated gene expression.  The authors suggest that this may be true for other genes in the more transcriptionally active euchromatin as well.

In the first set of experiments, Zhang and coworkers used a model system where the heat inducible gene HSP82 is flanked by the HMRE silencer from the HMR mating type cassette.  These silencers cause a 30-fold reduction in transcription of this hsp82-2001 transgene.   

Using chromatin immunoprecipitation (ChIP) the authors show that their transgene is indeed embedded in heterochromatin.  They see a lot of Sir3p around the promoter, a high density of histones that lack any of the telltale modifications of euchromatin, and very little RNA polymerase II (Pol II) or the mRNA capping enzyme Cet1p around the promoter.  These are all hallmarks of heterochromatin in yeast.

Things change when the yeast is subjected to heat shock.  Consistent with the observed 200-fold increase in transcription, they suddenly see lots of Pol II and Cet1p around.  But there is not a big change in the number of histones around the gene nor in their modifications.

When HSP82 is in its normal place in the genome, its activation is accompanied by specific acetylation and methylation of H3 and H4 histones.   In heterochromatin, despite significant induction, there is none of this.  The histones remain looking the same whether there is significant transcription or not. 

One trivial explanation for this might be that the chromatin is unaffected because the levels of transcription are lower than normal.  In other words, the lower final activity in the induced state is affecting histone modification. 

Zhang and coworkers rule this out by using a TATA-less HSP82 gene in euchromatin and show that all the appropriate histone modifications still happen.  This is true even though the damaged gene has 5-fold less activity compared with their transgene.  The low level of transcription does not appear to explain activation in the absence of histone modification.

Of course another reason for this unexpected observation might be that this pretty artificial construct isn’t representative of natural genes.  This doesn’t change the fact that its transcription is activated in the absence of histone modification, but it does question its relevance in the real world.

To address this issue, the authors looked for an inducible gene in natural heterochromatin and with a little bit of detective work, found the subtelomeric YFR057W gene.  No one knows what this gene does, but a close look showed a possible Stb5p binding site in its promoter. 

When Stb5p heterodimerizes with Pdr1p, the resulting dimer activates genes involved in pleiotropic drug resistance.  Indeed the authors found that YFR057w was induced 150-fold with a small amount of cycloheximide.  And when they used ChIP to compare the induced and uninduced states, they again found almost no changes in the chromatin around this gene despite an increase in the amount of Pol II and Cet1p.

Taken together these results suggest that activation doesn’t always have to come with chromosomal changes.  Which, while a bit surprising today, wouldn’t have turned any researchers’ heads a few decades ago.

In the old days (1980’s and 1990’s), a lot of focus was on how transcriptional activators might affect the ability of Pol II to load onto the DNA and to pry it open and start transcribing.  A lot of this was based on prokaryotic work where there really isn’t very much in the way of chromatin and a lot of activation depends on improving the ability of the polymerase to transcribe.

These days when people think about turning up a gene, they think about changing nearby chromatin.  Various enzymes work to modify histones at specific places, which both loosens up the chromatin to allow access by Pol II and serves as a way for various coactivators to recognize the DNA. 

As usual, reality is probably a combination of the two.  Activators can activate transcription in lots of different ways, some of which probably include chromatin changes while in others chromatin changes are simply a consequence of activation.  Not all transcription activation needs stinkin’ histone modifications.  

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* This is actually a misquote that may have come from the Mel Brooks film Blazing Saddles.

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

Categories: Research Spotlight

Tags: heterochromatin, histone modification, Saccharomyces cerevisiae, transcription

Measure Twice, Cut Once

April 23, 2014

As any seasoned carpenter knows, if you are going to cut a piece of wood, you want to do it right the first time!  There is no second chance.

This means that good carpenters are very, very careful.  They use a clamp to hold the wood in place and measure where to cut not once but twice.  Now they have a good shot at getting a length of lumber they can use.

As shown in a new paper in Molecular Cell by Liang and coworkers, it turns out that our cells do something similar when cutting their RNA.  The yeast enzyme Rnt1p measures a piece of double stranded RNA twice to make sure it cuts in the right place.  And, like a second-rate carpenter, if it measures the RNA only once it often cuts the RNA in the wrong place.   

This is almost certainly not just a yeast thing.  Rnt1p is a member of the conserved RNAse III family, which is present in all domains of life except Archaea.

In higher organisms, RNAse III enzymes such as Dicer produce the small interfering RNAs (siRNA) and microRNAs (miRNA) that have important roles in gene regulation via RNA interference. S. cerevisiae doesn’t use RNA interference, but Rnt1p is still important for maturation of small nuclear RNAs, small nucleolar RNAs, and ribosomal RNA, and also for degradation of some specific mRNAs.

