New & Noteworthy

Resistance is Not Futile: How Yeasts Avoid Getting Ill from IILs

September 24, 2018


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In the Batman comics, Poison Ivy is resistant to all natural toxins and diseases (from deviantart)

Resistance to poisoning is a good thing in the cartoon world, where both good and bad guys and gals often have superpowers that render them resistant to all sorts of toxins and poisons. But does that happen in the “real” world that we live in?

Well, some people would say that Keith Richards and cockroaches are the ultimate examples of earthly organisms resistant to every known toxin! But there are other more mundane examples of humans that are resistant or tolerant to certain chemical or organic compounds.

For example, almost everyone knows someone (or themselves) who can’t drink milk, because if they do, they feel really sick, with uncomfortable or even severe gastrointestinal symptoms. These people are said to be “lactose intolerant” (which is NOT an allergy to milk), because their gut can’t digest a sugar called lactose found only in milk and milk-based products.

Interestingly, all humans have the “lactase” gene that allows their body to digest lactose, but lactose intolerant folks have a particular “switch” sequence in their DNA that causes the gene to get totally turned off once they’re not babies anymore.

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The lactase enzyme splits the disaccharide lactose into galactcose (1) plus glucose (2) (from wikimedia)

However, people who CAN drink milk as adults (“lactose tolerant” people) have a change in their DNA sequence that inactivates the switch. So these people have the lactose-digesting gene turned on and functioning for their whole lives and can consume milk and dairy products even as adults.

This is an example of genomic diversity among human beings, where people’s individual genome sequences have differences that can make them resistant to the negative effects of some foods or chemicals, while other people have little or no tolerance at all.

This type of DNA sequence variation across all the members of a species is called “standing genetic variation” and it’s a very good thing. Why? Because when a changing or novel environment challenges a population, standing genetic variation increases the chance that some individuals can adapt to new types of foods (like what happened with the lactase gene) or have other characteristics that make them better suited to survive in the new environment…or even be resistant to new diseases!

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In the Planet of the Apes series, “simian flu” kills almost all humans except for those few who are resistant (from deviantart)

But what does all of this have to do with yeast? Well, just like lactose intolerant humans, yeast can get sick when they try to “eat” certain things too, and will fail to grow well in the presence of such compounds. But could there be some yeast individuals somewhere in the world that have tolerance to certain normally-toxic compounds?

This is exactly the question that Higgins and co-workers asked, and in the September issue of Genetics they describe how they found naturally occurring yeast that can tolerate high levels of compounds called “ionic liquids.” And they also discovered the underlying naturally-occurring genetic variations in two genes that make the yeasts more tolerant to these compounds!

So what are ionic liquids and why were these investigators interested in them? Ionic liquids are a type of salt that can exist in a liquid state at temperatures under the boiling point of water or even at room temperature.

One type in particular, “imidizolium ionic liquids” (IILs) are often used in production of biofuels because they efficiently solubilize plant biomass cellulose and help turn it into glucose. The glucose can then be fermented (often by our yeast friend Saccharomyces cerevisiae) into bioethanol or other biofuels.

However, most commonly used S. cerevisiae strains, when grown in the presence of IILs, get very ill. But if IIL-tolerant yeasts could be found, they could help improve the production of cellulosic ethanol and other bioproducts.

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Cellulosic biofuels may help decrease worldwide carbon emissions by reducing fossil fuel use (from wikimedia)

Higgins and co-workers decided to make use of the standing genetic variation within the S. cerevisiae species by taking hundreds of different strains of S. cerevisiae, isolated from around the world, and seeing if any of them could grow better in the presence of IILs.

And indeed they found some strains that were extremely IIL-tolerant compared to other S. cerevisiae strains such as beer, wine or lab strains, or even those commonly used in biofuel production! The yeast strain that was the most IIL-tolerant was, surprisingly, a clinical isolate from Newcastle, England.

The researchers then took genomic DNA from this IIL-tolerant strain and chopped it up into large pieces and put the pieces (carried on special plasmids called fosmids) into the common S288C lab strain (which is intolerant to high IILs) to see which regions could allow the lab strain to now grow in the presence of high levels of IILs.

They found two genes that conferred tolerance to the IILs: the SGE1 gene, plus a previously unnamed gene, YDR090C, which had not been studied very much. The proteins made from both genes appear to be located in the plasma membrane of the cell. The authors propose that the tolerant version of the SGE1 protein, already known to be a multidrug efflux pump that exports toxic cationic dyes out of the cytoplasm, is directly involved in the pumping the IILs out of the cells to help yeast tolerate these toxic compounds.

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The Sge1 protein is a multidrug transporter. Similar proteins in humans can cause resistance to anti-cancer drugs (from wikimedia)

The researchers were not able to exactly figure out what YDR090C is doing in the membrane to help cells be resistant to the bad effects of IILs, but they did find out that cells with a deletion of the gene were less tolerant to IILs. They thus named this gene “ILT1” for “Ionic Liquid Tolerant”.

The authors also found that the SGE1 gene from the IIL-resistant “wild” strain from England had a change in its protein sequence relative to the lab strain, but rather than making the protein more efficient at pumping, it looks like the changed protein is more abundant in the cell and thus can just pump out more of the obnoxious IILs.

Whenever they put this resistant gene version into an IIL-intolerant yeast, it did the trick of allowing the strain to grow better in high IILs concentration. This discovery might allow greater use of IIL-treated biomass for the production of biofuels, as it shows one powerful method of increasing the tolerance of biofuel-producing yeast to toxic IILs.

So this is indeed a case where looking at the “standing genetic variation” of a species has helped discover new and useful biotechnological functions in yeast, and also shown us that resistance (to IILs at least) is NOT futile, and may indeed help us make yeast more efficient at making more biofuels!

Resistance might be futile if you’re up against the Borg, but at least yeast can resist IILs!

by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: lignocellulosic biomass, biofuels, variation

Chaperone Wars: The Last Co-chaperone

August 22, 2018


In Star Wars: The Force Awakens, the evil First Order rises up and threatens to eliminate the New Republic. The protagonists, who join forces of the Resistance, must find the reclusive Jedi Master Luke Skywalker so that he can take up arms against the First Order and save the galaxy.

(Warning: Star Wars spoilers below!)

One of the protagonists, Rey, keeps precious cargo with her as she searches for Luke: the lightsaber of Anakin Skywalker, Luke’s father. She must deliver the lightsaber to Luke in order to galvanize his joining the Resistance and to help motivate him to resume his role as a Jedi Master.

Just like how the galaxy would be doomed if Rey could not reach Luke, budding yeast cells would be doomed if a Hsp70 protein chaperone could not interact with another chaperone, Hsp90. But Hsp70 isn’t trying to deliver a lightsaber to Hsp90—instead, Hsp70 is trying to deliver “client” protein substrates, so that Hsp90 can help fold and mature these proteins.

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Like how R2-D2 helped Rey find Luke, Sti1p helps Hsp70 and Hsp90 interact. Image from http://starwars.wikia.com/wiki/Star_Wars:_Episode_VII_The_Force_Awakens.

In Star Wars, the trusty droid R2-D2 was ultimately there to help Rey find Luke with a holographic map to Luke’s location. But is our friend Saccharomyces cerevisiae just as fortunate? In fact, it just so happens that S. cerevisiae also has an “R2-D2” of its own: the co-chaperone Sti1p. Just like how R2-D2 helps Rey find Luke, Sti1p helps bring Hsp70 and Hsp90 together so that Hsp90 can receive its protein substrates and save the galaxy (or at least help fold proteins and save the yeast cell).

To give some background, Hsp90 (encoded by HSP82 and HSC82 in yeast) is a molecular chaperone that assists in the folding and maturation of specific protein substrates, or “clients”. It functions as a homodimer that undergoes ATP-regulated cycles of “opening” up to receive clients, closing, and then opening again. Many clients of Hsp90 first bind to a chaperone of the Hsp70 family, such as Ssa2p, which assists in the early stages of protein folding before interacting with Hsp90 and passing on the client.

The co-chaperone Sti1p comes into the picture by bridging Hsp70 and Hsp90 and helping them interact, so that the Hsp70-bound client can be delivered to Hsp90. Although this function of Sti1p has long been known, the exact mechanistic details have been obscure, and mutational studies have suggested that Sti1p does more than just bridge the two chaperones together.

Thanks to a recent GENETICS study by Reidy and coworkers, we now know more about how Sti1p helps save the galaxy: it not only helps Hsp90 interact with Hsp70, but also prepares Hsp90 to receive its client protein and advance in its reaction cycle.

To uncover the role of Sti1p in the Hsp90 cycle, the authors examined Hsp90 mutants that were dependent on Sti1p for viability. They mapped both previously-known and newly found Sti1-dependent mutants and found that all of the mutations clustered in just two sites. They designated the sites Sti1-dependent N and C-terminal domain proximal, or “SdN” and “SdC”, respectively.

Because previous studies showed that some Hsp90 SdN mutants don’t interact well with Hsp70, and that analogous SdC mutations in E. coli weaken interactions with Hsp90 client proteins, the authors hypothesized that Sti1p assists Hsp90 with these functions in particular.

To clarify how Sti1p and Hsp90 cooperate, the authors utilized a combination of mutational studies, pull downs with purified proteins, mass spectrometry, and more. They observed that SdN mutations in Hsp90 reduce interactions with Hsp70, while SdC mutations do not. Further, they found that the Sti1p dependency of SdN mutants could be cured through a novel suppressor mutation (E402R), which increases the interaction of Hsp90 with Hsp70. These results suggest that Hsp90 interacts with Hsp70 through the SdN region, and that Sti1p is needed to bring the two chaperones together if they aren’t able to do so well enough on their own.

Importantly, although the E402R suppressor mutation was able to “cure” SdN mutants of their Sti1p dependency, it was unable to do so in SdC mutants. This indicated that SdC mutants are defective in a function that Sti1p assists with, but one that’s separate from having Hsp70 interact with Hsp90.

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Luke needed a little motivation from R2-D2, as does Hsp90 from Sti1p. Image from http://starwars.wikia.com/wiki/Luke_Skywalker

To uncover why SdC mutants depend upon Sti1p for viability, the authors investigated suppressor mutations. The authors isolated multiple SdC suppressors and also found that a previously characterized mutation, A107N, is able to ameliorate the effects of SdC mutations. Previous studies on A107N show that this mutation promotes closure of the open-state Hsp90 heterodimer, which is an important step of the Hsp90 reaction cycle. The authors found that other SdC suppressor mutations were consistent with A107N and could relieve the Sti1p dependence of SdC but not SdN mutants. These results indicate that Sti1p not only promotes Hsp90-Hsp70 interaction, but also has an additional function in promoting Hsp90 heterodimer closure and progression of the Hsp90 cycle.

So it turns out that Sti1p is like R2-D2 in more ways than one. In its first role, Sti1p helps Hsp70 and Hsp90 interact, much like how R2-D2 helps Rey find Luke in The Force Awakens. In its second role, Sti1p helps Hsp90 accept its substrates, progress through its reaction cycle, and perform its function. This is similar to what R2-D2 does in Star Wars: The Last Jedi. In the movie, Luke initially shows reluctance to resume his role as a Jedi Master and help save the galaxy, despite being found by Rey. But thankfully, R2-D2 was there to motivate Luke to return to his heroic duties, much like how Sti1p is there to “motivate” Hsp90 to capture client proteins and do its job.

Thanks to the efforts of Reidy and coworkers, how Sti1p helps save the galaxy yeast cell is that much clearer. Not only does Sti1p help Hsp70 interact with Hsp90 and deliver lightsabers client proteins, but it also helps Hsp90 do its job as a Jedi Master chaperone by promoting progression of the Hsp90 reaction cycle!

by Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: chaperones

(Almost) All Hands on Deck for Calcineurin

July 31, 2018


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Multiplex flag of countries in the 2018 World Cup (from pixabay.com)

If there was a World Cup soccer championship for cellular proteins, it’s a pretty sure bet that calcineurin wouldn’t make the team. That’s because this protein is one of those players that just can’t help but use their hands! And as pretty much everyone knows, that’s a big no-no for soccer players (except goalies, of course).

Conserved across virtually all eukaryotic organisms — from plants and protozoa to fungi and humans — calcineurin is a very abundant calcium-binding protein. In fact, it’s so abundant that it makes up 1% of the total protein content in a cow’s brain!

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Moo! (from pngimg.com)

But what does this ubiquitous protein do? Well, it’s “merely” responsible for regulating many diverse, fundamental life processes… things such as fertilization, development, behavior, life span, responses to environmental cues, immune responses, cell death, and so on.

For eukaryotes, certain environmental and developmental cues, such as hormones or nutrient availability, can initially signal their presence by causing a change in the cell’s internal calcium levels. Calcineurin helps detect these calcium level changes and then passes the signal on in a chain of events.

Calcineurin is a calcium-regulated protein phosphatase, meaning that when it is activated by calcium level changes, it removes a phosphate group(s) from other proteins, particularly transcription factors. When these transcription factors have their phosphate group(s) chopped off by calcineurin, they travel into the cell’s nucleus and turn on a specific set of genes needed to make the cell respond appropriately to the original environmental or developmental cue.

Calcineurin is actually made up of two different proteins that bind together. One is a catalytic protein subunit (called CNA) and the other is a regulatory subunit (CNB). In our yeast friend Saccharomyces cerevisiae, the regulatory CNB protein subunit of the calcineurin complex is encoded by the CNB1 gene.

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Talk to the (EF) hand! …named for Helices E and F (image from Wikimedia Commons)

The Cnb1 protein contains a set of four almost-identical short amino acid domains that are conserved in the CNB proteins of all other organisms. These motifs are called “EF hand” domains because each of them looks like a spread thumb and forefinger of a human hand. And crucially, each “EF hand” can grab and hold onto calcium ions (Ca2+). The four EF hands of the Cnb1 protein are called EF1, EF2, EF3 and EF4, respectively.

But besides holding onto calcium ions, what else do these hands do? Why are there 4 of them? Are each of the hands doing the same thing? Does the right hand know what the left (or the middle, or the other middle) hand is doing?

Well, in the July issue of GENETICS, Connolly and co-workers describe experiments they’ve performed that help figure out some of the answers to these questions for the yeast Cnb1 protein.

The authors made a series of 4 different mutant CNB1 genes, each one having a disabling mutation in one of the 4 EF hands so that the hand can’t grab calcium ions any more. They put these mutant-handed CNB1 genes into yeast cells that had their normal CNB1 gene completely removed. The yeast cell thus ends up depending on a Cnb1 protein with one mutant hand and three functional hands.  In a way, they are making Cnb1p have one of its 4 hands tied behind its back, and then seeing how well it can do its job!

How did they test how well each of the mutant-handed proteins works? Remember that the Cnb1 protein is the regulatory subunit of calcineurin, and calcineurin activates a transcription factor by dephosphorylating it. In yeast, the transcription factor regulated by calcineurin is encoded by the CRZ1 gene. The Crz1 protein recognizes and binds a certain DNA sequence (called CDRE) located just upstream of each one of its target genes and turns these genes on, ultimately changing the yeast cell’s behavior during calcium signaling. The authors put a special reporter gene into their yeast strains; this reporter gene has the special CDRE DNA sequence fused to an often-used bacterial gene called lacZ. The amount of lacZ protein produced by the yeast cells (which positively correlates with calcineurin function) can be sensitively monitored by an easy test tube assay.

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Totally tubular! (from pixabay.com)

Using this test tube assay, Connolly and co-workers tested how well each of the EF hand mutants worked. First they tested how well each could turn on the CRZ1-regulated genes, and also how well the mutant proteins detected calcium. Then they also tested how well each of the mutant-handed Cnb1 proteins worked in high salt environments, during the mating response, under oxidative stress conditions, and even in the presence of immunosuppressive drugs! Why the latter? Calcineurin is a target of immunosuppressive drugs, which are used when people get organ transplants to stop their own bodies from attacking the “foreign” organ. Yeast calcineurin is so similar to human calcineurin that it too is affected by these drugs!

The results were clear (well, actually yellow in the assay)—in all cases, the Cnb1 protein was able to have its EF4 hand disabled and still function perfectly or almost as well as the intact Cnb1 protein! But whenever one of the other EF hands was disabled, the function of calcineurin suffered, and this was true for each of the many ways it was tested, from salt to mating to immunosuppressive drugs.

It appears that when any of the useful hands (EF1, 2 or 3) were mutated, it causes Cnb1p to improperly change its shape in response to calcium, and this misshapen protein can’t do its job of activating Crz1p and ultimately getting the cell to respond to calcium-mediated signals properly.

And (#APOYG alert!) these yeast genetic results for the 4 EF hands match very closely to what’s been seen for mammalian calcineurin EF hand mutants in test tube (“in vitro“) experiments, giving an even stronger confirmation to these mammalian results. Maybe yeast will help develop new strategies for calcineurin-related diseases!

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You’ve gotta “hand” it to calcineurin! (from Wikimedia Commons and Pixabay)

So now back to soccer… As we’ve found out, calcineurin HAS to use its hands, so it’s not a good pick for a regular soccer position player. But maybe it could be a super-awesome goalie since it has 4 hands! It even seems that you can tie its EF4 hand behind its back and Cnb1p can still guard the goal just fine with its remaining 3 hands – the EF4 hand seems to be a totally useless appendage! But if you disable any of the other hands, then it causes the Cnb1 protein to bend awkwardly and not do its job anymore.

