September 28, 2016
Awards season just kicked off with the Emmys a couple of weeks ago. Julia Louis-Dreyfus won her fifth Emmy in a row for her work on Veep, and Tatiana Maslany finally got an Emmy for her incredible work on Orphan Black. (If you haven’t seen Orphan Black, it’s a fascinating look at the ramifications of human cloning and genetic engineering.)
For a lot of people, the awards themselves are secondary to what everyone is wearing and who shows up with whom. You can learn a lot about the evolution of a performer’s career through these subtle (and not so subtle) cues.
Turns out that PLOS just had their own awards. Not quite as glamorous as the Emmys or the Oscars but almost certainly more significant.
The PLOS Genetics Research Prize for 2016 went to a fascinating paper from December of 2015 that uses yeast to explore evolution and provide ways to get at the underlying mechanisms of polygenic inheritance. And it does this by studying the subtle cues of what happens when two different yeast species show up on a yeast plate together (and mate).
The study uses a technique called a sign test. This is a way to find a set of genes that have been up- or down-regulated in response to some sort of selection pressure.
The first step is to mate two different strains or species. In this case Naranjo, Smith and coworkers used two different yeasts – Saccharomyces cerevisiae and Saccharomyces paradoxus.
The next step is to take the result of this mating, the F1 hybrid, and to compare the expression from each species’ alleles to the other’s. In other words, how does the expression of gene A compare in the two species? And gene B? And so on, through all of the genes.
What they are looking for in this allele specific expression (ASE) is a set of genes in the same pathway that are all affected in the same way. In this case they found a set of eleven genes linked to resistance to the toxin citrinin that was upregulated in S. paradoxus, but not in S. cerevisiae, in the absence of citrinin. This suggested that there was some sort of evolutionary pressure on S. paradoxus to become resistant to citrinin.
An obvious prediction from this is that their strain of S. paradoxus, CBS432, is more resistant to citrinin than is their strain of S. cerevisiae, S288C. They tested this and their S. paradoxus strain did indeed do better than S288C when citrinin was around.
They next did RNA-seq on the F1 hybrid yeast in the presence and absence of citrinin to find the up-regulated genes responsible for the ability of S. paradoxus to better tolerate citrinin. They ultimately settled on five genes that were both more highly expressed in the absence of citrinin, and more strongly induced by citrinin.
To figure out which of these genes is critical for resistance, they next deleted each gene individually and tested each deletion strain for its ability to grow in citrinin. Four of the five genes – GPX2, FRM2, RTA1, and CIS1 – made it through this test.
They next checked to see if making more product from all of these genes at once increased the strain’s resistance to citrinin. To pull this off they turned to everyone’s favorite genetic tool, CRISPR/Cas9.
Unless you’ve been hiding under a rock, you already know that CRISPR/Cas9 uses a guide RNA to get the protein Cas9 to the specific spot in the genome you want to edit. But in this case they aren’t editing a gene. Instead they are activating genes by using a version of Cas9 with two important changes: it can’t cut DNA anymore and it has a transcription activation domain added to it.
The idea is to activate all four genes at once by providing the yeast with guide RNAs that can lead this Cas9 to each of the four genes. What a powerful and simple way to easily activate all four genes at once.
They found that this overexpression strain was able to better tolerate citrinin but that it came at a cost – the strain grew more poorly in the absence of citrinin.
They next set out to see if the mutations that distinguish S. paradoxus from S. cerevisiae were in the promoters of these four genes. First, they replaced the S. cerevisiae promoter with the S. paradoxus promoter for each gene in the citrinin-sensitive S. cerevisiae strain. This created four new strains, each with one of the promoters swapped.
They found that in all cases the S. paradoxus promoter led to increased gene activity. Expression from these genes increased by anywhere from 1.6-6.9 fold.
Their final experiment was a competition between the original S. cerevisiae parent and the four strains in which the native S. cerevisiae promoter had been swapped out with the S. paradoxus one. They found that except for the strain overexpressing RTA1, these strains did better than the original S. cerevisiae strain in the presence of citrinin, but worse in its absence. Each of the three strains alone did not provide as much advantage as all three together did.
This is pretty powerful stuff! They used the sign test and CRISPR/Cas9 to nail down the three differences between S. paradoxus and S. cerevisiae that help to explain the polygenic trait of citrinin resistance.
And this isn’t just some cool yeast experiment either (although it is definitely that – #APOYG!). Sign tests may provide a new way for all those geneticists dutifully doing genome-wide association studies (GWAS) to find the set of genes responsible for polygenic traits that have so far eluded them. This is the kind of work that definitely deserves an award!
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight