New & Noteworthy
April 22, 2015
A study published a few years ago made a big splash in the health news by showing that obesity is socially contagious. If one person gains weight, their friends tend to gain weight too—even if they don’t live in the same town! This works the opposite way too: thinner people are more likely to be socially connected with thinner people.
You might think this is because people tend to make friends with others of a similar size, but this doesn’t seem to be the case. The researchers concluded that there is actually a cause-and-effect relationship: we all influence the weight of our friends.
Well, S. cerevisiae cells are not so different. They may not have social lives, but since they can’t move on their own, they do tend to live together in colonies. And within these colonies, they influence each other: not in terms of weight, but in terms of the effect that calorie intake has on the length of their lives.
Turns out that like nematodes, fruit flies and even mice, living on a meager diet makes yeast live longer. And in a new study published in PLOS Biology, Mei and Brenner found that yeast cells actually share the life-extending benefits of calorie restriction with their neighbors, probably via a still-unidentified small molecule.
Yeast are normally grown in the lab on medium containing 2% glucose. To a yeast cell, this is like an all-you-can eat buffet that goes on for its entire lifetime. Media with a glucose content of 0.5% or less represent a meager diet. But that deprivation comes with a benefit, in the form of an extended lifespan.
Mei and Brenner already had some hints from previous studies that yeast cells might excrete a substance that promoted lifespan extension. To study this systematically, they devised an experiment to test whether mother cells change the media surrounding them as they divide.
The researchers placed individual mother cells in specific spots on Petri plates containing an all-you-can-eat buffet (2% glucose), a restrictive diet (0.5% glucose), or a near-starvation diet (0.2% glucose). They watched as the cells budded, and removed each new daughter cell as it separated from the mother, counting the buds. The lifespan of a mother yeast cell, termed the replicative lifespan, is measured as the number of times she can bud during her lifetime.
After the mother cells had budded 15 times, half of them were physically moved to fresh parts of the same plate, while the other half were left in place. For the mothers on the 2% glucose plates where calories were abundant, the move didn’t change anything. The mothers that were moved had exactly the same replicative lifespan as those that stayed put.
On the plates where calories were restricted, it was a different story. The cells that stayed in place had extended lifespans, as expected under these low-calorie conditions. But the cells that were moved to new locations lost most or all of the life extension—even though calories were still restricted in their new locations. This suggested that the mother cells had secreted a “longevity factor” into the medium surrounding them, which then extended their lifespan when they got older.
There were a couple of metabolites that were prime candidates for the longevity factor: nicotinic acid (NA) and nicotinamide riboside (NR). NA and NR are precursors to nicotinamide adenine dinucleotide (NAD+), a compound that acts as an essential cofactor for many important enzymes. They had already been implicated in lifespan extension because mutating genes involved in their metabolism can affect how long various creatures live.
When the scientists tried supplementing calorie-restricted cells that had been moved to fresh medium with either NA or NR, they found that supplying these metabolites could restore the longevity benefit. This finding strengthens the idea that NAD+ metabolism is involved.
But was the longevity factor actually NA or NR? To test this, Mei and Brenner grew yeast in liquid media with the different glucose concentrations and then tested for NA and NR in the medium using liquid chromatography-mass spectrometry analysis. They found that under all the conditions, the amount of NA secreted by the cells didn’t change and secreted NR was undetectable, suggesting that neither was the factor induced by calorie restriction.
To ask directly whether there is a diffusible longevity factor, the researchers grew cells in liquid medium containing 2% or 0.2% glucose until all the glucose was used up, then separated out the cells and freeze-dried the remaining liquid. They suspended the dried “conditioned” medium in water and spread it on plates to repeat the cell-moving assay.
Just like before, cells grown in 2% glucose had the same lifespan after being moved to a fresh spot, and the addition of resuspended conditioned medium to the plate didn’t change that. However, the starved cells grown on 0.2% glucose not only kept their lifespan extension when moved to conditioned media, but actually lived 10% longer compared to starved cells on un-conditioned media that were not moved.
When the researchers dialyzed the conditioned medium so that molecules smaller than 3.5 kDa were lost, the longevity factor was lost too. So it looks to be a small molecule, and of course they are actively pursuing its identity. Intriguingly, this would explain why other scientists have been unable to detect calorie restriction-induced lifespan extension in yeast using microfluidic technology, where immobilized yeast cells are grown with a constant exchange of growth medium. Under these conditions, a small molecule that promotes longevity would be washed away.
So, even though they don’t have Facebook friends, yeast cells influence the health of their peers. Rather than spreading the influence through social interactions as we humans do, they broadcast a chemical that is the key to long life.
It’s tempting to think that the identity of this chemical will tell us something about human aging. But if this mysterious molecule worked in humans the same way as it does in yeast, people would still have to eat just enough food to stay alive to get the benefits. Still, perhaps the molecule can point us towards finding a treatment that will let us live longer while enjoying lots of good food. We could have our cake and eat it too!
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
April 21, 2015
This will be the 45th year that the legendary summer Yeast Genetics & Genomics course has been taught at Cold Spring Harbor Laboratory. (OK, the name didn’t include “Genomics” in the beginning…) The list of people who have taken the course reads like a Who’s Who of yeast research, including Nobel laureates and many of today’s leading scientists.
The application deadline is May 15th, so don’t miss your chance! Find all the details and application form here.
This course (July 21 – August 10) provides a comprehensive education in all things yeast, from classical genetics through up-to-the-minute genomics. Things you’ll learn include:
- How to Find and Analyze Yeast Information Using SGD
- Transformation of Plasmids & Integrating DNAs
- Looking at Yeast Cells using Light Microscopy and Fluorescence Microscopy
- Manipulating Mating-Type and Epigenetic Transcriptional Silencing
- Meiosis & Tetrad Dissection
- Isolation and Characterization of Mutants
- Working with Essential Genes
- Synthetic Genetic Array Analysis
- Measuring Mutation Rates and Studying Human Genetic Variation in Yeast
- Detecting Copy Number Variants using Comparative Genomic Hybridization
- Mutation Detection using Whole Genome Sequencing and Linkage
- Barcode Sequencing and Comparative Functional Genomics
All these techniques will be summarized in a completely updated course manual, which will be published by CSHL Press.
Scientists who aren’t part of large, well-known yeast labs are especially encouraged to apply – for example, professors and instructors who want to incorporate yeast into their undergraduate genetics classrooms; scientists who want to transition from mathematical, computational, or engineering disciplines into bench science; and researchers from small labs or institutions where it would otherwise be difficult to learn the fundamentals of yeast genetics and genomics. Significant stipends (in the 30-50% range of total fees) are available to individuals expressing a need for financial support and who are selected into the course.
Besides its scientific content, the fun and camaraderie at the course is also legendary. In between all the hard work there are late-night chats at the bar and swimming at the beach. There’s a fierce competition between students at the various CSHL courses in the Plate Race, which is a relay in which teams have to carry stacks of 40 Petri dishes (used, of course). There’s also a sailboat trip, a microscopy contest, and a mysterious “Dr. Evil” lab!
April 15, 2015
In the science fiction novel Dune, the most precious thing in the universe is spice. It is harvested from the sands of the planet Dune under very dangerous conditions—every time people start to mine it, a gigantic worm comes to kill them. The spice is so valuable, though, that the miners harvest it until the very last second. As time runs out, a carryall whisks them away as the worm rises out of the desert.
While nothing quite so exciting happens in the nucleus of a yeast cell, one of the closest situations may be at the rDNA locus. Here the precious commodity is ribosomes and yeast cells need them to be made almost constantly. The only pauses in production are when this part of the genome needs to be replicated and when it needs to be segregated into a daughter cell. In both cases, as something akin to giant sandworms comes crashing onto the scene, harvesting stops and the machinery is removed with a cellular carryall.
Of course there aren’t harvesters extracting whole ribosomes from the yeast nucleus! Instead, it is RNA polymerase I (Pol I) and RNA polymerase III (Pol III) making the raw stuff of ribosomes, the 35S and 5S rRNAs, respectively. These polymerases need to stop transcribing and clear the way for the rDNA replicating replisome during S phase and for condensins at the end of anaphase. If they don’t, the rDNA locus becomes unstable, resulting in the mother yeast cell living a shorter life and in its daughter not getting the rDNA locus (and the rest of the chromosome it is on) at all.
In a new study in Nature Communications, Iacovella and coworkers have identified the carryall that helps to remove Pol I from the rDNA locus. Surprisingly, it is a kinase, named Rio1, that was previously known to be involved in rRNA processing and building ribosomes in the cytoplasm.
Rio1 does not physically remove Pol I from the 35S gene. What this study suggests is that it phosphorylates one of the 14 subunits of Pol I, Rpa43, so that Pol I no longer interacts as strongly with the transcription factor Rrn3. The end result is the untethering of the polymerase and its release from the DNA. Now condensins can glom onto the rDNA locus at the end of anaphase and DNA polymerase can barrel through the region in S phase without wreaking genomic havoc.
The first key finding in this study was that Rio1 isn’t just active in the cytoplasm, but also in the nucleus. In fact, it was most active in the nucleolus, a small moon shaped section of the nucleus that is formed around the rDNA locus.
The researchers went on to show that Rio1 is present in the nucleolus only at certain times in the cell cycle (S phase and anaphase). Using chromatin immunoprecipitation (ChIP) assays, they were able to show that Rio1 was enriched specifically at the rDNA’s 35S promoter and coding sequence.
They next created a conditional Rio1 mutant that could not enter the nucleus in the presence of galactose. When Rio1 was kept out of the nucleus, nucleoli became fragmented, there were no condensins on the rDNA locus at anaphase, and the mother yeast did not pass the replicated nucleolus to her daughter. Obviously Rio1 is a critical housekeeper for the rDNA locus!
They next used ChIP assays to show that when Rio1 was kept out of the nucleus, there was around 3-fold more Pol I at the 35S promoter and gene during anaphase than when Rio1 was allowed to go nuclear. This resulted in a 5-8 fold increase in 35S rRNA levels—implying that Pol I was still there cranking out rRNA.
The most likely explanation is that all the hyperactive Pol I transcribing the rDNA locus prevents condensins from binding the DNA and that removal of Pol I by Rio1 allows the condensins to bind. The condensins then shrink the rDNA region such that it can move though the tiny bud opening into the daughter.
They got similar results in S phase, where lack of nuclear Rio1 caused an increase in Pol I occupancy at the rDNA and an increase in 35S transcription as well. Here the lack of Rio1 has more devastating consequences to the genome. Its absence most likely causes the replisome to collide head-on with the transcribing Pol I, resulting in double strand DNA breaks. Because the rDNA locus is such a repetitive region, the cell makes mistakes when it repairs the break using homologous recombination. The end result is nucleolar fragmentation and a shorter life span.
The final set of experiments showed that Rio1 most likely affects Pol I occupancy by phosphorylating one of its subunits, Rpa43. First the authors used Western blot analysis to show that Rpa43 is less phosphorylated when Rio1 is kept out of the nucleus and when a mutant version of Rio1 lacking its kinase function is used. They also showed that Rio1 could phosphorylate Rpa43 in vitro.
They postulate that this phosphorylation causes the interaction between Pol I and the transcription factor Rrn3 to weaken, allowing Pol I release. Alternatively, Rpa43 phosphorylation could lead to the disintegration of the Pol I enzyme itself. Confirmation of one or the other will require more research.
Taken together, these studies paint a fascinating picture of the rDNA locus. Here Pol I is frenetically transcribing as much 35S rRNA as it possibly can to keep up with the yeast cell’s unquenchable thirst for ribosomes. The only time it stops is when continuing could harm the cell, during DNA replication in S phase and DNA transmission in anaphase. And even then, like spice hunters on Dune, they stay until the very last minute. A spicy tale, indeed.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Rise, replisome, rise!
April 9, 2015
Have you ever heard of the Baader-Meinhof phenomenon? (Don’t worry, we hadn’t either!) But you’ve surely experienced it.
The phenomenon describes the experience of suddenly seeing something everywhere, after you’ve noticed it for the first time. For those of us in Northern climates, it’s like like robins coming back in the springtime: one day, you see a single robin hopping on the grass; the next day, you look around and realize they’re all over.
Something similar happened to Godin and colleagues as they investigated the S. cerevisiae Shu2 protein. People knew about the protein and its SWIM domain but no one had looked to see just how conserved it was.
When the researchers looked for homologous proteins that contained the characteristic SWIM domain, they found homologs in everything from Archaea through primitive eukaryotes, fungi, plants, and animals. They were practically everywhere (in the evolutionary sense).
In a new paper in GENETICS, Godin and coworkers described their studies on this relatively little-studied, unsung protein. They found that it is an important player in the essential process of DNA double-strand break repair via homologous recombination, and its SWIM domain is critical to this function. Not only that, but being able to compare the SWIM domains from so many different homologs allowed them to refine its consensus sequence, identifying a previously unrecognized alanine within the domain was both highly conserved and very important.
Double-strand DNA breaks (DSBs) can happen because of exposure to DNA damaging agents, but they are also formed normally during meiotic recombination, in which a nuclease actually cuts chromosomes to start the process. Homologous recombination to repair DSBs is a key part of both mitosis and meiosis.
During homologous recombination, one strand of each broken DNA end is nibbled back to form a single-stranded region. This region is then coated with a DNA-binding protein or proteins, forming filaments that are necessary for those ends to find homologous regions and for the DSB to be repaired.
Shu2 is one of the proteins that participates in the formation of these filaments in S. cerevisiae. It was known that it was part of a complex called the Shu complex, and a human homolog, SWS1, had been identified. But the exact role of Shu2 and the significance of the SWIM (SWI2/SNF2 and MuDR) zinc finger-like domain that it contains were open questions.
One of the first questions the authors asked was whether Shu2 was widely conserved across the tree of life. Genes with similar sequences had been seen in fission yeast (Schizosaccharomyces pombe) and humans, but no one had searched systematically for orthologs. They used PSI-BLAST, a variation of the Basic Local Alignment Search Tool (BLAST) algorithm that that is very good at finding distantly related proteins, to search all available sequences.
Querying with both yeast Shu2 and human SWS1, the researchers found hits all across the tree of life—both in “lower” organisms such as Archaea, protozoa, algae, oomycetes, slime molds, and fungi, and in more complex organisms like fruit flies, nematode worms, and plants. The homologous proteins that they found across all these species also had the SWIM domain, suggesting that it might be important.
The sequence similarity was all well and good, but did these putative Shu2 orthologs actually do the same job in other organisms that Shu2 does in yeast? One way to test this is to do co-evolutionary analysis. Proteins that work together are subject to the same evolutionary pressures, so they tend to evolve at similar rates. Godin and colleagues found that evolutionary rates of the members of the Shu complex in fungi and fruit fly did generally correlate with those of other proteins involved in mitosis and meiosis.
The awesome power of yeast genetics offered Godin and coworkers a way to look at the function of Shu2. They first tested the phenotype of the shu2 null mutation, and found that it decreased the efficiency of forming filaments of the Rad51 DNA-binding protein on the single-stranded DNA ends that are created at DSBs. Formation of these filaments is a necessary step in repairing the DSBs by homologous recombination.
The comparison of SWIM domains from so many different proteins highlighted one particular alanine residue. This alanine hadn’t previously been considered part of the domain’s consensus sequence, but it was conserved in all the domains.
When the researchers changed the invariant alanine residue in yeast Shu2, the mutant protein bound less strongly to its interaction partner in the Shu complex, Psy3. When they mutated the analogous residue in the human Shu2 ortholog SWS1, this also decreased its binding to its partner, SWSAP1.
Other mutations within the SWIM domain of Shu2 also affected its interactions with other members of the Shu complex, and made the mutant cells especially sensitive to the DNA-damaging agent MMS. Diploid cells with a homozygous mutation in the Shu2 SWIM domain had very poor spore viability, suggesting that the SWIM domain is important for normal meiosis.
As one more indication of the SWIM domain’s importance, Godin and colleagues took a look in the COSMIC database, which collects the sequences of mutations found in cancer cells. Sure enough, a human cancer patient carried a mutation in that invariant alanine residue of the SWIM domain in the Shu2 ortholog, SWS1.
There’s still much more to be done to figure out exactly what Shu2 and the Shu complex are doing during homologous recombination. Yeast obviously provides a wonderful experimental system, and the discovery of Shu2 orthologs in two other model organisms that also have awesomely powerful genetics and happen to be multicellular, Drosophila melanogaster and Caenorhabditis elegans, expands the experimental possibilities even further.
There’s also a lot to be learned about the SWIM domain in particular. The discoveries that it affects the binding behavior of these proteins and that it is mutated in a cancer patient show that it’s very important, but just what does it do in Shu2? It will be fascinating to find out exactly how this domain works to help cells recover from the lethal danger of broken chromosomes. And it is amazing what you can see, once you start looking.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD