October 11, 2016
As anyone who has tinkered with the inside of a living cell or looked at one of those daunting and overwhelming biochemistry poster knows, life is complicated. Like thousands of gears all interconnected in some vast steampunk machine, a cell has thousands of genes making thousands of proteins that come in a variety of flavors all interacting in overlapping, complicated ways to keep the cell alive. Toss in those important RNAs and a few other bells and whistles and you begin to understand why scientists have had to just focus in on individual parts of the machine to study.
Until now that is. In a new study out in Science led by University of Toronto Professors Brenda Andrews and Charles Boone, and Professor Chad Myers of the University of Minnesota-Twin Cities, Costanzo and coworkers were able to get a first approximation of how all those genes are connected in a yeast cell. And it is as complicated as you imagined.
This was a daunting undertaking that took over 15 years to pull off but it looks like it was well worth it. They have already learned an incredible amount and people will continue to learn more and more as scientists mine this treasure trove of data for a long time to come (Stay tuned for the data to be available here at SGD). This is the sort of study that will provide an invaluable reference guide that will keep giving year after year after year.
And it isn’t just a tour de force of basic biology either. This study will, among other things, reveal what genes of unknown function are actually doing in cells, identify new targets to go after in diseases like cancer and may even help geneticists find that “missing heritability” in their genome-wide association studies (GWAS). Given that these three examples are just a subset of what we’ll get out of this study, I think you can see why it is such a game changer.
It would not have been possible without all of the yeast work that has been done and carefully curated over the years. And there is no way it could have been done in another organism, like humans. Human cells are simply too complicated, we don’t yet have the necessary tools, and we don’t have as deep a knowledge as we do with yeast. It is only in yeast that we could begin to get this amazing peek at life happening inside of a cell.
Yeast as a model organism is alive and well and helping us better understand life and ourselves. This sort of study drives that point home in a way that benefits many, many scientists and, eventually, patients the world over.
When 1+1 Does Not Equal 2
So how did these researchers pull this off? They basically looked at what happens when they knock out two genes at once in yeast. They did this for every possible gene pair for 5416 out of the 6,000 or so yeast genes.
Of course this strategy won’t work with the 1000 or so yeast genes that are essential—the yeast dies when these are knocked out. For these genes, the researchers used temperature sensitive mutations under conditions where the gene is tweaked but not dead.
What they were looking for were combinations that did either better or worse than you might predict based upon each individual deletion on its own. And they found plenty.
They generated 23 million of these double knockout yeast (Yes, you read that right!) and found ~550,000 negative interactions and ~350,000 positive interactions—a huge amount of data about the inner workings of a cell. The next step was to take all of this data and try to make sense of it.
They did this by clustering the genes together based on who was interacting with whom. If deleting gene A with either gene B or gene C had a larger negative effect than expected, then these three genes were clustered together. And if deleting gene A had an effect on gene D, but deleting gene B or C did not have an effect on D, then A was involved in both a cluster with B and C as well as a separate cluster with D. Now repeat this over and over thousands and thousands of times.
When they did this, many things about living cells quickly came into focus. I only have the space to touch on a few.
First off, while negative interactions usually happened between genes in the same biological processes or in the same subcellular compartments, the same was not true for the positive interactions. These were much more spread out and often genes like chaperones.
They also found about 1000 genes that weren’t involved with many other genes at all. These are most likely genes that would show more interactions under different growth conditions. For example, deleting a gene critical for utilizing galactose might not show a lot of interactions if a cell is grown in glucose. Repeating this experiment under different conditions will probably uncover where these genes fit in.
This is just a taste of what they found and of what is to come in the next few years. Here is a bit more:
Finding Out What a Gene Does
You’d think having a genome sequence for over 20 years might mean you’d pretty much know what all those genes are doing, but you’d be wrong.
There are still plenty of genes in yeast with unknown function. This kind of study can help uncover what a gene might be doing by seeing which genes it clusters together with. If gene X is clustered with known DNA repair genes, it is probably involved in something to do with DNA.
One such gene they looked at in this study was YJR141W. Not a lot was known about this gene other than it was essential.
What they found was that this gene clustered with genes in the cleavage polyadenylation factor (CPF) and cleavage factor 1A (CF1A) protein complexes suggesting this gene might have a role in mRNA 3’-end processing and polyadenylation. After showing experimentally that the protein from this gene physically interacted with CPF complex members Mpe1p and Ysh1p and that the temperature sensitive mutant had trouble processing mRNA in vitro, the researchers felt confident enough to give this gene a name based on its function. They named it IPA1 (Important for cleavage and PolyAdenylation 1).
Helping Find New Cancer Treatments
This study also has important implications for human health in lots of different ways. For example, it might help scientists find better cancer treatments.
Cancer happens when mutations in key genes cause cells to grow uncontrollably or to refuse to die. In an ideal world we’d treat these cancers by going after these genes directly. Unfortunately many of them are not particularly great targets to go after with pharmaceuticals.
This is where synthetic lethals can help. A synthetic lethal is when mutations in two separate genes do not kill a cell on their own, but they do when a single cell has both mutant genes.
The idea then is to find a pair of mutant genes that kills a cell with one of the genes being involved in cancer and the second one some other gene. You can go after the second gene and by affecting its activity kill the cancer cells that have the other gene in this pair mutated.
And of course noncancerous cells are fine with you targeting that second gene—mutating only that gene isn’t lethal. Chemotherapy with many fewer side effects!
If you think this sounds like something this study might uncover, you’d be right. This study found around 10,000 or so double knockouts that were lethal which might provide a lot of possibilities for cancer researchers.
Cancer is almost certainly just one of many possible applications to human health. And we will learn so much about the basic biology of a cell from such an exhaustive and detailed analysis of the inner workings of a cell.
This holistic portrait of the genetic interactions of a yeast cell is elegant, beautiful and useful. Not bad for the beast that gives us bread and booze.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
April 06, 2016
In the book Dune, the mentat Thufir Hawat is captured by the evil Harkonnens and given a residual poison. He can only stay alive by getting a constant dose of the antidote. Once it is withdrawn, he will die.
A new study in the journal GENETICS by Dodgson and coworkers shows that the same sort of thing can happen to yeast that carry an extra chromosome. In this case, certain genes on the extra chromosome turn out to be like the residual poison. And a second gene turns out to be the antidote.
Once that second gene is deleted, the yeast cell dies. It has been deprived of its antidote.
This synthetic lethal phenotype isn’t just a cool finding in yeast either. Cancer cells invariably have extra and missing chromosomes. If scientists could find similar “antidote genes” in specific types of cancers and target them, then the cancer cell would die. And this would happen without damaging the other cells of the body that have a typical number of chromosomes.
The first thing these researchers did was to make separate yeast strains each with an extra chromosome I, V, VIII, IX, XI, XII, or XVI. The next step was to see what happens when every gene was deleted individually, one at a time, from each strain.
As expected, these yeast did pretty well when a gene on the extra chromosome was deleted. So, for example, a strain with an extra chromosome I tolerated a gene deleted from chromosome I. This makes sense as this just brings that gene back to its normal copy number.
But this was not the case with chromosomes VIII and XI. Here deleting genes on the extra chromosome often had a negative effect. This suggested that the screen probably had a high number of false positives and these researchers later confirmed this.
Likely reasons for the high number of false positives include the strain with the extra chromosome being W303 and the deletion strain being S288C, errors in the deletion collection itself, and what they refer to as neighboring gene effects. Basically this last one is the effect that deleting a gene has on nearby genes.
Once Dodgson and coworkers corrected for these problems, they found two broad sets of phenotypes – general and chromosome specific.
The general ones were the ones shared by most or all of the strains. These were deletions that affected the yeast no matter which chromosome they had an extra copy of.
For the most part, these genes were enriched for the Gene Ontology (GO) term vesicle-mediated transport, indicating that they have something to do with the transportation of substances in membrane-bounded vesicles. For example, deletion of MNN10, HOC1, and MNN11, genes all involved in protein transport and membrane-related processes, had a negative effect on many of the yeast strains with an extra chromosome. Consistent with this, brefeldin A, a drug that targets protein trafficking, negatively affected most of the strains.
Another gene that affected many of these strains when deleted was TPS1. This gene encodes a subunit of trehalose-6-phosphate synthase, a key enzyme for making trehalose, a molecule that helps yeast deal with stress. Perhaps not surprisingly, having an extra chromosome is stressful!
In addition to the genes that affect many strains with an extra chromosome, there were also genes that were chromosome specific. The best characterized of these was the EDE1 gene in the strain with an extra chromosome IX. Deleting EDE1 in this strain increased its doubling time by more than 80 minutes while only causing an increase of 5 minutes in the doubling time of wild type yeast. This was a severe phenotype in their assay.
They next tried to find which gene on chromosome IX might be responsible for the severe effect of deleting EDE1. Since EDE1 is known to be involved in endocytosis, they looked for genes involved in the same process. And they found one – PRK1.
The strain with a deleted EDE1 gene and an extra chromosome IX was rescued by deleting one copy of the PRK1 gene. The extra PRK1 gene was the poison and the EDE1 gene was the antidote.
If a similar pair could be found in cancers that often have the same set of extra chromosomes, then perhaps scientists could develop drugs that target an antidote gene. Now the cancer cells would die and the “normal” cells would be fine. Thanks again, yeast, for pointing us toward new ways to treat human disease.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
October 24, 2013
Current cancer treatments are a lot like trying to destroy a particular red plate by letting a bull loose in a china shop. Yes, the plate is eventually smashed, but the collateral damage is pretty severe.
Ideally we would want something a bit more discriminating than an enraged bull. We might want an assassin that can fire a single bullet that destroys that red plate.
One way to identify the assassin that can selectively find and destroy cancer cells is by taking advantage of the idea of synthetic lethal mutations. “Synthetic lethal” is a genetic term that sounds a lot more complicated than it really is. Basically the idea is that mutating certain pairs of genes kills a cell, although mutating each gene by itself has little or no effect.
A synthetic lethal strategy seems tailor made for cancer treatments. After all, a big part of what happens when a cell becomes cancerous is that it undergoes a series of mutations. If scientists can find and target these mutated genes’ synthetically lethal partners, then the cancer cell will die but normal cells will not.
This is just what Deshpande and coworkers set out to do in a new study in the journal Cancer Research. They first scanned a previous screen that looked at 5.4 million pairwise interactions in the yeast S. cerevisiae to find the best synthetic lethal pairs. They found 116,000 pairs that significantly affected cell growth only if both genes in the pair were mutated.
A deeper look into the data revealed that 24,000 of these pairs had human orthologs for both genes. In 500 of these pairs, at least one of the partner genes had been shown to be mutated in certain cancers. Using a strict set of criteria (such as the strength and reproducibility of the synthetic lethal effect, and the presence of clear one-to-one orthology between yeast and human), the authors narrowed these 500 down to 21 pairs that they decided to study in mammalian cell lines.
When the authors knocked down the expression of both genes in these 21 gene pairs in a mammalian cell line, they found six that significantly affected growth. They focused the rest of the work on the strongest two pairs, SMARCB1/PMSA4 and ASPSCR1/PSMC2. These mammalian gene pairs correspond to the yeast orthologs SNF5/PRE9 and UBX4/RPT1, respectively.
The authors identified two separate cancer cell lines that harbored mutated versions of the SMARCB1 gene. When this gene’s synthetic lethal partner, PMSA4, was downregulated in these cancer lines, the growth of each cell line was severely compromised. The same was not true for a cell line that had a wild type version of SMARCB1—this cell line was not affected by downregulating PMSA4. The authors used a synthetic lethal screen in yeast to identify a new cancer target which when downregulated selectively killed the cancer without killing “normal” cells.
This proof of principle set of experiments shows how the humble yeast may one day speed up the process of finding cancer treatments without all those nasty side effects (like vomiting, hair loss, anemia and so on). Yeast screens can first be used to identify target genes and then perhaps also to find small molecules that affect the activity of those gene products. Yeast may one day tame the raging bull in a china shop that is current cancer treatments.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
May 04, 2012
When is a temperature sensitive mutation not a temperature sensitive mutation? When the process being studied is affected by temperature too.
Mutations conferring temperature sensitivity – that is, a phenotype that only appears at higher- or lower-than-normal growth temperature due to the loss or alteration of function of a gene product at that temperature – have for decades been an invaluable tool in dissecting many biological processes in yeast and other model organisms. Now there is reason to question whether some temperature sensitive phenotypes might actually be the result of a more complex interaction between multiple genes.
This synthetic genetic interaction is easy to mistake for the effect of a single mutation. A scientist starts out with a mutation that weakens a gene in a certain process. Unfortunately for the scientist, the process being studied is itself weakened by higher temperatures. The effect of the higher temperature combines with the effect of the mutation to shut down the process. The mutation looks temperature sensitive even when it isn’t.
And this does not appear to be merely a theoretical concern. As Paschini and coworkers show in a new study out in GENETICS, something similar may have happened with key mutations used to study telomere function in yeast.
These researchers looked at several mutations but we’ll focus on the work they did with cdc13-1. A key experiment that had been previously done with this mutation dealt with the effects of the loss of cdc13 function in the absence of RAD9.
Basically, researchers had found that prolonged incubation of a cdc13-1/Δrad9 strain at 36° severely compromised viability. These results were used to infer CDC13 function based on its loss at 36°. However, Paschini and coworkers provide compelling data that cdc13-1 behaves equally poorly at 23° and 36°.
First off, they showed that prolonged incubation of wild type yeast at the temperatures used in these studies (36°) resulted in shorter telomeres. They found very little effect on telomere length at 32°.
Next they showed that biochemically, cdc13-1 didn’t behave like a temperature sensitive mutation. Strains with cdc13-1 produced around 4-fold less protein at both 23° and 36° and the protein that was made bound telomeres equally well at both temperatures.
They argue from these two pieces of data that the loss of viability comes from a combination of the compromised cdc13-1 mutation and the effects of higher temperature on telomere function. Something is going on in the rad9 experiment but it is not due to an increased loss of CDC13 function at higher temperatures. There is some other factor involved that is being inhibited.
Of course it could be that cdc13-1 still confers temperature sensitivity, but that they didn’t have the right biochemical assay to see it. To address this issue, they generated five new mutations in cdc13 that behaved more like traditional temperature sensitive mutations.
They focused on one, cdc13-S611L, that was compromised for protein production at temperatures of 32° and above. They then repeated the rad9 double mutant experiment at 32° and 36°. They found that viability was compromised only at 36° even though Cdc13p was equally absent at both 32° and 36°. These results suggest that the loss of viability at 36° is not only the result of cdc13-1.
If this and other results hold up, scientists will need to rethink what previous experiments meant and they may need to modify their models. This should also get other researchers thinking about their temperature sensitive mutations. It is important to confirm biochemically that a mutation indeed makes a specific gene product temperature sensitive. Because sometimes even if it quacks, it isn’t a duck…
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight