Mining gene expression data for pollutants (dioxin, toluene, formaldehyde) and low dose of gamma-irradiation

Abstract

General and specific effects of molecular genetic responses to adverse environmental factors are not well understood. This study examines genome-wide gene expression profiles of Drosophila melanogaster in response to ionizing radiation, formaldehyde, toluene, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. We performed RNA-seq analysis on 25,415 transcripts to measure the change in gene expression in males and females separately. An analysis of the genes unique to each treatment yielded a list of genes as a gene expression signature. In the case of radiation exposure, both sexes exhibited a reproducible increase in their expression of the transcription factors sugarbabe and tramtrack. The influence of dioxin up-regulated metabolic genes, such as anachronism, CG16727, and several genes with unknown function. Toluene activated a gene involved in the response to the toxins, Cyp12d1-p; the transcription factor Fer3's gene; the metabolic genes CG2065, CG30427, and CG34447; and the genes Spn28Da and Spn3, which are responsible for reproduction and immunity. All significantly differentially expressed genes, including those shared among the stressors, can be divided into gene groups using Gene Ontology Biological Process identifiers. These gene groups are related to defense response, biological regulation, the cell cycle, metabolic process, and circadian rhythms. KEGG molecular pathway analysis revealed alteration of the Notch signaling pathway, TGF-beta signaling pathway, proteasome, basal transcription factors, nucleotide excision repair, Jak-STAT signaling pathway, circadian rhythm, Hippo signaling pathway, mTOR signaling pathway, ribosome, mismatch repair, RNA polymerase, mRNA surveillance pathway, Hedgehog signaling pathway, and DNA replication genes. Females and, to a lesser extent, males actively metabolize xenobiotics by the action of cytochrome P450 when under the influence of dioxin and toluene. Finally, in this work we obtained gene expression signatures pollutants (dioxin, toluene), low dose of gamma-irradiation and common molecular pathways for different kind of stressors.

Figure 1. Principal components biplot on variance stabilized data, color-coded by condition-sex.

PCA scores plot obtained from analysis of gene expression profiles. Proportion of the variance explained is 97.1% for PC1 and 1.1% for PC2.

Figure 2. Diagrams representing the quantity of shared genes between the different treatments.

Figure 3. Heat map of up-regulated biological processes based on Gene Ontology IDs for treated males (m) and females (fm).

The color scale indicates the number of genes in a group.

Figure 4. Heat map of down-regulated biological processes based on Gene Ontology IDs for treated males (m) and females (fm).

The color scale indicates the number of genes in a group.

Figure 5. Gene-concept networks by gene ontology analysis for formaldehyde treated males and females.

Figures 5–8 represent gene-concept networks by gene ontology analysis using the Bioconductor GeneAnswers Package. Yellow nodes are gene ontology terms, gray nodes correspond to differential expressed genes from the RNA-Seq data. The sizes of the centroid nodes reflect p-values of the gene associations as calculated by GeneAnswers. Genes, up-regulated in males: Upregulated genes were characterized by their importance for such functions as RNA splicing, aggressive behavior, spermatogenesis, exit from mitosis. Genes, down-regulated in males: All genes, enriched with unknown and those with low quality annotation, were related mostly to single-organism metabolic process functional category. Genes, down-regulated in females: Only a few multifunctional genes were characterized with significant downregulation, including Vm32E, involved in extracellular matrix organization and assembly, Npc2c, important for sterol and lipid transport, Cad88C and Nach, important for fluid transport and cell adhesion. The AttB gene, involved in defense response to Gram-positive bacterium, was also downregulated.

Figure 6. Gene-concept networks by gene ontology analysis for dioxin treated males and females.

Genes, up-regulated in males: Most of upregulated gene the involved in biological regulation, G-protein coupled receptor signaling pathway and regulation of cell differentiation. Smaller functional groups of genes, involved in the regulation of oskar mRNA translation, spermatogenesis, regulation of reproductive process and phospholipase C-activating G-protein coupled receptor signaling pathway were also upregulated. Genes, down-regulated in males: The genes characterized with decreased expression were mostly known to be involved in the cellular respiration and related functional category of mitochondrial ATP synthesis coupled electron transport. Small independent cluster of carboxylic acid metabolic process was also downregulated. Genes, up-regulated in females: Most of genes were annotated as involved in the processes of the proteolysis, defense response, and response to biotic stimulus. The smaller clusters of cellular response to heat and humoral immune response were revealed. Genes, down-regulated in females: The most of downregulated genes were annotated as involved in cell communication. Related functional categories, such as homophilic cell adhesion, were also revealed.

Figure 7. Gene-concept networks by gene ontology analysis for toluene treated males and females.

Genes, up-regulated in males: Upregulated genes were known to be involved in the protein folding, circadian sleep/wake regulation, positive regulation of transcription from RNA polymerase II promoter and proteolysis regulation. Genes, down-regulated in males: The large number of downregulated genes in this treatment were functionally clustered to four main groups: response to stress (including related functional category of response to heat), response to biotic stimulus, proteolysis and oxidation-reduction process. Genes, up-regulated in females: The overexpressed genes were functionally clustered into four main clusters: oxidation-reduction process, proteolysis, response to stress, response to biotic stimulus and the smaller cluster of cellular response to heat. Genes, down-regulated in females: The cell communication functional category was extremely downregulated in this treatment. Smaller gene clusters, involved in the phototransduction and regulation of response to external stimulus, were also revealed.

Figure 8. Gene-concept networks by gene ontology analysis for radiation treated males and females.

Genes, up-regulated in males: The most representative functional groups of genes were annotated as involved in the cell differentiaion, develpmnetal process involved in reproduction, cell cycle phase and cellular response to stimulus. The JAK-STAT cascade regulators were also revealed. Genes, down-regulated in males: Mostly downregulated genes were known to be involved in primary metabolism, including amino acid biosynthesis. The smaller cluster of immune response genes was also downregulated. Genes, up-regulated in females: Upregulated genes were clustered into three main functional groups: genes, annotated as involved into nucleic acid metabolic process, nucleosome assembly and regulation of transcription from RNA polymerase II promoter. Genes, down-regulated in females: The main functional gene groups, downregulated in this set, were known to be involved in proteolysis, immune response and chitin metabolism.

Figure 9. Heat map of molecular pathways based on KEGG analysis IDs for treated males (m) and females (fm), color-coded by enrichment-score.