From phenologs to silent suppressors: Identifying potential therapeutic targets for human disease

Abstract

Orthologous phenotypes, or phenologs, are seemingly unrelated phenotypes generated by mutations in a conserved set of genes. Phenologs have been widely observed and accepted by those who study model organisms, and allow one to study a set of genes in a model organism to learn more about the function of those genes in other organisms, including humans. At the cellular and molecular level, these conserved genes likely function in a very similar mode, but are doing so in different tissues or cell types and can result in different phenotypic effects. For example, the RAS-RAF-MEK-MAPK pathway in animals is a highly conserved signaling pathway that animals adopted for numerous biological processes, such as vulval induction in Caenorhabditis elegans and cell proliferation in mammalian cells; but this same gene set has been co-opted to function in a variety of cellular contexts. In this review, I give a few examples of how suppressor screens in model organisms (with a emphasis on C. elegans) can identify new genes that function in a conserved pathway in many other organisms. I also demonstrate how the identification of such genes can lead to important insights into mammalian biology. From such screens, an occasional silent suppressor that does not cause a phenotype on its own is found; such suppressors thus make for good candidates as therapeutic targets.

Keywords: rare disease; resiliency; suppression screen.

Figure 1. Phenologs are orthologous phenotypes

A visual explanation of phenologs. Organism 1 has mutant phenotype 1, which is caused by mutations in genes A, B, C, and D. Mutations in orthologs of genes B, C, and D in Organism 2 cause Phenotype 2. Mutations in gene A in Organism 1 cause Phenotype 1, so mutations in orthologs of gene A in Organism 2 might also cause Phenotype 2; the reverse would be true for gene E′ as well. The number of candidate genes, and confidence in their role in their specific biological process, is further strengthened by incorporating data from a third organism (Organism 3). For example, Orthologs B, D, and F (not shown) might cause Phenotype 3 in Organism 3, thereby reinforcing the evidence for the phenologs between Organisms 1 and 2, and adding gene F to the regulatory network. Adapted from McGary et al. (2010) and www.phenologs.org.

Figure 2. The RTK-RAS-ERK pathway is evolutionarily conserved

The canonical pathway by which an extracellular growth factor (GF) binds a receptor tyrosine kinase (RTK) to activate transcription in the nucleus is depicted. TX’L, transcriptional, ERK, Extracellular signal-regulated kinase; Ets, E26 transformation-specific transcription factor; GAP, GTPase-activating protein; GEF, Guanine nucleotide exchange factor; MEK, Mitogen-activated protein kinase (MAP)/ERK kinase. Reprinted, with permission, from Sundaram (2013).

Figure 3. The phenologs of the activated RAS gene

Three examples where gain-of-function mutations in RAS disrupt vulval development in C. elegans, eye development in D. melanogaster, and cell proliferation in cultured mammalian cells. WT, wild-type. Images reprinted, with permission, from González-Pérez et al. (2010) (C. elegans); Therrein et al. (1995) (D. melanogaster); and Lee et al. (2009) (mammalian cells).

Figure 4. Genetic suppressor screening

(a) In a typical C. elegans genetic screen, L4 larvae are mutagenized with a chemical mutagen. These mutagenized animals lay eggs (F1 generation), and these F1 then self-fertilize and lay eggs to generate the F2 generation. The F2 generation is screened for suppression of the original phenotype. Candidate suppressed animals are then isolated and retested for suppression. (b) A screen with a conditional (temperature-sensitive) allele is similar to the typical screening cascade, except that the first steps of the screen are carried out at the permissive temperature, usually 15–16°C. The F2 animals are then shifted to the non-permissive temperature, usually 24–25°C to screen for suppression of the original phenotype. In both approaches described, the suppressor screen utilizes non-null alleles; hypomorphic missense alleles are most frequently used.

Figure 5. Suppressors restore gene activity over some threshold

(a) A model for how suppressors interact genetically. For the majority of genes, only one wild-type copy (Δ/+; hemizygous) is often enough for normal development, resulting in only 50% gene activity over some threshold for the function of a specific gene in a specific biological process. The homozygous mutant (mut/mut) genotype can be associated with a “compromised” gene product, where the mutant has some activity, but not enough to exceed the threshold required for normal function. In the suppressed strain (sup/sup), the desired gene activity can achieve above-threshold levels, thereby suppressing the original phenotype. (b) Depiction of how a suppressor mutation might restore gene function to one cell type or tissue, but not to other cell types or tissues. Such cell-type or tissue-specific suppression supports the hypothesis that the threshold for gene function varies among different cell types. In both panels, the suppressor mutation (sup) is shown as homozygous recessive, although dominant suppressor mutations may also exist.