New & Noteworthy

Green Monster Evolution to Hulk

February 25, 2022

A clever new study has used a modified yeast strain “ABC₁₆-Green Monster” with fewer export pumps (i.e. more susceptible to drugs) to exert powerful selection pressure on yeast cells. By exposing this strain to multiple libraries of chemical compounds with potential use as antifungal agents, Ottilie et al. in a recent issue of Communications Biology identified an evolved set of 25 genes with frequent mutations.

In comparing the set of mutations to those frequently found upon extended growth without selection, they noted that intergenic mutations were comparatively rare, presumably due to the heavy selection pressure for functional resistance. For another confirmation of the screen’s effectiveness in isolating useful variants, i.e. not passenger mutations, they introduced 61 of the altered alleles back into the unevolved strain by CRISPR/Cas9 integration and verified that 45 variants across 37 genes restored resistance.

Looking more closely at the mechanisms of action for the observed resistances, they noted mutations clustered in the active sites of target molecules for which the target was previously known. For example, the antifungal drug tavaborole (a type of benzoxaborole) led to identification of four active-site mutations in Cdc60p (the yeast leucyl-tRNA synthetase) that are predicted to interfere with binding to the drug. In a related example, the chemotherapy drug camptothecin inhibits topoisomerase and they isolated two mutations in TOP1 that would be expected to fall in the binding pocket.

Their isolation of mutations in TOR2 and FPR1 (both associated with TOR signaling) in strains with evolved resistance to rapamycin reproduce the findings of a related study looking at rapamycin resistance in yeast. In fact, both studies identified mutations in the same S1975 residue of TOR2. In other interesting findings, they isolated mutations leading to yeast resistance to drugs used typically against soil-transmitted helminths (worms), trypanosomatid parasites, and malarial Plasmodium.  Each mutant variant in yeast affords insight into how these pathogens might likewise evolve resistance or could be alternately targeted.

The set of identified mutant genes was highly enriched for transcription factors and among these were two—YRR1 and its paralog YRM1—that together mediated resistance for nearly 25% of the compounds tested.  These two zinc transcription factors had previously been shown to activate genes involved in multidrug resistance. In this study, they were mutated 100 times in screens against 19 diverse chemical compounds. Interestingly, deletion of these genes does not confer resistance and the sum of the data suggests the screen identified gain-of-function alleles. In support of this idea, integration of the L611F allele of YRR1 into a susceptible strain by CRISPR/Cas9 reconstructed resistance to a suite of compounds. The researchers hypothesize that modified proteins lead to constitutive activation of genes aiding resistance.

In sum, this study demonstrates the awesome power of yeast genetics (#APOYG) for revealing insight into the molecular underpinnings of medical chemistry. Studies like this one provide not only data but intriguing clues about where to look next—most of which will be easy to test in yeast.

Categories: Research Spotlight

Tags: Drug resistance, Multidrug resistance, Saccharomyces cerevisiae, yeast model for human disease

C-Circles, an ALT Plan for Telomere Restoration

February 18, 2022

The telomerase ribonucleoprotein complex is the primary means by which yeast cells maintain telomeres. However, it turns out that cells lacking functional telomerase have a backup plan to restore telomere length by “alternative lengthening of telomeres” (ALT). ALT employs recombination via extrachromosomal telomere elements called C-circles. In a process for which the reasons remain unclear, C-circles get paired with eroded telomeres at the nuclear pore complex on the nuclear membrane. This pairing requires the SAGA/TREX2 complex and, once paired, the recombination between C-circles and telomeres appears to be effected by Rad59p, the paralog of Rad52p.

This interesting model is described in a recent paper in The EMBO Journal, in which Aguilera et al. adapt a method developed in human cancer studies to detect ALT and C-circles in yeast. In humans, ~10% of cancers depend on ALT for unchecked growth. In yeast, cells with ALT were able to be detected as survivors among telomerase mutant (est2∆) cells.

As other types of extrachromosomal DNA circles were previously reported to associate with the nuclear pore complex, the authors addressed the possibility that C-circles bind the NPC and demonstrated it clearly. They also showed the circles interact with the SAGA/TREX2 complex, which favors telomere recombination.

The novel finding that ALT in yeast so closely mirrors that of some human cancer cells is a boon to study of these cancers. The ability to develop ALT inhibitors in yeast would provide a new set of potential anticancer therapies, making this an ideal model system.

Categories: Research Spotlight

Tags: cancer, cell aging, Saccharomyces cerevisiae, senescence, telomeres, yeast model for human disease

Memories of Deceptive Courtship

February 10, 2022

Yeast courtship provides an excellent model for how a simple organism manages to remember past events. Recent yeast studies reveal how this memory can involve prion-like proteins that build up in a cell and persist as a form of memory. In a recent online issue of Current Biology, Lau et al. show how Whi3p self-templates into large assemblies upon perception of “deceptive courtship,” i.e. when pheromone is perceived but no mating partner appears. The super-assembly state allows escape from the G1 arrest triggered by potential courtship, but also prevents any further response to pheromone. While this pheromone refractory state is stable for the remainder of this mother cell’s life, daughter cells do not inherit this state and are fully pheromone responsive.  

The organization of domains in Whi3p and the ability to self-template into large assemblies are features shared with prions. The lack of infectious heritability, though, distinguishes Whi3p as a mnemon rather than a prion. Fascinatingly, Lau et al. show that assembled Whi3p becomes heritable in cells with defective diffusion barriers, where the physical barriers at the bud neck normally restrict the super-assembled protein to the mother cell.

In studies of diffusion barrier disruption, the authors observed that the main super-assembly of Whi3p remains in the mother cell, while the daughter cells get “seeds” of Whi3^mnem upon which to assemble further. They note that transmission of seeds is most prevalent in the first few divisions following escape from pheromone arrest, which suggests there is a limiting factor to this diffusion. They propose a model in which the assembled Whi3^mnem “matures” to a form that no longer propagates seeds to daughter cells, even in the absence of a barrier.

Thus, remarkably, prion-like behavior appears to be one of the most rudimentary forms of memory, and may have implication for understanding cell memory in higher organisms.

Categories: Research Spotlight

Tags: mnemons, prion-like proteins, Saccharomyces cerevisiae, yeast cell memory, yeast mating

New ncRNA Represses Zinc Regulator

February 04, 2022

In a twist to an established story, the termination of noncoding RNAs by the NNS complex (NRD1 snoRNA termination complex) appears to be dependent on the phosphorylation of a regulatory component. In the latest issue of Nucleic Acids Research, Haidara et al. show how the NNS-complex component Sen1p acts to repress transcription of the zinc master regular ZAP1 when Sen1p is phosphorylated, which appears to happen in response to excess zinc.

The NNS complex had previously been shown to terminate transcription of PHO84 via antisense RNA. In this current paper, the authors identify the previously unannotated noncoding RNA ZRN1 as lying directly upstream of ZAP1 and, when transcribed without termination, repressing the downstream gene. Termination of ZRN1 transcription by dephosphophorylated Sen1p derepresses ZAP1 mRNA levels via removal of the interfering RNA. As evidence of this relationship, a Sen1p phospho-mimetic mutation (T1623E) results in stable repression of ZAP1 transcription.

The model proposed is that zinc excess leads to phosphorylation of Sen1p by an unidentified kinase, which then causes the level of ZRN1 transcript to increase because termination is impaired. This in turn represses ZAP1 mRNA levels by interference, thereby repressing genes responsible for increased zinc uptake and storage.  

Interestingly, the same system (i.e. Sen1p as a component of the NNS complex) represses PHO84 expression via interference, and PHO84 encodes a low-affinity Zn transporter that also contributes to zinc homeostasis. Might RNA interference play an expanded regulatory role over what is currently known?

Categories: Research Spotlight

Tags: noncoding RNA, Saccharomyces cerevisiae, transcription, zinc homeostasis

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

September 24, 2018

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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.

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!

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.

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.

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: biofuels, lignocellulosic biomass, variation

Chaperone Wars: The Last Co-chaperone

August 22, 2018

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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.

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.

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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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!

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.

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.

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!

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: protein structure, signal transduction

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

July 02, 2018

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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”!).

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.

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.

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

Mixing Mitochondria Makes Magic

May 31, 2018

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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.

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.

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”.

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: environmental stress, mitochondria, recombination

Trouble with Triplets

April 06, 2018

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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.

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.

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

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