New & Noteworthy
May 4, 2017
If the movie WarGames is anything to go on, the US government does not make it easy to launch a nuclear missile. Two soldiers have to do many things simultaneously and in the right order before that missile can take flight.
This makes perfect sense as you do not want to launch a nuclear attack unless you absolutely have to. The continued existence of the human race depends on these fail safes being in place and working. The same goes for a cell that is heading into meiosis.
Meiotic fail safes are in place to ensure the survival of a cell during the dangerous, early part of meiosis, when there are lots of double-strand breaks in the DNA. These all need to be resolved before a cell is allowed to continue through meiosis to create gametes. If the cell moves on while the breaks are still there, gamete production will fail and the cells will die.
While the exact sequence of events needed to launch World War III is known (at least by a few people), the exact details of getting a cell safely through meiosis are a bit murkier. With the help of good old Saccharomyces cerevisiae, we have the broad outlines, but are still investigating the finer points.
A new study by Prugar and coworkers in GENETICS has helped clear up a bit of the murk in yeast. They have uncovered a connection between the meiosis-specific kinase Mek1p and the transcription factor Ndt80p that may explain how a cell “knows” when it is safe enough to emerge from prophase and keep progressing through Meiosis I.
Mek1p is known to be active when there are lots of these double-strand breaks around and to lose activity as these breaks are resolved. Ndt80p, on the other hand, is inactive when there are lots of these breaks and active when they are resolved. So it makes sense that their activities might be related to each other.
In this study, the authors show that once Mek1p activity falls below a certain level, it can no longer keep tamping down Ndt80p activity. Once unleashed, Ndt80p can go on to activate many genes, including the polo-like kinase CDC5 and the cyclin CLB1. This round of gene activation allows the cell to progress through meiosis.
The key to teasing this out was a set of experiments where Prugar and coworkers were able to control the activities of Mek1p and Ndt80p independent of the cell’s DNA state. It is like circumventing the set of protocols to get those missiles launched.
To independently control Ndt80p activity, they used a form of the protein that requires estradiol to be active. And they controlled the activity of Mek1p by using a mutant, mek1-as, that is sensitive to the purine analogue 1-NA-PP1. In the presence of this inhibitor, Mek1p stops working.
They looked at the targets of these two proteins to infer activity. For example, they determined if Ndt80p was active by looking for the presence of CDC5. And to see if Mek1p was active, they looked for phosphorylated Hed1p.
In the first experiment, they showed that in the absence of both estradiol and 1-NA-PP1, Hed1p stayed phosphorylated. Mek1p was constitutively active in the absence of Ndt80p even as double-strand breaks were resolved. (They used phosphorylated Hop1p as an indirect measure of double-strand breaks.)
When Ndt80p was activated through the addition of estradiol, CDC5 was turned on and Hed1p lost its phosphorylation. This loss of Mek1p activity did not happen as quickly as with 1-NA-PP1.
These results suggest a negative interaction between Mek1p and Ndt80p. When Ndt80p is active, Mek1p is not and when Mek1p is active, Ndt80p is not. The resolution of the DNA breaks as indicated by the loss of phosphorylated Hop1p was not sufficient to shut off Mek1p activity. It took the activation of Ndt80p for this to happen.
Well, Ndt80p did not directly cause Mek1p’s inhibition. A second set of experiments suggested that a target of Ndt80p, CDC5, was responsible.
For this they made Cdc5p activity independent of Ndt80p induction by making it dependent on estradiol, similar to what they did with Ndt80p. Using a strain deleted for NDT80, they found that inducing Cdc5p activity was enough to eliminate Mek1p activity.
I don’t have the space to go into the rest of the experiments in this study, but I urge you to read it if you want to learn about more of the details of the cell’s protocol for know when it is OK to progress through meiosis.
With the help of the awesome power of yeast genetics (#APOYG), Prugar and coworkers have added to our knowledge about the safeguards that are in place to keep a cell from launching into meiosis too soon. Turns out they are even more complicated than the ones that prevent accidental thermonuclear war.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
April 20, 2017
In the original Star Wars, Luke destroys the Death Star with a precise strike of proton torpedoes down a small thermal exhaust port. For him it was as easy as bullseyeing “womp rats in my T-16 back home.”
Luke and the rest of the Rebel Alliance learned of this engineered fatal flaw from Jyn and her friends in the prequel Rogue One. With this information the Rebel Alliance was able to keep the rebellion alive long enough to finally bring down the Empire by the end of Return of the Jedi.
It turns out that our friend Saccharomyces cerevisiae has taught us about a fatal flaw in mitochondria. Like proton torpedoes in an exhaust port, when the gene YME1 is inactivated, mitochondria become unstable. But instead of bits of Death Star raining down on nearby planets, mitochondrial DNA (mtDNA) is released into the cytoplasm.
Sometimes this mtDNA can end up in the nucleus and find its way into nuclear DNA. And if the conclusions of a new study in Genome Medicine by Srinivasainagendra and coworkers turns out to be right, this numtogenesis (as the authors call this process) can have profound consequences when it happens in people. Their data suggests that it might lead to cancer or possibly cause cancers to spread.
These researchers searched through whole genomes of colon adenocarcinoma patients and found that these cancer cells had 4.2-fold more mtDNA insertions compared to noncancerous cells from the same patient. They also found that patients with more of these insertions tended to do worse (although the sample sizes were too small to say this definitively).
Why is this happening in the cancer cells? What has caused the mitochondria to give up their DNA?
Srinivasainagendra and coworkers turned to previous work that had been done on the YME1 gene in the yeast S. cerevisiae to find one possible reason. YME1 had been shown to be an important suppressor mtDNA migration to the nucleus. Perhaps this was true in mammalian cells as well.
A search through the genomes of cancers suggested that this seemed to be the case. Around 16% of the colorectal tumors they looked at had a mutated YME1L1 gene, the human homologue of YME1. And mutated YME1L1 genes were found in other tumors as well.
If only destroying gene function was as fun.
They used CRISPR/Cas9 to directly test the effects of knocking out YME1L1 in the breast cancer cell line MCF-7. The knock out cells had a 4-fold increase in the amount mtDNA in the nuclear fraction compared to cells that still had working YME1L1.
As a final experiment, they used a yeast strain, yme1-1, in which YME1 function was inactivated, to show that the human homologue, YME1L1, could suppress the migration of mtDNA to the nucleus.
This yme1-1 strain has a TRP1 gene encoded in the mtDNA instead of the nucleus. Since the gene cannot be read by the mitochondrial transcription machinery, the only way this yeast strain can survive in the absence of tryptophan is if the TRP1 gene moves from the mitochondrion to the nucleus.
In their experiment, with vector alone, they got around 1000 TRP+ colonies with yme1-1. When they added back yeast YME1, this number dropped to less than 50 compared to the 100 or so they got when they added the human homologue, YME1L1. So YME1L1 can suppress mtDNA migration to the nucleus.
Given that YME1L1 was mutated in just a subset of the cancers, it is unlikely that it is the only player in the mtDNA these authors found in the nuclei of cancer cells. But it does look like it is one way this can happen.
And it would have been very hard to fish out the human gene without the critical work that had been done in yeast previously. Yeast shows us the way again. #APOYG
April 17, 2017
With the construction of a global genetic interaction network in S. cerevisiae, it’s not hard to see why yeast genetic interactions remain a treasure trove for biological discovery. When combined with tools for visualization and analysis, these data can be used to draw powerful functional maps of the cell and infer potential functions for uncharacterized genes.
In a recent paper published in G3: Genes|Genomes|Genetics, Usaj et al. describe a web-based resource for exploring the global yeast genetic interaction network: TheCellMap.org. TheCellMap.org is an online database and visualization tool for quantitative yeast genetic interaction data. It provides an interactive version of the global yeast genetic interaction similarity network described by Costanzo et al., enabling users to scroll through and zoom in on different clusters of functionally related genes within the network. Users can search for specific genes or alleles, extract and re-organize sub-networks for genes of interest, functionally annotate genetic interactions, and more. Further, if more details about a gene are needed, users can even double-click on the gene to be taken to its respective locus summary page at SGD!
