March 23, 2016
Diagnosing why something has gone wrong in a complicated system can be difficult. There are so many bells and whistles that you can easily get lost.
That’s why it can sometimes help to turn to a simple system and then apply what you have learned to the more complicated one. This will, of course, sound familiar to any scientists studying that marvel of a model organism, Saccharomyces cerevisiae.
For example, it is amazing what you can glean from this yeast about human brain and blood diseases. Even though, of course, baker’s yeast has neither blood nor a brain!
This becomes very clear in a study out in PLOS Genetics by Fernandez-Murray and coworkers. In this study they use yeast to help figure out why mutations in the SLC25A38 gene in people leads to something called congenital sideroblastic anemia. And even better, their work hints at a possible treatment.
People with sideroblastic anemia make too little hemoglobin in their red blood cells and have too much iron in the mitochondria close to the nucleus (perinuclear mitochondria). The current treatment for this condition is not ideal, involving lots of transfusions and iron chelation.
Sometimes people are born with this anemia and sometimes people get it later in life. One subset of the inherited version happens because a gene with an unknown function, SLC25A38, isn’t working correctly. This group of patients is the focus of this study.
Fernandez-Murray and coworkers started out by using yeast to figure out what the yeast homolog, HEM25, does in a yeast cell. When the gene was deleted, the cells made about 50% less heme than wild type yeast and adding back the human gene, SLC25A38, to this deletion strain restored heme levels. Looks like they had made a yeast model of this inherited anemia.
Previous work had suggested that SLC25A38 might be a glycine or serine transporter and the next set of experiments confirmed it as a glycine transporter in a couple of ways. In both, they took advantage of cases in which yeast can use glycine as their sole nitrogen source if the glycine can make it into the mitochondria.
In the first case, they showed that yeast cells deleted for HEM25 grew poorly on plates where glycine was available as the only nitrogen source. In the second case, they showed that cells deleted for both SER1 and HEM25 grew poorly on plates where again glycine was the only nitrogen source. This last result confirms HEM25 as a glycine transporter since yeast deleted for the SER1 phosphoserine aminotransferase can only grow in the absence of serine if they can get glycine into their mitochondria. (There isn’t space to go into it here, but they also showed that HEM25 was not a serine transporter.)
OK so now they had created a yeast cell that mimicked the effect of sideroblastic anemia and figured out why people with a mutated SLC25A38 gene had the condition. Now it was time to find a treatment.
The researchers came up with three possibilities. The first treatment was just to give the yeast extra glycine, the second was to drive glycine synthesis within the cell by adding a lot of serine, and the third was to add a downstream precursor of heme synthesis, 5-aminolevulinic acid (5-Ala).
They tested each scenario on yeast cells deleted for HEM25 and found that both glycine and 5-Ala worked to restore heme synthesis, but that added serine had no effect. Both glycine and 5-Ala returned heme levels to that seen in wild type.
Of course we aren’t yeast, so they next tested their treatment on something a bit more complicated — zebrafish. By using morpholino technology to knock down both copies of the zebrafish SLC25A38 homolog, SLC25A38a and SLC25A38b, the researchers managed to lower a zebrafish’s heme levels to about 50% of normal.
When they gave these zebrafish extra glycine or 5-Ala, their heme levels did not improve. They were still anemic!
After a bit of thought, the researchers realized that folate might be what the zebrafish were missing. In work that we didn’t have time to go over before, the researchers had shown that a folate dependent pathway was critical for getting heme levels up to normal.
Yeast could get away without added folate in these experiments because they make their own. However, zebrafish, like people, do not.
So the final step was to try to add both glycine and folate to these fish. Now the zebrafish’s heme levels returned to about 80% of normal.
These results suggest a better treatment for some people with sideroblastic anemia — added folate and glycine. And it all came from studying the problem in the simpler, bloodless S. cerevisiae. Nice work again yeast.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
April 09, 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
Categories: Research Spotlight
February 28, 2013
Remember when sequencing the human genome was going to help us better understand and treat complex diseases like Type 2 diabetes or Parkinson’s? Well, ten years later, we’re still waiting.
Sure we’ve made some progress. Using genome wide association studies (GWAS), scientists have uncovered markers here and there that explain a bit about how a genetic disease is inherited. But despite a seemingly never-ending stream of these assays, scientists simply can’t explain all of the genetics behind most of these diseases.