Thanks to the efforts of Connolly and coworkers, we now know that it’s not quite “all hands on deck” for Cnb1p, but rather “EF 1, 2, and 3 hands on deck” in order to carry out its “goal” of regulating cellular responses to calcium!

by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: signal transduction, protein structure

Too Much of a Good Thing Can Be Hazardous to Your Health

July 02, 2018


When someone says they can “read you like a book”, they probably aren’t saying that they know your entire genome sequence. (…or for you Westworld fans, you certainly hope they aren’t saying they’ve got access to the Delos Incorporated “library”!).

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Can someone read you (or your genome) like an open book?
(from maxpixel.net)

But in fact everyone’s genome CAN be thought of as a book, sort of like a giant cookbook. Within your genome are many “recipes” for gene products — DNA sequences that each give instructions on how, when, and where to make a protein or RNA molecule. The recipes of the genome are used to “cook up” a person!

When a parent cell divides and creates another cell, it makes a copy of its genome “cookbook” and passes it down to the new cell. It’s extremely important to make sure that there aren’t any errors in the newly copied text, as mistakes in recipes for crucial enzymes can have disastrous results for the cell. Indeed, our cells (as well as yeast cells, and in fact, all organisms) have enzymes that are dedicated to scrupulously inspecting the new copies of our genome cookbooks that are made during cell division, and then helping to correct any errors.

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No typos here!
(from pixabay.com)

A certain class of these enzymes are known as DNA mismatch repair (MMR) proteins. These enzymes carefully proofread the newly made copy of the genome, and if something doesn’t match the original version, they will help fix the error.

You might think that having more of these MMR proteins would be a good thing, because there would be more thorough checking and repairing of the new copy of the genome. But the situation isn’t so simple, especially when it comes to human cancer.

Previous studies show that some cancers and cancer predisposition syndromes have less of the MMR proteins. This makes sense because cancers almost always arise due to genome mutations, and it is known that if a cell has less of the error-checking MMR proteins, there are more genome mutations. However, other studies report a puzzling discrepancy: in some cancers, there are in fact MORE of the MMR proteins rather than less!

In their recent GENETICS study, Chakraborty and coworkers decided to investigate this discrepancy further. They analyzed data from two databases of human cancer genome information, The Cancer Genome Atlas (TCGA) and the cBioPortal for Cancer Genomics, specifically looking at how much the genes encoding MMR proteins were turned on or off in cancer.

They observed that many types of human cancer cells make more of two particular MMR proteins: MSH2 and MSH6. It turns out (as it often does!) that our good buddy Saccharomyces cerevisiae has MMR proteins very similar to the human ones, also encoded by genes called MSH2 and MSH6. So Chakraborty and coworkers made yeast cells that overexpress the yeast genes MSH2 and MSH6 and used the awesome power of yeast genetics (and genomics) (#APOYG!) to investigate whether this might lead to cellular and genome changes like those seen in human cancers.

Using various yeast genetic assays to measure rates of genome alterations such as homologous recombination, mutations, and loss of large chromosome regions, Chakraborty et al. observed increased rates for all of these measures of genome instability in cells when both MSH2 and MSH6 were overexpressed, but not for overexpression of either one alone (or of other MMR proteins). So even though there are more of the “good guy” MMR proteins in these cells, this actually ends up making the genome of the cell MORE likely to get damaging mutations of the same types seen in cancer cells.

So why is too much of a good thing such a bad thing in this case? The authors hypothesize that both Msh2p and Msh6p act together as a joined pair to go to the spot where the chromosomal DNA is actively being copied (“replicated”). When they are both overexpressed, too many of the Msh2p-Msh6p pairs can go to the replication spot and actually interfere with the copying process.

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Medieval scribe and author Jean Miélot at his desk, late 15th century (from Wikimedia Commons)

It’s as if you were a medieval scribe carefully copying an illuminated manuscript of a genome cookbook, and instead of one supervisor occasionally checking your work, there are a bunch of people constantly looking over your shoulder and maybe even bumping into your arm, causing you to make mistakes in your writing. They may even make you drop the book you’re copying, scattering pages so that you might leave some out or put them back in the wrong order, seriously messing up your work!

Here’s hoping our cells don’t overdo it with their MMR proteins, so that they can be careful with their cookbook copying job and do it “write”!

by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: MSH6, DNA replication, DNA mismatch repair, MSH2, cancer

Mixing Mitochondria Makes Magic

May 31, 2018


In the Harry Potter universe, there are two materials that make up a wand: the wood, which comes from trees like cedar and holly, and the core, which is a magical substance such as the feather of a phoenix or a unicorn hair.

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A variety of wizarding wands. From Flickr.

Every wand has unique properties that depend mainly on the combination of its wood and core. Different wood-core pairings give different characteristics that can either antagonize or synergize with the wizard using it. When a wand meets up with its ideal owner, it will begin to learn from and teach its human partner. Such auspicious pairings can continuously improve the wizard’s spell-casting, helping the wizard perform better and better under ever more varied circumstances. However, a poor pairing between a wand and a wizard can be devastating, enfeebling the wizard’s magic or even causing it to backfire.

And just like wizards and wands, it turns out that mitochondrial DNA and nuclear DNA in a cell need to be properly paired to perform the “magic” of running a cell in the most efficient way.

Mitochondria are dynamic structures inside eukaryotic cells that provide much of the energy to keep a cell humming along.

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A mitochondrion. These sub-cellular organelles produce much of the energy for a cell in the form of ATP. From Wikimedia Commons.

Mitochondria contain their own DNA, encoding genes necessary for the organelle to do its work. Although mitochondrial DNA is physically separate from nuclear DNA, it turns out that the two need to work together if the cell is to make functioning mitochondria.

Like the wand-wizard pairing in Harry Potter’s world, the combination of a specific mitochondrial genome (the wand) with a particular nuclear genome (the wizard) is important for making a healthy mitochondrion. Some mitochondrial-nuclear combinations work well and others not so much, but not a lot is known about where different mitochondrial DNAs come from and how they end up paired with their favored nuclear genomes.

Knowing more about this may help us understand how mitochondrial genomes evolve during interspecific hybridizations, such as in lager beer yeast and certain other fermentation yeasts.

A new study in GENETICS from Wolters et al. shows that when S. cerevisiae yeast cells go through the mating process, there is often mixing of mitochondrial genomes to give new combinations of mitochondrial genes — almost as if lots of new wood-core combinations of wands were being created.

How do these new mitochondrial combinations arise? When two haploid yeast cells mate, they merge to form a single diploid cell that contains mitochondria from both of its parents. Sometimes, these mitochondria exchange pieces of DNA, mixing-and-matching genes in a process known as mitochondrial recombination.

The authors found that a surprising proportion of mated yeast cells (~40%) had recombinant mitochondrial DNA. And in many cases, the recombined mitochondrial genomes work even better with the nuclear genome to make a super healthy cell. Often these optimal pairings allowed the cells to develop new powers, tolerating higher temperatures and more oxygen-stressed conditions than the original parent cells — in other words, the cell has found its optimal “wand”!

But other pairing combinations were inauspicious, giving sickly or dead mitochondria that can harm the cell, especially when it is growing under stressful conditions. For instance, when the authors swapped the mitochondrial-nuclear pairing for two different but very fit cell types, these new pairings gave unhealthy cells, meaning that the original fit cells had already found their perfect “wand”.

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Harry Potter first meeting his perfectly-paired wand. From harrypotter.wikia.com.

So just like when Harry Potter was in Ollivander’s wand shop and finally found his holly-phoenix feather wand and felt unified with its amazing magic, yeast cells can acquire new super powers when their nuclear and mitochondrial genomes are perfectly paired!

by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.

…and we wish a fond farewell to Barry Starr, Ph.D. who has left the Stanford Department of Genetics for new horizons. We miss you Barry and wish you well!

Categories: Research Spotlight

Tags: recombination, mitochondria, environmental stress

Trouble with Triplets

April 06, 2018


This Research Spotlight also comes as a video animation. Be sure to check it out!

In the Star Wars movie Attack of the Clones, Padme and Anakin end up fighting Genosians on an automated assembly line. Padme manages to survive because the assembly line is set up in an ordered and predictable way. She knows when to jump, when to pause and so on to survive. If there was more chaos in the line, she might have been killed and then there’d be no Luke or Leia!

If the Genosian assembly line weren’t predictable, Padme might’ve ended up a pancake.

The same kind of thing can happen when cells read genes. An enzyme called RNA Polymerase II (Pol II) slides along the gene, spewing out a long string of messenger RNA behind it.

Just like the assembly line on Genosis, the gene is set up in an ordered and predictable way. Nucleosomes (barrel-shaped clusters of proteins) are spaced along the gene’s DNA to help keep it from getting tangled up. But when Pol II comes hustling along, the nucleosomes need to be carefully removed in front and then replaced after it passes.

Similar to the plight of poor Padme getting hurt if the assembly line were not predictable, genes can likewise get damaged if the nucleosome removal/replacement process goes haywire. In Star Wars, the result of a defective assembly line is damaged droids and TIE fighters. In cells, the result is a changed assembly line—the gene can end up longer! This change can be harmful for both the gene and the cell.

In a new study in GENETICS, Koch and coworkers use our favorite beast, the yeast Saccharomyces cerevisiae, to show that the ISW1 gene is a key player in making sure that the nucleosomes get back on DNA after being taken off. When yeast lack the ISW1 gene, nucleosomes don’t always return after being removed to make way for Pol II. And for some genes, this spells even more trouble.

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“CAG” starts to spell “trouble” if genes get too many of them. from Wikimedia Commons.

The genes that have the most problems are those that have something called triplet repeats. Basically, this means the same 3 bases (e.g. CAG) are repeated many times in a row in the gene.

Using PCR, Koch and coworkers showed that a gene with triplet repeats ended up with more of them if the ISW1 protein (Isw1p) wasn’t there to shepherd the nucleosomes properly. And too many repeats in a gene can be harmful–not just to the yeast, but to us as well.

In fact, Triplet Repeat Expansion Disorders happen when the number of repeats increases from one generation to the next.  These diseases are some of the most devastating ones around.

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Woody Guthrie succumbed to a triplet repeat disorder, Huntington’s Disease. from Wikipedia.

They mostly cause slow but steady degeneration of nerve and brain cells, and the cruel symptoms (loss of body movement control, dementia) often only show up in mid-life. The most well-known is probably Huntington’s disease, which killed folk-singer Woody Guthrie.

So understanding how Isw1p helps keep the Pol II assembly line running smoothly in yeast might help us understand how triplet repeat expansion happens in humans, and may eventually give us ideas how to keep it from happening in the first place. This is especially likely because humans have proteins that are similar to Isw1p and probably do something similar.

To determine how Isw1p regulates nucleosome reassembly, Koch and coworkers used Southern blots to show that yeast that lacked Isw1p couldn’t replace their nucleosomes after Pol II had passed as efficiently as yeast that had the protein. This means that when cells are missing the ISW1 gene, a long stretch of the DNA is left bare after Pol II has passed by.

This stretch of nucleosome-free DNA can end up forming new structures called hairpins that can cause cells to send in DNA repair machinery to deal with it. Unfortunately this machinery isn’t always that great at fixing the DNA. Like the Three Stooges adding more pipe to try to fix a leak, the cell can end up adding more DNA to deal with the hairpin.

Like adding extra DNA, throwing extra pipes at a plumbing leak is a great way to make a bad problem worse.

But for the cell, the results are not as hilarious as they were for Moe, Larry, and Curly. That extra DNA can lead to deadly genetic diseases.

A yeast cell needs Isw1p to keep the cell from bringing in the Three Stooges to mess up its DNA and potentially cause devastating genetic diseases. If it turns out the same is true in people, then once again yeast will have shown us how human genetic diseases might happen. And perhaps provide a target for us to go after to prevent these diseases from happening. #APOYG!

by Barry Starr, Ph.D., Barbara Dunn, Ph.D., and Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: DNA repair, ISW1, RNA polymerase II, nucleosome, chromatin remodeling

Don’t Ignore the Scullery Maid, Hobbits, or Pol III

February 13, 2018


In the Disney story, Cinderella is ignored as the scullery maid. Most of the attention falls on her step-sisters.

CinderellaMaid

If the prince ignored Cinderella, he would not have a happily ever after. Nor might we if we ignore Pol III. (strwalker)

This is, of course, a mistake. The best person by far is Cinderella and she should not be ignored because of her position in life.

The RNA polymerases in a eukaryotic cell are similar except that one is the star and two are the scullery maids. RNA polymerase II (Pol II), the reader of protein encoding genes, gets most of the attention. Pol I and Pol III, the enzymes that make RNAs that aren’t usually translated into proteins, are mostly relegated to supporting roles.

In a study in Nature, Filer and coworkers show that ignoring Pol III can be as big a mistake as ignoring Cinderella. If you want to live a longer life that is.

Just as there were hints of Cinderella’s specialness, so too there were hints that perhaps Pol III plays an important role in longevity. That hint goes by the name of TORC1.

Previous work had shown that inhibiting TORC1 can increase the lifespan of beasts ranging from yeast to mice. Given that TORC1 is a strong activator of Pol III, Filer and coworkers set out to determine if inhibiting Pol III could do the same thing.

As any good researcher should, they started out by looking in our favorite model organism, the yeast Saccharomyces cerevisiae.

Pol III is pretty complicated—it is made up of 17 different subunits encoded by 17 different essential genes. These authors went after the largest subunit, C160, encoded by the RPO31 (RPC160) gene.

To control its level of expression, they fused an auxin-inducible degron to the protein. This fusion protein is degraded in the presence of indole-3-acetic acid (IAA) and E3 ubiquitin ligase from the rice Oryza sativa.

This means that there should be less Pol III in a cell when IAA is around. Western blot analysis confirmed that this was indeed the case.

The authors found that yeast with less Pol III had a longer chronological lifespan. In other words, each yeast cell survived longer. Having less Pol III did not significantly increase replicative lifespan though; they did not bud more often over their lifetime.

These authors did not get as significant a result when they targeted the largest subunit of Pol II, RPO21 (RPB220). The strain appeared to do better in the presence of IAA compared to the control strain, suggesting that targeting Pol II had a small effect on longevity; but it was nothing like was seen with Pol III.

They next turned to a couple of model organisms with more cells—nematodes, also known as C. elegans, and fruit flies, or Drosophila.

When Filer and coworkers used RNA interference (RNAi) to knock down the amount of the largest subunit of Pol III, rpc-1, in C. elegans, the worm lived longer. And when they generated a strain of Drosophila which had only one working copy of dC53 (CG5147), a gene that encodes a Pol III subunit, these fruit flies lived longer too. Lowering the amount of Pol III is a good thing if you want to live longer!

Sauron

Sauron would still be alive if he didn’t ignore those hobbits. We might live longer too if we stop ignoring Pol III! (Colony of Gamers)

Previous work had shown that the gut often controls longevity in C. elegans. These authors showed that the same holds true for Pol III reduction. Targeting just the Pol III in the gut was sufficient to give this little worm a longer life.

The same turned out to be true in the fruit fly. Using RNAi against two different Pol III subunits in the Drosophila gut was enough to have extend these flies’ lives. Targeting Pol III in the fat body did not have the same effect, and targeting Pol III in neuronal cells extended life only a little.

So reducing Pol III in the gut is good enough for flies and worms to live longer. In fact, reducing Pol III activity had as much effect on longevity as does inhibiting TORC1. Scientists ignore this hidden gem at their own risk!

Since Pol III is ignored, but dangerous for lifespan, perhaps a better analogy than Cinderella might be Sauron and the hobbits of the Shire from the Lord of the Rings Trilogy. Sauron ignores the hobbits who hold the key (or the ring) to Sauron’s shortened lifespan.

If Sauron had paid attention, he’d still be lording it over Middle Earth at the end of the third book. Just as we might live longer if we start paying more attention to that hidden danger—excessive Pol III activity, possibly in the gut.

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

Categories: Research Spotlight

Tags: Longevity, TORC1, RNA polymerase III

Strung Out Chromosomes and Coyotes

January 29, 2018


As Wile E. Coyote knows, when setting a bear trap, you need to make sure the tension is just right. As you can see in this video, if there isn’t enough, then the Road Runner gets away:

And of course, Wile E. ends up getting caught in the trap!

Like our super-genius coyote friend, Saccharomyces cerevisiae cells need to make sure there is the right amount of tension sometimes, too. One of those times, for example, is tension on their chromosomes at the metaphase stage of cell division.

If the chromosomes aren’t attached to the spindle in just the right way, the cell will not let mitosis move from metaphase to anaphase. The cell won’t put down the birdseed until it “knows” the trap is set properly.

While Wile E. Coyote probes the tension by eyeballing it, yeast does it in a much more thorough way. Two key pieces of the sensing machinery in yeast are the tension sensing motif (TSM) from histone H3, 42KPGT, and the Shugoshin 1 protein, Sgo1p.

The current model is that the TSM of histone H3 acts as an anchoring point or a landing pad for Sgo1p, the protein responsible for sensing that there is the right amount of tension between chromosomes and the spindle/kinetochore. When the TSM is mutated with, for example, a G44S allele, the idea is that Sgo1p can no longer bind causing chromosomes to sometimes segregate incorrectly.

Previous work had shown that mutating Gcn5p can partially rescue the effects of the G44S allele. In a new study in GENETICS, Buehl and coworkers provide evidence that the reason it can has to do with the tail of histone H3. Basically, this tail can act as a secondary anchor if the TSM, the first anchor point, is mutated. But the tail can only work if it is not acetylated by Gcn5p.

CoyoteRoadrunner

Bad things can happen when plans go astray for Wile E. Coyote and for chromosome segregation in yeast. (Wikimedia)

It is as if Wile E. Coyote’s bear trap is broken so he has to fashion a new trigger to get it to snap. When the TSM of H3 is gone, yeast can fashion a new sensor by eliminating the acetylation on the tail of the H3 histone.

Buehl and coworkers focused on the tail because it is a known target of Gcn5p acetylation. In particular, there are four lysine residues in the tail—K9, K14, K18, and K23—that are acetylated by Gcn5p to varying degrees. The most acetylated is K14 followed by K23 and K18. K9 is hardly acetylated at all.

When they deleted the histone tail, they found that the effects of the TSM mutation, G44S, were more severe. But when they mutated the four lysines all at once, the effects of G44S weren’t as bad. This is consistent with the unacetylated tail serving as a landing pad for Sgo1p in the absence of the TSM. No tail, no binding, leading to chromosome mis-segregation.

As might be expected if this was linked to Gcn5p, each individual lysine mutant had effects that corresponded to their level of acetylation by Gcn5p. So K14A did the best job of partially rescuing the G44S allele, K18A and K23A did an okay job and K9A had very little effect.

Buehl and coworkers provide three additional sets of experiments to show that G44S causes chromosome mis-segregation and that K14A can partially rescue it. There is only time to discuss one of these, but you can read the paper for the other two.

This strategy uses the idea that diploids cannot mate because of a repression function on each copy of chromosome III (chrIII). If a diploid loses one of its chrIII’s, then it will be able to mate.

As expected, the authors found that both wild type and the K14A mutant had very few diploids that could mate. But it was a different story for the G44S mutant—it had many more diploids able to mate, presumably due to the loss of one chrIII. The double mutant K14A/G44S had more diploids mate than wild type but less that G44S on its own.

This is what we would expect if K14A can only affect tension sensing in a TSM mutant. It would do little on its own but could partially rescue the TSM mutant G44S.

ROADrunner

If Wile E. Coyote had a backup plan like yeast does for chromosome segregation, he might get to dine on the Road Runner. (Rosenfeld Media)

The authors next used chromatin immunoprecipitation (ChIP) assays to directly show that the K14A mutation in histone H3 allowed Sgo1p to bind when the TSM was mutated. There was one hitch though—Sgo1p bound most everywhere and not just at the pericentromeres where it usually does.

In a final set of experiments, the authors used GST-pulldowns to show a direct interaction between the H3 tail and Sgo1p. To their surprise, they found that acetylating the tail did not affect Sgo1p binding. They suggest that perhaps the acetylated tail interacts with something that blocks Sgo1p from binding.

Yeast has a backup just in case its main way of sensing tension between the spindle/kinetochore and chromosome, the TSM of histone H3, breaks down. In that case, it can use the tail of histone H3, assuming it is not acetylated.

If only Wile E. Coyote included backups like this in his crazy plans! Then he could finally dine on that tasty Road Runner he is always chasing.

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

Categories: Research Spotlight

Tags: acetylation, Histone H3, chromosome segregation, metaphase

Exploding Yeast to Protect the Wild

January 22, 2018


In the first Kingsman movie, Samuel L. Jackson plants little bombs into the necks of people who will follow him to his “ark”. If they disobey him, Jackson can simply blow up their heads.

In an amazing scene from the movie, our heroes get a hold of the “detonator” and blow up the heads of every one of Jackson’s followers. As you can see below, the director uses cool colors to make it more fabulous than gruesome:

In a new study out in Nature Communications, Maselko and coworkers explode misbehaving yeast instead of people’s heads. (Click here for an incredibly cool video of it happening…so cool!)

These researchers have created a strain of yeast that can only form diploids within the same strain. If instead it mates and forms a diploid with a yeast outside of its strain, the resulting diploids explode.

These authors aren’t being strainist—they are creating a proof of principle organism that could potentially solve the problem of gene transfer from genetically modified organisms (GMOs) to wild organisms.

Assuming a similar idea works in plants, then the idea is that a GMO like an herbicide-resistant GM plant could not pass its herbicide-resistance to its wild cousin. The resulting plants would explode (or perhaps die in a bit less spectacular fashion). The herbicide-resistant gene would not escape into the wild population because none of the resulting plants could survive.

And that isn’t this approach’s only possible uses. It might be used to kill off disease carrying mosquitoes or controlling invasive species. Watch out sea lamprey!

The basic idea is that the engineered species makes a “poison” that it is immune to. If it mates with a wild species, it passes that poison on to the resulting “children.” These progeny are not immune and so die.

The poison in this case is a CRISPR/Cas9-inspired transcription activator. This activator turns a gene up so much that it kills the yeast. But the catch is that the activator’s binding site is only found in the wild type genome and not the engineered genome.

So the engineered strain carries around this deadly poison that does not affect it. But when this yeast mates with a strain that has the binding site, the resulting diploid yeast has the activator binding site, meaning that the silent killer can now bind and kill the diploid.

For their proof of principle experiments, Maselko and coworkers used a well-tested and available CRISPR/Cas9 transcriptional activator that involves dCas9-VP64 and a single guide RNA aptamer binding MS2-VP64. Previous work has shown that this is a very powerful activator.

Next they turned to the Saccharomyces Genome Database (SGD) to find genes that have been shown to be lethal when overexpressed. They created at least one guide RNA for each of the 8 genes and found that one of the guide RNAs that targeted the ACT1 promoter was lethal. The yeast made actin until it exploded (think the man in Monty Python’s Meaning of Life who eats so much he explodes).

Now they were ready to create their engineered strain. They used CRISPR/Cas9 to engineer a single nucleotide change such that their activator could no longer bind near the ACT1 gene. This strain survived when the activator was introduced.

But when they mated this strain with a wild type strain, they got almost no survivors. The resulting diploids grew fat on overproduced actin and exploded!

Note that there were a few survivors…as Jeff Goldblum famously said in Jurassic Park, “Life will find a way.” The survivors appeared at a frequency of 4.83 × 10−3 with the most common reason found being a mutation in the activator binding site.

This survival rate is one area of obvious concern. Given the genetic variability of wild populations, there may be a few individuals that happen to have a mutation that is resistant to the activator. These immune individuals would then go on to found a new population.

Hawat

Like the engineered yeast in this strain, Hawat can survive only because he has both the poison and the antidote. (Dunepedia)

The authors suggest that targeting a species that has gone through a recent bottleneck to decrease genetic variability might help. Another possibility might be to target multiple genes making the likelihood of an individual having and/or developing multiple mutations exceedingly unlikely. (This is the strategy used to target HIV, a virus with a high mutation rate.)

These authors have created an elegant system reminiscent of the one created by Baron Harkonnen in the novel Dune. In the book, a character named Thufir Hawat is given a slow acting poison by the Baron. Hawat can be controlled because he needs the antidote each day to survive. Once the antidote is removed, he dies.

Here the researchers have given the poison and engineered the antidote into their new strain. When this strain mates with a strain lacking the antidote, like a wild strain, most of the progeny die. Like Hawat, the yeast die for the good of mankind. And hopefully those pesky weeds can be prevented from picking up transgenes too.

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

Categories: Research Spotlight

Tags: synthetic biology, horizontal gene transfer, CRISPR

Moonlighting Proteins Do Double Duty

January 11, 2018


While most people only know her as the friendly waitress at the local diner, it is by night that Louise gets to live her passion. At night, she is a mime.

Mimes

Like a waitress who moonlights as a mime, some proteins have more than one job too. (Max Pixel)

If she loses her voice, Louise will struggle as a waitress but do fine as a mime. It is a different story when she gets walloped with a nasty flu and can’t get out of bed. Now she can’t wait tables by day or be stuck in an imaginary box by night.

In a new study out in GENETICS, Espinosa-Cantu and coworkers get to explore which enzymes in our devoted pal Saccharomyces cerevisiae are like Louise in that they do two unrelated jobs. They set out to systematically identify enzymes that have both a catalytic job and a job unrelated to its catalytic function. In other words, they set out to find enzymes that moonlight at second jobs.

Other researchers have stumbled upon these moonlighting enzymes in the course of their regular studies. Something along the lines of stumbling upon Louise struggling against an imaginary wind at a local park.

The difference here is that Espinosa-Cantu and coworkers systematically looked for dual-function enzymes. They did this by comparing the effects of knocking out the entire protein vs. just knocking out the catalytic activity of the enzyme with a single point mutation. They compared Louise having laryngitis vs. her being bedridden with illness.

They found that 4 out of the 11 enzymes they looked at did indeed have additional jobs unrelated to their catalytic function. These enzymes were both mimes and waitresses.

They started out looking at enzymes involved in amino acid biosynthesis mostly because it is easy to test them for loss of function. The yeast can’t grow in the absence of the amino acid the enzyme helps synthesize!

Next, like every good yeast researcher should, they turned to the Saccharomyces Genome Database (SGD) to identify 86 genes that when knocked out, caused the mutant yeast strain to not grow in the absence of an amino acid. They winnowed this list to 18 by focusing only on those enzymes with well-characterized catalytic sites and that were annotated with a single molecular function.

Then, they needed to have versions of these enzymes that were essentially identical to wild type except that they could not catalyze their reaction. They focused on single amino acid mutants that wiped out catalytic activity. The authors got this to work in 15 of their candidate enzymes.

They were now ready for the big experiment. They started out with a strain deleted for one of the enzymes and then added back either a wild type version or a catalytically inactive mutant version on a low copy (CEN-ARS) plasmid. They also included a third strain that had just an empty plasmid (their knockout strain).

They compared the growth of these three strains under 19 different growth conditions and looked for conditions where the knockout strain had a larger effect on growth compared to the strain with the catalytic mutation. The idea here is that if an enzyme does more than one job, knocking it out should have a larger effect than just disabling its enzymatic activity.

PennyGorilla

If Penny the waitress from Big Bang Theory is sick, both the Cheesecake factory and her movie set will suffer. (Wikia)

Before analyzing their results, the authors wanted to make sure that their wild type strain, the knockout strain with the deleted gene added on a CEN-ARS plasmid, grew equivalently to a true wild type strain. This was only true in 11/15 of the strains and so their analysis focuses on these 11 enzymes.

Espinosa-Cantu and coworkers found no difference in growth rate with all three strains for 38.6% of the growth rate comparisons. Under these conditions, the enzyme isn’t doing much for the growth rate of the cells.

In 233 growth conditions, the enzyme did matter for growth and in 70% of these, there was no difference between the knockout and catalytically inactive strains. Presumably under these conditions the enzymes have a single job—their catalytic function.

But this left 30% of cases where there was a difference suggesting that under these conditions, the enzymes have more than one job. Most of these involved 4 of the 11 genes—ALT1, BAT2, ILV1, and ILV2.

They decided to focus in more detail on one of these, ILV1. To figure out what else Ilv1p might be doing besides its threonine deaminase job, they generated two separate strains of double knockouts with 3,878 non-essential genes. The first set of 3,878 strains had a deletion of ILV1 while the second set had a catalytically inactive form of ILV1.

When they looked at the cases where the ILV1 knock out strain was more severely affected compared to the catalytically inactive strain, they found that 187 out of 345 negative interactions had at least some non-catalytic component.  The non-catalytic interactions were enriched for gene regulation with chromatin modification being one of the most striking.

These interactions put the non-catalytic role of ILV1 in a category more similar to stress response, protein sorting and chromatin remodeling genes than to other metabolic genes. This suggests that Ilv1p is both involved in amino acid metabolism and stress response. Quite a different moonlighting gig compared to its day job!

So researchers need to be careful when drawing conclusions from their knockout experiments. Sometimes the effect you are seeing is unrelated to the function you are studying. Sometimes when a waitress falls ill, the mime performance is affected as well.

No this isn’t a show about ILV1.

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

Categories: Research Spotlight

Tags: protein moonlighting, pleiotropy, systems genetics

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