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. 2016 Nov 17:7:200.
doi: 10.3389/fgene.2016.00200. eCollection 2016.

Functional Toxicogenomic Profiling Expands Insight into Modulators of Formaldehyde Toxicity in Yeast

Matthew North  1 , Brandon D Gaytán  1 , Carlos Romero Jr  2 , Vanessa Y De La Rosa  1 , Alex Loguinov  1 , Martyn T Smith  2 , Luoping Zhang  2 , Chris D Vulpe  1
Affiliations

Functional Toxicogenomic Profiling Expands Insight into Modulators of Formaldehyde Toxicity in Yeast

Matthew North et al. Front Genet. .

Abstract

Formaldehyde (FA) is a commercially important chemical with numerous and diverse uses. Accordingly, occupational and environmental exposure to FA is prevalent worldwide. Various adverse effects, including nasopharyngeal, sinonasal, and lymphohematopoietic cancers, have been linked to FA exposure, prompting designation of FA as a human carcinogen by U.S. and international scientific entities. Although the mechanism(s) of FA toxicity have been well studied, additional insight is needed in regard to the genetic requirements for FA tolerance. In this study, a functional toxicogenomics approach was utilized in the model eukaryotic yeast Saccharomyces cerevisiae to identify genes and cellular processes modulating the cellular toxicity of FA. Our results demonstrate mutant strains deficient in multiple DNA repair pathways-including homologous recombination, single strand annealing, and postreplication repair-were sensitive to FA, indicating FA may cause various forms of DNA damage in yeast. The SKI complex and its associated factors, which regulate mRNA degradation by the exosome, were also required for FA tolerance, suggesting FA may have unappreciated effects on RNA stability. Furthermore, various strains involved in osmoregulation and stress response were sensitive to FA. Together, our results are generally consistent with FA-mediated damage to both DNA and RNA. Considering DNA repair and RNA degradation pathways are evolutionarily conserved from yeast to humans, mechanisms of FA toxicity identified in yeast may be relevant to human disease and genetic susceptibility.

Keywords: alternative models; formaldehyde; functional genomics; yeast.

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Figures

Figure 1
Figure 1
Determining the FA IC20 for functional screens. (A) Representative growth curves in YPD media for the wild-type BY4743 strain exposed to FA. For clarity, the 0.2 mM FA curve is not shown. (B) The area under the curve (AUC) was determined for each FA dose from three independent growth curve experiments, expressed as the mean and SE, and plotted as a percentage of the untreated control. The FA IC20 was calculated to be about 0.6 mM (600 μM).
Figure 2
Figure 2
Network mapping identifies biological processes required for FA tolerance. The Cytoscape software tool was used to map fitness data for FA-sensitive strains onto the S. cerevisiae BioGRID interaction dataset (3.4.130 release). The jActiveModules plugin identified a genetic subnetwork (n = 204) enriched with fitness data, consisting of genetic and physical interactions between sensitive, non-sensitive, and essential genes. Using the subnetwork of 204 genes as input, the BiNGO plugin discovered significantly overrepresented Gene Ontology (GO) Biological Processes (p-value cutoff of 0.001). For clarity, GO categories at a p-value cutoff of 1E-5 (0.00001) are displayed, with all BiNGO output shown in Table S2. The BiNGO output node color (orange to yellow) and size correspond to p-values and number of genes, respectively. Edge arrows illustrate GO term hierarchy. Genetic subnetworks for selected GO categories are shown, where node color (green to white) corresponds to strain fitness score and edge indicates the type of interaction (physical/genetic) between the genes.
Figure 3
Figure 3
SKI mutants are sensitive to FA. The AUC was calculated for each strain after 24 h of exposure to the indicated concentrations of FA. Bars display mean AUC as a percentage of the untreated strain AUC with standard error (SE) for three independent replicates. Statistical significance between the wild-type and mutant strains was calculated with Student's t-test, where **p < 0.01 and *p < 0.05.
Figure 4
Figure 4
Osmoregulation and stress response mutants are sensitive to FA. The AUC was calculated for strains treated for 24 h with indicated concentrations of FA and expressed as a percentage of the AUC for the untreated strain. Bars show the mean and SE for three independent cultures. Statistical significance between the wild-type and mutant strains was calculated with Student's t-test, where **p < 0.01 and *p < 0.05.
Figure 5
Figure 5
Mre11 complex mutants are sensitive to FA. The AUC was calculated for each strain treated for 24 h with various FA concentrations and expressed as a percentage of the AUC for the untreated strain. Bars show the mean and SE for three independent cultures. Statistical significance between the wild-type and deletion strains was calculated with Student's t-test, where ***p < 0.001, **p < 0.01, and *p < 0.05.
Figure 6
Figure 6
Mutants defective in DSB repair via homologous recombination are sensitive to FA. Strains were treated for 24 h with indicated FA concentrations and the AUC was calculated. Shown is the mean AUC as a percentage of the AUC for the untreated strain and SE for three independent cultures. Statistical significance between the wild-type and mutant strains was calculated with Student's t-test, where ***p < 0.001, **p < 0.01, and *p < 0.05.
Figure 7
Figure 7
Mutants defective in DSB repair via single strand annealing are sensitive to FA. The AUC was determined for each strain after 24 h treatment with the indicated FA concentrations. Graph bars express AUC as a percentage of the AUC for the untreated strain and show the mean and SE for three independent experiments. Statistical significance between the wild-type and mutant strains was calculated with Student's t-test, where **p < 0.01 and *p < 0.05.
Figure 8
Figure 8
Mutants defective in postreplication repair are sensitive to FA. Strains were exposed for 24 h to various FA concentrations and AUCs were calculated. Bars express AUC as a percentage of the untreated strain AUC and display the mean and SE for three independent replicates. Statistical significance between the wild-type and mutant strains was calculated with Student's t-test, where ***p < 0.001, **p < 0.01, and *p < 0.05.
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