Hexavalent chromium-induced epigenetic instability and transposon activation lead to phenotypic variations and tumors in Drosophila

Abstract

Developmental robustness represents the ability of an organism to resist phenotypic variations despite environmental insults and inherent genetic variations. Derailment of developmental robustness leads to phenotypic variations that can get fixed in a population for many generations. Environmental pollution is a significant worldwide problem with detrimental consequences of human development. Understanding the genetic basis for how pollutants affect human development is critical for developing interventional therapies. Here, we report that environmental stress induced by hexavalent chromium, Cr(VI), a potent industrial pollutant, compromises developmental robustness, leading to phenotypic variations in the progeny. These phenotypic variations arise due to epigenetic instability and transposon activation in the somatic tissues of the progeny rather than novel genetic mutations and can be reduced by increasing the dosage of Piwi - a Piwi-interacting RNA-binding protein, in the ovary of the exposed mother. Significantly, the derailment of developmental robustness by Cr(VI) exposure leads to tumors in the progeny, and the predisposition to develop tumors is fixed in the population for at least three generations. Thus, we show for the first time that environmental pollution can derail developmental robustness and predispose the progeny of the exposed population to develop phenotypic variations and tumors.

Keywords: canalization; developmental robustness; epigenetic inheritance; epigenetic instability; hexavalent chromium; tumor.

Figure 1:

Cr(VI) exposure leads to developmental defects in the progeny. (a) A flowchart representing our working hypothesis. (b) Eye developmental defects (white arrows) observed in the progeny of flies exposed to various concentrations of Cr(VI). (c) Quantitation of developmental defects. ‘n’ indicates the number of flies scored for developmental defects. (d) Immunoblot results showing the levels of indicated proteins in various Cr(VI)-exposure conditions

Figure 2:

Cr(VI) exposure–dependent developmental defects in the progeny are not due to genetic mutations. (a) Eye phenotypes in the progeny upon exposure of only mothers to 10 μM Cr(VI) for 6 days. (b) Quantitation of phenotypes in the progeny upon either maternal or paternal exposure to 10 μM Cr(VI). Maternal exposure has a significant effect when compared to paternal exposure. (c) Results from bioinformatic analysis of single-fly whole genome sequencing. An image of the fly eye with phenotypic variation used for whole genome sequencing is shown in a highlighted box in A. A summary of small variations and large structural variations in the genome is also shown. Mutations that would directly affect the function of a gene directly did not occur upon exposure to 10 μM Cr(VI)

Figure 3:

Cr(VI) exposure induces epigenetic instability in F1 somatic tissues. (a) An experimental scheme to identify differentially expressed transcripts. (b) Evidence for epigenetic instability in F1 somatic tissues. We measured epigenetic instability by comparing transcriptomes of indicated fly heads and plotting the correlation coefficient (r). (c) Motifs enriched in promoter elements of downregulated genes are shown. Potential transcription factors that bind to these motifs are also shown. The number in parentheses represents the number of differentially expressed genes that possess a match to the respective motif. (d) The mRNA levels of potential transcription factors that can bind to the motifs are shown in C. Fold-change represents log2 (FPKM + phenotype/FPKM − phenotype). FPKM values were obtained from mRNA-seq analysis using RSEM (see Methods). FPKM from two biological repeats were averaged prior to fold-change calculation

Figure 4:

The relationship between Cr(VI) exposure and Piwi. (a) A column plot showing the relationship between the level of Piwi mRNA in the mother’s germline and F1 phenotypic variations. The number of flies assessed for phenotypic variations are shown. (b) A scatter plot showing the levels of transposon mRNAs in fly heads with and without phenotypic variations. Reads were normalized as counts per million mapped reads. (c) An illustration summarizing the findings in A and B

Figure 5:

Cr(VI) exposure–dependent transgenerational predisposition to cancer. (a) Drosophila eyes (ey-Gal4 > Delta) showing tumors (black arrows) when exposed to Cr(VI). In addition to tumors, we also noted bristle phenotypes. Follow the black arrows. (b) Quantitation of phenotypes observed in A. The experiment was performed in triplicates. (c) A flowchart showing the crossing scheme used to test if Cr(VI) exposure–induced epigenetic instability is fixed in a population for multiple generations. (d) F4 fly eyes exhibiting tumors and bristle phenotype (black arrows). (e) Quantitation of phenotypes observed in D

Figure 6:

a model showing the effect of Cr(VI) exposure on the robustness of epigenetic inheritance in the offspring