November 11, 2015
Imagine what our email inboxes would look like if we didn’t have spam filters! To find the meaningful emails, we’d have to wade through hundreds of messages about winning lottery tickets, discount medications, and other things that don’t interest us.
When it comes to sorting out meaningful mutations from meaningless variation in human genes, it turns out that our friend S. cerevisiae makes a pretty good spam filter. And as more and more human genomic sequence data are becoming available every day, this is becoming more and more important.
For example, when you look at the sequence of a gene from, say, a cancer cell, you may see many differences from the wild-type gene. How can you tell which changes are significant and which are not?
SuperBud to the rescue! Because many human proteins can work in yeast, simple phenotypes like viability or growth rate can be assayed to test whether variations in human genes affect the function of their gene products. This may be one answer to the increasingly thorny problem of variants of uncertain significance—those dreaded VUS’s.
In a new paper in GENETICS, Hamza and colleagues systematically screened for human genes that can replace their yeast equivalents, and went on to test the function of tumor-specific variants in several selected genes that maintain chromosome stability in S. cerevisiae. This work extends the growing catalog of human genes that can replace yeast genes.
More importantly, it also provides compelling evidence that yeast can help us tell which mutations in a cancer cell are driver mutations, the ones that are involved in tumorigenesis, and which are the passenger mutations, those that are just the consequence of a seriously messed up cell. Talk about a useful filter!
The researchers started by testing systematically for human genes that could complement yeast mutations. Other groups have done similar large-scale screens, but this study had a couple of different twists.
Previous work from the Hieter lab had identified genes in yeast that, when mutated, made chromosomes unstable: the CIN (Chromosome INstability) phenotype. Reduction-of-function alleles of a significant fraction (29%) of essential genes confer a CIN phenotype. The human orthologs of these genes could be important in cancer, since tumor cells often show chromosome rearrangements or loss.
So in one experiment, Hamza and colleagues focused specifically on the set of CIN genes, starting with a set of 322 pairs of yeast CIN genes and their human homologs. They tested functional complementation by transforming plasmids expressing the human cDNAs into diploid yeast strains that were heterozygous null mutant for the corresponding CIN genes. Since all of the CIN genes were essential, sporulating those diploids would generate inviable spores—unless the human gene could step in and provide the missing function.
In addition to this one-to-one test, the researchers cast a wider net by doing a pool-to-pool transformation. They mixed cultures of diploid heterozygous null mutants in 621 essential yeast genes, and transformed the pooled strains with a mixture of 1010 human cDNAs. This unbiased strategy could identify unrecognized orthologs, or demonstrate complementation between non-orthologous genes.
In combination, these two screens found 65 human cDNAs that complemented null mutations in 58 essential yeast genes. Twenty of these yeast-human gene pairs were previously undiscovered.
The investigators looked at this group of “replaceable” yeast genes as a whole to see whether they shared any characteristics. Most of their gene products localized to the cytoplasm or cytoplasmic organelles rather than to the nucleus. They also tended to have enzymatic activity rather than, for example, regulatory roles. And they had relatively few physical interactions.
So yeast could “receive messages” from human genes, allowing us to see their function in yeast. But could it filter out the meaningful messages—variations that actually affect function—from the spam?
The authors chose three CIN genes that were functionally complemented by their human orthologs and screened 35 missense mutations that are found in those orthologs in colorectal cancer cells. Four of the human missense variants failed to support the life of the corresponding yeast null mutant, pointing to these mutations as potentially the most significant of the set.
Despite the fact that these mutations block the function of the human proteins, a mutation in one of the yeast orthologs that is analogous to one of these mutations, changing the same conserved residue, doesn’t destroy the yeast protein’s function. This underscores that whenever possible, testing mutations in the context of the entire human protein is preferable to creating disease-analogous mutations in the yeast ortholog.
Another 19 of the missense mutations allowed the yeast mutants to grow, but at a different rate from the wild-type human gene. (Eighteen conferred slower growth, but one actually made the yeast grow faster!)
For those 19 human variants that did support life for the yeast mutants, Hamza and colleagues tested the sensitivity of the complemented strains to MMS and HU, two agents that cause DNA damage. Most of the alleles altered resistance to these chemicals, making the yeast either more or less resistant than did the wild-type human gene. This is consistent with the idea that the cancer-associated mutations in these human CIN gene orthologs affect chromosome dynamics.
As researchers are inundated by a tsunami of genomic data, they may be able to turn to yeast to help discover the mutations that matter for human disease. They can help us separate those emails touting the virtues of Viagra from those not-to-be-missed kitten videos. And when we know which mutations are likely to be important for disease, we’re one step closer to finding ways to alleviate their effects.
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD