Xenobiotic-induced activation of human aryl hydrocarbon receptor target genes in Drosophila is mediated by the epigenetic chromatin modifiers

Abstract

Aryl hydrocarbon receptor (AHR) is the key transcription factor that controls animal development and various adaptive processes. The AHR's target genes are involved in biodegradation of endogenous and exogenous toxins, regulation of immune response, organogenesis, and neurogenesis. Ligand binding is important for the activation of the AHR signaling pathway. Invertebrate AHR homologs are activated by endogenous ligands whereas vertebrate AHR can be activated by both endogenous and exogenous ligands (xenobiotics). Several studies using mammalian cultured cells have demonstrated that transcription of the AHR target genes can be activated by exogenous AHR ligands, but little is known about the effects of AHR in a living organism. Here, we examined the effects of human AHR and its ligands using transgenic Drosophila lines with an inducible human AhR gene. We found that exogenous AHR ligands can increase as well as decrease the transcription levels of the AHR target genes, including genes that control proliferation, motility, polarization, and programmed cell death. This suggests that AHR activation may affect the expression of gene networks that could be critical for cancer progression and metastasis. Importantly, we found that AHR target genes are also controlled by the enzymes that modify chromatin structure, in particular components of the epigenetic Polycomb Repressive complexes 1 and 2. Since exogenous AHR ligands (alternatively - xenobiotics) and small molecule inhibitors of epigenetic modifiers are often used as pharmaceutical anticancer drugs, our findings may have significant implications in designing new combinations of therapeutic treatments for oncological diseases.

Keywords: PcG epigenetic complexes; aryl hydrocarbon receptor; drosophila; xenobiotic.

Figure 1. Phenotypic effects of endogenous and exogenous ligands of the human AHR on Drosophila growth and morphogenesis

(A) Ubiquitous expression of UAS-AhR leads to developmental lethality. The majority of tub>AhR animals die at the embryonic stage, with very few escapers that die at early larval stages, showing arrest in growth and development. Two four-day old larvae are shown, the larger one is the control (UAS-AhR/+; UAS-GFP/+, yellow arrow), the smaller green larva (red arrow) is tub>AhR, with ubiquitous expression of transgenic AHR (UAS-AhR/+; UAS-GFP/Tub-GAL4). The expression pattern of tub-GAL4 is visualized by GFP expression (green). (B–C). Drosophila leg phenotypes of Dll>AhR flies. (B) control (UAS-AhR/+). (C) Dll>AhR (Dll-GAL4/UAS-AhR). Flies developed on standard medium with no exogenous ligands. (D–I). Drosophila leg (D–F) and wing (G–I) phenotypes of dpp>AhR flies. (G) control (UAS-AhR/+). (D–F, H–I) dpp>AhR (dpp-GAL4/UAS-AhR). Flies developed on standard medium without exogenous ligands (G–H) or with indirubin (D), indinol (E, I) and beta-Naphthoflavone (F). At least 80 legs, 40 wings and more than 20 flies were analyzed for each genotype. Leg segments are indicated. Note the loss of tarsal segments in (C–D).

Figure 2. Activation of AHR in germline and nervous systems causes different abnormalities during Drosophila oogenesis and neurogenesis

(A–D) Confocal sections of the normal ovariole of MTD-GAL4/UAS-AhR female reared on standard medium (A); degraded egg chamber of MTD-GAL4/UAS-AhR female fed with beta-Naphthoflavone (arrows point on picnotic nuclei) (B); egg chamber with disordered follicular layer (arrow) from MTD-GAL4/UAS-AhR female reared on medium with indinol (C); follicle with 32 trophocytes (arrow) from MTD-GAL4/UAS-AhR female fed with indirubin (D). Ovaries were stained with SytoxGreen (green) for DNA visualization and Phalloidin (red) for cytoskeleton visualization. Asterisks, T and FC indicate oocytes, trophocytes, and follicular cells, respectively. (E–F). Confocal sections of the central nervous system of UAS-mCD8-GFP; UAS-AhR/+; Elav-GAL4/+ larvae merged into a single 3D-image. Brains of late third instar larvae developed on standard medium (E) or on the medium containing beta-Naphthoflavone (F). Control brain (E) is significantly bigger than the brain with AhR expression (F). Elav-GAL4 (green, visualized by GFP expression) drives pan-neuronal expression of transgenic UAS-AhR in brain hemispheres (Br) and the ventral nerve cord (VNC). Magnification scale bars represent 100 μm in A, E, F and 20 μm in B, C, D.

Figure 3. Drosophila eye phenotypes of GMR>AhR flies

Flies developed on standard medium without exogenous ligands (A, A′), on medium with indinol (B, B′), beta-Naphthoflavone (C, C′) or indirubin (D, D′). Ommatidia are arranged in a highly regular pattern in control flies (A–A′), while flies reared on medium with exogenous ligands develop roughened eye phenotypes with irregular pattern and decreased number of mechanoreceptors (B–D, B′–D′).

Figure 4. Activation of AHR target genes in leg imaginal discs of Dll>AhR larvae in the absence of exogenous ligands

mRNA levels in leg imaginal discs of Dll-GAL4/UAS-AhR larvae (red) was compared to control UAS-AhR/+ larvae (blue) developed in the same conditions. The relative level of mRNA expression was measured using real-time PCR. Data are shown as representative of two independent experiments. The error bars represent the measurement error. Asterisk means the significant change in gene expression compared to the control.

Figure 5. The increase of AHR target genes expression in heads of GMR-GAL4/UAS-AhR; Pc⁴/+ imagoes

Flies developed from larvae grown on medium with added indirubin (green), beta-Naphthoflavone (purple), or standard medium without additives (blue). mRNA levels were analyzed by real-time PCR in heads dissected from GMR-GAL4/UAS-AhR; Pc⁴/+ imagoes. Data are shown as representative of two independent experiments. The error bars represent the measurement error. Asterisk means the reliable change in gene expression compared to the control.

Figure 6. The increase of AHR target genes expression in heads of GMR>AhR imagoes

Flies developed from larvae grown on medium with UNC1999 (A), Belinostat (B), and ligands, indirubin (IR, green) and beta-Naphthoflavone (BNF, purple), or standard medium without additives (blue). mRNA levels were analyzed by real-time PCR in heads dissected from GMR-GAL4/UAS-AhR imagoes. Data are shown as representative of two independent experiments. The error bars represent the measurement error. Asterisk means the reliable change in gene expression compared to the control.