New & Noteworthy

Yeast Winnows Down GWAS Hits in Autism

October 10, 2013

Cheap and easy genome sequencing has been both a blessing and a curse. We are able to find an incredible wealth of variation, but for the most part we have no easy way to tell whether a difference might contribute to a disease or not.

The poster child for this problem is autism. Lots of genome wide association studies (GWAS) have been done and lots of rare variants in lots of different genes have been found – unfortunately, way too many to pick out the ones that really matter.

Luckily our friend yeast can help. Various researchers have identified a number of variants in the human cation/proton antiporter gene NHE9 that associate with autism. In a new study, Kondapalli and coworkers used the NHE9 ortholog NHX1 from S. cerevisiae as an initial screen to identify which variants impact the activity of the NHE9 protein. They found that two of the three mutations they looked at compromised the activity of yeast Nhx1p.

They then set out to confirm these results in mammalian cells.  When they looked at protein activity in glial cells, they found that all three mutations compromised the activity of NHE9.  This is obviously different from what they found in yeast.

Now this doesn’t mean that yeast is useless for this approach (God forbid!).  No, instead it means that it is probably only useful for a subset of autism mutations.  Kondapalli and coworkers had suspected this, but apparently the subset is smaller than they initially thought.

The first thing they did was to generate a rough three dimensional map of the NHE9 protein in order to see which parts the two proteins shared.  The idea is that they could then do a quick screen in yeast with mutations that affect the shared structure.

While the structure of NHE9 has not been solved, we do have the structure of its distant bacterial relative, NhaA.  Kondapalli and coworkers aligned the two along with the yeast ortholog Nhx1p and identified conserved regions.

Three of the NHE9 mutations associated with autism—V176I, L236S, and S438P—were all predicted to be in shared, membrane-spanning parts of the protein.  The researchers introduced the equivalent mutations into NHX1—V167I, I222S, and A438P. 

 A yeast deleted for NHX1 grows poorly in high salt and low pH and also has increased sensitivity to hygromycin B, as compared to a yeast with a functioning NHX1.  Two of the mutant genes, carrying A438P or I222S, failed to rescue these growth defects.  The other mutant gene, with the V167I change, worked as well as wild type NHX1 at rescuing the yeast.  So at least in yeast, two of the three mutations appear to impact protein activity.

The next step was to see if the same was true in mammals.  Easier said than done!  Ideally they would want to investigate whether these mutations affected the protein in the cells where NHE9 is usually active.  Too bad no one knows this protein’s natural habitat.  This is why the researchers starting slicing mouse brains to figure out when and where the protein is expressed.

While we don’t have time or space to go into all the details here, Kondapalli and coworkers found that when and where in the brain NHE9 was expressed made sense as far as a possible contribution to autism.  They also found that glial cells had about 1.2 fold more NHE9 transcripts than did neuronal cells.  They therefore did their assays of protein activity in a type of glial cells called astrocytes.

While they couldn’t completely knock out NHE9 in mouse astrocytes, they were able to knock down its expression by over 80%.  When they added back the mutant NHE9 genes, they found that all three failed to mimic the effect of adding back wild type NHE9 to these cells.  This is different than what they found in yeast, where only two of the mutations impacted protein activity. 

When they went back to their 3D model, they saw that the mutation that differed, V167I, affected a less defined part of the structure.  This points to the fact that for the quick yeast screen to work, they need to be looking at parts of the protein where the structure is shared between the yeast and the human version.  In a perfect world they would have had crystal structures of each to work off of instead of having to kludge together a model.

In any event, this is the first step towards validating yeast as a quick screen for identifying mutations that can impact protein activity and so are good candidates for being involved in disease.  Yeast may help scientists separate the wheat from the chaff of GWAS and so help figure out how diseases happen and maybe help find treatments or even cures.  Well done yeast.

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

Randy Schekman and the Awesome Power of Yeast!