Most RNase III enzymes recognize the RNA they are to cut by certain secondary structures like loops.  Liang and coworkers used X-ray crystallography on Rnt1p in complex with an RNA substrate to learn how Rnt1p recognizes its substrate and “knows” where to cut it. The RNA had a double-stranded stem capped by a 4-nucleotide loop, a so-called tetraloop, that had a conserved G residue at the 2^nd position.

Rnt1p cleaves this RNA at a fixed distance from the tetraloop, and it cleaves the two strands unequally so that they have 2-nucleotide 3’ overhanging ends. The crystal structure showed that two of the five RNA-binding motifs (RBMs) in Rnt1p form a pocket that clamps down on the conserved G residue in the tetraloop. This clamp is fastened so tightly that the RNA structure is changed.  It is like the clamp distorting the carpenter’s piece of wood.

When Liang and coworkers deleted one of these two Rnt1p RBMs, or mutated the conserved G in the substrate, the substrate was no longer held or cleaved.  Clamping the RNA was critically important for the reaction. 

They also showed both by structural modeling and by mutational analysis that other parts of Rnt1p interact with the RNA stem structure. Clamping the RNA and interaction with the rest of the substrate puts the cleavage site at a fixed position relative to the Rnt1p active site.

This tight binding and measurement by protein-RNA interactions would seem to be good enough to ensure accurate cleavage. But it’s not the whole story.

Another domain of Rnt1p, the N-terminal domain (NTD), was known to contribute to substrate selection, but it was unclear exactly how it did this. Surprisingly, the crystal structure showed that it, too, contacts the tetraloop. When Liang and colleagues deleted the NTD, the RNA substrate was still cleaved but there was a mixture of products, cleaved at several different sites. So it too is needed for precise cleavage.

The overall conclusion is that two different domains contact the tetraloop, each acting like a ruler. The protein-protein and protein-RNA interactions stiffen each ruler such that the cleavage site is always precisely measured before cutting.  Just like our carpenter friend, to get the right cut, Rnt1p needs to measure twice before cutting. The same knowledge that is handed down through generations of carpenters is also deeply ingrained in our biochemistry! 

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

Categories: Research Spotlight

Tags: crystal structure, RNAse III, Saccharomyces cerevisiae

How Yeast Populations Make the Cut

April 15, 2014

Imagine that your dream is to be a professional basketball player.  Unfortunately for you, you are only five feet six inches tall and you can’t jump very high.  No matter how much you practice and work out, it is exceedingly unlikely you will be a starter for the Miami Heat.

Now imagine instead that you are six feet tall with a reasonable vertical jump.  Here, with enough effort you have a shot at beating out the guy with the genetic advantage of being six foot six inches high who doesn’t work as hard as you do.  Keep practicing and you might be passing the ball to LeBron James instead of him! 

In a new study in GENETICS, Frenkel and coworkers show that something similar can happen in yeast too.  If a population of yeast has some overwhelming advantage over a second population, the first will quickly outcompete the second every time.  But if the first population is just a bit better than the second, then the second can sometimes end up with a mutation that gives it an even better advantage than the first.  Now the first population is outcompeted and the second takes over.

Of course, when presented in a general way this is sort of obvious.  But Frenkel and coworkers set up their experiments in such a way that they got some hard numbers for just how much of an advantage one population needs to overcome to have a chance at winning.  If six feet is tall enough, what about five feet eleven inches?

The first step was to generate a number of mutants with different measured fitness advantages.  They selected mutant populations with advantages of 3, 4, 5, or 7%.  These populations were all tagged with a fluorescent marker.

They then seeded these mutants individually into 658 replicate reference populations that were tagged with a different fluorescent marker.  The mutants were seeded at a high enough level to prevent genetic drift from wiping them out.  The authors then followed each population for hundreds of generations by determining the levels of each population every 50 or so generations.

Their first finding was that mutants with a 7% advantage won out every time.  The reference population had no chance at getting a good enough mutation to beat it out.  No one is going to beat LeBron James out for his starting position with the Miami Heat.

Once the advantage was only 5%, around 16% of the time the second population won out.  As the advantage got smaller and smaller, the second population won out more and more often.   Even a genetically less gifted player has a shot at beating out the 12^th guy on the Heat’s roster!

These results can tell us quite a bit about the mutational landscape of haploid Saccharomyces cerevisiae.  For example, from these data Frenkel and coworkers figured out that only populations that get mutations that give at least a 2% advantage have a chance at outcompeting other populations.  By assuming a mutation rate of 4X10^-3, around 1 in 1000 mutations fit this bill, which might seem surprisingly high but is consistent with previous studies.  With a bit more hand waving, the authors hypothesize that disruption of something like 1 in 100 yeast genes is actually beneficial!

So yeast have a surprisingly level playing field.  Unless they are up against the equivalent of Kobe Bryant or Michael Jordan, they have a good shot at stumbling on a mutation that gives them an edge over their peers.    

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

Categories: Research Spotlight

Tags: evolution, mutation, population genetics, Saccharomyces cerevisiae

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