April 4, 2017
Sometimes in life you need to take risks to survive and prosper. Maybe you need to take a leap of faith like Bruce Wayne does to escape that pit in The Dark Knight Rises. Or you need to try a risky business strategy to put your company out in front.
While these are critical things to do at the time, chronic risk-taking is not usually a good idea. Even Bruce Wayne retires to Florence at the end of The Dark Knight Rises, his risky life choices done now that he has saved his beloved Gotham City. He can now settle down with Selina Kyle (Catwoman) and live happily (and safely) ever after.
Something similar can happen in the budding yeast, Saccharomyces cerevisiae. In the right environment, certain yeasts can build up mutations like mad, hoping to hit on one that lets them make it out of that pit.
But then, over time, yeasts with the successful mutation lose that frenetic mutation rate—they take fewer risks with their DNA. One way they can do this is by ending up with a mutation that suppresses the high mutation rate. This is like Bruce Wayne retiring to a nice villa on the Arno.
Another way they can reestablish their old mutation rate is to mate with a nonmutator, a yeast strain that does not risk its DNA with a high mutation rate. Now, the next generations have the beneficial mutation and a lowered mutation rate as well. It is as if Bruce Wayne settled down with an accountant instead of Catwoman and had risk-averse children to carry on the Wayne name.
Bui and coworkers investigate this phenomenon in yeast in a new study out in GENETICS. They show that natural isolates exist that can sporulate into one of these mutator strains. And that at least one strain predicted to have a high mutation rate has a number of suppressor mutations that tamp down that higher rate.
Previous work had shown that a high mutation rate can happen with mutations in two genes in the mismatch repair (MMR) pathway, MLH1 and PMS1. This was discovered when researchers saw that some of the haploid strains from a mating of two laboratory strains, S288C and SK1, showed this mutator phenotype. A closer look revealed that S288C has a mutation in MLH1, and SK1 has a mutation in PMS1.
Bui and coworkers show that these mutator haploid strains outcompete other strains in high salt media. The strain hits upon mutations that allowed for better survival in high salt faster than its slower mutating brethren. But this advantage does not last.
After 120 generations these mutator strains start to lag behind strains with more typical mutation rates. Their DNA becomes as beat up as Bruce Wayne’s body by the third movie.
The researchers next looked at the MLH1 and PMS1 genes of 1,010 natural isolates of S. cerevisiae to see if there were any that might be able to sporulate into a mutator strain. Or that were already mutator strains themselves.
Since the mutations that lead to the mutator phenotype are recessive, strains that are heterozygotes for mutations in both genes should be able to form haploid spores with the higher mutation rate. They found 18 that fit the bill.
They also found one strain, YJM523, that was homozygous for both mutations and so would be predicted to have a high mutation rate. They further investigated this strain.
The first thing they did was to test the YJM523 mutant genes in an S288C background. They found that these two mutant genes did indeed give S288C a mutator phenotype. In fact, it had a higher mutation rate than the original genes from S288C and SK1 did, suggesting there were other mutations in one or both YJM523 genes that further increased the mutation rate.
These researchers next wanted to determine if YJM523 had a higher mutation rate or not, but needed to first develop a new assay that could be used in natural isolates. They came up with a reporter system that is not dependent on the typical markers used in yeast experiments, but instead relies on two antibiotic markers.
The reporter uses the NatMX antibiotic resistance marker for selection and the KanMX marker to identify revertant mutants. By scoring the number of colonies resistant to both antibiotics, they could determine the mutation rate.
When the researchers did this experiment, they found that YJM523 did not have a particularly high mutation rate. Sporulation experiments showed that this was most likely due to multiple suppressor mutations.
So out in the wild, the potential for mutator strains exists. And a close look at the genome of YJM523 showed that if it did have a mutator phenotype, it was for a short time. This yeast at least quickly lost its high mutation rate (assuming it ever had one).
This study helps to explain how yeast can obtain that mutator phenotype that researchers have seen before. And how yeast can escape that mutation pit to get back to the safety of a reasonable DNA repair pathway. To quote the prisoners in The Dark Knight, “Deshi Basara”, or ‘rise up’ yeast!
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics