New & Noteworthy

Move Along, Transcription Factor

December 07, 2016

Sumo wrestlers throw each other out of the ring. Just like sumoylation throws Gcn4p off DNA. Image from Wikimedia Commons.

For an election to go smoothly, people cannot stay too long in the voting booth. If a lot of people stayed in the booth and answered emails, sent texts, etc., after they finished voting, then the whole process would grind to a halt.

There is some evidence that activating genes may work similarly. The transcription factors (TFs) that bind DNA and turn up the expression of nearby genes can’t stay too long. If they do, the activation starts to peter out.

What is thought to happen with these sorts of TFs is that they bind their preferred DNA, and then once they have attracted the cellular machinery needed to read the gene, they are targeted for destruction. Then a new TF can bind and repeat the process.

In a new study in GENETICS, Akhter and Rosonina set out to investigate the process by which the yeast transcription activator Gcn4p is removed after it has bound DNA and done its job. Gcn4p activates a number of genes in response to amino acid starvation.

They found that a key step in the process is the addition of SUMO proteins to DNA-bound Gcn4p, which gets the ball rolling on the destruction of Gcn4p. Imagine a sumo wrestler settling in next to a voter once he enters the booth and then throwing him out if he tarries too long.

Their model is that once Gcn4p binds DNA, it is sumoylated. Then the DNA-bound, sumoylated GCN4 is further modified by kinases like Cdk8p, a component of the mediator complex which acts as a bridge between TFs and the cellular machinery responsible for reading a gene. This modified TF is then sent off to the 26S proteasome where it is degraded making room for an unmodified Gcn4p.

Previous research had shown that sumoylation of GCN4 required DNA binding. The first thing these authors did in this study was to determine if Gcn4p had to bind to its target DNA sequence in order to be sumoylated. It did not.

When they fused a mutant Gcn4p that could not bind DNA to the DNA binding domain of Gal4p, they found that this molecule was sumoylated at the correct places on the Gcn4p part of the fusion protein, lysines 50 and 58, when bound to a Gal4p binding site. Therefore, Gcn4p does not need to occupy its own DNA binding site in order to be sumoylated.

Another set of experiments showed that while DNA binding was required for sumoylation, interaction with RNA polymerase II (RNAP II), the enzyme that reads the genes that Gcn4p activates, does not appear to be necessary. For one of these experiments they used a temperature sensitive mutant of the largest subunit of RNAP II, Rpb1p, and showed that even at higher temperatures when RNAP II is inactive in these cells, DNA-bound Gcn4p is still sumoylated. In the other experiment they showed that DNA-bound
Gcn4p was still sumoylated when they used the “anchor away” technique to drag Rpb1p out of the nucleus and into the cytoplasm.

So DNA binding is sufficient, and the specific site is not important. And Gcn4p doesn’t have to be activated in order to be sumoylated.

Of course, turnover like this is a delicate thing. If Gcn4p is pulled off too soon, then it can’t activate as much as it might otherwise be able to do. This might affect the cell’s response to starvation just as much as Gcn4p staying put too long. Sort of like the sumo wrestler throwing a voter out of the voting booth before they could finish their voting can muck up the election.

Akhter and Rosonina created a fusion protein of Gcn4p and the yeast SUMO peptide Smt3p. Unlike Gcn4p, this protein is sumoylated before it binds DNA.

They found that yeast expressing this fusion protein fared less well under starvation conditions compared to yeast cells that expressed the wild type version of GCN4. And using chromatin immunoprecipitation (ChIP) analysis they showed that at least at the ARG1 gene, this was because there was less of the fusion protein bound under activating conditions.

So cells need for TFs to stay at the right place for the right amount of time. If they are pulled off too early or stay too long, the levels of activation can fall below what is best for the cells.

Unfortunately, we don’t have time to go over other experiments that tease out which kinases are important and when, but I urge you to read about them for yourselves. They take full advantage of the genetic tools available in yeast to make this sort of study possible…#APOYG!

Integrating all of this gives the following model:

Gcn4p model

Gcn4p is only a dimer when bound to DNA and this dimerization may be the signal for sumoylation by Ubc9p. A preinitiation complex forms through its interaction with the DNA-bound, sumoylated Gcn4p which brings in the enzyme RNAP II to transcribe the gene. Once the polymerase has left the nest, the kinase Cdk8p comes in and phosphorylates Gcn4p which signals Cdc4p/Cdc34p to ubiquitinate Gcn4p. The ubiquitinated Gcn4p is then degraded by the 26S proteasome opening the upstream activator sequence (UAS) up to a fresh, new Gcn4p.

Here, with the help of our super hero Saccharomyces cerevisiae, Akhter and Rosonina have dissected out what happens to a transcription factor once it binds to DNA (at least ones that bind for short times). It will be fascinating to see if this translates to other TFs in other beasts. While I love yeast for all it can do for us for bread, wine, beer, human health, helping solve world problems like climate change, and so on, I think my favorite use is still that it allows us to better understand the basic biology of how our cells work.

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

Categories: Research Spotlight

Tags: Cdk8 , transcription , gene activation , Gcn4 , sumoylation

Unleashing the Awesome Power of Yeast Transcription

October 07, 2015

With the right mutations, yeast can activate transcription over long distances—just as with the right social media, distance is meaningless for finding and organizing like-minded people. Image via

In the old days, before the internet, planes or even mass publishing, it was hard to spark a quick, worldwide movement. You simply couldn’t reach out to likeminded people who lived far away.

Nowadays things are very different. With the advent of social media, it is now trivially easy to spread the word. Using Twitter and Facebook, organizers can easily and effectively organize people who live on the other side of the world.

In terms of transcription activation, our friend Saccharomyces cerevisiae seems to be stuck in the old world. Its transcription factors can only turn up nearby genes. This is different from most other eukaryotic beasts, where activation at a distance is routine.

Except maybe yeast isn’t as backward as we think. It may be that yeast has the potential to activate transcription at a distance, but keeps that potential locked away.

This idea is supported in a new study out in GENETICS by Reavey and coworkers. These authors found that they could mutate away yeast’s inability to activate transcription at a distance.

In other words, this ability is there but is just prevented from being used. Yeast is keeping social media out of the hands of its genes.

Getting around social media/internet controls is not easy. Pressure might need to be applied at multiple points before people power is finally released through social media. And even when it does happen, it can sometimes be hard to figure out exactly why certain events tipped the balance.

Turns out that both of these are also true for long range transcription activation in yeast. Mutating a single gene was not enough—it took mutations in multiple genes to see any significant effect.

As you can imagine, it would be very tricky to hit all of the right mutations for a polygenic trait in one fell swoop. This is why Reavey and coworkers started their mutant hunt with a strain that could already weakly activate transcription from a distance, a strain in which the SIN4 gene was deleted. Now they just needed additional mutations to make the effect stronger.

In their screen they used a reporter in which the GAL4 upstream activating sequence (UAS) was placed 799 base pairs upstream of the HIS3 reporter. This reporter gives very low levels of activity in a wild type strain. They included a second reporter, the URA3 gene under control of the same upstream sequences, because Reavey and Winston had discovered in previous work using a single reporter that cis acting mutations and chromosomal rearrangements were a frequent source of false positive results.

The researchers put the reporter strain through multiple rounds of mutation with UV light and selection with increasing levels of 3AT, a competitive inhibitor of His3. After each round of selection, they measured mRNA levels for HIS3 and URA3 and chose strains that not only had higher 3AT resistance but also showed more transcription of the reporter genes.  In the end they found three strains that survived in the presence of galactose (to turn on the activator) and 10 mM 3AT.

As expected, each strain had multiple mutations. One strain had acquired mutations in the GRR1 and MOT3 genes. To confirm that these were the most important mutations, Reavey and colleagues engineered a fresh strain with just the original sin4 null mutation and the selected grr1-1 and mot3-1 mutations. The fresh strain completely recapitulated the selected strain, showing that these three mutations could unlock yeast’s potential for long-range transcriptional activation.

It makes sense that a grr1 mutation could affect transcriptional activation. Grr1 is a ubiquitin ligase that destabilizes Med3 (also known as Pdg1), a key component of the Mediator complex involved in transcription activation. The researchers provided evidence that this is how the grr1-1 mutation affects the process, by showing that mutating MED3 mimicked the effects of mutating GRR1.

It’s also not too hard to imagine how a mutation in Mot3, a sequence-specific transcriptional activator, could affect transcriptional activation, presumably by changing the expression of a gene under its control.

If you stay away from social media, you’ll lose opportunities for impromptu pillow fights. Who knows what yeast is missing without long-range transcription activation? Image via Wikimedia Commons

The results were not so clear-cut for two other strains that were selected. They arose from the same lineage, and each had acquired the ptr3-1 mutation on top of the original sin4 null mutation. One strain went on to further pick up the mit1-1 mutation, while the other got an msn2-1 mutation.

Again it isn’t too surprising that mutations in genes that encode sequence-specific transcriptional activators like Mit1 and Msn2 arose in these strains. But the selection of the ptr3 mutation in these lineages is something of a mystery.

It is hard to imagine how the usual job of Ptr3 in nutrient sensing and transport would be involved in keeping long range transcription activation down. Perhaps the researchers have uncovered a novel function for this gene.

And re-creating these two strains only partially restored the levels of transcription activation at a distance that were seen in the original strains. A little genetic detective work showed that a big reason for this was that both of the selected strains had acquired an extra copy of the chromosome that had the HIS3 reporter, chromosome III.

Reavey and colleagues deleted each of their identified genes to see which ones caused their effect through a loss of function mutation. Deleting GRR1, PTR3, and MSN2 all had the same effect as the original isolated mutations.

The same was not true for MOT3 and MIT1. Deleting either gene actually weakens long range transcription activation, suggesting that these two had their effect through gain of function mutations.

Finally, the researchers showed that the increase in long-distance transcriptional activation was not simply due to a general increase in transcription activation in the selected strains, by showing that their mutants did not have increased activity of a reporter with the GAL4 UAS placed 280 base pairs upstream of HIS3. In fact, if anything, the strains showed decreased activation with this reporter.

So this experimental strategy allowed Reavey and coworkers to identify some of the key genes involved in keeping transcription activation at a distance under control in yeast. In particular, they found compelling evidence that the Mediator complex is an important player. But there is still plenty of work to do. For example, which of the genes regulated by Mit1, Msn2, and Mot3 are important in long range activation? And what on Earth is Ptr3 doing in all of this?

The success of this approach also confirms that doing repeated rounds of selection in yeast is a viable way to select multiple mutants and study polygenic traits. This strategy may prove a boon for studying the many human diseases that are the result of polygenic traits.

Not only can we use yeast to uncover its activation potential, but we can also now potentially use it to uncover new treatments for human disease. Unleashing another awesome yeast power… 

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

Categories: Research Spotlight

Tags: transcription , Saccharomyces cerevisiae , polygenic trait

A Scientist Sees Transcription

September 23, 2015

While Horton uses his sensitive ears to hear a single Who, researchers need to use optical tweezers to see a single gene being transcribed. Image by Dave Parker via Flickr

In the classic Dr. Seuss tale Horton Hears a Who, the elephant Horton thinks he hears voices coming from a speck of dust. He gets into all sorts of trouble over this until all the Whos in Whoville prove they are alive when they all shout at once. Now Horton’s jungle compatriots believe him and Horton can hang out with his new friends.

Horton’s companions never get to hear an individual Who. They are not blessed with Horton’s big elephant ears and so have to just hear all the Whos shouting at once.

Up until recently, we have been in the same situation as the kangaroo and everyone else in the jungle when it comes to transcription in a cell. We can use all sorts of tools to get at what goes on when RNA polymerase II (pol II) gets ready and then starts to transcribe a gene, but we can only get an aggregate picture of lots of cells where it is happening. We can’t hear the Mayor of Whoville amidst all of the other Who voices.

In a new study out in Nature, Fazal and coworkers use the equivalent of elephant ears, optical tweezers, to study the initiation of transcription by purified pol II machinery from Saccharomyces cerevisiae on single molecules. And what they find is that at least for one part of the process, our having looked at things in the aggregate may have fooled us about how the process worked. It was important that we be able to pick out individual voices from the cacophony of the crowd.

Not surprisingly, transcribing a gene is tricky work. It is often split into three steps: initiation, elongation, and termination. And each of these can be subdivided further.

Fazal and coworkers focused on transcription initiation. Previous work had suggested that the process goes something like this:

Top image via Wikimedia Commons

Basically, an alphabet soup of general transcription factors and pol II sit down on a promoter. This complex then pries open the DNA and looks for a signal in the DNA to start transcribing. The polymerase then transcribes short transcripts until it shifts into high gear when it escapes the promoter and enters elongation phase.

This theory comes from the study of transcription in bulk. In other words, it derives from looking at many cells all at once or many promoter fragments in a test tube.

Fazal and coworkers set out to look at how well this all holds up when looking at single genes, one at a time. To do this they used a powerful technique called optical tweezers.

Optical tweezers can “see” what is going on with moving enzymes by measuring the change in force that happens when they move. For this study, the preinitiation complex bound to a longish (2.7 kb) piece of DNA was attached to one bead via pol II, the moving enzyme. The other end of the DNA was attached to a second bead. Each bead is then immobilized using lasers (how cool is that!) and the DNA is stretched between the two beads. Watch this video if you want more details on the technique.

Depending on where you attach the DNA to the bead, you can either track polymerase movement or changes in DNA by precisely measuring changes in the forces keeping the beads in place. Using this technique the researchers found that the bulk studies had done pretty well for most every step. Except for the initial transcribing complex.

The earlier studies had suggested that an open complex of around 15 nucleotides was maintained until elongation began. This study showed that in addition to the 15 base pairs, an additional 32 to 140 base pairs (mean of about 70 base pairs) was also opened before productive elongation could begin. And that this whole region was transcribed.

This result paints a very different picture of transcription initiation. Rather than maintaining a constant amount of open DNA, it looks like the DNA opens more and more until the open DNA collapses back down to the 12-14 base pair transcription bubble seen during elongation.

It turns out that this is consistent with some previous work done in both yeast and fruit flies. Using KMnO4, a probe for single stranded DNA, scientists had seen extended regions of open DNA around transcription start sites but had interpreted it as a collection of smaller, opened DNA. In other words, they thought they were seeing different polymerases at different positions along the DNA.

These new results suggest that they may have actually been seeing initial transcribing complexes poised to start processive elongation. Seeing just one complex at a time changed how we interpreted these results.

Fazal and coworkers were also able to see what happened to some of the 98% of preinitiation complexes that failed to get started. Around 20% of them did end up with an extensive region of open DNA of around 94 +/- 36 base pairs but these complexes were independent of transcription, as they didn’t require NTPs.

But since this opening did require dATP, they propose that it was due to the general transcription factor TFIIH, a helicase. It looks like in these failed complexes, TFIIH is opening the DNA without the polymerase being present.

A clearer picture of what might be going on at the promoter of genes starts to emerge from these studies. Once around 15 base pairs of DNA are pried open to form the appropriately named open complex, TFIIH unwinds an additional 70 or so base pairs. The polymerase comes along, transcribing this entire region. The whole 85 or so base pairs stays open during this process.

Eventually the polymerase breaks free and the opened DNA collapses back down to around 12-14 base pairs. Now the polymerase can merrily elongate to its heart’s content. Until of course something happens and it stops…but that is another story. 

Categories: Research Spotlight

Tags: transcription , RNA polymerase II , Saccharomyces cerevisiae , optical tweezers

Runaway Polymerases Can Wreak Havoc in Cells

October 16, 2014

A train without working brakes can cause a lot of destruction if it careens off the tracks. And it turns out that a runaway RNA polymerase II (pol II) can cause a lot of damage too.  But it doesn’t cause destruction, so much as disease.

Working brakes are important for both large and small machines, including RNA polymerase. Image from Wikimedia Commons

Unlike a train, which has its brakes built right in, pol II has to count on outside factors to stop it in its tracks. And one of these brakes in both humans and yeast is a helicase: Sen1 in yeast and Senataxin, the product of the SETX gene, in humans. 

Mutations in SETX are associated with two devastating neurological diseases: amyotrophic lateral sclerosis type 4 (ALS4) and ataxia oculomotor apraxia type 2 (AOA2), both of which strike children and adolescents.  One idea is that these mutations may short circuit the brakes on pol II, causing it to keep on transcribing after it shouldn’t. And this is just what Chen and colleagues found in a new paper in GENETICS.

The researchers used the simple yet informative yeast model system to look at some of these mutations, and found that they disrupted the helicase function of Sen1 and caused abnormal read-through of some transcriptional terminators.  Looks like bad brakes may indeed have a role in causing these devastating diseases.

Some human proteins can function perfectly well in yeast. Unfortunately, Senataxin isn’t one of those; it could not rescue a sen1 null mutant yeast, so Chen and coworkers couldn’t study Senataxin function directly in yeast. But because Senataxin and Sen1 share significant homology,  they could instead study the yeast protein and make inferences about Senataxin from it.

First, they sliced and diced the SEN1 gene to see which regions were essential to its function. They found that the most important part, needed to keep yeast cells alive, was the helicase domain. But this wasn’t the only key region.

Some flanking residues on either side were also important, but either the N-terminal flanking region or the C-terminal flanking region was sufficient. Looking into those flanking regions more closely, the researchers found that each contained a nuclear localization sequence (NLS) that directed Sen1 into the nucleus. This makes perfect sense of course…the brakes need to go where the train is!  If we don’t put the brakes on the train, it won’t matter how well they work, the train still won’t stop.

These flanking sequences appeared to do more than direct the protein to the nuclear pol II, though.  When the authors tried to use an NLS derived from the SV40 virus instead, they found that it couldn’t completely replace the function of these flanking regions even though it did efficiently direct Sen1 to the nucleus.

Next the researchers set out to study the disease mutations found in patients affected with the neurological disease AOA2.  They re-created the equivalents of 13 AOA2-associated SETX mutations, all within the helicase domain, at the homologous codons of yeast SEN1.

Six of the 13 mutations completely destroyed the function of Sen1; yeast cells could not survive when carrying only the mutant gene. When these mutant proteins were expressed from a plasmid in otherwise wild-type cells, five of them had a dominant negative effect, interfering with transcription termination at a reporter gene. This lends support to the idea that Sen1 is important for transcription termination and that the disease mutations affected this function.

The remaining 7 of the 13 mutant genes could support life as the only copy of SEN1 in yeast. However, 5 of the mutant strains displayed heat-sensitive growth, and 4 of these showed increased transcriptional readthrough.

Taken together, these results show that the helicase domains of Senataxin and Sen1 are extremely important for their function. They also show that Sen1 can be used as a model to discover the effects of individual disease mutations in SETX, as long as those mutations are within regions that are homologous between the two proteins.

It still isn’t clear exactly how helicase activity can put the brakes on that RNA polymerase train, nor why runaway RNA polymerase can have such specific effects on the human nervous system. These questions need more investigation, and the yeast model system is now in place to help with that.

So, although it might not be obvious to the lay person (or politician) that brainless yeast cells could tell us anything about neurological diseases, in fact they can. Yeast may not have brains, but they definitely have RNA polymerase. And once we learn how the brakes work for pol II in yeast cells, we may have a clue how to repair them in humans.

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

Categories: Research Spotlight Yeast and Human Disease

Tags: transcription , RNA polymerase II , helicase , ALS , 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.

The histones in yeast heterochromatin don’t need no stinkin’ modifications for gene activation. Image from Wikimedia Commons

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.  

* 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: histone modification , transcription , Saccharomyces cerevisiae , heterochromatin

Silly Sod’s Two Jobs

March 27, 2014

Most SGD users are probably too young to remember Saturday Night Live’s early years.  One very funny commercial parody involved Gilda Radner and Dan Aykroyd arguing over a product called Shimmer.  Gilda argues that it is a floor wax while Dan says it is a dessert topping.  In comes Chevy Chase to tell them that it is both.  Not quite as funny as Bassomatic, but still hilarious.

Not quite as weird as if this whipped cream were also a floor wax, but Sod1p being an enzyme AND a transcription factor was unexpected. Image from Wikimedia Commons

In a new study, Tsang and coworkers show something similar for the enzyme Sod1p.  Most people know Sod1p as an enzyme that protects the cell and its DNA by directly deactivating harmful reactive oxygen species (ROS) like superoxide.  Turns out that it may also be a transcription factor.

Now these two jobs aren’t quite as disconnected as a dessert topping and floor wax.  When Sod1p acts as a transcription factor, it is regulating genes that affect a cell’s response to ROS.  It is actually using its two functions to attack the same problem on multiple fronts.

Tsang and coworkers started out by looking at what happens to nuclear DNA under oxidative stress, using the Comet and TUNEL DNA damage assays. They found that endogenous and exogenous ROS caused DNA damage that was much worse in the sod1 null mutant – in other words, Sod1p protected the cells’ DNA. Using immunofluorescence, they also showed that Sod1p quickly went into the nucleus in the presence of ROS.  But if they restricted Sod1p to the cytoplasm by adding a nuclear export signal, the protein no longer protected the DNA.  In fact, it did no better than a strain deleted for SOD1.

In the course of these experiments one of the ways the researchers induced nuclear localization was with a burst of hydrogen peroxide.  But since hydrogen peroxide isn’t a substrate of the enzyme Sod1p, Tsang and coworkers next wanted to figure out how Sod1p got its signal to go nuclear.

Previous work had shown that SOD1 genetically interacted with MEC1, a yeast homolog of ATM kinases which sense oxidative stress.  They deleted MEC1 and found that Sod1p was trapped in the cytoplasm, unable to protect the cell’s DNA from damage.  This result was confirmed in human cells by showing that Sod1p only went nuclear if the cell made ATM kinase.

Tsang and coworkers suspected that this interaction might happen through a protein kinase called Dun1p, whose human homolog is a Mec effector. They confirmed a previous mass spectrometry result that showed Sod1p interacted physically with Dun1p.  And indeed, when DUN1 was deleted, Sod1p was again stranded in the cytoplasm.  Further work showed that Dun1p does its job by phosphorylating Sod1p on two serine residues, S60 and S99. When both these serines are mutated to alanine, preventing phosphorylation, less of the mutant Sod1p makes it into the nucleus. 

Using DNA microarrays, Tsang and coworkers next showed that SOD1 was required to activate 123 genes needed by the cell to respond to hydrogen peroxide.  These genes fell into five categories: oxidative stress, replication stress, DNA damage response, general stress response and Cu/Fe homeostasis.  The final experiment used chromosomal immunoprecipitation (ChIP) to show that in the presence of hydrogen peroxide more Sod1p was bound at the promoters of two of these genes, RNR3 and GRE2, but not the control gene ACT1

Of course, the authors have only looked at two of the 123 genes and an obvious next step is to figure out how many of the 123 have more Sod1p bound to their promoters in the presence of hydrogen peroxide.  Still, if these results can be confirmed and expanded they will suggest that Sod1p is able to combat oxidative damage in two completely different ways. 

In the first it uses its enzymatic activity to directly inactivate the ROS superoxide, while in the second it helps the cell respond to other ROS apparently by acting as a transcription factor.  While the jobs themselves are not as different as a floor wax and a dessert topping, how Sod1p goes about getting each job done is.  “Calm down you two, Sod1p is an enzyme AND a transcription factor.”

In addition to these two roles, we’ve written before about yet another regulatory role for Sod1p: it regulates glucose repression by binding to two kinases and stabilizing them. This is truly an overachiever of a protein!

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

Categories: Research Spotlight

Tags: transcription , oxidative stress , Saccharomyces cerevisiae

Passing the Hog: How a Long Noncoding RNA Helps Yeast Respond to Salt

February 25, 2014

Lucky Incans already had bridges to run over. Hog1p has to build its own bridge to get from one end of a gene to the other. Photo courtesy of Rutahsa Adventures via Wikimedia Commons

Most people know that Incans relied on human runners to get messages across their empire.  Basically they had runners stationed at various places and one runner would hand the message off to the next.  This relayed message could then quickly travel across the country.

As shown in a new study by Nadal-Ribelles and coworkers, it turns out that something similar happens in yeast when the CDC28 gene is turned up in response to high salt.  In this case, the runner is the stress activated protein kinase (SAPK) Hog1p and it is stationed at the 3’ end of the gene.  When the cell is subjected to high salt, the message is relayed from the 3’ end of the CDC28 gene to its 5’ end by the Hog1p kinase.  The end result is about a 2-fold increase in the amount of Cdc28p made, which allows the cell to enter the cell cycle more quickly after the salty insult.

Unlike the Incans who had their paths all set up in front of them, poor Hog1p has to build its own path.  It does this by activating a promoter at the 3’ end of the CDC28 gene that produces an antisense long noncoding RNA (lncRNA) that is needed for the transfer of the Hog1p.  It is as if our Incan runner had to build a bridge over a gorge to send his message.

This mechanism isn’t peculiar to the CDC28 gene either.  The authors in this study directly show that something similar happens with a second salt sensitive gene, MMF1.  And they show that a whole lot more lncRNAs are induced by high salt in yeast as well.

Nadal-Ribelles and coworkers started off by identifying coding and noncoding regions of the yeast genome that respond positively to high salt.  The authors found that 343 coding regions and 173 noncoding regions were all induced at 0.4 M NaCl.   Both coding and noncoding regions required the SAPK Hog1p for activation. 

The authors next focused on CDC28 and its associated antisense lncRNA.  After adding high salt, Nadal-Ribelles and coworkers found that Hog1p was both at the start and end of the CDC28 gene – as would be expected, since both CDC28 and the antisense lncRNA required this kinase for transcriptional activation. 

Things got interesting when they were able to prevent the lncRNA from being made.  When they did this, Hog1p was missing from both the 5′ and 3′ ends of the CDC28 gene and as expected, activation was compromised.  But Nadal-Ribelles and coworkers showed that expressing the lncRNA from a plasmid did not allow for CDC28 activation. It appears that where the lncRNA is made is just as important as whether it is made.

Through a set of clever experiments, the authors showed that not only does the lncRNA need to be made in the right place, but it needs to be activated in the right way.  When they set up a system where the lncRNA was induced in the right place using a Gal4-VP16 activator, CDC28 was not induced by high salt.  A closer look showed that this was most likely due to a lack of Hog1p at the start of the CDC28 gene.

The situation was different when they activated the lncRNA with a Gal4-Msn2p activator which uses Hog1p to increase expression.  In this case, CDC28 now responded to high salt and Hog1p was present at both the start and end of the CDC28 gene.  But this activation went away if they added a terminator which prevented the full length lncRNA from being made. 

Phew, that was a lot!  What it means is that for there to be a Hog1p at the business end of the CDC28 gene, there needs to be one at the 3’ end.  It also means that for the Hog1p to get to the start of the CDC28 gene, the antisense lncRNA needs to be made.

This would all make sense if maybe the lncRNA was involved in DNA looping, which could get the Hog1p from the end of CDC28 to the start where it can do some good.  Nadal-Ribelles and coworkers showed that this indeed was the case, as CDC28 activation required SSU72, a key looping gene.  When there was no Ssu72p in a cell, salt induction of CDC28 was severely compromised.

So it looks like an antisense lncRNA in yeast is being used as part of a looping mechanism to provide the cell with a quick way to start dividing once it has dealt with its environmental insult.  The authors show that yeast that can properly induce their CDC28 gene enter the cell cycle around 20 minutes faster than yeast that cannot induce the gene.  The cells are poised for a quick recovery.

And this is almost certainly not merely a yeast phenomenon.  Some recent work in mammalian cells has implicated lncRNAs in recruiting proteins involved in controlling gene activity through a looping mechanism as well (reviewed here).  Now that the same thing has been found in yeast, scientists can bring to bear all the powerful tools available to dissect out the mechanism(s) of lncRNA action.  And that’s far from a loopy idea…

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

Categories: Research Spotlight

Tags: transcription , DNA looping , Saccharomyces cerevisiae , lncRNA

Yeast, Smarter than a Train Wreck

January 30, 2014

Imagine you run a railroad that has a single track. You need for trains to run in both directions to get your cargo where it needs to go.

Not the best way to run a genome either. Image from the Cornell University Library via Wikimedia Commons

One way to regulate this might be to have the trains just go whenever and count on collisions as a way to regulate traffic. Talk about a poor business model! Odds are your company would quickly go bankrupt.

Another, more sane possibility is to somehow keep the trains from running into each other. Maybe you schedule them so their paths never cross. Or maybe you have small detours where a train can wait while the other passes. Anything is better than regulation by wreckage!

Turns out that at least in some cases, nature is a better business person than many people previously thought. Instead of trains on a track, nature needs to deal with nearby genes that point towards one another, so-called convergent genes. If both genes are expressed, then the RNA polymerases will barrel towards one another and could collide.

A new study in PLoS Genetics by Wang and coworkers shows just how big a deal this issue is for our favorite yeast Saccharomyces cerevisiae. An analysis of this yeast’s genome showed that not only did 20% of its genes fit the convergent definition but that in many cases, each gene in a pair influenced the expression of the other gene. Their expression was negatively correlated: when one of the pair was turned up, the other went down, and vice versa.

One way these genes might regulate one another is the collision model. When expression of one gene is turned up and a lot of RNA polymerases are barreling down the tracks, they would crash into and derail any polymerases coming from the opposite direction. A prediction of this model is that orientation and location matter.  In other words, the negative regulation would work only in cis, not in trans.  Surprisingly, the authors show that this is clearly not the case.

Focusing on four different gene pairs, Wang and coworkers showed that if the genes in a pair were physically separated from one another, their expression was still negatively correlated.  This was true if they just flipped one of the genes so the two genes were pointed in the same direction, and it was still true if they moved one gene to a different chromosome.  Clearly, collisions were not the only way these genes regulated one another.

Using missense and deletion mutation analysis, the authors showed that neither the proteins from these genes nor the coding sequence itself was required for this regulation.  Instead, the key player was the overlapping 3’ untranslated regions (UTRs) of the transcripts.  The authors hypothesize that the regulation is happening via an anti-sense mechanism using the complementary portions of the 3’ UTRs.

This anti-sense mechanism may be S. cerevisiae’s answer to RNAi, which it lost at some point in its evolutionary history.  Given the importance of RNA-mediated regulation of gene expression in other organisms, perhaps it shouldn’t be surprising that yeast has come up with another way to use RNA.  

Instead of RNAi, it relies on genomic structure and overlapping 3’ UTRs to regulate genes.  This may be a bit more cumbersome than RNAi, but at least yeast came up with a more clever system than polymerase collisions to regulate gene expression.  

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

Categories: Research Spotlight

Tags: UTR , transcription , RNA polymerase II , Saccharomyces cerevisiae

The Brazor of Biology

January 08, 2014

The janitor on the U.S. comedy series Scrubs is always coming up with terrible inventions. One of his worst was the knife-wrench. It is what it sounds like—a tool with a knife at one end and a wrench at the other.

Just like this brush-razor, or brazor, Cet1p has two distinct, but related, functions.

Of course not all dual purpose tools have to be so useless.  Imagine a tool like the one at the right with a razor at one end and a toothbrush on the other.  Now you can easily brush your teeth and shave in the shower or at your bathroom sink (as long as you are careful not to cut your cheek).

Turns out that biology has these dual purpose tools too except that they are almost always more useful.  For example, Lahudkar and coworkers show in the most recent issue of GENETICS that Cet1p doesn’t just help out with capping mRNA.  No, these authors found that it also helps clear RNA polymerase II (RNA pol II) away from promoters.  And what’s most interesting is that this second function has little to do with its job in mRNA capping.

Basically the two functions are probably in the same protein because they both happen in the same place, at the start site of a promoter.  Just like our brazor is useful because both jobs happen in the bathroom.

The first step was to show that in the absence of Cet1p, RNA pol II was more likely to be found near the start of transcription.  The authors showed that this was the case by using a temperature sensitive mutant of Cet1p and a chromatin immunoprecipitation (ChIP) assay targeted at RNA pol II—there was more RNA pol II crowded near the promoter at the nonpermissive temperature. 

The next set of experiments showed that merely messing with the cap is not sufficient to cause the polymerase to pause. Lahudkar and coworkers found that RNA pol II occupancy was unchanged in strains carrying mutations in STO1 (also known as CBP80) or CEG1, two components of the capping machinery. Cet1p apparently has a separate, unrelated function in helping to clear polymerases away from the start site of transcription.

The final set of experiments showed that the unpausing activity of Cet1p was found in a different part of the protein from its capping function.  Cet1p be can be broadly divided into three regions—a poorly characterized N-terminal domain (amino acids 1-204), a Ceg1p interaction domain (aa 205-266), and a triphosphatase domain (aa 265-549).  The last two domains are critical to its capping function.

Lahudkar and coworkers found that deleting the 1-204 aa domain from Cet1p caused polymerase stalling at the promoter without affecting its capping ability.  And conversely, that when they impaired the ability of Cet1p to perform its capping function while retaining its 1-204 aa domain, RNA pol II escaped the promoter at the same rate as it did in the presence of wild type Cet1p.  A final experiment showed that just expressing the first 300 amino acids of Cet1p was sufficient to get the polymerases moving. 

All in all these experiments provide strong evidence that Cet1p has two separate functions—an enzymatic role in capping mRNA and an unrelated activity that helps clear RNA pol II from the regions around the promoters of genes.  Which all goes to show that even when you think you have a handle on a protein, it can still surprise you with something new.  Turn it around and you just might find a toothbrush at the end.

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

Categories: Research Spotlight

Tags: transcription , mRNA capping , Saccharomyces cerevisiae , bifunctional protein

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.

Too much noise is bad for individuals.

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

Old Genes, New Tricks

March 14, 2013

You can’t teach an old dog new tricks, or so the saying goes. But imagine you found that your old dog knew a complicated trick and had been doing it all her life, right under your nose, without your ever noticing it! You’d be surprised – about as surprised as the Hinnebusch group at NIH when they discovered that some long-studied S. cerevisiae genes had an unexpected trick of their own.

old dog

Don’t underestimate old dogs or well studied genes. Sometimes they’ll surprise you!

They were working on the VPS* (vacuolar protein sorting) genes. While known for a very long time to be important in protein trafficking within the cell, Gaur and coworkers found that two of these genes, VPS15 and VPS34, play an important role in RNA polymerase II (pol II) transcription elongation too. Now there is an unexpected new trick…like your dog learning to use a litter box!

There had been a few hints in recent years that the VPS genes, especially VPS15 and VPS34, might have something to do with transcription. Following up on these, the researchers tested whether vps15 and vps34 null mutants were sensitive to the drugs 6-azauracil and mycophenolic acid. Sensitivity to these drugs is a hallmark of known transcription elongation factors. Sure enough, they were as sensitive as a mutant in SPT4, encoding a known transcription elongation factor. Further experiments with reporter genes and pol II occupancy studies showed that pol II had trouble getting all the way to the end of its transcripts in the vps mutant strains.

There was a bit of genetic interaction evidence that had suggested that there might be a connection between VPS15, VPS34, and the NuA4 histone acetyltransferase complex. This is important, since NuA4 is known to modify chromatin to help transcription elongation. Looking more closely, the researchers found that Vps34p and Vps15p were needed for recruitment of NuA4 to an actively transcribing reporter gene.

Other lines of investigation all pointed to the conclusion that these VPS proteins have a role in transcription. They were required for positioning of several transcribing genes at the nuclear pore, could be cross-linked to the coding sequences of transcribing genes, and could be seen localizing at nucleus-vacuole junctions near nuclear pores.

One appealing hypothesis to explain this has to do with what both genes actually do. Vps34p synthesizes phosphatidylinositol 3-phosphate (PI(3)P) in membranes, while Vps15p is a protein kinase required for Vps34p function. The idea is that when Vps15p and Vps34p produce PI(3)P at the nuclear pore near transcribing genes, this recruits the NuA4 complex and other transcription cofactors that can bind phosphoinositides like PI(3)P. There are hints that this mechanism may also be at work in mammalian and plant cells.

There’s a lot more work to be done to nail down the exact role of these proteins in transcription. But this story is a good reminder to researchers that new and interesting discoveries may always be hiding in plain sight.

* These genes were also called VPL for Vacuolar Protein Localization and VPT for Vacuolar Protein Targeting

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

Categories: Research Spotlight

Tags: transcription , VPS genes , RNA polymerase II , Saccharomyces cerevisiae

Puzzling Out Gene Expression

February 21, 2013

Have you ever put together a million piece puzzle that was all blue? That is sort of what it sometimes feels like figuring out how genes are turned on or off, up or down.

jigsaw puzzle

There are hundreds or even thousands of proteins called transcription factors (TFs) controlling gene expression. And there is a seemingly simple but frustratingly opaque string of DNA letters dictating which TFs are involved at a particular gene. Figuring out which sets of proteins bind where to control a gene’s expression can be a baffling ordeal.

Up until now most of the ways of identifying which TFs are bound at which genes have been incredibly labor intensive to do on a large scale. With all of the current techniques, researchers need to construct sets of reagents before they even get started. For example, to be able to immunoprecipitate TFs along with the DNA sequences they bind, you need to insert epitope tags in all the TF genes so an antibody can pull them down. Other techniques are just as involved.

What the field needs is a quick and dirty way to find where TFs bind in the genome. And now they just might have one.

In a new study, Mirzaei and coworkers used a modification of the well-known technique mass spectrometry (mass spec) to identify TFs that bind to a specific piece of DNA. With this technique, called selected reaction monitoring, the mass spec looks only for specific peptide sequences. This not only makes it much more sensitive and reproducible than ordinary mass spec, but it should also be relatively straightforward to do if a lab has access to the right sort of mass spec. They haven’t worked out all the bugs and it is definitely still a work in progress, but the technique looks promising.

Mirzaei and coworkers set up assays to detect 464 yeast proteins that are known or suspected to be involved in regulating RNA polymerase II transcription. Then they tested their assay on a 642 base pair piece of DNA known to contain signals that affect the levels of FLO11 transcription. They found fifteen proteins (out of the 222 they searched) that bound this piece of DNA. Of these, only one, Msn1p, had been previously identified as regulating the FLO11 gene. The other fourteen had not been found in any previous assays.

The authors next showed that two of these fourteen proteins, Mot3p and Azf1p, represented real regulators of the FLO11 gene. For example, deletion of MOT3 led to a threefold increase in FLO11 expression under certain conditions. And when AZF1 was deleted, FLO11 could not be activated under a different set of conditions. So Mot3p looks like a repressor of FLO11 and Azf1p looks like an activator.

This was a great proof of principle experiment, but much more work needs to be done before this will become a standard assay in the toolkit of scientists studying gene expression. They need to figure out why some known regulators of FLO11 (Flo8p, Ste12p, and Gcn4p) were missed in the assay and whether the other twelve proteins they discovered play a role in the regulation of the FLO11 gene.

Having said this, it is still important to note that even this early stage model of the assay identified two proteins that scientists did not know controlled FLO11 gene expression. At the very least this is a quick and easy way to quickly identify candidates for gene expression. We may not be able to use it to see the whole picture on the puzzle, but it will at least get us a good start on it.

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

Categories: Research Spotlight

Tags: transcription , RNA polymerase II , Saccharomyces cerevisiae

When Polymerases Collide

October 25, 2012

Lots of recent studies are showing that transcription happens over way more DNA than anyone previously thought. For example, the ENCODE project has shown that most of a genome gets transcribed into RNA in humans, fruit flies and nematodes. This transcriptional exuberance was recently confirmed in the yeast S. cerevisiae as well.

There is also a whole lot of antisense transcription going on. Taken together, these two observations suggest that there are lots of opportunities for two polymerases to run headlong into each other. And this could be a big problem if polymerases can’t easily get past one another.

Goats butting heads

What happens when RNA polymerases meet head-on?

Imagine that the two polymerases clash in the middle of some essential gene. If they can’t somehow resolve this situation, the gene would effectively be shut off. Bye bye cell!

Of course this is all theoretical at this point. After all, smaller polymerases like those from T3 and T4 bacteriophages manage to sneak past one another. It looks like this isn’t the case for RNA polymerase II (RNAPII), though.

As a new study by Hobson and coworkers in Molecular Cell shows, when two yeast RNAPII molecules meet in a head on collision on the same piece of DNA, they have real trouble getting past each other. This is true both in vitro and in vivo.

For the in vivo experiments, the authors created a situation where they could easily monitor the amount of transcription close in and far away from a promoter in yeast. Basically they pointed two inducible promoters, from the GAL10 and GAL7 genes, at one another and eliminated any transcription terminators between them. They also included G-less cassettes (regions encoding guanine-free RNA) at different positions relative to the GAL10 promoter, so that they could use RNAse T1 (which cleaves RNA at G residues) to look at how much transcription starts out and how much makes it to the end.

When they just turned on the GAL10 promoter, they saw equal amounts of transcription from both the beginning and the end of the GAL10 transcript. But when they turned on both GAL10 and GAL7, they saw only 21% of the more distant G-less cassette compared to the one closer to the GAL10 promoter.

They interpret this result as meaning the two polymerases have run into each other and stalled between the two promoters. And their in vitro data backs this up.

Using purified elongation complexes, they showed that when two polymerases charge at each other on the same template, transcripts of intermediate length are generated. They again interpret this as the polymerases stopping dead in their tracks once they run into one another. Consistent with this, they showed that these stalled polymerases are rock stable using agarose gel electrophoresis.

Left unchecked, polymerases that can’t figure out how to get past one another would obviously be bad for a cell. Even if it were a relatively rare occurrence, eventually two polymerases would clash somewhere important, with the end result being a dead cell. So how do cells get around this thorny problem?

King Arthur and the Black Knight

To get past the Black Knight, Arthur had to destroy him. Hopefully the cell has more tricks up its sleeve than that!

One way is to get rid of the polymerases. The lab previously showed that if a polymerase is permanently stalled because of some irreparable DNA lesion, the cell ubiquitinates the polymerase and targets it for destruction. In this study they used ubiquitin mutants to show that the same system can work at these paused polymerases too. Ubiquitylation-compromised yeast took longer to clear the polymerases than did their wild type brethren.

The authors think that this isn’t the only mechanism by which polymerases break free though. They are actively seeking factors that can help resolve these crashed polymerases. It will be interesting to see what cool way the cell has devised to resolve this dilemma.

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

Categories: Research Spotlight

Tags: transcription , RNA polymerase II , ubiquitin-mediated degradation , Saccharomyces cerevisiae

Yeast with Sticking Power

July 16, 2012

Stickier yeast might make beer brewing easier.

Most strains of Saccharomyces cerevisiae don’t stick together very well. And hardly any of them form biofilms. But it would be very useful to have a better understanding of why some strains like to stick together and others do not. 

Stickiness helps in any process where you want the yeast to do something and then get rid of it. An obvious example is ethanol production either for energy or to make our beer and wine. After sticky yeast are done with their job of making the alcohol, they simply fall to the bottom of the fermentor or float on the surface in a biofilm (the “flor”). This makes the step of separating the yeast from the finished product that much easier.

Understanding more details of yeast stickiness would also be useful for studying harmful yeast. Adhesion to other cells and to substrates is an important factor in pathogenesis. It would be nice to investigate this phenomenon in the more tractable brewer’s yeast.

The Ibeas lab has decided to figure out why most strains of S. cerevisiae can’t flocculate by comparing one of the few that can (the “flor” strain used to make sherry) to a reference strain that can’t.  They previously showed that a key gene in the process, FLO11 (also known as MUC1), is expressed at much higher levels in flor.  They were also able to show that a large part of this increased expression comes from a 111 base pair deletion in the FLO11 promoter in this particular strain.

In a recent paper in GENETICS, Barrales and coworkers set out to investigate why the loss of these 111 base pairs leads to increased gene expression.  They were able to conclude that the deletion does not significantly affect histone occupancy at the promoter.  What they could see was that histone placement was affected and that PHO23 may play a significant role in this.

The researchers had previously shown that the histone deacetylase complex (HDAC) Rpd3L was important for maximal FLO11 activity.  They next wanted to determine if this complex was the major player in explaining the increased activity of the 111 bp deletion FLO11 promoter (Δ111) over the wild type (WT) one.  They did this by comparing the level of mRNA made by each promoter in strains lacking either the Pho23p or the Rpd3p subunits of the Rpd3L complex.  They found that the Δ111 construct was much more severely compromised by the loss of PHO23 than was the WT one.  (A bit confusingly, neither was much affected by the loss of RPD3.) 

Given that PHO23 is part of a complex that affects chromatin, the next thing the researchers did was look at the histones in and around both FLO11 promoters.  They found that PHO23 was involved in maintaining an open chromatin structure at the FLO11 promoter but that deleting the 111 base pairs didn’t affect this process significantly.

Where they started to see subtle differences was when they looked at histone placement as opposed to occupancy.  Using micrococcal nuclease protection to map chromatin structure, they found a number of differences between the two promoters, centered on the deletion and the TATA box, and deleting PHO23 affected the two promoters in different ways.

It appears that FLO11 is upregulated in the flor strain because the deletion of 111 base pairs leads to an altered chromatin structure.  The next steps will be to figure out what this means and then to use that knowledge to create stickier yeast. We’ll end up with a better understanding of transcriptional regulation and adhesion, and beer and wine makers may end up with even better self separating yeast.

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

Categories: Research Spotlight

Tags: transcription , flocculation , Saccharomyces cerevisiae , biofilm