New & Noteworthy

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

February 13, 2018

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

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!

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, RNA polymerase III, TORC1

Strung Out Chromosomes and Coyotes

January 29, 2018

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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.

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.

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, chromosome segregation, Histone H3, metaphase

Exploding Yeast to Protect the Wild

January 22, 2018

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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.

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: CRISPR, horizontal gene transfer, synthetic biology

Moonlighting Proteins Do Double Duty

January 11, 2018

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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.

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.

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: pleiotropy, protein moonlighting, systems genetics

Retooling Yeast Factories

December 13, 2017

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Imagine you own a rifle factory and you want to retool it to make solar panels instead. Or vice versa.

It wouldn’t make a lot of sense to make small, incremental changes. Make one change, test to see how it works, make a second change, test to see how that works, and so on.

Not only is this inefficient and time consuming, but you may not end up with the best factory for making your new product. The best approach is to rip out the old machinery and replace it all at once. Or at the very least make many changes at a time.

The same sorts of problems exist for cellular factories—microbes we engineer to churn out useful products. Ideally we would want to get to an optimized, retooled cellular factory as soon as possible by making as many changes as we can at once. And yet, that is not how we currently do it.

Most of the time when we repurpose a microbial organism like Saccharomyces cerevisiae to make something new, we do it the slow way. We either do a screen to find genes that affect the process or change a known gene and analyze the results. We then use this mutant strain as a starting point to repeat the process. Over many cycles, we hopefully get to a yeast strain that can make something new or more efficiently.

Not only is this time consuming and inefficient, but we might miss some synergies that can happen when two or three genes are tweaked at once. This is no way to retool a factory!

In a new study out in Nature Communications, Lian and coworkers came up with a clever way to make many changes all at once in yeast. And they aren’t limited to just deleting genes either. They can transcriptionally activate, transcriptionally interfere, or delete specific genes. Which is where they got their clever acronym for their process—CRISPR-AID.

As you can tell from the name, their system uses the seemingly ubiquitous gene-editing tool CRISPR. And they use it in some very cool ways to get all three processes happening in a cell at the same time with different genes.

Conventional CRISPR works through a nuclease being guided by an aptly named guide RNA (gRNA) to a precise place in cell’s DNA through base pairing between the DNA and the gRNA.  Once there, the nuclease makes a double-stranded break and at least in yeast, the cell invariably repairs it using homologous directed repair (HDR). (Other eukaryotes tend to “fix” their DNA with the more error prone non-homologous end joining [NHEJ] pathway. Yes, yeast is superior in yet another way!)

Scientists have been able to tweak this system to activate or repress gene transcription by knocking out the nuclease activity of the CRISPR protein and adding back either an activation or repression domain. Now they have an RNA-guided, sequence specific transcription activator or repressor.

This is a great tool chest of gene editing tools but there is one problem—how to get the right protein to the right spot when all three use a gRNA for specificity. Like Mork from Ork turning to Mindy to help guide him on Earth, Lian and coworkers turned to a PAM to help guide these proteins to the right place in the yeast genome.

The protospacer adjacent motif (PAM) is a small part of the gRNA that binds the DNA and helps to pry it open so the rest of the gRNA can bind. Every gRNA has to have a PAM or the CRISPR protein won’t bind.

It turns out that different bacteria have different CRISPR systems that use different PAMs. The idea then is to use a CRISPR protein from different bacteria for each of the three different functions of the CRISPR-AID system.

The most common form of CRISPR protein is Cas9 from Streptococcus pyogenes (Sp), which has NGG for its PAM sequence. Lian and coworkers used a nuclease deficient form of this protein attached to a repressor domain to create their transcription interference tool dSpCas9-RD1152. (The d means the nuclease in inactivated, the Sp is the bacteria it came from, and the RD1152 is the repression domain.)

For their deletion tool they turned to the Staphylococcus aureus (Sa) Cas9, SaCas9, which has NGRRT or NGRRN for its PAM sequence.

They turned to a different CRISPR protein, Cfp1 from Lachnospiraceae bacterium ND2006 (Lb), for their activation tool. This protein recognizes TTN as its PAM. They attached an activation domain to a nuclease-deficient form to generate dLbCfp1-VP.

All three proteins were stably integrated into a yeast cell. Now that their toolbox was full and their yeast cell ready, Lian and coworkers set out to show that these tools were effective.

They first used it on a known system, making beta-carotene in yeast. Previous work had shown that increasing the expression of the HMG1 gene, decreasing the activity of ERG9, and deleting ROX1 led to increased beta-carotene production.