So now scientists need to try to explain this missing heritability. If they can find out why they aren’t getting the answers they need from GWAS, then maybe they can restructure these assays to give better results.
As usual, when things get dicey genetically, scientists turn to the yeast Saccharomyces cerevisiae to help sort things out. And in a new study out in Nature, Bloom and coworkers have done just that.
In this study, they mated a laboratory and a wine strain of yeast to get 1,008 test subjects from their progeny. They extensively genotyped each of these 1008 and came up with a colony size assay that allowed them to determine how well each strain grew under various conditions. They settled on 46 different traits to study genetically.
What they found was that none of these traits was determined by a single gene. In fact, they found that each of the 46 different traits had between 5 and 29 different loci associated with it, with a median of 12 loci. This tells us that at least in yeast, many genetic loci each contribute a bit to the final phenotype. And if this is true in people, it could be a major factor behind the missing heritability in GWAS.
If a trait is dependent on many genetic loci that each have a small effect, then researchers need large populations in order to tease them out. In fact, when Bloom and coworkers restricted their population to 100 strains, they could only detect a subset of the genetic loci. For example, the number of loci went from 16 to 2 when they looked at growth in E6 berbamine.
So it may be that scientists are missing loci in GWAS because there are simply too few participants in their assays. If true, then the obvious answer is to increase the size of the populations being studied. Thank goodness DNA technologies get cheaper every year!
Of course as the authors themselves remind us, we do need to keep in mind that humans are a bit more complex than yeast. There may be other reasons that we aren’t turning up the genetic loci involved in various traits. It may be that we can’t as accurately measure the phenotypes in humans or that human traits are more complicated than the yeast ones studied. Another possibility is that in humans, there are more rare alleles that can contribute to a given trait. These would be very hard to find in any population studies like GWAS.
Still, this study at the very least tells us that larger populations will undoubtedly uncover more loci involved in human disease. Thank you again yeast.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
January 04, 2012
Even though it doesn’t have a brain, yeast is teaching us a lot about Alzheimer’s. Researchers are using this simple eukaryote to figure out what previously identified Alzheimer’s-related genes may be doing in humans as well as to identify new genes that might be involved in this terrible disease. Studies like this may even one day help scientists find better treatments.
Alzheimer’s is a form of dementia that hits about 50% of people over 85. The video below has a great summary of the how the disease progresses:
As the video states, plaques and tangles are linked to the memory loss that is associated with Alzheimer’s. Scientists know that the plaques are amyloids of misfolded AΒ peptides and that AΒ peptides that come from the amyloid precursor protein (APP). What they don’t know is how AΒ peptides cause their damage and if it can be stopped. And so far, genome wide association studies (GWAS) in humans have not shed much light on this problem either.
That isn’t to say that GWAS have been a waste of time. They haven’t. These studies have identified a number of alleles of a few genes that impact a person’s risk for ending up with Alzheimer’s. They just haven’t been able to link the build up of plaques with the identified genes. This is where yeast comes in.
Treusch and coworkers created a strain of yeast in which the AΒ peptide was sent to the endoplasmic reticulum. This mimics what happens to the peptide in the cells of Alzheimer’s patients. These yeast grew more slowly and developed protein complexes reminiscent of plaques.
They then added each of 5532 yeast open reading frames to this strain to identify genes that specifically affected its growth rate. Of the 40 different yeast genes they found, two (YAP1802 and INP52) were yeast homologs of human genes (PICALM and SYNJ1) that had already been identified to be important in Alzheimer’s risks. These results validated the screen and gave the researchers the confidence to dive deeper into their results.
The researchers decided to focus on the 12 genes that had very close human homologs. Of these 12 genes, 10 dealt with endocytosis and the cytoskeleton and at least three had been implicated in previous genome wide association studies in humans. Further work by these authors validated four of these genes by showing that they had similar effects on AΒ cell toxicity in the worm model C. elegans.
In one of the most interesting parts of the study, the researchers used the yeast strain to show why the GWAS-identified gene PICALM affects Alzheimer’s patients. Rather than modifying APP trafficking as had been previously proposed, their results support a model where PICALM lessens the impact of misfolded AΒ plaques on the cell.
This study is another example of the awesome power of yeast genetics. Who would have thought that a brainless yeast could teach us so much about Alzheimer’s?
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics