New & Noteworthy

Unlocking Chromatin

February 10, 2016

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In Die Hard, Hans Gruber and associates need to break through seven locks in the right order on a safe to get to bearer bonds worth 640 million dollars. Of course the hero John McClane foils the plot and beats the villains.

Nothing so exciting in yeast, but some genes are nearly as hard to turn on as that safe was to open. One of the most stubborn is the HO gene. It requires three locks or gates be opened in the right order to start making the HO endonuclease.

A new study in GENETICS by Yarrington and coworkers shows that the second lock for HO is a set of nucleosomes that blocks the binding of the transcription activator SBF. When they rejiggered this promoter so that these nucleosomes were removed, the HO gene needed fewer steps to get activated.

It is as if Hans Gruber and his gang only had five or six locks to get through to open their safe. And the 7th, hardest one was removed.

The HO gene is usually turned on in three sequential steps. First the Swi5p activator binds to a region called URS1, which recruits coactivators that then remodel the chromatin at the left half of URS2 (URS2-L). This allows SBF to bind its previously hidden binding sites which then remodels the chromatin again. Now a second set of SBF sites is revealed in the right half of URS2 (URS2-R).

These authors set out to provide direct proof that nucleosome positioning over URS2-L is the key to the second lock. They did this by making a set of chimeric promoters between HO and CLN2.

Both of these promoters are activated by SBF. A key difference between the two is that the CLN2 promoter, like 95% of yeast promoters, is in a nucleosome depleted region (NDR).

The idea then is to make an HO promoter in which the usual URS2-L is replaced with the NDR region of CLN2. If the nucleosomes matter over URS2-L, then this construct should be activated in two instead of three steps.

Or, to put it another way, Swi5p binding to URS1, the first lock, will no longer be needed to open the second lock. HO activation will now be Swi5p independent. This is what the authors found.

When they looked at their chimeric protein that lacked nucleosomes over URS2-L, they found that using a strain deleted for SWI5 had very little effect on activity. There was only around a 2-fold difference in activity with this construct in the wild type and SWI5-deleted strains. This is very different than the wild type HO promoter where there was around a 15-fold difference between the two strains.

The authors then did an additional experiment where they took their chimeric reporter and mutated the nucleosome depleted region such that nucleosomes could bind there. This construct was now more Swi5p-dependent: there was around a 5-fold difference in activity between the wild type and SWI5 deletion strains. They had at least partially rebuilt that second lock.

Yarrington and coworkers continued with ChIP experiments to confirm that their chimeric construct was indeed depleted for nucleosomes, as well as other experiments to tease out more subtle details about the regulation.

Given that it switches a yeast cell’s mating type, it isn’t surprising that the HO gene is under such lock and key. The yeast cell wants to make sure it only turns on when needed. Just as the Nakatomi Corporation wanted to make sure only the right people could get to that fortune in bearer bonds.

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

Categories: Research Spotlight

Tags: chromatin remodeling, HO endonuclease, nucleosomes, transcription regulation

Pulsating Yeast

November 19, 2015

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Sometimes when you get a minor injury, doctors will recommend alternating heat and cold as a therapy. The heat opens things up and the cold shuts them back down again.

Now obviously it would be pretty useless to apply both at the same time. Adding a bit of lukewarm water to an injury is not going to be very helpful at all.

The same thing holds true for many genes. If activators and repressors all turned on at the same time, there wouldn’t be much of an effect on the expression of a gene regulated by both. It is no way to respond to something in the environment!

Instead, if you want a gene to go up and then go back down again, you’d have the activator turn on first, followed by the repressor. Another way to put this is you’d have a pulse where all of the activators activate their genes at once and then stop working followed by a pulse where all of the repressors work at once.

This is exactly what Lin and colleagues found in their recent study in Nature. There they looked at the effect of certain external stimuli on the timing of when the activator Msn2p activated genes and when the repressor Mig1p repressed genes in our favorite yeast S. cerevisiae. These transcription factors coregulate many of the same genes.

The authors found that in the presence of either lowered glucose concentrations or 100 mM NaCl, most of the Msn2p in the cell turned on first followed closely by the Mig1p repressors. In the absence of either stimulus, there was no coordination.

So there does seem to be a carefully choreographed dance between these two transcriptional regulators with these signals. But of course gene regulation is a bit more complex than a sprained ankle.

There may be situations where a cell wants both regulators to do their jobs at the same time. Sometimes lukewarm water may be just what the doctor ordered.

And this is what Lin and colleagues found with 2.5% ethanol. Under this condition, the pulses of the two regulators overlapped—both were on at the same time. Apparently different stimuli call for different responses which means different timing of transcription factor pulses.

The authors next wanted to get at why Mig1p repression lagged behind Msn2p activation. Since both transcription factors can only enter the nucleus and do their job after they lose a few key phosphate groups, the authors reasoned that perhaps Mig1p dephosphorylation lagged behind that of Msn2p.

They decided to look at the PP1 phosphatase, Glc7p, as previous work had shown that it can indirectly regulate both Msn2p and Mig1p. And indeed, when the authors lowered the expression of GLC7, Msn2p and Mig1p no longer pulsed one after the other at lower glucose concentrations. It looks like Glc7p is a key player in controlling the pulsing of these two regulators.

Even though much of this work was done with synthetic promoters with Mig1p and Msn2p binding sites, the results were not restricted to these artificial constructs. Lin and colleagues found that around 30 endogenous targets also responded to lowered glucose concentrations in a coordinated way just like their synthetic construct. Yeast regulates genes by controlling when activators and repressors pulse.

Finally, all of these studies were done using fluorescent proteins and filming single cells in real time. (Is biology cool or what?) This makes sense because subtle signs of synchronization can be lost when averaged over a large population.

This also allowed the authors to investigate what happens in unstimulated cells. In other words, what happens when both regulators enter the nucleus at the same time? Or if a repressor gets in first?

The first thing they found was that even in the absence of stimulation, there were still pulses. So at seemingly random times, suddenly all of the Msn2p would swoop into the nucleus at the same time and then all leave a short time later. Or the same thing would happen with Mig1p.

If by chance the two entered the nucleus at the same time, both the synthetic reporter and an endogenous gene, GSY1, were not activated. But if Msn2p happens to get in there first, both were activated.

And if the repressor Mig1p managed to get into the nucleus at least 4-5 minutes before Msn2p, activation by Msn2p was muted. The presence of Mig1p beforehand seemed to keep Msn2p from activating coregulated genes to as high a level.

Taken together these results confirm that just like a synchronized swim team, yeast regulates genes by controlling when activators and repressors can work. First there is a pulse where the all of the molecules of a certain activator are primed to do their job and then, after a short time, they all stop doing their job. This can then be followed later by a pulse of repressors shutting it all down.

And this isn’t just in yeast either. For example, these kinds of pulses are important in neuroscience as well.

This work suggests that in dissecting regulatory pathways, researchers may need to pay more attention to the timing of pulses. Then they can see that hot followed by cold makes much more sense than both together.

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

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, transcription regulation

Dealing With Alcohol, a Messy Business

September 16, 2014

Different people can respond to alcohol differently because of their genes.  For example, many Asians flush or even become ill from alcohol because of a mutation in their ALDH2 gene. (This is not just a minor annoyance—these unpleasant side effects come with a significant increase in esophageal cancer rates.)

This is a simple example where one gene has a significant effect. But of course, not everything to do with people and alcohol is so simple at the genetic level! 

For example, some people can drink you under the table while others are lightweights.  Some of this has to do with their lifestyle (how often they drink, how much they weigh, etc.), but a lot undoubtedly has to do with the variations they carry in multiple genes.

Well, it turns out this is also the case for yeast (our friend in the alcohol business). A new paper in GENETICS by Lewis and coworkers confirms that different strains of the yeast Saccharomyces cerevisiae tolerate high levels of alcohol differently because of their specific genetics. And at first the response seems…shall we say…incapacitatingly complex.

The results are interesting in that they help parse out how yeast responds to ethanol, but the implications are more far-reaching than that.  This analysis helps to form the framework for investigating how natural variation in gene expression can affect the traits of individuals and their responses to certain environmental stimuli.

Lewis and coworkers used three strains in their study: a lab strain that came from everyone’s favorite workhorse S288c, the strain M22 from a vineyard, and the oak soil strain YPS163.  They had previously shown that thousands of genes in each strain responded differently to 5% ethanol.  In this study they set out to find out what was behind these differences.

First off they wanted to confirm their previous results.  Using six biological replicates, they found that 3,287 genes out of a total of 6,532 were affected in at least one strain when treated for 30 minutes with 5% ethanol.  This is over half the genes in the genome!

To try to get a handle on what is causing such widespread effects, they next performed eQTL mapping in 45 F2 crosses of S288c X M22 and S288c X YPS163 (these particular matings were chosen because much of the variation they saw was in S288c).  This analysis was designed to try to find “hotspots” in the genome:  loci that affected many different transcripts, or that could account for all the variation they saw.

When they did this analysis they found 37 unique hotspots. Each hotspot represented 20-1,200 different transcripts, with a median of 37 transcripts.  Of these, 15 were seen in both crosses, 12 in just the S288c X M22 and 10 in the S288c X YPS163 matings. No silver bullet, but 37 is certainly easier to work with than 3,287!

Lewis and coworkers next set out to find the key gene(s) in the hotspots responsible for affecting multiple transcripts in the presence of ethanol.  Some were easy to find.  For example, HAP1 in S288c and CYS4 in M22 X S288c.  But the big prize in this analysis probably goes to MKT1, which affected over 1,000 transcripts in this study.

Now MKT1 is not one of the usual suspects, in that it is not a transcription factor.  However, MKT1 has been implicated in lots of observed differences between strains, including alcohol tolerance in one Brazilian overproduction strain.  Given this, the authors set out to explore whether there were any differences in Mkt1p activity in response to ethanol in the different strains.

This analysis revealed that Mkt1p localizes to P-bodies upon ethanol stress in S288C but not YPS163. And this wasn’t some general defect in Mkt1p, since it is known to colocalize with P-bodies in both strains in response to hypo-osmotic stress.

With this discovery, things were starting to make a bit more sense!  Since P-bodies are involved in mRNA turnover, it follows that a P-body component might affect so many transcripts. One potential explanation might be that Mkt1p serves as a regulator by translationally silencing specific mRNAs at P-body loci. This would be consistent with its known role in translational regulation of the HO transcript.

This study reveals how difficult it is to get to the bottom of determining exactly how massive differences in gene expression lead to differences in traits.  But it also shows that while daunting, it is doable.  And perhaps yeast can show us how best to interrogate our own differences in gene expression to help figure out why we are the way we are—not only in terms of whether we dance on the tables or fall to the floor after a few drinks, but in many other respects as well.

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

Categories: Research Spotlight

Tags: eQTL mapping, ethanol response, Saccharomyces cerevisiae, transcription regulation

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