They used CRISPR-AID to accomplish all three in one fell swoop. Not only did expression from HMG1 go up, expression from ERG9 go down, and ROX1 get deleted, but the resulting yeast also made more beta-carotene. They got a 2.8-fold increase in beta-carotene compared to only a 1.7 fold increase by hitting just one of the genes.

They next set out to increase expression of recombinant Trichoderma reesei endoglucanase II (EGII). Previous work had identified 14 genes that increased expression when activated, 17 that increased expression when inhibited and 5 that increased expression when deleted. Each of these was done as a single gene experiment.

Lian and coworkers created a library of gRNAs that could target each of the 1620 possible combinations to see if any combination would lead to a synergistic increase in activity. They found a combination that did just that—increased expression of PDI1, decreased expression of MNN9, and deletion of PMR1 led to the highest yield of EGII. The increased expression of EGII was beyond what any of the three did on their own.

So the system appears to be working well. Multiple targets can be upregulated, downregulated or deleted all at once.

While we are getting closer to being able to retool microbial factories as efficiently as brick and mortar ones, we aren’t there yet. That will require a deeper understanding of how a particular microbial organism works. And there is no better understood eukaryote than our old friend S. cerevisiae.

Like a good PAM sequence, Mindy helps Mork find his place in the world.

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

Categories: Research Spotlight

Tags: CRISPR, gene editing, microbial factory

A SRTain Surprise in a Lipid Droplet

December 04, 2017

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Barney Stinson from the sitcom “How I Met Your Mother” is right—the accidental curly fry in your plate of fries is a wonderful thing. It is unexpected but adds immensely to the French fry eating experience.

In a new study in GENETICS, Hoffmann and coworkers show that spore wall lipid droplets, organelles used to move neutral lipids to the cell wall in the yeast Saccharomyces cerevisiae, have a surprise component, too. Instead of an errant curly fry in an order of fries, the greasy droplet has long chain polyisoprenoids made by Srt1p and its partner, Nus1p.

And in an unexpected twist, they are not used to glycosylate a protein as most polyprenols do. No, they upregulate the activity of a key protein involved in spore wall synthesis—Chs3p. Polyprenols may have more varied roles than scientists thought. A pleasant surprise indeed.

When a diploid yeast cell hits hard times, it undergoes sporulation to make sturdy spores to ride them out. And the spores’ sturdiness comes from the spore cell walls.

Yeast need many factors to make these resistant walls. One is Chs3p, the chitin synthase that makes the chitin that is used to make one of the four spore cell wall layers. Another is the lipid droplet that brings a variety of lipids and proteins to the site of the expanding cell wall.

Hoffmann and coworkers used transmission electron microscopy (TEM) to show that spores from strains deleted for SRT1 lack the chitosan layer of their cell walls. Additional experiments showed that this strain is missing this layer because it fails to make chitin as opposed to failing to be able to assemble chitin properly.

The Srt1p/Nus1p complex is a cis–prenyltransferase—it adds 5-carbon isopentyl groups to farnesyl diphosphate to make polyprenols. In the next set of experiments, these researchers showed that an enzymatically inactive mutant of Srt1p, Srt1p-D75A, did not make the chitosan layer as well. This suggested that it is the long chain polyisoprenoids that Srt1p/Nus1p synthesizes that matters for spore wall formation.

In the final set of experiments, Hoffmann and coworkers showed that extracts lacking Srt1p had decreased chitin synthase activity. In other words, they showed that the activity of Chs3p is dependent on Srt1p.

Taken together, these results suggest that polyprenols like long chain polyisoprenoids may do more than simply facilitate the glycosylation of proteins. Here, they upregulate the activity of a protein involved in cell wall synthesis.

And we aren’t just talking yeast either. Most every living thing, including people, have polyprenol-derived dolichols which carry sugars to facilitate protein glycosylation. It may be that these types of molecules also have hidden, unexpected roles. Researchers may want to give these molecules a second look to see if they missed that extra curly fry.

Taking a man’s bonus curly is inexcusable.

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

Categories: Research Spotlight

Tags: cell wall, chitin, chitosan, spore, SRT1

Squeezing Out a Tiny Bubble of DNA

November 20, 2017

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Have you ever tried to walk back to the lab with too many plates between your thumb and middle finger? And had them be so compressed that they shoot out of your hands, spilling all over the floor? Yeah, me too.

While this situation is bad for researchers, it turns out that the same sort of force may be helpful in cells by forcing the DNA open just a bit to get the ball rolling on transcribing a gene into messenger RNA (mRNA). The DNA is squeezed between two points so that a bubble of DNA pops open, leaving some single stranded DNA for RNA polymerase II (RNAP II) to get a hold of.

In a new study in Nature Structural & Molecular Biology, Tomko and coworkers show that Ssl2p, the double-stranded DNA translocase subunit of the basal transcription factor TFIIH, hydrolyzes dATP or ATP to force open around 5 or 6 base pairs of DNA. Once pried open, this small DNA bubble is expanded to 13 base pairs in the presence of NTP hydrolysis, presumably RNAP II beginning to transcribe the DNA. This stable open complex is now ready for promoter clearance and elongation.

These authors teased apart this mechanism using single-molecule magnetic-tweezers (henceforth referred to as tweezers). The idea is to stretch DNA out between two anchor points, add various components of the RNAP II machinery and various reagents, and to measure the changes in the stretched out DNA. Under the right conditions, these length changes directly reflect DNA unwinding.

Getting a gene read in a eukaryote like Saccharomyces cerevisiae is no easy task. A complicated mass of proteins called the preinitiation complex needs to form on the promoter first.

Tomko and coworkers loaded a subset of this complex, which included the TATA binding protein (TBP), TFIIB, TFIIF, TFIIH, and RNAP II, onto a piece of DNA with a single TATA box-containing followed by a strong initiation site. The authors arranged it so the 2.1 kilobases of DNA they used in their experiments was negatively supercoiled as is found in vivo.

In the first set of experiments, they added NTPs and dATP to their reactions. Most of the DNA did not show any changes as measured by the tweezers, but around 5% did.

There were two distinct populations of DNA in the subset that were active. Around 1/3 of the DNA showed  clear open and closed DNA transitions with the open DNA stretching over about 13 base pairs. The other 2/3 of the DNA showed longer-lived, smaller bubbles of around 5 or 6 base pairs of DNA. The authors interpreted the first as stable open complexes and/or elongation complexes and the second set as bubbles of DNA forced open by the preinitiation complex.

They repeated the experiment in the absence of NTPs and saw only the smaller bits of open DNA. And when they left out both NTPs and dATP, they saw no opened DNA at all.

So the small regions of opened DNA are dependent only on dATP hydrolysis making the Ssl2p subunit of TFIIH, a polypeptide known to hydrolyze dATP, the prime culprit for causing them. Which also makes sense, as Ssl2p is a DNA translocase that has been implicated in previous experiments in forcing the DNA to shorten between the preinitiation complex and downstream DNA. These experiments also showed that NTPs needed to be hydrolyzed to proceed to the next step, a stable open complex.

Tomko and coworkers next wanted to determine if these smaller bits of open DNA play a meaningful role in transcription. They tackled this question with a couple of different experiments that both rely on the idea that opening the 5-6 base pairs of DNA is a necessary first step on the way to transcription.

In the first experiment, the authors saw that these smaller regions of open DNA had longer lifetimes at lower NTP concentrations. Remember, NTP hydrolysis is required to proceed to the next step, the 13 bp, more stable open complex. By decreasing the concentration of NTPs, the authors slowed this step down, causing the preceding step of the smaller 5-6 base pairs of opened DNA to hang around longer.

In the next set of experiments, they engineered DNA bubbles into the DNA that varied between 3 and 12 base pairs and tested what size bubble would allow transcription to proceed in the absence of TFIIH, the basal or general transcription factor they implicated in forcing these DNA bubbles open. Previous work had shown that TFIIH is unnecessary if the DNA has a 12 base pair region that is already opened.

They found that while 3 base pairs of opened DNA was insufficient to allow transcription in the absence of TFIIH (and dATP), an open region of 4 base pairs was. This is consistent with the idea that the 5-6 base pairs of opened DNA they saw in their experiments are relevant and that they are caused by the action of TFIIH.

Reading a gene is nontrivial in eukaryotes like us, and our friend yeast. The DNA is squeezed by TFIIH within the preinitiation complex to open just enough of the DNA for RNAP II to get a toe hold and get transcription rolling. A much better result than when a similar force gets your stack of plates rolling on the lab floor.

Don Ho probably isn’t singing about tiny, Ssl2p-mediated DNA bubbles but they too are fine.