October 7, 2013

Congratulations to Randy Schekman, James Rothman, and Thomas Südhof, who have been awarded the 2013 Nobel Prize in Physiology or Medicine for their work in understanding how the cell organizes its transport system. Randy Schekman used the awesome power of yeast to identify and characterize genes required for vesicle traffic. James Rothman characterized these and other proteins in mammalian cells, and Thomas Südhof showed the critical role of vesicle trafficking in nerve cells. You can read summaries of their Nobel winning work at Nature, The Scientist, and The New York Times, or search SGD to see how each of these researchers has used our model organism in their research: Randy Schekman, James Rothman, Thomas Südhof.

Yeast Muad’Dib

October 2, 2013

Remember in Dune when Paul Muad’Dib took a sip of the “Water of Life” and needed weeks in a coma to turn it into something that let him survive and emerge even more powerful than before?  Turns out yeast sometimes have to do something similar.

Now of course the yeast aren’t consciously moving molecules around to deal with a poison like Paul did.  No, instead they sometimes need to transcribe low levels of a mutated gene over a long period of time to survive in a new environment.  

This process is called retromutagenesis.  The idea is that a cell gets a mutation that would allow it to survive and prosper in a new environment if only it could replicate its DNA.  Unfortunately the new environment is so unforgiving that the cell can’t replicate. 

The cell escapes this catch-22 by transcribing the gene with the mutation so that the mutant protein can get made.  Once enough of this protein is made, the cell manages to get up enough steam to power through a cell cycle.  Now the mutation is established and the yeast can make lots of mutant protein and happily chug along.

In a new study in the latest issue of GENETICS, Shockley and coworkers hypothesize that something like this is happening in their experiments.  They were studying oxidative damage to DNA and found that some of their mutants required many days before they could grow in the absence of tryptophan (trp).  They argue that these late arising revertants were due to the cells having to wait until retromutagenesis allowed enough functional Trp5p to be made so the cell could replicate.

The authors have created strains of yeast with various mutations in the TRP5 gene that cause the yeast to be unable to grow in the absence of trp.  What makes these strains so useful is that they are set up in such a way that six different, specific point reversions can result in a functional TRP5 gene.  They can then analyze any Trp+ revertants to see what types of damage lead to which type of mutations.

One of the first things the authors discovered was that oxidative damage caused all six different reversions.  While this was interesting, the specific mutation they wanted to focus on was a G to T transversion which occurs when G is converted to 8-oxoguanine.  This is why they focused on the trp5-A149C strain.

The main way that yeast cells deal with 8-oxoguanine is by removing it with the Ogg1 protein, a DNA glycosylase.  When Shockley and coworkers deleted this gene in their strain, the number of revertants increased by 20-fold.  From this they concluded that most of the revertants were the result of the misreplication of an 8-oxoguanine. 

This is where the yeast run into a problem.  In the absence of trp, the trp5 mutants do not replicate at all…they do not go through even one cell cycle.  But to revert to a functional TRP5 gene, this strain needs to go through a cell cycle.  This is why the authors think that the first step towards reversion is a mutation in the TRP5 transcript.

Consistent with this idea is the fact that the mutated G in this strain is on the transcribed strand and that this is important for high revertant frequencies.  It also helps to explain why revertants took so long to appear.  Basically there had to be a buildup of enough functional Trp5p to allow a single cell cycle to happen.  Then the G could be converted to a T and the yeast could happily grow.  In this specialized case, it looks like reversion is dependent on retromutagenesis. 

But retromutagenesis, also called transcriptional mutagenesis, doesn’t happen only in yeast cells.  It’s being studied as a possible way that all kinds of quiescent cells, such those in the process of becoming tumor cells, or bacteria whose growth has been stopped by an antibiotic, can mutate and escape the conditions that are restricting them. Our little friend may not save the human race from destruction like Paul did, but once again yeast is proving pretty darn useful in getting results that make a difference for human health.

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