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

Of Medieval Market Townes and Wasp Guts

February 3, 2016

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Back before trains, planes and automobiles, people didn’t get around as much. And for the people of medieval Europe, this could be a real problem genetically.

At this time there were a lot of small, isolated villages scattered across Europe. If people in these villages stayed put, inbreeding might have gotten as bad as the poor Spanish Hapsburgs. Their last king, Charles II, was infertile, riddled with genetic diseases and his royal line died out with him.

One reason (among many) that this didn’t happen to people all over Europe was market towns. These were centrally located places where villagers came to sell goods. And where they also found partners to bring home to freshen up the gene pool.

Turns out that out in the wild, our friend yeast is in an even worse predicament than medieval Europeans. Because they are all clones of each other, they exist in isolated colonies with almost no genetic diversity.

Yeast are also way less mobile than people. They do have spores but these don’t tend to travel very far without help.

And yet, looking at yeast DNA shows that yeast definitely get around. There are all sorts of signs of various DNA mixing over time. So where are all these yeast hooking up?

A new study by Stefanini and coworkers in PNAS suggests that yeasts’ market towns are in the guts of wasps. It is there that various yeasts can meet and mate before heading back to their “villages.”

This makes sense in a lot of ways. First off, as we described in an earlier blog, there is good evidence that yeast winter in wasp guts.

So there are definitely a variety of yeast hanging around for months, waiting for warmer weather. The gut is also the kind of harsh place where spore dissolution, the first step in yeast mating, can happen.

When the authors looked at the yeast isolates from a wasp’s gut they saw a lot more outbreeding compared to other sources. This suggests that a lot of mating is indeed going on there.

The next step was to directly test how much mating can actually happen in a wasp gut. Stefanini and coworkers tested this by having the wasps eat five different yeast strains and then analyzing the isolates genetically over time. They compared the results from this experiment to the amount of mating that happens in wine must and under ideal lab conditions.

What they found was a whole lot of mating going on.

After two months, around 1/3 of the yeast in the wasp’s gut were outcrossed. This is OK but pretty comparable to what is found in wine must.

It was a different story after four months. Now 90% of the yeast were outcrossed. This is an even better result than scientists typically get in the lab. Clearly the wasp gut is a great place for a yeast to find a partner.

The authors also found that the S. paradoxus strain had to mate to survive in the gut. The only time they found this strain in yeast isolates was in hybrids with S. cerevisiae.

The next steps will be to see if this kind of mating actually has a big effect on yeast diversity in the wild. And of course what, if anything, the wasp gets out of hosting these cavorting yeast.

A market town was great for both the town and the visitors. People met up, sold goods, found partners and the towns prospered from all of this traffic. I can’t wait to find out if the wasp/yeast situation is so mutually beneficial as well.

Jerry Lee Lewis has a whole lot of shakin’ going on, just like a wasp’s gut has a whole lot of matin’ going on.

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

Unfrying An Egg

January 20, 2016

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Eggs start out as slimy and awful, but can end up warm, firm and wonderful. All it takes is some heat to denature the egg proteins and voilà, a tasty breakfast.

Not that anyone would want to do it, but of course it is impossible to do the reverse. You can’t take a fried egg and turn it back into a raw one. The denaturation is pretty much permanent.

When a cell is hit with high temperatures, its proteins start to denature as well. And scientists thought that most of the denaturation of many of these proteins was as irreversible as the eggs. The thought was that many or most of these denatured proteins were “eaten” through proteolytic degradation. Although cellular chaperones are capable of disaggregating and refolding some heat-denatured proteins, it wasn’t known which aggregated proteins met which fate in a living cell.

A new study out in Cell by Wallace and colleagues shows that at least in yeast, most eggs get unfried. After a heat shock, aggregated proteins in the cell return to their unaggregated form and get back to work.

Now those earlier scientists weren’t crazy or anything. The proteins they looked at did indeed clump up and get broken down by the cell after a heat shock. But these were proteins introduced to the cell.

In the current study, Wallace and colleagues looked at normal yeast proteins being made at their normal levels. And now what happens after a brief heat shock is an entirely different story.

The first experiment they did looked at which endogenous yeast proteins aggregated after they were shifted from their normal 30 to 46 degrees Celsius for 2, 4, or 8 minutes. The researchers detected aggregation using ultracentrifugation—those proteins that shifted from the supernatant to the pellet after a spin in the centrifuge were said to have aggregated.

Using stable isotope labeling and liquid chromatography coupled to tandem mass spectroscopy (LC-MS/MS), they were able to detect 982 yeast proteins easily. Of these, 177 went from the supernatant to the pellet after the temperature shift. (And 4 did the reverse and went from the pellet to the supernatant!)

After doing some important work investigating these aggregated proteins, the researchers next set out to see what happened to them when the cells are returned to 30 degrees Celsius. Are they chewed up and recycled, or nursed back to health and returned to the wild?

To figure this out they did an experiment where proteins are labeled at two different times using two different labels. The researchers first grew the yeast cells at 30 degrees Celsius in the presence of arginine and lysine with a “light” label. This labels all of the proteins in the cell that have an arginine and/or lysine.

Then the cells are washed and a new media is added that contains “heavy” labeled arginine and lysine. The cells are shifted to 42 degrees Celsius for 10 minutes and then allowed to recover for 0, 20, or 60 minutes.

After 60 minutes of recovery, the ratio of light to heavy aggregated proteins looked the same as proteins that hadn’t aggregated. In other words, aggregation did not cause proteins to turn over more quickly.

It looks as if aggregated proteins are untangled and allowed to go about their business. So after a heat shock the cell doesn’t throw its hands in the air and simply start things over.

Other experiments done by Wallace and coworkers in this study, that we do not have the space to tackle here, suggest that the cell has an orderly process for dealing with heat stress. After a heat shock, certain proteins aggregate with chaperones in specific areas of the cell. Once the temperature returns to normal, these stress granules disassemble and the aggregated proteins are released intact.

None of this will help us unfry an egg — a denatured egg protein is obviously significantly different than an aggregated protein protected by chaperones in a stress granule. But this study does help us better understand how our cells work. And that’s a good thing.

Unlike Mr. Bill’s dog, most aggregated yeast proteins can return from a heat shock.

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

Join SGD at The Allied Genetics Conference

January 12, 2016

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SGD will be attending The Allied Genetics Conference (TAGC) in Orlando, Florida, July 13–17, 2016! For the first time ever, the meetings of the yeast, C. elegans, ciliate, Drosophila, mouse, and zebrafish model organism communities will be united under one roof, along with a new meeting on population, evolutionary, and quantitative genetics.

Submit your abstracts now! Abstract submission will be open until March 23, 2016. If you want GREAT science and access to the leaders of the field, then TAGC is the place for you. SGD will be there, will you?