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

Categories: Research Spotlight

Tags: open complex, Preinitiation, RNA polymerase II, SSL2, TFIIH

Yeast Reveals a Vicious Circle Between Sugar and Cancer

November 10, 2017

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The housing crisis that started in 2006 is a classic example of a vicious circle. As prices went down, some people couldn’t afford their mortgages and so were foreclosed upon. These foreclosed houses entered the market and caused prices to drop further which caused more foreclosures and so on. Only a Herculean effort by the government and the Federal Reserve was able to break this cycle.

Vicious circles can happen in biology too.

In a new study in Nature Communications, Peeters and coworkers have used our friend Saccharomyces cerevisiae to uncover one of these in cancer cells. And it turns out that this yeast is the perfect model organism for this study. (Funny how often that is true…)

Both S. cerevisiae and cancer tend to utilize their sugar through fermentation instead of respiration. This process in yeast is obviously great news for beer and wine drinkers. However, it is not such great news for people with cancer.

In the Warburg effect (named after the scientist who discovered it), the more aggressive a cancer is, the more sugar it ferments. This new study suggests that the consumption of sugar via fermentation and the aggressiveness of the cancer are related in a vicious circle.

Fermentation creates the byproduct fructose-1,6-bisphosphate (Fru1,6bisP) which, according to this study, activates the oncogene Ras which causes the cell to grow faster. As it grows faster, it ferments sugar faster creating more Fru1,6bisP which activates more Ras and so on. The cancer cells grow out of control until doctors apply some Herculean treatment to put a stop to it.

While yeast and cancer cells have a lot in common, wild type yeast isn’t quite up to cancer’s standards when it comes to cancer’s unbridled intake of glucose into the glycolysis pathway. These authors turned yeast a bit more cancer-like in this regard by deleting the TPS1 gene. Tps1p is a yeast hexokinase that tightly regulates yeast’s glucose intake into glycolysis.

Yeast cells without TPS1 deal poorly with glucose and need to be grown in galactose. When Peeters and coworkers added glucose to tps1Δ cells that had been previously grown in galactose, the cells activated Ras, a potent oncogene. This activation caused the cell to undergo apoptosis as assayed by cytochrome c release from the mitochondria, exposure of phosphadityl serine on the plasma membrane and generation of reactive oxygen species.

Deletion of hexokinase 2 eliminated this activation of Ras and suppressed the apoptosis. This suggests that perhaps some buildup of an intermediate metabolite in the glycolytic pathway might be to blame for the Ras activation.

The authors tested this hypothesis by removing the cell wall of wild type yeast cells and adding glycolysis metabolites to the resulting spheroplasts. They found that one metabolite, Fru1,6bisP, activated Ras. Supporting this finding, they found that deletion of both PFK1 and PFK2, two genes that encode phosphofructokinase 1, the enzyme responsible for making Fru1,6bisP, eliminated Ras activation in tps1Δ cells. Together, these data support the idea that Fru1,6bisP is the culprit behind Ras activation in yeast.

All well and good, but what about human cancer cells? Is something similar going on there? You have probably guessed that the answer is yes.

The authors first depleted the level of Fru1,6bisP in two different cell lines by starving them of glucose for 48 hours. They then added back glucose, which has been shown to increase the levels of Fru1,6bisP in cells, and measured the activation level of Ras and two of its downstream targets, MEK and ERK. All three were transiently activated in both HEK293T and Hela Kyoto cell lines. Looks like Fru1,6bisP activates Ras in cancer cells as well.

So this might explain the Warburg effect that I mentioned earlier. The more glucose a cancer cell uses during fermentation, the more Fru1,6bisP it generates. And the more Fru1,6bisP that is made, the more Ras gets activated prompting the cell to grow faster. Which of course results in more glucose getting fermented and so on.

I don’t have time to go into the work these authors did to try to uncover the mechanism by which Fru1,6bisP activates Ras, but suffice it to say that it does not appear to be a direct interaction with Ras. Instead, it appears to work at least partially by disrupting the interaction between Ras and one of its guanine nucleotide exchange factors, Cdc25p (or Sos1 in humans).

And the story is not yet complete. There is still a lot of work to do in pinning down the specifics of this vicious circle. Yeast will undoubtedly be instrumental in helping us work through the rest of the details.

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

Categories: Research Spotlight

Tags: cancer, fermentation, PFK1, PFK2, TPS1, Warburg Effect

Something from Nothing: A New, Structured Protein from Noncoding DNA

November 01, 2017

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In the Rick and Morty episode Mortynight Run, a gaseous life form pulls off what alchemists had been trying to do for centuries. It literally creates gold out of thin air!

Our ever faithful friend, Saccharomyces cerevisiae can’t do that (at least not yet). But what it has done is create a protein, Bsc4p, out of noncoding intergenic DNA. And while not gold, it is a fully functioning protein.

In a new study, Bungard and coworkers show that this recently evolved gene, BSC4, encodes a protein that can fold into at least a partially defined structure. This matters as there was some debate about whether newly generated proteins could attain a defined structure or if they would remain as intrinsically disordered proteins (IDPs). A reasonable debate, given how rare structure is among amino acid sequences and how plentiful IDPs are in a cell.

Bsc4p is a great protein to study in this regard as there is very strong evidence that it has evolved relatively recently in S. cerevisiae, but not in other closely related species. And it definitely does work in a S. cerevisiae cell. While not essential, some genetic studies (including this one) indicate that it plays an important role in DNA damage repair pathways.

Here is an example from S. paradoxus of the noncoding sequence that the S .cerevisiae BSC4 gene almost certainly sprang from:

gtg TCT GTA ATT CTA CGG AAA AGT AAA CAA AAA AAC TGT AAT TGC ATA ACG AGC AAT TTA TAT ACA ATA CAC ATA GAA AGA CTT TCG CTC tga TGT CCG AAC TGC CAT TGT CAT TGG AGA AAA TCC TTA TGT GGA GTG GAG TTC CCT GCA GGT TAT TTT CAG AGA AAA CGT GGT TAC AAA AAG GGA CCA GAT TCG CCC tag CTT ACA ACT CGC TTG AAT CAT CTT TAT GCC AGA CCT TTC AAC GCC GCG ACC CCA AAA ACA taa ATG CTG AGT CAC CAT GGT GCT GGG CGC TGT CGC TGT CGC GCT GTT CCT TTC CGA GAA AAG CAC GGC AAC AAC AAC AAC AGT CCA TAT GAC CAA AAA AAA AAT AAC CGC AAA TGG CAG tga AAT GCA ATT ATC ATT GTA TAC GA?

In order for this sequence to become a gene coding a protein, at a minimum the first lowercase, dark orange codon needs to be mutated to ATG, a start codon, and the rest of the lowercase, dark orange codons need to be mutated away from being stop codons.

This seems to be part of what happened in S. cerevisiae:

ATG TCT ATT GTG CTA CGG AAG AGT AAC AAA AAA AAC AAA AAC TGC ATA ACA AGC AAG TTT TAT ACA ATA CAC ATT ATA AAA ATT TCT ACT CCG GTG TTC CGA GCT CCC ATT GCC ATT GGA GAA AGC CCT TAT GTG GAG TGG AGC TGC CTA CAG GTT GTT TTC AGG AAA GAC ATG GTT ACA AAA AAG ACG ACA TTC GCC CAA CTT ATC ACT CGC TTG AAC CAC TTT TTA TGC CAA GCC CTT AAA CGC CGC GAC TCA AAA ACA TAC ATA CTG TGC CGC ACG GCA GTT TTT GGC GCT ATG ACA CCC TTT TCC CCA AGA AAA TCG CAT ATT AAC AAC AAA TTA CCC ATG CAA CCC AGG AAA AAA AAA ATA GTC ATT ATA TAC GTA GTG CGC TTT CAT TGA

Through a few small changes, we now have a 131 amino acid polypeptide where before we had some noncoding DNA between LYP1 and ALP1.

Bungard and coworkers use a variety of techniques to show that this newly evolved protein has structure. Not as much structure as many proteins that have been around longer, but more than many of those IDPs.

Consistent with protein structure, Bsc4p forms compact oligomers under native conditions that are partially resistant to proteolysis, has a far UV circular dichroism (CD) spectra consistent with beta sheets, has a buried tryptophan, Trp47, that becomes solvent accessible under denaturing conditions, as measured by tryptophan fluorescence, and has a near UV spectra consistent with a hydrophobic core. However, they found no evidence of any significant interactions between the secondary structures to form a single three dimensional shape, in the protein.  In other words, no evidence of a tertiary structure.

And that wasn’t the only sign that Bsc4p wasn’t a mature, fully structured protein. For example, that near UV CD that showed a hydrophobic core, was weak in intensity, which is consistent with at least “partially molten character.” And Bsc4p bound certain dyes: Congo red, Thioflavin T, and ANS, in a way consistent with some molten globule and/or amyloid character.

So we have a bit of a mixed bag with Bsc4p. One way to think about it is as a young protein still developing its ultimate three dimensional structure. Or, it could be that for the job it does, this is all the structure it needs.

In any event, it is definitely a newly evolved protein with at least some structure which shows that this can indeed happen. Sometimes Mother Nature can make structured proteins from noncoding DNA. Like that gaseous being on Rick and Morty, producing gold out of thin air.

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

Categories: Research Spotlight

Tags: BSC4, newly evolved gene, noncoding DNA, protein structure

Transcription Factors: Kings of the Genome Jungle

October 20, 2017

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Imagine that Jane is in trouble on the edge of the jungle. She needs to be saved soon or she will be sent back to Europe (which she does not want).

Tarzan knows he can’t get there in time by running along the jungle floor. But of course he has a trick up his sleeve—swinging from vine to vine!

He gets there in time, fends off her would-be kidnappers, and saves the day. All because he transferred from one strand of vine to another instead of “sliding” along the jungle floor.

A new study by Wollman and coworkers shows that at least two transcription factors in Saccharomyces cerevisiae, Mig1p and Msn2p, seem to share a lot in common with Tarzan. In a process termed intersegment transfer, they get to where they need to in the genome by “swinging” from DNA segment to DNA segment instead of just sliding along the DNA.

And transcription factors like these need to get to the right place in time to save the cell from outside threats. Just like Tarzan had to swing from vine to vine to save Jane.

This sort of approach would not necessarily work well with just a single transcription factor with a single DNA binding domain. It would sort of be like a one-armed Tarzan—it is hard to take advantage of swinging from vine to vine without at least two arms!

The authors argue that Mig1p gains its “extra arms” through joining together into a cluster. Now each Mig1p can bind DNA and drag the other transcription factors with them. A neat solution to the one arm problem.

Their basic approach is to use fluorescence microscopy to follow a GFP-Mig1p fusion protein in a single cell, something that has only become possible recently. What they found was that there were two populations of transcription factors—a diffuse set of smaller molecules and distinct, larger clusters made up of multiple Mig1p’s.

The clusters appeared to be the ones doing the work in the nucleus. Kinetic studies showed that they stayed in one place in the nucleus for over 100 seconds which is consistent with the clusters and not the monomers being bound to the DNA.

OK so this transcription factor tends to clump up into clusters and it looks like these clusters are the ones regulating gene expression. They also showed that a second transcription factor, Msn2p, did the same thing.

The authors next set out to see if this approach made sense for genetic regulation by running simulations of Mig1p finding its sites in the nucleus as either monomers or as clusters. It made sense to form clusters.

A lot of previous work has been done in S. cerevisiae in terms of the three dimensional map of the genome and where Mig1p DNA binding sites were located in this mesh of DNA. And in the course of their studies, Wollman and coworkers were able to estimate how many Mig1p molecules were in yeast cells and how many were in clusters.

They now had all the information they needed to run their simulations. When they crunched the numbers, they found that clusters fit their data much better than monomers (R² = 0.75 vs. R² < 0).

The final step was to work out what part of Mig1p was involved in forming the clusters. To do this, the authors compared Mig1p and Msn2p, the second transcription factor they studied that also formed into clusters, and looked for structural regions they might have in common.

What they found was both proteins had a highly disordered region. For Mig1p, it was at the C-terminus and for Msn2p it was at the N-terminus.

The hypothesis is that these disordered regions, which are both at the opposite end of the protein from the DNA binding domain, interact and form ordered structures that enable clusters to form.  Wollman and coworkers used circular dichroism to show that when Mig1p was put in conditions that favor cluster formation, there was a transition consistent with unstructured protein becoming structured.

What we seem to have is a cluster of transcription factors connected in the middle with their DNA binding domains pointing out. This rolling cluster can more easily hop from DNA strand to DNA strand to find the right spots to bind.

Without this mechanism, Mig1p couldn’t get to where it needs to in time. It is as if Tarzan had 6-9 arms circling his body so he could get to Jane even more quickly.

With the wide range of tools available and our deep understanding of how yeast works and how a yeast cell is organized, our trusted ally S. cerevisiae again teaches us something fundamental about how our biology works.

Mig1p may be able to swing like Tarzan but it can’t yell like him. Or like Carol Burnett!

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

Categories: Research Spotlight

Tags: free diffusion, intersegment transfer, MIG1, Saccharomyces cerevisiae, transcription factor

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